1

6th International BAU Drug Design Congress

13-14-15 DECEMBER 2018

CONGRESS BOOK

2

INVITED TALKS

3

IL-1

How many bits of information can be transferred within a protein and how fast?

Burak Erman

Chemical and Biological Engineering Department, Koc University, Istanbul Turkey Binding a ligand at some point on a protein leads to conformational changes in other parts. The locations where these changes take place may include regions where the protein performs its function, which is called the active site. In this talk, we will discuss how ligand binding affects the active site. A possible effect will take place if the ligand-binding site communicates with the active site, in other words, if information flows from the ligand to the active site. This process takes place stochastically as a result of the coupling of the fluctuations of amino acids at the two sites. If the changes take place deterministically, like the movements of machine parts, designing an effective drug would be as easy as machine design. What makes life difficult here is the presence of uncertainties in the system. If ligand binding reduces the uncertainties in the system, it will be easier to control the system. Entropy is a measure of uncertainty. The problem reduces to measuring the amount of entropy or information transferred from the ligand-binding site to the active site. In this talk, we determine how many bits of information can be transferred between two residues in a protein. We use the widely studied protein, the third PDZ domain from the synaptic protein PSD-95, (Protein Data Bank code 1BE9) as our example. We show that it is possible to transfer gigabytes of information per second between specific residue pairs in 1BE9.

4

IL-2

Targeting immunosuppressive myeloid derived suppressor cells (MDSCs): A critical molecular target in metastatic process

Eunmi Lee, Raziye Piranlioglu, Mustafa Guzel, Daniela Marasco, Omercan Durmus,

Feyzanur Bayraktar, Hande Uslu, Hasan Korkaya Georgia Cancer Center, Augusta University, Augusta, GA

We recently demonstrated that infiltration of granulocytic MDSCs in distant organs is a critical step in metastatic process. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model and patient samples revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) are regulated by distinct set of genes. Two genes, S100A8 and S100A9 were highly upregulated in gMDSCs isolated from mice with metastatic 4T1 tumors compared to mice with non-invasive tumors. Using the recombinant single S100A8, S100A9 proteins or the S100A8/S100A9 heterotetramer (calprotectin), we demonstrated that calprotectin is a critical player in gMDSC induction and infiltration in distant organs particularly in the lungs. Furthermore, injection of recombinant calprotectin but not single S100A8 or S100A9 proteins via tail vein enhanced the metastatic ability of nan-invasive EMT6 tumors in mice. We therefore performed computational screen of NCI compounds against the crystal structure of calprotectin and identified over 100 lead compounds. In vitro gMDSC differentiation assay identified 3 compounds with strong gMDSC inhibitory activity. We therefore designed a pilot experiment to test these compounds in preclinical mouse models. In detaile preclinical testing, one of the compounds (#10) showed a potent activity against gMDSCs in 4T1 tumor-bearing mice and significantly inhibited pulmonary metastasis. Moreover, when our compound was combined with cyclophosphamide (standard of care), animals were free of cancer and showed no sign of disease for several months. Furthermore, our compound also enhances the efficacy of immunotherapy which unfortunately have limited activity in breast cancer despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. In summary, we have identified a molecular target which regulates gMDSC induction and play critical role in metastatic process. Therefore, our findings from in vitro screens and preclinical testing provide a strong rationale for targeting MDSCs alone or in combination of chemotherapies or immunotherapies in metastatic breast cancer patients.

5

IL-3

Pathway based analysis of Whole Exome sequencing data reveals novel therapy candidates in Rare Diseases

O. Ugur Sezerman

Acibadem University, School of Medicine, Biostatistics and Medical Informatics Department Recent advancements in the next generation sequencing technologies and in the “omics” technologies makes it possible to look for the coordinated behavior among different levels of biochemical activity. The current challenge is the integrative analysis of sequencing data to better understand disease development and progression mechanisms. In contrast to isolated molecules, network and pathway oriented analyses are thought to better capture pathological perturbations and hence, better explain predisposition to disease.This also enables us to understand individualized disease development mechanisms and identify potential therapy candidates. In this talk go over the methods we developed to identify individualized therapy candidates and their applicaiton to several Rare diseases such as Behçet and Primary Ciliary Diskinesia.

6

IL-4

Dynamic and Static Differences Between Conformations of HLA-B51 and HLA-B52: Implications for the Pathogenic Mechanisms of HLA-B51

in Behçet’s Disease

Mert Gur1*, Ahmet Gul2 and Burak Erman1* 1 Mechanical Engineering Department, Istanbul Technical University Istanbul, Turkey

2Division of Rheumatology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey

3Center for Computational Biology and Bioinformatics, Koc University, Istanbul, Turkey

*Correspondence: Mert Gur and Burak Erman E-mail: gurme@itu.edu.tr berman@ku.edu.tr

Behçet’s disease, an inflammatory disorder of unknown etiology, is characterized mainly by recurrent exacerbations of oral aphthous ulcers, genital ulcers, skin lesions and uveitis. Genetic tendency has an important role in its pathogenesis, and HLA-B51, a class I MHC antigen, has been recognized as the strongest susceptibility factor for Behçet’s disease. Despite the confirmation of the association of HLA-B51 with Behçet’s disease in different populations, its pathogenic mechanisms remain elusive. HLA-B51 differs in only two amino acids from the HLA-B52, which is the other split antigen of HLA-B5 along with –B51, but is not associated with Behçet’s disease, at positions 63 and 67 of the alpha1 helix. We, therefore, investigated the structure of both HLA antigens by the unbinding process of the YAYDGKDYI peptide, which is one of the catabolic products of HLA-B51 and also known to bind well to HLA-B52, using molecular dynamics simulations. For HLA-B51, alpha1 helix fluctuated to larger extent than for HLA-B52. Free energy profiles showed that unbinding from HLA-B52 results in greater free energy differences than for HLA-B51. Coarse grained thermodynamic analysis supported these observations and also showed that the correlation of CYS164 with those of all other residues occurs in HLA-B52, but not in HLA-B51. This results in excessive fluctuations of HLA-B51, including the fluctuations of the peptide binding residues, while they are more stable in HLA-B52. Further comparative calculations on six additional peptides known to be presented by HLA-B51 showed that the floppiness of HLA-B51 is a generic property of the HLA-B51 protein itself, independent of the type of the peptides. This floppiness in the overall structure of the HLA-B51 may contribute significantly to its pathogenic role in Behçet’s disease by binding a larger and possibly more promiscuous repertoire of low-affinity peptides and also affecting the strength of its interaction with different receptors.

7

IL-5

Towards characterizing the factors tuning the specificity of biomolecular complexes

Ezgi Karaca

Computational Structural Biology Lab Izmir Biomedicine and Genome Center

The interplay between specificity and affinity is a significant drive in biomolecular recognition. Since the experimental determination of both has been a challenge for many systems, there is a growing interest in their computational prediction. There are numerous algorithms present, which are poised to determine the binding affinity of biomolecular complexes. On the other hand, we have only a handful of tools tuned to predict the specificity of binding. To that end, we have developed novel and simple specificity-related metrics through structural and dynamical analysis of two model systems. The complementarity metric used in our first model system has delivered the factors guiding the selective binding of transmembrane receptor tyrosine kinase TAM (Tyro3, Axl, Mer) proteins to different ligands. Structural investigation of our second system has pinpointed the distance-based measures explaining the specificity of de novo DNA methyltransferase 3 towards its non-CpG DNA binding site(s). Expanding on these findings, our short-term goal is to probe the generic applicability of our metrics in dissecting the specificity of different types of biomolecular systems.

8

IL-6

Mechanism of Action of Benzazoles As Topoisomerase II Enzyme Inhibitors

Esin AKI-YALCINa, Ozum OZTURKa, Ismail YALCINa, Renate GRIFFITHb aPharmaceutical Chemistry, Ankara University, Faculty of Pharmacy Pharmaceutical

Chemistry Research Laboratory Tandogan, 06100, Ankara, Türkiye bSchool Of Medical Sciences, Unsw Sydney, Wallace Wurth Building, Unsw Sydney Nsw 2052

Australia, 2052, Sydney, Australia E-mail: esinaki@ankara.edu.tr

Background: It can be described as one of the mechanisms of cancer therapy is the inhibition of DNA-Topoisomerase enzymes, which change the topology of DNA by causing replication, transcription, chromosome condensation and segregation. It is known that topoisomerase inhibitors block the ligation step of the cell cycle, generating single- and double-stranded breaks that harm the integrity of the genome. Introduction of these breaks subsequently leads to apoptosis and cell death. The DNA binding and cleavage domain is one of the active sites of this enzyme1. Methods: This work covers the interactions between DNA topoisomerase II and a series of compounds2 inhibiting this enzyme were examined in detail by molecular modeling studies such as molecular docking and pharmacophore analysis (Figure 1) performed by using Discovery Studio3 and LigandScout4. Results: Docking studies and complex-based pharmacophore analysis were clarified binding modes of these compounds to the topoizomerase II enzyme, then virtual screening studies were revealed lead compounds against topoisomerase II enzyme from databases. Conclusions: This study also provides a model to design novel and more potent antitumor agents as human topoisomerase II inhibitors. Keywords: Topoisomerase II, benzazole, molecular docking, complex-based pharmacophore

9

IL-7

Spatiotemporal Regulation of Chromosome Segregation in Asymmetrically Dividing Yeast Cells

Ayse Koca Caydasi1,2,3, and Gislene Pereira2,3 1Department of Molecular Biology and Genetics, Koç University, Turkey

2DKFZ-ZMBH Alliance, German Cancer Research Centre (DKFZ), Germany 3Centre for Organismal Studies (COS), University of Heidelberg, Germany

Corresponding Author: aykoca@ku.edu.tr

Position of the mitotic spindle with respect to the polarity cues is a key determinant of asymmetric cell division in many polarized cells. The asymmetrically dividing budding yeast, Saccharomyces cerevisiae, relies upon alignment of the mitotic spindle along the mother-to-daughter cell polarity axis for the fidelity of chromosome segregation. A surveillance mechanism named the spindle position checkpoint (SPOC) monitors the orientation of the mitotic spindle in budding yeast and prevents mitotic exit until correct spindle positioning is achieved. Similar checkpoint mechanisms might exist in other asymmetrically dividing cell types to provide stem cell maintenance and tissue homeostasis. Here, I present molecular mechanisms underlying the SPOC with a focus on recent advances in the field.

10

IL-8

Development of Imidazolone Based Small Molecule as Chemotherapeutic Agent for Cell Cycle Inhibition

Timuçin Avşar1, Berfu Nur Yiğit2, Gizem Turan2, Deniz Altunsu2, Fatma Aybüke Mazı3, Alihan

Sürsal2, Bahar Kurt4, İpek Bulut5, Türker Kılıç6, Serdar Durdağı7, Melih Acar1 1Bahcesehir University, School of Medicine, Medical Biology Department, Istanbul, Turkey 2Bahcesehir University, Health Sciences Institute, Neuroscience Program, Istanbul, Turkey

3Bahcesehir University, School of Medicine, Neuroscience Research Laboratory Istanbul, Turkey 4Bahcesehir University, School of Medicine, Turkey

5Yıldız Technical University, Science and Letters Faculty, Molecular Biology and Genetics Department, Turkey 6Bahcesehir University, School of Medicine, Neurosurgery Department, Istanbul, Turkey

7Bahcesehir University, School of Medicine, Biophysics Department, Istanbul, Turkey

e-mail: timucinavsar@gmail.com

Background: Preclinical and clinical development of cell cycle inhibitor agents, have brought opportunity to discovery of wide range molecules that are relatively more selective to cancer cells. G2/M phase arresting molecules are widely used in chemotherapies, because agents can block division of fast dividing cells. Here we discovered the effects of novel imidazolone based molecule 19D, which causes G2/M arrest in various cell lines and has potential as chemotherapic agent, further we clarified the underlying mechanisms of the molecule. Methods: Seven similar structured new molecules were tested with different concentrations to analyze cytotocix effects on cell lines; growth inhibitory effect and possible apoptotic findings of 19D were evaluated. For understanding of cell cycle arrest mechanism, six cell lines; HUVEC, MCF-7, HCT, HEK, RFL6 and U87-MG were exposed to the different doses of 19D for 24 and 48 hours, then cell cycle progressions were investigated by flow cytometry with propidium iodide staining. Cellular and nuclear morphological alternations were investigated based on hematoxylin-eosin and DAPI staining techniques. In order to obtain deeper perspective on agent induced cells’ visual corruptions, multiplex fluorescence staining applied which was carried out lamin and tubulin antibodies. The target molecule of cell cycle arrest and apoptosis was investigated through caspase3/7 fluorescence staining. Finally, agent causing cell growth inhibition was determined by colorimetric MTT assay. Results: After 24-hour 19D treatment, the growths of different cell lines were notably inhibited in a dose dependent manner ranging from 25 µM to 100 µM. Flow cytometry results revealed that 19D inhibited proliferation by inducing dose dependent G2/M phase arrest, especially at high dose treatments. At the same time, we discovered that this novel molecule 19D, caused same level arrested cell population with highly used anticancer drug Paclitaxel. Furthermore, we have found that 19D disrupted nuclear envelope of cells resulting small multiple nuclear pieces which represent subG1 population including fragmented small DNA and activated apoptosis mechanism. According to the morphological changes and staining results, 19D led to apoptosis with IC50 values, following significant increase of caspase 3/7. MTT colorimetric analysis; proved that agent stop cells’ division with the relation to their division time.

11

Conclusions: Our results showed that 19D induces cell growth inhibition by G2/M phase arrest and accelerates apoptotic events in various cancer and healthy cell lines. Suppression of cell division by G2/M arrest in cell cycle is widely used approach to treat cancer cells, in our study; this novel imidazolone based small molecule has great potential as an anticancer therapy agent.

Keywords: Drug discovery, G2/M arrest, apoptosis, cancer

12

IL-9

Discovery of Novel GLP-1 Modulators for The Treatment of Diabetes

Mustafa Guzel 1,2

1 İstanbul Medipol University, Regenerative and Restorative Medicine Research Center (REMER), Kavacik Campus, Kavacik-Beykoz/ISTANBUL 34810, Turkey

2 Istanbul Medipol University, International School of Medicine, Department of Medical Pharmacology, Kavacik Campus, Kavacik-Beykoz/ISTANBUL 34810, Turkey

mguzel@medipol.edu.tr

Diabetes is one of the well-known diseases silently leading to death. Incretin hormones were postulated following the observation that oral administration of glucose leads to higher and more sustained insulin levels than an equivalent glucose infusion. Two such hormones were subsequently discovered, named glucagon-like peptide 1 (GLP-1) and gastric inhibitory peptide (also sometimes referred to as glucose-dependent insulinotropic peptide), and found to stimulate insulin release from pancreatic beta cells. The main circulating form of GLP-1 is a 30-amino acid product of the proglucagon gene, GLP-1[7-36]NH2 (1). It is produced in L-cells in the ileum and colon in response to nutrients in the gut and acts on the GLP-1 receptor (GLP-1R) on pancreatic beta cells to stimulate insulin release, suppress glucagon production, and promote beta cell proliferation. GLP-1 has multiple metabolic effects that are attractive for an anti-diabetic agent. A key function of GLP-1 is to activate its receptor, GLP-1R, on the pancreatic β-cell to enhance glucose-dependent insulin secretion. Additional positive metabolic benefits of GLP-1 include suppression of excessive glucagon production, decreased food intake, delayed gastric emptying and improvement of β-cell mass and function. Unfortunately, the rapid proteolysis of GLP-1 in blood limits its use as a therapeutic agent.

There are currently several marketed GLP-1 mimetics (biologic agents). These agents have demonstrated notable glucose lowering in addition to weight loss. However, their widespread use is hindered by the route of administration (injection), and by the high incidence of gastrointestinal side effects (nausea and vomiting). A new molecule that has been identified as an orally bioavailable, potent, non-peptide agonist of GLP-1R for the treatment of type 2 diabetes. This molecule is the second generation from our path-finder leading molecule, and it is anticipated to provide excellent glycemic control and an attractive safety profile for the treatment of type 2 diabetes. The drug candidate has completed a 3 month phase 2B study in patients with T2DM where it demonstrated a statistically significant reduction in HbA1c. İt is was well-tolerated, with negligible incidences of nausea and vomiting across all arms of the study. Trends towards weight loss were also observed.

The author will outline the discovery of this drug candidate from computational modelling, target validation, lead optimization to IND submitted clinical development of these novel series. References: 1) Annual Reports in Medicinal Chemistry, Volume 48, 2013 Elsevier Inc. Page: 119-130, ISSN 0065-7743

13

IL-10

Allosteric Action in Proteins

Turkan Haliloglu Department of Chemical Engineering & Polymer Research Center,

Boğaziçi University, Istanbul, Turkey Proteins are complex machines that are highly dynamic in nature within a complex interrelation of sequence, structure and function paradigm. Proteins bind other molecules and communicate within and across structures in a dynamic framework adopted to their functionality. Dissecting functional motion from local to global fluctuations and casual dynamic infrastructure is of challenging interest to disclose the key events to modulate or control the function. Elastic Network Models with Molecular Simulations provide insights into the mechanisms of protein-protein/ligand interactions, a plausible dynamic order in allosteric regulation underlying signaling traits. Exemplary cases such as İnsulin Receptor, ATPcase, and ABC transporters will be visited. Understanding and exploiting intrinsic dynamics of proteins are thus likely to open new ways for design and discovery.

14

IL-11

Fragment-Based Discovery of Chemical Probes for New Targets

Prof. Paul E. Brennan Target Discovery Institute

Structural Genomics Consortium Nuffield Department of Medicine

University of Oxford paul.brennan@sgc.ox.ac.uk

Chemical probes are potent and selective small molecule inhibitors that can be used in cellular assays to induce a phenotype. We are developing chemical probes to enable target discovery in disease relevant cellular models. Direct crystallographic screening of fragments in target proteins (XChem) provides structure enabled hits for rapid development into chemical probes. We will describe out latest efforts to develop chemical probes for novel protein families such as the Nudix hydrolases.

15

IL-12

Understanding Dynamics of G Protein-Coupled Receptor Oligomers for Development of Heterobivalent Ligands: As Novel Therapeutic Candidates

for Treatment of Parkinson’s Disease

Samman Mansoora, Gulru Kayikb, Serdar Durdagib, Ozge Sensoya

aSchool of Engineering and Natural Sciences, Istanbul Medipol University, 34810, Turkey bComputational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of

Medicine, Bahcesehir University, Istanbul, Turkey

The onset of Parkinson disease, which is the second most common neurodegenerative disorder, depends on disruption of cells which provide dopamine to the striatum region of the brain. The main approach for the treatment of the disease relies on increasing dopaminergic signaling. The currently used therapeutics have been developed for i) supplying additional dopamine in the form of L-dopa, ii) increasing dopaminergic receptor signaling by means of dopamine agonists, and iii) preventing dopamine breakdown via monoamine oxidase enzymes. Dopamine agonists act well by per se in the initial phase of the disease, but they become inadequate as the disease progresses and additional L-dopa is needed. Even though L-dopa is known as the most effective drug for treatment of the disease; its efficacy falls down as the time passes. Both L-dopa and dopamine agonists target Dopamine 2 receptor (D2R), which is one of class A G-protein-coupled receptors (GPCR). D2R forms a heteromer with another class A GPCR, namely Adenosine 2A receptor (A2AR) at the post-synaptic striatal cells. In such circumstances, heteromeric structures of GPCRs should be considered since the function of the monomeric GPCR changes in the presence of other receptors due to allosteric interactions occurring within the heteromer. From this perspective, ligands, which are capable of binding two receptors simultaneously - namely heterobivalent ligand-, are considered as more effective for modulating the function of a given receptor than that is exerted when they are provided as a combination drug therapy. Recently, the stoichiometry of the oligomer has been determined to be tetramer, which is composed of a pair of A2AR/D2R dimer in striatal cells [1]. It has been also shown that simultaneous binding of A2AR agonist and antagonist to the tetramer can increase D2R signaling more than that is exerted when an antagonist binds alone [1]. These findings suggest that heterobivalent ligands, which are composed of an A2AR which are currently used for the therapy, for treatment of the disease, and thus decreasing the risk of onset of motor complications such as motor fluctuations and dyskinesia. Here, we first model the tetramer, dock a library of heterobivalent ligands to A2AR dimer, and analyze their effect on the dynamics of the tetramer by means of accelerated molecular agonist and an antagonist, provide a potential for using lower dose of L-dopa, lower than that dynamics simulations. Preliminary results show that heterobivalent ligands with different chemical properties have elicited distinct dynamics on the target D2R receptor in the tetramer. References: 1) Bonaventura et.al. “Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer”, PNAS, 2015, E3609-E3618

16

IL-13

Structure-Activity Relationship Analysis of Some New Benzothiazole and Benzoxazole Derivatives as hGST P1-1 Enzyme Inhibitors

Ismail Yalcina, Esin Aki-Yalcina, Kayhan Bolellia, Tugba Ertan Bolellia, Yaman Musdalb, Bengt Mannervikb

aAnkara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry TR-06100 Tandogan, Ankara, Turkey; b Stockholm University, Department of Neurochemistry, SE-10691

Stockholm, Sweden E-mail: yalcin@ankara.edu.tr

Background: Glutathione S-Transferases (GST) are a family of enzymes that play an important role in detoxification by catalyzing the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione. [1] However, human GST P1-1 enzyme leads to resistance against chemotherapeutic agents. Consequently, it is clinically named multi-drug resistance (MDR) and nowadays it becomes a challenge in cancer treatment. GST is overexpressed in many cancers and contributes to MDR by directly conjugating to chemotherapeutic agents including cisplatin, adriamycin, etoposide, thiotepa, and chlorambucil. [2] Therefore, hGST is considered a promising target for inhibition in cancer treatment. Methods: Pharmacophore models were established using HipHop method and docking study was carried out by using CDocker [3] for predict of binding properties between the hGST P1-1 enzyme H-side and the heterocyclic compounds, which were found active as the in vitro GST enzyme inhibitors. Results: According to the results of the observed hGST P1-1 enzyme inhibitory activities, the most active compounds were used as references during the HipHop study and the best pharmacophore model was generated. Additionally, in order to explain the interactions with hGST P1-1 enzyme, the docking studies were performed.

Conclusions: For the 2-substituted benzothiazoles, the para position of the phenyl ring in the benzamide moiety at the 2nd position on the benzothiazole system was found as significant for the inhibitory activity and structure-activity relationship analysis revealed that increasing the aromaticity of the benzothiazole ring system enhances the activity. On the other hand, inhibitory activity of newly synthesized sulphonamido-substituted benzoxazole compound found as showing better activity than the previously synthesized benzothiazole analog, which includes a NO2 group instead of CF3 at the para position of the benzene ring, bound to sulphonamide moiety. According to the docking results, the SO2 group of the benzoxazole compound revealed two H bonds with the GSH and the CF3 group possessing a halogen bond with Cys101 differently from the benzothiazole derivative. Therefore, we can consider that these data make a sense and can explain its higher activity.

17

Keywords: anticancer, benzothiazole, benzoxazole, docking, glutathione transferase, pharmacophore References 1. Kano T, Sakai M, Muramatsu M. Structure and expression of a human class pi glutathione S-transferase

messenger RNA. (1987) Cancer Res.; 47(21), 5626-5630. 2. Tsuchida S, Sato K. Glutathione transferases and cancer. (1992) Crit. Rev. Biochem. Mol. Biol.; 27(4-5), 337-

384. 3. Accelerys Inc., (2012) Discovery Studio 3.5.

18

IL-14 Translational research for neuromuscular disorders: From genetic mutations

to personalized medicine

Sebahattin Cirak1,2,3

1Center for Molecular Medicine Cologne, Cologne, Germany; 2Department of Pediatrics, University Hospital of Cologne, Cologne, Germany;

3Center for Rare Diseases, Cologne 50937, Germany. Electronic address: sebahattin.cirak@uk-koeln.de

Neuromuscular disorders are a diverse group of inherited disorders that lead to progressive muscle wasting and disability. Many forms are lethal. These inborn disorders are caused by Mendelian genetic mutations. In recent years, a number of disease-causing gene mutations have been uncovered but unfortunately in about 30% of the patients the underlying disease genes are unknown. Despite advances in gene therapy and genome editing therapies currently there is no curative treatment available. There have been disease-ameliorating therapies very recently successfully approved for Duchenne Muscular Dystrophy (Cirak et. al., 2011; Novak et al., 2017, Akpulat et al., 2018) and Spina Muscular Atrophy (Hua et al., 2011). Unfortunately, for other rare forms, the underlying disease-associated genes and mechanisms are not well understood. The lack of knowledge of disease-causing genes leads to absence of understanding of underlying disease mechanisms and hampers ultimately the development of specific therapies. To ease this unmeet medical need, we developed a unique rational approach in our center. We are using the latest genetic technologies to find the disease-causing mutations in unsolved families/cases with neuromuscular disorders (Cirak et al., 2013; Wang et al., 2017; Haliloglu et al., 201. This enables us to provide genetic diagnosis and counseling for the patients and families. Moreover, to create genetically defined patient cohorts for the development of clinical outcomes and for clinical trial readiness. Because, gene therapy and gene edition require the use of viral vectors which is a costly, technically challenging and very risky from the patients safety perspective; we are using rather a rational approach by focusing on RNA-therapeutics (Cirak et. al., 2011; Novak et al., 2017, Akpulat et al., 2018) and Chemical chaperones. Successfully delivered and published contributions towards drug development in neuromuscular disorders will be demonstrated. Akpulat U, Wang H, Becker K, Contreras A, Partridge TA, Novak JS, Cirak S. Shorter Phosphorodiamidate Morpholino Splice-Switching Oligonucleotides May Increase Exon-Skipping Efficacy in DMD. Mol Ther Nucleic Acids. 2018 Oct 10;13:534-542. PMID: 30396145; Novak JS, Hogarth MW, Boehler JF, Nearing M, Vila MC, Heredia R, Fiorillo AA, Zhang A, Hathout Y, Hoffman EP, Jaiswal JK, Nagaraju K, Cirak S, Partridge TA. Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle. Nat Commun. 2017 Oct 16;8(1):941. PubMed PMID: 29038471. Cirak S, Arechavala-Gomeza V, Guglieri M, Feng L, Torelli S, Anthony K, Abbs S, Garralda ME, Bourke J, Wells DJ, Dickson G, Wood MJ, Wilton SD, Straub V, Kole R, Shrewsbury SB, Sewry C, Morgan JE, Bushby K, Muntoni F. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet. 2011 Aug 13;378(9791):595-605. PubMed PMID: 21784508;

19

Hua Y, Sahashi K, Rigo F, Hung G, Horev G, Bennett CF, Krainer AR. Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature. 2011 Oct 5;478(7367):123-6. PubMed PMID: 21979052; Cirak S, Foley AR, Herrmann R, Willer T, Yau S, Stevens E, Torelli S, Brodd L,Kamynina A, Vondracek P, Roper H, Longman C, Korinthenberg R, Marrosu G, Nürnberg P; UK10K Consortium, Michele DE, Plagnol V, Hurles M, Moore SA, Sewry CA, Campbell KP, Voit T, Muntoni F. ISPD gene mutations are a common cause of congenital and limb-girdle muscular dystrophies. Brain. 2013 Jan;136(Pt1):269-81. PubMed PMID: 23288328. Wang H, Salter CG, Refai O, Hardy H, Barwick KES, Akpulat U, Kvarnung M, Chioza BA, Harlalka G, Taylan F, Sejersen T, Wright J, Zimmerman HH, Karakaya M, Stüve B, Weis J, Schara U, Russell MA, Abdul-Rahman OA, Chilton J, Blakely RD,Baple EL, Cirak S, Crosby AH. Choline transporter mutations in severe congenital myasthenic syndrome disrupt transporter localization. Brain. 2017 Nov 1;140(11):2838-2850. PMID: 29088354. Haliloglu G, Becker K, Temucin C, Talim B, Küçükşahin N, Pergande M, Motameny S, Nürnberg P, Aydingoz U, Topaloglu H, Cirak S. Recessive PIEZO2 stop mutation causes distal arthrogryposis with distal muscle weakness, scoliosis and proprioception defects. J Hum Genet. 2017 Apr;62(4):497-501. PubMed PMID: 27974811.

20

IL-15

Nanotechnology and Drug Delivery

Thomas Mavromoustakos National Kapodistrian University of Athens Chemistry Department, Laboratory of Organic Chemistry,

Panepistimiopolis, Zografou 11571 Drug delivery is an important aspect of the drug discovery. A drug is usually an organic molecule that has to be delivered in the organ target intact and capable to exert its beneficial to human health action. However, some highly lipophilic drugs either to not reach the organ target or are metabolized to toxic materials that are detrimental to human health. Nano- drug delivery systems have been developed to overcome the obstacle of the high lipophilicity. Examples of our drug research related to AT1 antagonism and thus antihypertensive drugs (Figure 1), as well as anticancer drugs formulated in supramolecules and nanoparticles with calixarenes will be discussed.

Figure 1: Irbesartan is a commercial drug with well known antihypertensive properties. It can be encapsulated in membrane bilayers through different formulations and exert its antihypertensive action. References 1.Α. S. Liossi , D. Ntountaniotis, T.F. Kellici, M.V. Chatziathanasiadou, G. Megariotis, M. Mania, J. Becker-Baldus, M. Kriechbaum, A. Krajnc, E. Christodoulou, C. Glaubitz , M. Rappolt, H. Amenitsch, Gregor Mali, D.N. Theodorou, G. Valsami, M. Pitsikalis, H. Iatrou, A. Tzakos, T. Mavromoustakos. Exploring the interactions of irbesartan and irbesartan–2-hydroxypropyl-β-cyclodextrin complex with model membranes. Biochimica et Biophysica Acta 1859, 1089–1098 (2017). 2. T.F. Kellici, M. V. Chatziathanasiadou, D. Diamantis, A. V. Chatzikonstantinou, I. Andreadelis, E. Christodoulou, G. Valsami, T.Mavromoustakos, A. G. Tzakos, Mapping the interactions and bioactivity of quercetin—(2-hydroxypropyl)-b-cyclodextrin complex. International Journal of Pharmaceutics 511 (2016) 303–311. 3.T.F.Kellici, D. Ntountaniotis, G. Leonis, M. Chatziathanasiadou, A.V. Chatzikonstantinou, J. Becker-Baldus, C. Glaubitz, A.G. Tzakos, K. Viras, P. Chatzigeorgiou, S. Tzimas, E. Kefala, G. Valsami, H. Archontaki, M.G. Papadopoulos, T. Mavromoustakos. Investigation of the interactions of silibinin with 2-hydroxypropyl-β-cyclodextrin through biophysical techniques and computational methods. Molecular Pharmaceutics 12(3), 954-965 (2015).

21

IL-16

Protein-Protein Interactions in Live Cells: Reinventing the Wheel

Çağdaş Devrim Son ODTÜ Biyolojik Bilimler Bölümü, Moleküler Biyoloji ABD, Çankaya Ankara, 06800

cson@metu.edu.tr G protein-coupled receptors (GPCRs) are membrane proteins that mediate physiological response to a diverse array of stimuli. In humans, they mediate the action of hundreds of peptide hormones, sensory stimuli, odorants, neurotransmitters, and chemokines. GPCRs also are targets for ~40% of all currently marketed pharmaceuticals. These receptors traditionally been thought to act as monomeric units. However, recent evidence suggests that GPCRs may form dimers as part of their normal trafficking and function. While the formation of GPCR dimers/oligomers have been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how GPCR dimers interact with other proteins such as G-proteins and Arrestin. We are interested in studiying the interactions between GPCRs and effect of this dimerization on G-protein dimerization. Our group also focus on the mechanisms of receptor-arrestin binding in live cells using Föster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays. We designed and developed tagged receptors, G-proteins and Arrestin proteins using Green florescent protein variants that can be used in imaging studies. These constructs are suitable for testing drug candidates and/or analyze protein-protein interfaces for GPCRs or G-protein dimers.

22

IL-17

Using Simulation to Improve Drug Design

Phil Biggin Oxford University

Department of Biochemistry The calculation of the absolute binding free energy (ABFE) of a small molecule for a protein target has long been seen one of the major challenges in computational chemistry. Being able to do this reliably is seen as an extremely useful tool in the context of structure-based drug-design, particularly if this can be done rapidly as well as accurately. A related challenge is to be able to predict whether bound water molecules in the binding pocket are likely to be displaced or not by an incoming drug molecule. In this talk I will show how computing the ABFE of drug molecules for protein targets can be computed to an accuracy that is considered useful for drug design and how the “displaceability” of waters in binding pockets can also be predicted. I will also demonstrate how these approaches can be used alongside conventional MD simulations to provide direction and a higher level of rationality to an ongoing drug-discovery program.

23

IL-18

Differential requirement for centriolar satellites in cilium formation and ciliary signaling among different vertebrate cells

Elif Nur Karalar

Koc University Department of Molecular Biology and Genetics

Centriolar satellites are an array of membrane-less granules that localize and move around the mammalian centrosome/cilium complex. They play key roles in the biogenesis and function of centrosomes and cilia and mutations affecting satellite components cause ciliopathies. However, the mechanisms underlying their regulatory functions and disease phenotypes are poorly understood. Although centriolar satellites has been implicated in regulating various centrosome/cilium-mediated processes including protein targeting to the centrosome/cilium complex, ciliogenesis and ciliary recruitment of the BBSome complex, their precise contribution to these processes remains poorly understood. To address these controversies, we generated multiple vertebrate cell lines that lack centriolar satellites by genetically ablating the satellite scaffolding component PCM1 and studied the phenotypic consequences of satellite loss on cilium-related cellular processes. While satellites are essential for primary cilium formation in retinal pigmental epithelial cells, their loss resulted in significant decreases in the efficiency of cilium formation in kidney epithelial cells. Importantly, loss of satellites caused defects in responding to Hedgehog signals, as well as ciliary recruitment of signaling receptors. Finally, comparison of the transcriptome and proteome of wild-type and satellite-less uncovered potential mechanisms for the phenotypes associated with loss of satellites. Focusing on one of these mechanisms, we identified regulatory roles for satellites in dynamic recruitment of proteins to the centrosome and cilium. Together, our findings identify important roles for centriolar satellites in primary cilium formation and ciliary trafficking in part through regulation of dynamic behavior of centrosomal and cilia proteins, and importantly show that their roles in these processes are regulated in a cell-type specific manner.

24

IL-19

Systems level identification of drug targets overcoming resistance in breast cancer

Özgür Şahin1,2

1Department of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC, USA

2Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women worldwide with an estimate of more than 2 million new cases and nearly 630,000 deaths in 2018. It is a heterogeneous disease exhibiting a high degree of diversity between and within tumors. BC is classified into at least 6 major molecular subtypes: normal-like, luminal A, luminal B, HER2-enriched, basal-like and claudin-low. Luminal A and B are collectively known as Estrogen Receptor positive (ER+) BCs, and claudin-low and basal-like are triple negative breast cancers (TNBCs). While TNBCs solely rely on chemotherapy, patients that are of luminal subtype can benefit from targeted therapies against the overexpressed ER e.g., tamoxifen. However, development of resistance to these agents, in most cases, is inevitable leading to relapse and patients’ death. Therefore, it is crucial to elucidate the molecular mechanisms underlying resistance and to identify novel targets overcoming resistance. In this line, systems level approaches combining sequencing, network modeling, functional genomics, drug screening, medicinal chemistry and cancer cell biology provide new avenues for identifying and validating novel drug targets. In the first part of my talk, I will first present how we (1) develop chemotherapy resistant TNBC mouse models and measure transcriptome level changes in resistance to identify potential targets; (2) integrate expression profiling data of BC patients to filter these targets and validate them in in vitro, in vivo and organoid experiments; and (3) perform compound library screens to identify new hits targeting our candidates and overcoming chemotherapy resistance. In the second part, I will present how we (1) utilize pathway enrichment tools combined with patient data to identify targets overcoming tamoxifen resistance in ER+ BC; (2) solve the mechanism of drug sensitization combining cell biology, biochemistry and functional genomics; and (3) perform gene expression-based drug re-purposing to identify already available drugs to overcome tamoxifen resistance in BC. Overall, our efforts led to identification and pre-clinical validation of several novel drug targets, lead compounds and re-purposed drugs overcoming resistance in treatment refractory BC. Our future efforts will be to develop these compounds further with the help of medical chemists for potential clinical trials.

25

IL-20

The use of systems biology in treatment of liver diseases

Adil Mardinoglu1,2,3,*

1Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden 2Centre for Host–Microbiome Interactions, Dental Institute, King's College London,

London, SE1 9RT, United Kingdom 3Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg,

Sweden *Email: adilm@scilifelab.se

To develop novel strategies for prevention and treatment as well as to gain detailed insights about the underlying molecular mechanisms of liver associated diseases fatty liver disease, cirrhosis, type 2 diabetes and hepatocellular carcinoma, it is vital to study the biological functions of liver and how liver interacts with other human tissues as well as with the gut microbiota. Biological networks including metabolic, transcriptional regulatory, protein-protein interaction, signaling and co-expression networks can provide a scaffold for studying biological pathways operating in the liver in connection with disease development in a systematic manner. In my presentation, I will present our recent work where biological networks have been employed to identify the reprogramming in liver physiology in response to complex diseases including NASH/NAFLD and liver cancer. I will further discuss how this mechanistic modelling approach can contribute to identification of new mechanisms and discovery of novel drugs which may lead to design of targeted and effective treatment strategies.

26

IL-21

Pathological Modifications and Intrinsically Disordered Proteins in Alzheimer´s Disease: Towards Antibody Design

Orkide Coskuner Weber

Turkish-German University, Molecular Biotechnology, Beykoz, Istanbul Email: weber@tau.edu.tr

Alzheimer´s disease effects 45 million people worldwide. Currently, there is no cure or treatment to halt the progression of this severe disease. Intrinsically disordered proteins (IDPs), i.e., amyloid-β and tau are at the center of Alzheimer´s disease. These IDPs do not adopt stable structures at the monomeric and oligomeric levels and possess fast aggregation processes. Therefore, experiments face challenges in meausuring their chemical, physical and biological properties at the monomeric and oligomeric levels. Furthermore, pathological modifications resulting from genetics, mitochondrial dysfunction, oxidative stress and post-translational modifications impact the chemical, physical and biological features of these IDPs in Alzheimer´s disease. Here, we present the usefulness of quantum chemistry, statistical mechanics, thermodynamics and bioinformatics in the studies of IDPs in Alzheimer´s disease that are affected by pathological modifications. We also discuss the design of more efficient antibodies for the treatment of Alzheimer´s disease. A literature survey based on our own studies will be provided.

27

IL-22

Towards Modern Computational Drug Discovery –Integrated approaches to analyze protein drug targets from the perspectives of SNPs, allosteric regions

and dynamic residue networks

Özlem Taştan Bishop Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, Rhodes

University, Grahamstown 6140, South Africa E-mail:o.tastanbishop@ru.ac.za

Over the years, computer aided drug design technology has revolutionized the long process of drug research and development. Traditionally, computational drug discovery approaches include large scale structure based and/or ligand based in silico docking combined with molecular dynamics and binding free energy calculations. The identified hit compounds, then, can be tested via in vitro experiments, and the active compounds can be further studied via introducing modifications to get lead compounds. Although this approach often provides a faster, cheaper and more effective way of identifying potential hit and hit to lead compounds than screening of large-scale compound databases in vitro as a first step, unfortunately it misses a number of key factors to be considered in a rational drug design. For instance, continuously emerging resistant strains of various pathogens to current drugs present an immense challenge for the eradication of diseases. The effects of mutations on the structure and function of protein drug targets may lead to drug resistance; these effects should be understood in parallel to the traditional computational drug discovery approaches. Furthermore, the traditional way of targeting an enzyme is mainly to identify compound(s) that inhibit the active site. However, it is crucial to identify potential drug targeting sites other than the orthosteric ones, as it may be challenging to handle drug selectivity at the active site of an enzyme due to it being highly conserved within the protein family. In the last few years, Research Unit in Bioinformatics (RUBi) has been developing some novel protocols by merging computational chemistry, structural bioinformatics and biophysics approaches to address the issues mentioned above, e.g. SNP analysis in drug discovery, identification of allosteric sites as drug targeting sites (1-5); and combining with traditional methods (6-7). While doing so we have also developed new methods and tools (8-9). In this talk, I will be giving examples from a range of drug discovery research projects from the group. This will include analysis of SNPs in macrophage migration inhibitory factor (MIF) where the polymorphisms in the MIF gene have been linked with diseases including cancer; allosteric inhibition of heat shock proteins (Hsp), Hsp72 and Hsp90α, which have been strongly involved in various critical stages of cancer development; and identification of hit compounds for pteridine reductase 1 (PTR1) as potential drug target in Trypanosoma. Keywords: Drug discovery; Allosteric sites; Non-synonymous nucleotide polymorphism; MD-TASK; MODE-TASK

28

References:

1. Brown DK, Tastan Bishop Ö. Role of Structural Bioinformatics in Drug Discovery by Computational SNP Analysis: Analyzing Variation at the Protein Level. (2017) Glob Heart. Jun;12(2):151-161. doi: 10.1016/j.gheart.2017.01.009. Epub 2017 Mar 13. Review.

2. Brown DK, Sheik Amamuddy O, Tastan Bishop Ö. Structure-Based Analysis of Single Nucleotide Variants in the Renin-Angiotensinogen Complex. (2017) Glob Heart. Jun;12(2):121-132. doi: 10.1016/j.gheart.2017.01.006. Epub 2017 Mar 13.Ss

3. Penkler DL, Atilgan C, Tastan Bishop Ö. Allosteric Modulation of Human Hsp90α Conformational Dynamics. (2018) J Chem Inf Model. Feb 26;58(2):383-404. doi: 10.1021/acs.jcim.7b00630. Epub 2018 Feb 12.

4. Penkler D, Sensoy Ö, Atilgan C, Tastan Bishop Ö. Perturbation-Response Scanning Reveals Key Residues for Allosteric Control in Hsp70. (2017) J Chem Inf Model. Jun 26;57(6):1359-1374. doi: 10.1021/acs.jcim.6b00775. Epub 2017 Jun 12.

5. Penkler DL, Tastan Bishop Ö. Modulation of Human Hsp90α Conformational Dynamics by Allosteric Ligand Interaction at the C-Terminal Domain. (2018) BioRxiv. doi: https://doi.org/10.1101/386755

6. Musyoka TM, Kanzi AM, Lobb KA, Tastan Bishop Ö. Analysis of non-peptidic compounds as potential malarial inhibitors against Plasmodial cysteine proteases via integrated virtual screening workflow. (2016) J Biomol Struct Dyn. Oct;34(10):2084-101. doi: 10.1080/07391102.2015.1108231. Epub 2016 Jan 28.

7. Musyoka TM, Kanzi AM, Lobb KA, Tastan Bishop Ö. Structure Based Docking and Molecular Dynamic Studies of Plasmodial Cysteine Proteases against a South African Natural Compound and its Analogs. (2016) Sci Rep. 2016 Mar 31;6:23690. doi: 10.1038/srep23690.S

8. Brown DK, Penkler DL, Sheik Amamuddy O, Ross C, Atilgan AR, Atilgan C, Tastan Bishop Ö. MD-TASK: a software suite for analyzing molecular dynamics trajectories. (2017) Bioinformatics. Sep 1;33(17):2768-2771. doi: 10.1093/bioinformatics/btx349.

9. Ross C, Nizami B, Glenister M, Sheik Amamuddy O, Atilgan AR, Atilgan C, Tastan Bishop Ö. MODE-TASK: Large-scale protein motion tools. (2018) Bioinformatics. doi: 10.1093/bioinformatics/bty427. [Epub ahead of print]

29

IL-23

Effect of Oncogenic K-Ras4B dimerization on downstream MAPK and Akt signaling

Özlem Keskin Koç University

Chemical and Biological Engineering Dept. Ras activates effectors that transmit receptor-initiated signals. K-Ras4B may dimerize through the β- and α-interfaces, mapped to Switch I and effector binding regions and the allosteric lobe, respectively. We chose KRas4BG12D as control and its double mutants K101D/R102E and R41E/K42D to assess the impact of KRas4B mutants on dimerization and function. R41 and R102 are found in several adenocarcinomas in Ras isoforms. Site-directed mutagenesis, cellular localization experiments and molecular dynamics (MD) simulations are performed. α-interface K101D/R102E double-mutations reduced dimerization, but only slightly reduced pERK levels. β-interface R41E/K42D mutants did not affect dimerization but blocked pERK. Both mutants increased downstream pAkt levels in cells. They further altered ERK- and Akt-regulated expression of EGR1, JUN, and BCL2L11. The results underscore the role of the α- and β-interfaces in homodimerization and effector binding, respectively. MD simulations showed that the membrane and the hypervariable region interact with both interfaces, inhibiting homodimerization and effector binding. We conclude that dimerization is not necessary but enhances MAPK signaling.

30

IL-24

Proper interaction of canonical Wnt with its receptor complex at the cell surface is secured by the membrane lipid environment

Yagmur Azbazdar1,2, Erdinc Sezgin3, Ozgun Ozalp1,2, Xue Wen Ng4, Thorsten Wohland4,

Christian Eggeling3, Ezgi Karaca1,2 and Gunes Ozhan1,2 1 Izmir Biomedicine and Genome Center (IBG), Dokuz Eylul University Health Campus, Inciralti-Balcova,

35340 Izmir, Turkey 2 Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, Inciralti-Balcova,

35340 Izmir, Turkey 3 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford,

OX39DS, Oxford, United Kingdom 4 Department of Chemistry and Center for BioImaging Sciences, National University of Singapore, Singapore

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in binding of canonical Wnt to its receptors at the plasma membrane, a key step underlying initiation of the pathway. The plasma membrane contains ordered membrane nanodomains, conventionally referred to as membrane (lipid) rafts, that are highly dynamic membrane regions characterized by selective recruitment of saturated lipids, sterols and specific lipid-anchored proteins. These ordered structures generate compact transient platforms for ligand-receptor interaction and receptor clustering and are critical in signal transduction pathways including the canonical Wnt signaling. While the receptor Fz8 and the coreceptor Lrp6 of canonical pathway are distributed throughout the membrane, the membrane-bound Wnt pathway modulator Lypd6 becomes localized to ordered membrane nanodomains and ensures via direct physical interaction that Lrp6 is phosphorylated in these domains to activate signaling. Our recent work has unraveled the influence of the immediate plasma membrane environment on the canonical Wnt–receptor interaction by showing that canonical Wnt selectively binds to its pool of receptors in the ordered nanodomains and this domain-specific binding is necessary for downstream signaling activity. Next, we addressed the influence of the lipid modification on the ability of canonical Wnt ligand in binding the plasma membrane and activating signaling. Specifically, we investigated the effect of palmitoylation in zebrafish Wnt3 by generating a point mutation in a conserved serine residue. Our data support that this specific palmitoylation of Wnt ligand is necessary for its proper localization to the ordered membrane nanodomains. Moreover, mutant protein is unable to activate Wnt signaling as shown by reporter activity measurement, qPCR and in situ hybridization for Wnt reporter and target genes, indicating that proper acylation of Wnt is critical for its activity. Thus we believe that lipids are involved in regulation of Wnt signaling pathway not only as a posttranslational modification of the ligand and the receptors but also for their physical interactions at the plasma membrane, and may therefore be exploited as key regulators in development of new drugs that interfere with signaling activity.

31

IL-25

INTEGRATED BINARY QSAR MODELS, TEXT MINING AND TARGET-DRIVEN APPROACHES IN VIRTUAL SCREENING OF SMALL MOLECULES

Serdar DURDAGI

Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University

serdar.durdagi@med.bau.edu.tr durdagilab.com

The industry appears to be suffering from innovation costs and would benefit from new, more rational approaches to drug design which exploit the recent advances made in molecular and structural biology and computational chemistry. Computational chemistry provides a range of simulation tools for description of protein-ligand binding, statistical methods for analysis of the binding data that help to predict the optimal ligands, and molecular modeling tools that enable construction of novel ligands. In recent years, a growing number of defined crystal, as well as NMR and cryo EM structures and access to these structures from the protein database (PDB, protein data bank) have enabled these data to be used as template targets in molecular modeling studies. In this talk, examples of structure-based and ligand-based screening of small molecule databases for different targets will be highlighted. Filtered structures based on predicted binding energy results using high throughput virtual screening (HTVS) techniques are used in more sophisticated molecular simulations approaches (i.e., Glide/SP, Glide/XP, Induced Fit Docking- IFD, and Quantum Mechanics Polarized Ligand Docking- QPLD). Potent high binding affinity compounds that are predicted by molecular simulations are then tested by long molecular dynamics (MD) simulations. The molecular mechanism analysis, free energy calculations (i.e., MM/GBSA) using short/long multiple MD simulations for the identified compounds which show high predicted binding affinity against specific target structures, as well as structure-based pharmacophore development (E-pharmacophore) studies and drug re-positioning studies (Figure 1) will be summarized.1-5

Figure 1. Integrated structure- and ligand-based virtual screening techniques were applied in different projects for the identification of novel therapeutic small molecules.

32

References 1. Ramin Ekhteiari Salmas, Ayhan Unlu, Muhammet Bektaş, Mine Yurtsever, Mert Mestanoglu, Serdar

Durdagi: Virtual Screening of Small Molecules Databases for Discovery of Novel PARP-1 Inhibitors: Combination of in silico and in vitro Studies. Journal of Biomolecular Structure and Dynamics 2016

2. Shaher Bano Mirza, Ramin Ekhteiari Salmas, M. Qaiser Fatmi, Serdar Durdagi: Virtual Screening of Eighteen Million Compounds against Dengue Virus: Combined Molecular Docking and Molecular Dynamics Simulations Study. Journal of Molecular Graphics and Modelling. 2016

3. Serdar Durdagi, Ramin E. Salmas, Matthias Stein, Mine Yurtsever, Philip Seeman: Binding Interactions of Dopamine and Apomorphine in D2High and D2Low States of human Dopamine D2 Receptor (D2R) using Computational and Experimental Techniques. ACS Chem. Neuroscience 2016

4. Ramin Ekhteiari Salmas, Mert Mestanoglu, Mine Yurtsever, Sergei Y Noskov, Serdar Durdagi: Molecular Simulations of Solved Co-crystallized X-ray Structures Identifies Action Mechanisms of PDEδ Inhibitors. Biophysical Journal 2015

5. Ramin Ekhteiari Salmas, Murat Senturk, Mine Yurtsever, Serdar Durdagi: Discovering Novel Carbonic Anhydrase Type IX (CA IX) Inhibitors from Seven Million Compounds Using Virtual Screening and In Vitro Analysis. Journal of Enzyme Inhibition and Medicinal Chemistry 2015

6. Ramin Ekhteiari Salmas, Mine Yurtsever, Serdar Durdagi: Investigation of Inhibition Mechanism of Chemokine Receptor CCR5 by Micro-second Molecular Dynamics Simulations. Scientific Reports 2015

7. Tarek Kanan, Duaa Kanan, Ismail Erol, Samira Yazdi, Matthias Stein, Serdar Durdagi. Targeting the NF-κB/IκBα Complex via Fragment-Based E-Pharmacophore Virtual Screening and Binary QSAR Models. Journal of Molecular Graphics and Modelling 2018

8. Ramin Ekhteiari Salmas, Philip Seeman, Matthias Stein, Serdar Durdagi. Structural Investigation of the Dopamine-2 Receptor (D2R) Agonist Bromocriptine Binding to Dimeric D2HighR and D2LowR States. Journal of Chemical Information and Modeling 2018

9. Yusuf Serhat Is, Serdar Durdagi, Busecan Aksoydan, Mine Yurtsever. Proposing Novel MAO-B Hit Inhibitors Using Multidimensional Molecular Modeling Approaches and Application of Binary QSAR Models for Prediction of their Therapeutic Activity and Toxic Effects. ACS Chemical Neuroscience 2018

10. Serdar Durdagi, Busecan Aksoydan, Ismail Erol, Isik Kantarcioglu, Yavuz Ergun, Gulay Bulut, Melih Acar, Timucin Avsar, George Liapakis, Vlasios Karageorgos, Ramin E. Salmas, Barış Sergi, Sara Alkhatib, Gizem Turan, Berfu Nur Yigit, Kutay Cantasir, Bahar Kurt, and Turker Kilic: Integration of Multi-scale Molecular Modeling Approaches with Experiments for the in silico Guided Design and Discovery of Novel hERG-Neutral Antihypertensive Oxazalone and Imidazolone Derivatives and Analysis of Their Potential Restrictive Effects on Cell Proliferation. European Journal of Medicinal Chemistry 2018

11. Adriano Mollica,Gokhan Zengin, Serdar Durdagi, Ramin Ekhteiari Salmas, Giorgia Macedonio, Azzurra Stefanucci, Marilisa Pia Dimmito, Ettore Novellino. Combinatorial Peptide Library Screening for Discovery of Diverse α-glucosidase Inhibitors Using Molecular Dynamics Simulations and Binary QSAR Models. Journal of Biomolecular Structure & Dynamics 2018

12. Mehreen Zaka, Bilal Haider Abbasi, Serdar Durdagi. Novel Tumor Necrosis Factor-α (TNF-α) Inhibitors from Small Molecule Library Screening for their Therapeutic Activity Profiles against Rheumatoid Arthritis using Target-Driven Approaches and Binary QSAR Models. Journal of Biomolecular Structure & Dynamics 2018

33

ORAL PRESENTATIONS

34

OP-1

The role of multidrug resistance in PARP inhibitor-resistant triple negative breast cancer cells

Gamze Guney Eskilera, Gulsah Cecenerb, Unal Egelib, Berrin Tuncab

aDepartment of Medical Biology, Faculty of Medicine, Sakarya University, Sakarya, Turkey bDepartment of Medical Biology, Faculty of Medicine, Uludag University, Bursa, Turkey

E-mail: gamzeguney@sakarya.edu.tr

Background: Poly(ADP-ribose) polymerases (PARP) inhibitors, inhibit PARP activity in DNA-damage repair, is a promising strategy for targeting cancers with defective particularly BRCA mutation-associated breast cancer (1,2). However, multidrug resistance (MDR) mechanism, occurs in over 50% of cancer patients, remains one of major limitation for the successful treatment of breast cancer due to resulting in drug inactivation, minimal cell death and the development of drug-resistant cancer cells (3,4). For this purpose, the aim of this study was to investigate the role of MDR mechanism leading to BMN 673 (Talozoparib) resistance in triple negative breast cancer (TNBC). Methods: To identify the effect of MDR on BMN 673 resistance, we generated BMN 673 resistant HCC1937 TNBC cell line and then, HCC1937BRCA1-/- and HCC1937-R (BMN 673 resistant) TNBC cells were treated with different concentrations (0.01-10 nM) of BMN 673 for 12 days. To evaluate in vitro effects of BMN 673 on MDR mechanism, we performed WST-1, calcein, Rho123 and gene expression analysis in HCC1937 and HCC1937-R cells. Results: The WST-1 results indicated that the viability of HCC1937 and HCC1937-R cells treated with 10 nM of BMN 673 reduced to 31.0% and 87.0% for 12 days, respectively. BMN 673 significantly decreased intracellular accumulation of calcein and Rho123 in HCC1937 and HCC1937-R cells. We also analyzed the mRNA expression of P-gp, BCRP and MRP1 in these cells. Especially, high mRNA expression of BCRP (7.8-fold) and MRP1 (10.9-fold) was found in HCC1937-R cells at 10 nM of BMN 673 for 12 days. Thus, BMN 673 had dose and time-dependent inhibitory effect on HCC1937 cells. However, BMN 673-resistance resulted in increased expression of BCRP and MRP1 mRNA levels and also significantly reduced intracellular calcein and Rho-123 accumulation. Conclusions: BMN 673 had exhibited therapeutic potential in triple negative breast cancer cells. However, the overexpression BCRP and MRP1 caused BMN 673 resistance by decreasing the intracellular accumulation of calcein and Rho 123 in HCC1937 and HCC1937-R cells. Thus, further studies are warranted to develop new formulation of BMN 673 using drug delivery systems and to overcome BMN 673 resistance in treatment of TNBC. Keywords: Triple negative breast cancer, PARP inhibitors, Multidrug resistance (MDR). References:

1. Ashworth A. A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. (2008) J Clin Oncol.; 26, 3785-3790.

2. Lord CJ, Ashworth A. Targeted therapy for cancer using PARP inhibitors. (2008) Curr. Opin. Pharmacol.; 8, 363–369.

3. Fojo T, Bates S. Mechanisms of resistance to PARP inhibitors-three and counting. (2013) Cancer Discov.; 3, 20–23.

4. Lord CJ, Ashworth A. Mechanisms of resistance to therapies targeting BRCA-mutant cancers. (2013) Nat. Med.; 19, 1381–1388.

35

OP-2

Assessment of small molecule pKa prediction methods via the SAMPL6 blind challenge

Mehtap Işık a,b, Dorothy Levorsec, Andrea Rizzid,b, Bas Rustenburge,b, Ikenna N.

Ndukwef, Timothy Rhodesc, David L. Mobleyg, John D. Choderab a Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10065, United States; bComputational and Systems

Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, United States; cPharmaceutical Sciences, MRL, Merck & Co., Inc., 126 East Lincoln Avenue,

Rahway, New Jersey 07065, United States; dTri-Institutional PhD Program in Computational Biology and Medicine, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY

10065, United States; eGraduate Program in Physiology, Biophysics, and Systems Biology, Weill Cornell Medical College, New York, NY 10065, United States; fProcess and Analytical Research and Development, Merck & Co., Inc., Rahway, NJ 07065, United States; gDepartment of Pharmaceutical

Sciences and Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States

E-mail: mehtap.isik@choderalab.org Background: SAMPL (Statistical Assessment of the Modeling of Proteins and Ligands) is a series of annual blind prediction challenges for the computational chemistry community. It aims to advance computational tools for rational drug design. In addition to protein-ligand binding affinity predictions, evaluating methods for predicting relevant physicochemical properties like solvation free energies and distribution coefficients have been part of SAMPL. Physicochemical property prediction challenges have been helpful to pinpoint deficiencies in computational models that can lead to substantial errors in affinity predictions. Cyclohexane-water distribution constant (logD) prediction challenge of SAMPL5 in the previous year have indicated that neglecting protonation state effects leads to large errors in transfer free energies [1, 2]. Protonation state effects contributed to the error up to 6-8 kcal/mol for some compounds [2]. Similarly, any error in prediction of the protonation free energy of an ionizable ligand can directly add on to the error in binding free energy calculations for a protein-ligand complex. By organizing a computational acid dissociation constant (pKa) prediction challenge as a part of SAMPL6 we aimed to capture the performance of current pKa prediction methods and isolate potential detriment of inaccurate pKa estimates for predicting affinities of drug-like molecules. Methods: SAMPL6 pKa challenge consisted of predicting microscopic and macroscopic pKas of 24 small organic molecules. These fragment-like small molecules are selected for their similarity to kinase inhibitors and for experimental tractability. Macroscopic pKa values were collected experimentally with UV-absorbance based pKa measurements [3]. For several compounds in which the microscopic protonation states associated with macroscopic pKas were ambiguous, we performed follow-up NMR experiments to disambiguate the microstates involved in the transition. Experimental data was kept for 3 months to allow collection of blind computational predictions. 11 research groups participated in this challenge with 93 prediction submission sets that cover a large variety of contemporary pKa prediction methods.

36

Results: The analysis of computational predictions showed a huge variability in the accuracy of pKa prediction methods spanning RMSE values from 0.7 to 5 pKa units. Heterocycles and multiple titratable loci are found frequently in the set of molecules selected for this study, as in FDA-approved kinase inhibitors. These properties lead to many possible protonation and tautomerization microstates that provide challenging cases for pKa predictions for drug-like molecules. Conclusions: SAMPL6 blind community challenge highlighted that contemporary pKa prediction methods struggle with small molecules with drug-like properties. It is important to improve pKa prediction methods to prevent propagation of protonation and ionization state errors to molecular models that try to capture physicochemical and pharmaceutical properties. Keywords: acid dissociation constants, blind prediction challenge, macroscopic pKa, microscopic pKa, SAMPL

Figure 1: Protonation microstates of SM14, a drug-fragment-like molecule from SAMPL6 pKa prediction challenge set. Dominant microstates and protonation order was characterized by NMR. Macoscopic pKa values were determined by spectrophotometric pKa measurements with Sirius T3 [3]. References: 1. Bannan, Caitlin C., Kalistyn H. Burley, Michael Chiu, Michael R. Shirts, Michael K. Gilson, and David L. Mobley. “Blind Prediction of Cyclohexane–water Distribution Coefficients from the SAMPL5 Challenge.” Journal of Computer-Aided Molecular Design 30, no. 11 (November 2016): 927–44. https://doi.org/10.1007/s10822-016-9954-8. 2. Pickard, Frank C., Gerhard König, Florentina Tofoleanu, Juyong Lee, Andrew C. Simmonett, Yihan Shao, Jay W. Ponder, and Bernard R. Brooks. “Blind Prediction of Distribution in the SAMPL5 Challenge with QM Based Protomer and pKa Corrections.” Journal of Computer-Aided Molecular Design 30, no. 11 (November 2016): 1087–1100. https://doi.org/10.1007/s10822-016-9955-7. 3. Mehtap Işık, Dorothy Levorse, Ariën S. Rustenburg, Ikenna E. Ndukwe, Heather Wang, Xiao Wang, Mikhail Reibarkh, Gary E. Martin, Alexey A. Makarov, David L. Mobley, Timothy Rhodes, John D Chodera “pKa measurements for the SAMPL6 prediction challenge for a set of kinase inhibitor-like fragments.” bioRxiv 368787. https://doi.org/10.1101/368787

37

OP-3

Could Allometric Scaling Rules be Valid for Proteins?

Yekbun Adiguzela a Department of Biophysics, School of Medicine, Altinbas University, Istanbul, Turkey

E-mail: yekbun.adiguzel@altinbas.edu.tr

Background: The allometric scaling concept is the observation of associations between certain biological parameters and could be summarized with the following equation: y = aMb wherein M and y are the biological parameters of interest, b, the scaling factor and a, the constant. For instance, M could be the mass or cell volume and y the metabolic rate. The scaling factor is 0.75 then. This presence of such scaling relations can be investigated among the parameters of interest. Here, it is done for the protein size (or length) and we looked at its scaling relation with certain other parameters. Methods: The possible presence of allometric scaling relations regarding the protein length as the number of amino acids that can also be estimated from the mRNA length is investigated. In general, scaling relation of protein length with certain other parameters is looked for. This is performed both by using the literature values of the protein lengths, and by using the calculated values of the average protein lengths (in relation to the gene sizes, which we have started investigating previously with information theoretic approaches.) Results: The protein size and the biological parameters appear to have (allometric or non-allometric) scaling relations both in terms of the data from literature and the calculated values. The results imply the presence of scaling relation of certain parameters in relation to the calculated protein length. However, those relations were observed mostly different in case of biological data that is derived from literature values. Conclusions: Difference in the scaling relations with the same parameter is observed in the data from literature and the calculated values. This is possibly due to physical factors and the pressure of evolutionary forces, or such constraints that end up in deviations of the biological data from that of the calculated values. Biological data would be prone to such forces and likely deviate from expected models regardless of the validity of the assumptions in any calculations, unless they could be implemented in the models. This study also highlights the possible use and implementation of information theoretic approaches in the studies that investigate allometric scaling rules. Keywords: Allometric scaling, proteins, information communication theory

38

OP-4

New Imidazo[2,1-b]Thiazol Derivatives: Synthesis, Anticancer Activity And Molecular Docking

Başoğlu, F.a, Ece, A.b, Cimok, S.c, Ulusoy Güzeldemirci, N.c

aCyprus Health and Social Sciences University, Faculty of Dentistry, Department of Basic Medical Science, Mersin 10, Turkey; bBiruni University, Faculty of Pharmacy, Department of Pharmacetical

Chemistry, Istanbul, Turkey; cIstanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey

E-mail: faika.basoglu1990@gmail.com Background: In this study the synthesis of new compounds bearing an imidazo[2,1-b]thiazole moiety, 3-alkyl/aryl-2-[[[6-(4-bromophenyl)imidazo[2,1-b]thiazol-3-yl]acetyl]hydrazono]-5-methyl-4-thiazolidinones (5-13) were synthezised and their antcancer activities were evaluated. Additionally, the ligand interactions with a potential target were studied using molecular docking technique [1,2] Methods: In this study, new imidazo[2,1-b]thiazole derivatives were investigated by using both experimental and Schrödinger program packages. Both all of the synthesized molecules and X-ray the structure of cyclin-dependent kinase 2 (CDK2) (PDB ID: 1FVT) in complex with an oxindole inhibitor [3-5] were prepared by using related modules of Schrödinger. The docking calculations were performed by using the Glide SP (standard precision) module of Schrödinger Suite. Results: Molecular docking studies were performed to see possible binding modes of the synthesized compounds and also docking scores of the compounds 5–13 together with the native ligand, are obtained. Conclusions: In this study, the results obtained from experimental and in silico shed light on the synthesis and discovery of potential anticancer drug candidates. Keywords: Imidazo[2,1-b]thiazole, anticancer, molecular modeling

Figure 1: 2D ligand interaction diagram for inactive copound 5 (left), active compound 11 (middle), and native ligand (right)

39

References: 1. Ulusoy N. Synthesis and antituberculosis activity of cycloalkylidenehydrazide and 4-zza-1-thiaspiro[4.5]decan-3-one derivatives of imidazo [2,1-b]thiazole (2002) Arzneim-Forsch/ Drug Res; 52(7): 565-571. 2. Andreani A, Bonazzi D, Rambaldi M. Potential Antitumor Agents, VII. 5-Substituted 6-phenylimidazo[2,1-b]thiazoles (1982). Arch Pharm; 315: 451-456. 3. Ibrahim HS, Abou-Seri SM, Abdel-Aziz HA. 3-Hydrazinoindolin-2-one derivatives: Chemical classification and investigation of their targets as anticancer agents (2016). Eur J Med Chem; 122: 366-381. 4. Hongo F, Takaha N, Oishi M, Ueda T, Nakamura T. CDK1 and CDK2 activity is a strong predictor of renal cell carcinoma recurrence (2014). Urologic Oncology, 32: 1240-1246. 5. Zaman MS, Shahryari V, Deng G, Thamminana S, Saini S et al. Up-regulation of microRNA-21 correlates with lower kidney cancer survival (2012). Plos One; 7(4): e31060

40

OP-5 Discovery of New Sulfonyl Hydrazones with Potent Anticancer Agents in PC3

Cell Line Related to COX-2 Inhibition and Docking Studies

Sevil Şenkardeşa, İhsan Hanb, Özge Çevikc, Ş.Güniz Küçükgüzela

aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Haydarpaşa, İstanbul, TURKEY; bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Erciyes University,

Kayseri, TURKEY; cDepartment of Biochemistry, School of Medicine, Adnan Menderes University, Aydın, TURKEY.

E-mail: sevil.aydin@marmara.edu.tr Background: Cancer is a disease characterized by the formation and growth of abnormal cell caused by an uncontrolled cell proliferation. However, some drugs have been used for the cancer treatment. The side-effects, toxicity and the drug resistance of these drugs limit their use in the treatment. In our previous studies, some hydrazones were found to have remarkable anticancer activities [1-3]. Also, several studies have demonstrated the inhibition of COX-2 can prevent the tumor formation and COX-2 inhibitors could act as a chemotherapy agents. On the basis of these findings, we aimed to evaluate the biological activity of novel synthesized sulfonylhydrazones as antitumor agents related COX-2 inhibition. Methods: Fifteen 4-methyl-N'-(arylmethylidene)benzenesulfonyl hydrazides were synthesized by heating p-toluenesulfonylhydrazine with aromatic aldehydes. The characterization of the synthesized compounds were identified by the elemental analysis, IR, 1H-NMR and 13C-NMR spectral data while the purities of them will proved by TLC and HPLC. Novel synthesized sulfonylhydrazones in vitro using the MTT colorimetric method against prostate cell line PC-3 (androgen-independent human prostate cancer cell lines) and rat fibroblast cell L-929 at different doses for cell viability and growth inhibition for different doses. COX-2 (human) inhibitor screening assay kit was purchased from Cayman Chemical (MI, USA) and detected in comparison to celecoxib as a reference drug. Molecular docking studies were carried out to explore the possible binding modes of compound 14 in the active site of COX-2 enzyme (PDB code: 3LN1). Results: MTT assay demonstrated that compounds 12 (Ar=5-bromo-2-methoxyphenyl), 13 (Ar=4-fluoro-2-(trifluoromethyl)phenyl), 14 (Ar=2-chloro-3-methoxyphenyl) and 20 (Ar=2-(trifluoromethoxy)phenyl) showed anticancer activity (IC50=1.38-35.39 μM) on PC3 cells. Among them, compound 14 displayed the highest inhibitory activity against COX-2 in 50 �M dose (90% inhibition) and the highest anticancer activity against on PC3 cells (IC50=1.38 μM) compared with celecoxib (IC50=150 μM in PC3 cells and 100% COX-2 inhibition). The docking study suggested that compound 14 and celecoxib have similar COX-2 inhibitory effects in active region of the enzyme (Figure 1). The other compounds of the highest anticancer activity 12, 13 and 20 were tested for COX-2 inhibitory activity and showed good COX-2 inhibition (64-88% inhibition) in one dose. In addition, these potent compounds did not cytotoxicity towards L929 rat fibroblast cell.

41

Conclusions: These results indicated that there is a direct correlation between the COX-2 inhibition and anticancer activity potential of all active compounds as can be seen compound 14 displayed the highest COX-2 inhibitory effect and the best result in the MTT assay. As a result, compound 14 may be a promising lead candidate for further optimization. Keywords: Cancer, COX-2 enzyme, Molecular docking, MTT assay, Sulfonyl hydrazones. Acknowledgments: This work was supported by Research Fund of the Marmara University. Project number: SAG-K-120917-0495

Figure 1: General structure of synthesized compounds and interaction of compound 14 with COX-2 active site as 2D diagram. References:

1. Şenkardeş S, Kaushik-Basu N, Durmaz İ, Manvar D, Basu A, Atalay R, Küçükgüzel ŞG. Synthesis of novel diflunisal hydrazide-hydrazones as anti-hepatitis C virus agents and hepatocellular carcinoma inhibitors. (2016) Eur J Med Chem.; 108:301-308.

2. Küçükgüzel ŞG, Koç D, Çıkla-Süzgün P, Özsavcı D, Bingöl-Özakpınar Ö, et al. Synthesis of Tolmetin hydrazide–hydrazones and discovery of a potent apoptosis inducer in colon cancer cells. (2015) Arch Pharm.; 348: 730-742.

3. Çıkla P, Özsavcı D, Bingöl-Özakpınar Ö, Şener A, Çevik Ö, Özbaş-Turan S, Akbuğa J, Şahin F, Küçükgüzel ŞG. Synthesis, cytotoxicity, and pro-apoptosis activity of etodolac hydrazide derivatives as anticancer agents. (2013) Arch Pharm.; 346(5):367-379.

42

OP-6

Determination of drug targets in cancer cells with ATR-FTIR

Günnur Güler, Ercüment Karasulu

Center for Drug Research & Development and Pharmacokinetic Applications (ARGEFAR), Ege University, 35100, Izmir, Turkey

E-mail: gunnurgorucu@gmail.com

Background: Targeting specific molecules is the forefront of new drug development. Recently, targeted therapies have gained a great attention since drugs can exclusively interact with target molecules of cells, providing higher selectivity and lower toxicity [1,2]. Drug target structures can contains DNA, RNA, lipid, carbohydrates, peptide or a protein molecule, which play a key role in the intracellular signal transduction, involving directly in the mechanism of diseases [2,3]. In the present study, an infrared spectroscopic approach was used to find out drug targets (i.e, cellular metabolism, biochemical structures) in several cancer cells, which may promise novel strategies for efficient treatment. Methods: Fourier transform infrared spectroscopy combined with an attenuated total reflection (ATR-FTIR) unit was used for the analysis of several cancer cells types. Three independent cultures were grown for each cell type. The cells were washed three times with 0.9% NaCl for the ATR-FTIR analysis. A 1.8 μl amount of the cell suspension (about 1x106

cell/ml) was deposited on the ATR surface and dried at room temperature. The spectra were recorded in the range of 4000-800 cm-1. FTIR data processing and multivariate analysis were carried out using software Kinetics running under MATLAB. Results: ATR-FTIR data revealed that cancer cells exhibit significantly altered cellular biochemical structures when compared to their corresponding normal cells. These cellular changes are detected mainly in the lipid amount and composition, protein secondary structures, cell membrane dynamics and structures, altered nucleic acid conformation, carbohydrate composition or cellular metabolites, which can be crucial to improve the target specific therapies in cancer. Conclusions: A potential use of IR spectroscopy as a rapid and cost-effective technique in the pharmaceutical research area provided to gain new insights into the understanding of cancer cell features and metabolisms. Our study on probing of several cancer cells demonstrated that this IR technique is able to identify drug target candidates. Thus, this study may help to develop new therapeutic agents against cancer. Keywords: Drug targets, cancer, cell, ATR-FTIR spectroscopy, multivariate statistical analysis.

43

Figure 1: The ATR-FTIR spectrum of a cell in the mid-IR region, showing the signatures of cellular macromolecules (lipids, proteins, nucleic acids and carbohdyrates).

References:

[1] C. Marchetti, I. Palaia, M. Giorgini, C. De Medici, R. Iadarola, L. Vertechy, et al., Targeted drug delivery via folate receptors in recurrent ovarian cancer: a review, Onco. Targets. Ther. 7 (2014) 1223–36. doi:10.2147/OTT.S40947.

[2] H. Aktug, E. Acikgoz, A. Uysal, F. Oltulu, G. Oktem, G. Yigitturk, et al., Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells, Tumour Biol. (2016). doi:10.1007/s13277-016-5108-9.

[3] G. Güler, E. Acikgoz, N.Ü. Karabay Yavasoglu, B. Bakan, E. Goormaghtigh, H. Aktug, Deciphering the biochemical similarities and differences among mouse embryonic stem cells, somatic and cancer cells using ATR-FTIR spectroscopy, Analyst. 143 (2018) 1624–1634. doi:10.1039/c8an00017d.

44

OP-7

Plasma Membrane Changes of Human Monocytes Detected by Multicolor Flow Cytometry Coincide with FTIR Spectroscopy

Ayten NALBANT1† and Günnur GÜLER2

1 Molecular Immunology Laboratory, Department of Molecular Biology and Genetics, Izmir Institute of Technology, İzmir 35430, Turkey

2Center for Drug Research & Development and Pharmacokinetic Applications (ARGEFAR), Ege University, 35100, Izmir, Turkey

Email: aytennalbant@iyte.edu.tr Background: Flow cytometry is a high technology used to analyze the physical and chemical properties of liquid cells or particles as they pass through the laser. This technology can be used to study component of the cells that of interest including plasma membrane changes. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide better results to study plasma membrane changes of cells. In drug related studies, the influence of drugs on the plasma membrane structure and function is usually unnoticed since drugs are designed to target membrane proteins. The purpose of the study was to investigate the changes in the plasma membrane of human monocytes. THP-1 cells as a model for human monocytes has been used in this study since monocytes have several important functions to deal with pathogens in the immune system. Aberrant functions of monocytes can also contribute pathology of diseases. DNA topoisomerase I inhibitor Camptothecin (CPT) is used as drug to treat cells to induce apoptotic cell death. As a result plasma membrane changes of cells can be monitored. Methods: To do this, monocytes were grown to reach enough cell numbers. Washed and counted monocytes were cultured with 4μM Camptothecin (CPT) in RPMI (with 5% FCS) for 24 hours. CPT is used as drug to treat cells to induce apoptotic cell death. Therefore, plasma membrane changes of cells can be monitored. Immunoflourescence labeling of monocytes for Multicolor Flow cytometry was carried out with Annexin V concomitantly with 7-AAD. Same cells were also analyzed by Fourier Transform Infrared (FTIR) spectroscopy. Results: The presence of inner-membrane phosphatidylserine (PS) on the outer membrane of a cell is one of the earliest event in apoptosis. This process can be monitored by Annexin V, which binds to negatively charged PS, in conjunction with 7AAD, has strong affinity for DNA and is efficiently excluded by intact cells. According to the results of flow cytometry, CPTtreated monocytes were mostly Annexin V+/7AAD- phenotype compared to negative controls. This shows that the cells are in the early apoptosis stage. Moreover, since these cells are 7-AAD negative, the plasma membrane has not lost its integrity. Monocytes also displayed smaller forward light scatter (FSC) patterns, indicating the apperant decrease in cell size. Furthermore, FTIR results demonstrated pronounced alterations in the cell membrane components upon CPT treatment. The cell membrane phospholipids underwent dynamical and structural changes such as conformational disordering and increased mobility of the acyl chains (broad and upshift of the lipid CH2 signals). Additionally, the infrared signals of the CH2, ester C=O and PO2 groups of phospholipids are less intense.

45

These altogether indicates that both hydrophobic packing of tails and hydrophilic head groups of plasma membrane are considerably disturbed. Conclusions: Overall data showed that the quantitatively measured plasma membrane changes in human monocytes with Flow cytometry overlap with FTIR spectral signatures. (TÜBİTAK KBAG Grant # 215Z127 to Dr. Ayten Nalbant). Keywords: Human monocytes, plasma membrane changes, Flow cytometry and FTIR

46

OP-8

Repair of Rat Calvarial Bone Defect by Using Exosomes of Umbilical Cord Derived Mesenchymal Stem Cells Embedded in

Chitosan/Hydroxyapatite Scaffolds

Dilek Bahar a, Zeynep Burçin Gönen b, Menemşe Gümüşderelioğlu c, Mehmet Emin Önger d, Elvan Konuk c, Buket Banu Özkan a, Nur Seda Şahin a, Mustafa Çetin a,e

a Stem Cell Department Genome and Stem Cell Center (GENKOK), Erciyes University, Kayseri, Turkey b Oral and Maxillofacial Surgery, Genome and Stem Cell Center (GENKOK), Erciyes University, Kayseri, Turkey

c Department of Chemical Engineering, Hacettepe University, Ankara, Turkey d Department of Histology and Embryology, Medical School, Ondokuz Mayıs University, Samsun, Turkey

e Internal Medicine, Division of Hematology, Faculty of Medicine, Erciyes University, Kayseri, Turkey E-mail: ddilekbahar@gmail.com

Background: Bone is the second most frequently transplanted tissue with around one million procedures annually and the worldwide market of bone replacement material is currently estimated at five billion €. Although autogenous bone grafting is regarded as the gold standard, has limitations such as donor site morbidity, unpredictable graft resorption. Limited bone availability often impairs proper implant placement. Mesenchymal stem cells (MSCs) are appropriate to bone regeneration, based on its easy harvest and pluripotent capability. Exosomes are suggested to play as essential mediators of intercellular communication in physiological and pathophysiological processes. MSCs’ exosomes may have specific advantages to attenuate safety problems and have been recognized as new candidates for cell-free cellular therapy. The aim of this study was to seek out alternative off-the-shelf cellular therapy bone substitute for treating bone defects.

Methods: Human umbilical cord MSCs (hUC-MSCs) were cultured and characterized by using flow cytometry. Exosomes were purified by kit. The exosome morphology was verified by scanning electron microscopy (SEM), and size was determined by Zeta Sizer. Scaffolds were prepared by freeze-drying method. For chitosan scaffolds, chitosan (2%, w/v), was dissolved in 0.2M acetic acid solution and stirred for 2 days at room temperature, transferred into freeze-dryer at -80°C for 5 days. For chitosan/Hydroxyapatite (HA) scaffolds 0,75mg of powder form HA was added to 50mL chitosan solution. For chitosan/HA/exosome scaffold preparation, 30μL of exosome suspension with the exosome density of 2.5x105 exosome/scaffold were inoculated onto each stabilized chitosan and chitosan/HA scaffolds. SEM was used to observe the morphology, and Fourier transform infrared (FTIR) spectra were obtained for additives. The effects of the transplanted scaffolds were investigated in the repair of rat calvarial bone defect. A longitudinal incision of about 20mm was performed through sagittal suture. A diameter of 5mm calvarial bone defect was made bilaterally by standard trephin drill bit. The scaffolds were then transplanted. Twenty-five rats with bilateral defects were equally divided into five groups (group A: chitosan/exosome (n=10); group B: chitosan (n=10); group C: empty control (n=10); group D: chitosan/HA/exosome (n=10), group E: chitosan/HA (n=10)). All animals were euthanized at the postoperative sixth week. Samples were prepared using standard histological procedures.

47

The sections were photographed under a light microscope with an attached camera at x10 magnification. The Cavalieri method used to estimate the following parameters: volume of newly formed bone and volume of connective tissue. p<0.05 was considered statistically significant. Results: The SEM image showed that the exosomes were round-shaped vesicles with the membrane bounded and the diameter of exosomes was approximately 100 nm. Histologic analysis showed that mean new bone volumes were statistically significant with a mean bone formation of 1.50±0.14 (chitosan/exosome), 1.20±0.43 (chitosan), 1.06±0.10 (control), 1.83±0.54 (chitosan/HA/exosome) and 1.43±0.66 (chitosan/HA), respectively. The connective tissue volumes were also statistically higher in groups chitosan/HA/exosome. Conclusions: Chitosan/HA/Exosome combination is a novel for bone defect repair to induce bone formation. Chitosan scaffold can significantly promote bone regeneration in calvarial defects, and the combination with hydroxyapatite and exosomes is more promising for applications in bone tissue regeneration. Keywords: Bone Regeneration, Mesenchymal Stem Cells, Exosome, Chitosan/Hydroxyapatite, Scaffold

48

OP-9

Functional Modes of GluNR12 GluNR2A2 NMDA type Receptors

Muhammed Aktoluna, Ayhan Demira , Melis Gencela, Timothy S.Carpenterb , Sebnem Essiza aDepartment of Bioinformatics and Genetics, Kadir Has University, Istanbul, Turkey; bBiosciences and Biotechnology Division, Lawrence Livermore National Laboratory,

Livermore, CA, USA E-mail: sebnem.gokhan@khas.edu.tr

Background: Glutamate-bound cation selective channels are referred to as ionotropic glutamate receptors and mediate stimulating synaptic communication across the central nervous system. Heterotetrameric NMDA receptors (NMDARs), One of the subtype of ionotropic glutamate receptors are very important for neuronal communication [1]. heterotetramer structure consists of two dimers of dimers. There are seven different subunit types: NR1, NR2A-B-C-D and NR3A-B. Also The NMDA receptor is comprised of 4 regions named as; the amino terminal domain (ATD) and the ligand binding domain (LBD) outside the membrane, and the transmembrane domain (TMD) and the carboxyl terminal domain (CTD) within the cell (Figure 1) [2] . NMDAR have been associated with epilepsy, ataxia, migraine, schizophrenia, drug addiction, tolerance, and dependency and neurodegenerative diseases. For activation of the NMDA receptor, L-glutamate and glycine have to be bound to the ligand binding site. After this bounding, the clamshell-like LBD region closes and pushes the membrane-bound TMD to open the channel [3]. Although the structural studies of NMDA receptors are quite advanced in recent years, the gating mechanism is still not fully understood. So, A detailed atomistic understanding of the NMDAR activation, open-channel blockers’ binding site and its effects on channel dynamics will facilitate the design and development of improved inhibitors for excitotoxicity and neurodegeneration. Methods: The structural differences of two recently solved X-Ray structures of NMDA type glutamate receptors, [4, 5]are analyzed. Homology model of the human heterotetrameric NMDA receptor is modelled based on the template structures.1000 nanosecond Molecular Dynamics Simulations has been completed for halo and apo forms of the whole intact receptor. Results: Structural changes observed in amino terminal domain and ligand binding domain is reflected to ion channel region. The channel remained open for approximately 100 nanoseconds in the ligand-bound simulation. In the halo simulation, the LBD region rotate as more rigid bodies. the interaction between of ATD and TMD fragments has increased as a result of the movement of two lobes of LBD together due to ligand binding. Keywords: molecular dynamics, neurotransmitter, NMDA, membrane proteins, protein structure modeling

49

Figure 1: NMDAR subunits illustration. References:

1. Paoletti, P., C. Bellone, and Q. Zhou, NMDA receptor subunit diversity: impact on receptor

properties, synaptic plasticity and disease. Nat Rev Neurosci, 2013. 14(6): p. 383-400. 2. Ryan, T.J., et al., Evolution of NMDA receptor cytoplasmic interaction domains: implications for

organisation of synaptic signalling complexes. BMC Neurosci, 2008. 9: p. 6. 3. Traynelis, S.F., et al., Glutamate receptor ion channels: structure, regulation, and function.

Pharmacol Rev, 2010. 62(3): p. 405-96. 4. Karakas, E. and H. Furukawa, Crystal structure of a heterotetrameric NMDA receptor ion

channel. Science, 2014. 344(6187): p. 992-997. 5. Lee, C.H., et al., NMDA receptor structures reveal subunit arrangement and pore architecture.

Nature, 2014. 511(7508): p. 191-7.

R1

R2

D1

D2

1 432

ATD

LBD

TMD

GluNR1 GluNR2

50

OP-10

OmpG-16SL, an engineered porin, investigated using infrared spectroscopy

İrem Yılmaza, Özkan Yıldızb, Filiz Korkmazc a Middle East Technical University, Department of Physics, Ankara, Turkey

bMax Planck Institute for Molecular Biology, Max-von-Laue Str.1, Frankfurt am Main, Germany

c Atilim University, Physics Unit, Biophysics Laboratory, 06836, Ankara, Turkey E-mail: filiz.korkmaz@atilim.edu.tr

Background: Differing from other classical porins by being monomeric, Outer membrane protein G (OmpG) from E.coli has a 14-stranded beta barrel form. It allows molecule entrance up to 900 Da having a gating mechanism with changing pH. Below pH 6 (closed state), one of its loops (loop L6) closes onto the pore and blocks molecule trafficking while above pH 6 (open state), molecule uptake is allowed. These characteristics show that OmpG has a promising potential for nanopore biosensor and targeted drug delivery applications. Methods: The structural and functional differences between wild type (WT) and two mutants are investigated with Fourier transform infrared spectroscopy (FTIR). The comparison between proteins is made in terms of pH-dependent conformational changes, thermal stability and H/D exchange profiling. Three proteins are prepared in Na-phosphate buffer with pD 5.5/7.5 and infrared spectra are recorded at adjusted thermal and exchange conditions. Results: When pD is switched from acidic to neutral, the shifting and splitting of beta sheet signals point out that all proteins respond to pH change but at different degrees. At acidic environment, all proteins contain the same number of residues participated in beta sheet content. However, at neutral pH, no increase in beta sheet content implies that two mutants attain a new globular arrangement. Moreover, the thermal stability results show that mutants at neutral pH differs from WT. In addition, in two mutants for both pH values, H/D exchange rates are similar with WT at open state. This indicates that replaced arginines in the mutant OmpG-16SL did not create an electrostatic barrier to prevent excess water entering the pore. Conclusions: The intention of creating a larger pore having the same structural stability and gating mechanism is only partially fulfilled since the degree of changes is not enough to gain a fully closed form. However, the findings in two mutants provided deeper insight about the working principle of the WT porin. Keywords: porin, infrared spectroscopy, protein engineering, membrane protein

51

OP-11

In Silico Development of Phosphorylation-Independent Arrestins Via Small Molecules

Mehmet Hanifi Kurta, Giulia Morrab, Ozge Sensoyc

a Biomedical Engineering and Bioinformatics, Istanbul Medipol University; b Weill Cornell Medicine Department of Physiology and Biophysics;

c Department of Computer Engineering School of Engineering and Natural Sciences, Istanbul Medipol University

E-mail: mehmethanifikurt@gmail.com Background: G-protein-coupled receptors (GPCR) are responsible for maintaining communication between cell and its surroundings, which depends on the balance between proper signal initiation and termination. The latter function is fulfilled by Arrestin (Arr) via binding to activated and phosphorylated receptor (desensitization). In spite of being a small family (Arrestin1, 2, 3, and 4), the members display significant differences in their preferences towards the receptor phosphorylation for binding. In particular, Arr3`s requirement is lower than the others and it depends on the type of the receptor. For instance, it can bind to non-phosphorylated muscarinic 2 receptor in the absence but not to beta-2-adrenergic receptor (β2AR). An abrupt increase in agonist concentration, as in the case of congestive heart failure, can cause some anomalies in the phosphorylation pattern of the receptor, in particular β2AR. This leads to over-stimulation of the receptor, hence its down-regulation. Here, we aim at providing phosphorylation-independent binding of Arr3 to β2AR by means of small molecules to maintain receptor density on the membrane, hence preventing down-regulation. Methods: Firstly, atomistic molecular dynamics simulations of Arr3 is done. Then, representative structures are picked up from trajectories. This is followed by pocket search on the protein surface to which small molecules bind (See Figure 1). Subsequently, pharmacophore analysis is made on the pockets. Afterwards, candidate molecules are retrieved from ZINC database [1]. The candidate molecules are docked into the pockets and binding stability is investigated by calculating binding free energy via steered molecular dynamics simulations [2] with application of Jarzynski's equality [3]. Results: The pockets are found to be overlapped with regions which are involved in activation of Arr3. The binding constant of candidate molecules is found in the nanomolar range which suggests that they might be used as therapeutics if they succeed in vitro experiments

52

Figure.1: Pockets from selected structures Keywords: Arrestin Phosphorylation-independent, Cognestive heart failure, GPCR, Pharmacophore

References: [1] T. Sterling and J. J. Irwin, “ZINC 15 - Ligand Discovery for Everyone,” J. Chem. Inf. Model., 2015. [2] G. Scarabelli, G. Morra, and G. Colombo, “Predicting interaction sites from the energetics of isolated proteins: A new approach to epitope mapping,” Biophys. J., 2010. [3] S. Park and K. Schulten, “Calculating potentials of mean force from steered molecular dynamics simulations.,” J. Chem. Phys., 2004.

53

OP-12

Synthesis of A Novel Plant Origin Radiolabeled Compound and Its in vitro Determinations on Neuroblastoma Cell Lines

K. Busra KARATAYa, Fazilet Zumrut BIBER MUFTULERa, Ayfer YURT KILCARa

aEge University, Nuclear Application, Institute of Nuclear Sciences 35100 Bornova, Izmir/TURKEY E-mail: busrakaratay5@gmail.com

Background: Use of plants in the treatment of many diseases is increasing day by day. There are several plants that can be used for almost every type of disease diagnosis and treatment. One of the plants known to have positive effects on healthy nutrition is peanuts in the bean / legume family (Fabaceae). It is known that in the researches carried out, the peanut is rich in compounds such as resveratrol, phenolic acids, flavonoids and phytosterols, and contains high amounts of arginine1. Peanut sprouts; is an excellent source for resveratrol acting as a chemical preventive against cardiac disorders2, neurodegenerative diseases3 and various types of cancer1,4. Also, peanut sprouts are known to be richer in terms of phytochemicals, proteins, vitamins, minerals and resveratrol than peanuts5,6. Some of the radionuclides used in Nuclear Medicine can be directed to targeted cancerous tissues with the molecules which are selective for these tissues. In the literature, there are many scientific researches that examine the bioactivities of specific plant-derived compounds on specific organs and tissues by labeling them with appropriate radioisotopes on various cancer types7,8. Iodine-131 (131I) radioisotope is used for therapy and imaging with suitable half-life (8days) and gamma energy (364 keV)9. In present study, it is aimed to extract the main bioactive component (Resveretrol (RES)) of peanut sprouts, to radiolabel it with 131I and to determine its bioaffinity by using in vitro methods. Methods: In this study, peanut sprout seeds were germinated and sprouted. After that, collected peanut sprouts were dried and extracted10. Resveratrol (RES) which is one of the compound from peanut sprouts was isolated by High Performance Liquid Chromatography (HPLC) method and verified by using RES standard. The radioiodination of the RES was carried out with 131I radionuclide11. Stability and lipophilicity studies were performed by radiolabeled compound (131I-RES). Afterwards, quality control studies were done by using thin layer radio chromatography (TLRC) method to determine the radiolabeling potentials of 131I-RES. In vitro studies of 131I-RES were examined with human neuroblastoma (SY-SH5Y and SK-N-AS) cell lines. Results: As a result of extraction operation, RES which is one of the bioactive components in peanut sprouts, was successfully (98.6 %) purified from peanut sprouts. 131I-RES were obtained in high yields over 97 %. The % binding yield efficiencies of the 131I-RES at 0., 30., 60., 120., 240. and 1440 minutes were determined using the TLRC method and the time changes were examined. Experimental studies have shown that 131I-RES is lipophilic and the theoretical and experimental lipophilicity results were concordant. The binding activity of 131I-RES compound was investigated on SY-SH5Y and SK-N-AS neuroblastoma cell lines.

54

Conclusions: In recent years, the development of new brain imaging agents that can cross the blood brain barrier (BBB) has been of great interest because of the limited number of diagnostic agents for brain imaging. Studies have shown that resveratrol can cross the BBB and act as a neuroprotective agent against neurodegenerative disease12,13. Our study assesses whether the designed 131I-RES molecule has any potential for imaging and treatment of neurodegenerative disorders. ‘This project is supported by Ege University Scientific Research Projects Coordination (Project number is FYL2018-20081)’ Keywords: Peanut Sprouts, Resveratrol, Iodine-131 (131I), Neuroblastoma (SY-SH5Y, SK-N-AS) Cell Lines, Radiolabeled RES (131I-RES). References:

1. Arya SS, Salve AR, Chauhan S. Peanuts as functional food: a review. (2016) J Food Sci Technol.; 53(1): 31–41.

2. Juan ME, Vinardell MP, Planas JM. The daily oral administration of high doses of trans-resveratrol to rats for 28 days is not harmful. (2002) The J Nutr.; 132(2):257–260.

3. Chen J, Zhou Y, Mueller-Steiner S, Chen LF, Kwon H, Yi S, Mucke L, Gan L. SIRTI projects against microglia-dependent amyloid-b toxicity through inhibiting NF-kB signaling. (2005) J Biol Chem.; 280(48):40364–40374

4. Delmas D, Lançon A, Colin D, Jannin B, Latruffe N. Resveratrol as a chemopreventive agent: a promising molecule for fighting cancer. (2006) Curr Drug Targets.; 7(4):423–442

5. Kim HJ, Park KJ, Lim JH. Metabolomic analysis of phenolic compounds in buckwheat (Fagopyrum esculentumM.) sprouts treated with methyl jasmonate. (2011) J Agric Food Chem.; 59(10):5707-13.

6. Limmongkon A, Janhom P, Amthong A, Kawpanuk M, Nopprang P, Poohadsuan J, Somboon T, Saijeen S, Surangkul D, Srikummool M, Boonsong T. Antioxidant activity, total phenolic, and resveratrol content in five cultivars of peanut sprouts. (2017) Asian Pac J Trop Biomed.; 7(4): 332–338.

7. Bapat K, Chintalwar GJ, Pandey U, Thakur VS, Sarma HD, Samuel G, Pillai MRA, Chattopadhyay S, Venkatesh M. Preparation and in vitro evaluation of radioiodinated bakuchiol as an anti tumor agent. (2005) Appl. Radiat. Isot.; 62(3), pp. 389,393.

8. Yurt Kilcar A, Cekic B, Biber Muftuler FZ, Unak P, Medine I. In vitro evaluation of radiolabeled (125I) methanol extracts of yarrow in cell lines of MCF-7, PC-3, A-549 and Caco-2. (2013) J. Radioanal. Nucl. Chem.; 27(5), pp. 294,300

9. Saha GB. Fundamentals of Nuclear Pharmacy. (2004) New York, Springer.; 5. Edition, 383p. 10. Xiong Q, Zhang Q, Zhang D, Shi Y, Jiang C, Shi X. Preliminary separation and purification of resveratrol

from extract of peanut (Arachis hypogaea) sprouts by macroporous adsorption resins. (2014) Food Chem.; 145 (2014) 1–7.

11. Biber Muftuler FZ, Yurt Kilcar A, Unak P. A Perspective on Plant Origin Radiolabeled Compounds, Their Biological Affinities and Interaction between Plant Extracts with Radiopharmaceuticals. (2015) J Radioanal Nucl Chem.; 306:1-9.

12. Wang Q. Resveratrol protects against global cerebral ischemic injury in gerbils. (2002) Brain Res.; 958(2):439–447.

13. Sun AY. Resveratrol as a therapeutic agent for neurodegenerative diseases. (2010) Mol. Neurobiol.; 41 (2–3): 375–383.

55

OP-13

Drug Re-Positioning Studies for Novel HIV-1 Inhibitors Using Binary QSAR Models and Target-Driven In Silico Studies

Berna Doğan, Serdar Durdagi

Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey

E-mail: berna.dogans@hotmail.com

Background: Human immunodeficiency virus (HIV) which leads to Acquired Immunodeficiency Syndrome (AIDS) is still the cause of approximately one million deaths annually and currently more than 35 million people are living with HIV infections [1]. While these numbers have been decreasing thanks to many research studies on disease treatment methods, the quick resistance development nature of the virus to drugs is causing significant problems. Considering also the unexpected side effects of some existing drugs, there is always a need and search for new and preferably better drug molecules. Taking into account the expenses and the time necessary to develop a new drug molecule, drug repurposing which consists of giving existing approved/investigational drugs a new indication would be suitable [2]. In this study, we consider the small compounds that are already in use as drugs or in clinical trials against various types of diseases and screen them as potential anti-HIV compounds. Methods: NCGC Pharmaceutical Collection (NPC) is used as the source of approved and investigational drugs for high-throughput screening [3] and 7922 molecules downloaded. Using binary QSAR models of MetaCoreTM/MetaDrugTM [4] platform, their therapeutic activity against HIV are predicted. The potential hit compounds are then subjected to various ADME/toxicity tests using again MetaCoreTM/MetaDrugTM and approved HIV drugs are used as reference compounds. The compounds with acceptable levels of ADME/toxicity are considered for docking studies. Two target proteins that are important in the life cycle of HIV (shown in Figure 1) are considered here; HIV protease enzyme and chemokine co-receptor CCR5 which plays a vital part in the entry of virus to host cell. The protein-ligand docking studies performed using GOLD molecular docking platform [5]. The top hits for all considered molecules are then subjected to Molecular Dynamics (MD) simulations for 1 ns using Schrödinger Desmond Suite [6]. Using MMGBSA method, the binding free energies for each protein-ligand complex are calculated. The final molecules to be suggested are then subjected to long (200ns) MD simulations to determine the binding modes. Results: Current therapies against HIV, antiretroviral therapy (ART), involves the usage of at least two drugs that belong to the different classes, i.e. according to which stage of HIV life cycle they target (stages shown in Figure 1). This however most likely increases the side effects of the drugs and unwanted drug interactions. It would be preferable to have a multi-target drug candidate. Hence, we selected the molecules that have shown to interact with both protein targets; CCR5 chemokine co-receptor and HIV protease enzyme. The protein-ligand complexes are filtered based on their binding free energies as well as the docking scores of different fitness functions of GOLD docking algorithm. Conclusions: At the end, seven molecules of which two are approved drugs and five are investigational drugs are suggested for usage against HIV. The results of this study may open new avenues for the designing of new HIV-1 inhibitor scaffolds. (This study has been financially funded by Scientific and Technological Research Council of Turkey (TUBITAK) under the program of TUBITAK-2218) Keywords: HIV, protein-ligand docking, binding free energy, Molecular Dynamics simulations

56

Figure 1: The life cycle of HIV (taken from http://aidsinfonet.org/fact_sheets/view/106) References: 1. UNAIDS AIDSinfo–UNAIDS. http://aidsinfo.unaids.org/ (accessed 27.10.2018). 2. Mercorelli B, Palu G, Loregian A: Drug Repurposing for Viral Infectious Diseases: How Far Are We? (2018) Trends Microbiol; 26(10): 865-876. 3. Huang R, Southall N, Wang Y, Yasgar A, Shinn P, Jadhav A, Nguyen D-T, Austin C P: The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. (2011) Sci Transl Med; 3(80): 80ps16-80ps16. 4. Ekins S, Andreyev S, Ryabov A, Kirillov E, Rakhmatulin E A, Sorokina S, Bugrim A, Nikolskaya T: A combined approach to drug metabolism and toxicity assessment. (2006) Drug Metab Disposition; 34(3): 495-503. 5. Jones G, Willett P, Glen R C, Leach A R, Taylor R: Development and validation of a genetic algorithm for flexible docking. (1997) J Mol Biol; 267(3): 727-748. 6. Bowers K J, Chow E, Xu H, Dror R O, Eastwood M P, Gregerson B A, Klepeis J L, Kolossvary I, Moraes M A, Sacerdoti F D, Salmon J K, Shan Y, Shaw D E In Proceedings of the 2006 ACM/IEEE conference on Supercomputing, Tampa, Florida, ACM: Tampa, Florida, 2006; p 746.

57

OP-14

Cellular Uptake of Multi Wall Carbon Nanotubes, Protein Loading and Drug Loading Qualification

Ayhan Ünlüa, Bircan Dinçb, Muhammet Bektaşc

aTrakya University, Faculty of Medicine, Department of Biophysics, Edirne, Turkey bAltinbas University, Faculty of Engineering and Natural Sciences, Department of Basic Sciences,

Istanbul, Turkey cIstanbul University, Istanbul Faculty of Medicine, Department of Biophysics, Istanbul, Turkey

E-mail: bircan.dinc@altinbas.edu.tr Background: Carbon nanotubes (CNTs) are special nanomaterials grouped into single-walled (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). MWCNTs are used more frequently in industrial and medical areas than SWCNTs by virtue of their facile synthesis. Pristine MWCNTs have low solubility in aqueous medium. Surface functionalization changes dispersion in medium and gives better results for cellular uptake. When CNTs are produced suitable diameters and lengths, they are perfect carriers for drugs and molecules with needle like structure, wide surface area for molecular binding. Functionalized MWNTs suitable for cellular uptake and molecule transportation. Bovine serum albumin(BSA) and wide spectrum cancer drug, Doxorubicin are loaded MWCNTs, amount of binding and cellular uptake are shown in assays. Methods: MWCNTs were purchased from Nanografi and they are functionalized with acid treatment. MWCNTs are characterized by taking TEM images and using FTIR spectroscopy. Formation of BSA protein corona and loading of Doxorubicin were performed by non-covalent binding of molecules via π-stacking. Amount of binding BSA and Doxorubicin was analyzed with thermogravimetric analysis (TGA) and binding curve was obtained. FT-IR spectrum was obtained before and after binding. Cellular uptake of MWCNTs was displayed by fluorescent microscope images of nucleus and actin structure on MDA-MB-231 breast cancer cell line. Results: Characteristic peaks and change in the peaks according to -COOH groups, before and after functionalization were displayed in FTIR spectroscopy. We observed majör peaks after functionalizatin for carboxyl groups (O=C−OH and C−OH). Weight loss during TGA measurements was between 200-500 0C and it started degradation 220 0C, starting temperature for BSA. Doxorubicin degradation was found to happen along three steps that started at ~230, 320 and 400 0C. The TGA curve showed strong interaction between MWCNTs with BSA and Doxorubicin. Cellular uptake images showed penetration of CNTs even nucleus of the cells.

58

Figure 1: Cellular uptake of MWCNTs of MDA-MB-231 cells

Conclusions: Our results demonstrated capability of MWNTs for binding protein and drug molecules and provided insight for strategic design of nanotube-based molecule delivery sytems. Functionalized MWNTs can show strong interaction between Doxorubicin and Bovine Serum Albumin due to carboxyl groups on the surfaces. Carrying biomolecules at their surface have been provided necessary knowledge to make them an ideal material for targeted delivery and particular intracellular biosensing applications.

Keywords: MWCNT, BSA, Doxorubicin, Cellular Uptake References:

1. Farahanı B, Vasheghani B and Javadı N, Functionalized multi walled carbon nanotubes as a carrier for doxorubicin: drug adsorption study and statistical optimization of drug loading by factorial design methodology. J. Braz. Chem. Soc. ( 2016) 27 ( 4) : 694-705

59

OP-15

Engineering Plus-end-directed Dyneins

Mert Gur1, Sinan Can2,*, Samuel Lacey3,*Andrew P. Carter3, Ahmet Yildiz2,4 1Department of Mechanical Engineering, Istanbul Technical University, Istanbul 34437 Turkey

2Physics Department, University of California at Berkeley, Berkeley CA 94720 USA. 3Medical Research Council Laboratory of Molecular Biology, Division of Structural Studies,

Francis Crick Avenue, Cambridge, CB2 0QH, UK. 4Department of Molecular and Cellular Biology, University of California at Berkeley,

Berkeley CA 94720 USA. *These authors contributed equally to this work.

The complexity of eukaryotic cells requires intracellular organization, coordination, and locomotion. To overcome these challenges, cells utilize ATP-driven molecular motors, which transport intracellular components unidirectionally along cytoskeletal tracks. Kinesin and cytoplasmic dynein motors facilitate bidirectional transport of a variety of cargos by moving towards the plus- and minus-ends of microtubules (MTs), respectively. A comprehensive mechanistic understanding of major cellular events, such as mitosis and cell polarization, requires a detailed understanding of both classes of motors.

Dynein is currently the focus of much of the motor biophysics research as the mechanism of its movement is not well understood in comparison to kinesin. Experimental studies on S. cerevisiae dynein motility presented the first models of its stepping, processivity, force generation and the catalytic cycle. Yet, atomic details of how the ATP hydrolysis cycle triggers the structural rearrangements of the dynein motor domain, and these structural changes lead to release from the MT track, stepping forward, rebinding to MT, and generate force and unidirectional motility remain unknown. In order to understand the mechanism of dynein’s directionality, we sought to engineer a plus-end-directed dynein guided with molecular dynamics simulations and with cryo-electron microscopy. This talk will focus on the modeling part of this joint work while presenting how our experimental data are supporting our computational findings and vice versa. By changing the length and angle of the stalk, we successfully reversed the motility from the MT minus-end towards the MT plus-end. Our simulations suggest that altering the direction of the priming stroke generated by dynein’s linker relative to MT is key to reversing the dynein direction along MT.

60

OP-16

GASPIDs (Granule Associated Serine Peptidases in Immune Defence): From Pathophysiology to novel therapeutic targets

Jamshaid Ahmada, Saima Ikramb, Fawad Ahmadc, Irshad Ur Rehmand, Saeedullah Khattake

a,b,c,d,eCentre of Biotechnology and Microbiology, University of Peshawar, KPK, Pakistan. Email: jamshaidbiotech@yahoo.com

Immune system provides protection against diseases by identifying and killing pathogens, infected and tumorous cells. Variety of immune cells like; cytotoxic lymphocytes including cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells, mast cells and neutrophils, mediate this defence system. These immune defence cells express variety of serine proteases, known as GASPIDs (Granule Associated Serine Peptidases of Immune Defence) [1]. Best-characterised GASPIDs include; neutrophil elastase, cathepsin G, proteinase-3, granzyme A, B & H, mast cell chymase and tryptase, however little is known about other members. Although GASPIDs are highly related however they show different substrate specificities implying different biological functions. GASPIDs are synthesized as single domain serine proteases, encompassing a leader sequence, N-terminal pro-dipeptide, followed by a mature protein sequence. Being serine proteases, the active site of GASPIDs contains three important amino acids residues (His-57, Asp-102, Ser-195 chymotrypsin numbering) that constitute the catalytic triad. In some GASPIDs, in addition to the N-terminal pro-peptide, there is also a C-terminal pro-domain of variable length, that needs to be removed during activation of these proteases, however, the exact role of this C-terminal tail is not know. GASPIDs perform different intra– and extracellular functions. Some of the intracellular roles, that are dependent on perforin (pore forming protein)-mediated delivery into target cells, have been described as: cytotoxicity, accessory functions and cytokine signalling. In their cytotoxic role, they have the ability to initiate direct killing of target cells by activating intrinsic apoptotic pathways and is most potently induced by cytotoxic lymphocyte originated GASPID i.e. granzyme B [2, 3], while accessory functions are best described by granzyme-H, which degrades a virally encoded granzyme-B inhibitor, thus enabling granzyme-B to function [4]. In their cytokine-signalling role, they have the ability to facilitate the maturation and release of immuno-modulating cytokines thereby generating inflammatory response for the recruitment of other immune cells [5]. The extracellular functions, that are independent of perforin, mostly involve cleavage of extracellular matrix proteins and cell surface receptors, possibly to facilitate immune cells migration [6]. In addition, these extracellular proteases also activate cytokines. The extracellular GASPIDs are generated either constitutively by immune cells or when they escape from immunological synapse during granule exocytosis pathway. Initially thought as immune facilitator, these proteases act in a substrate specific manner. However in recent years the paradigm has now been changed and various studies have revealed over expression of these proteases in autoimmune, chronic inflammation, cardiovascular and other metabolic disorders [7-10]. The over expression of these proteases worsens disease conditions by creating highly proteolytic environment [10]. Screening novel inhibitors against these proteases can be promising and may open a new research interests.

61

The present study will review in detail the regulation of GASPIDs by endogenous inhibitors and their overexpression in various disease conditions with possible implications. Such understanding can be of paramount importance in designing and developing novel therapeutic inhibitors with low risk for immunogenicity in humans. Keywords: GASPIDs, Granzymes, Chymases, Neutrophilic proteases References: 1. Ahmad, J., P.I. Bird, and D. Kaiserman, Analysis of the evolution of granule associated

serine proteases of immune defence (GASPIDs) suggests a revised nomenclature. Biol Chem, 2014. 395(10): p. 1253-62.

2. Goping, I.S., et al., Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition. Immunity, 2003. 18(3): p. 355-65.

3. Sutton, V.R., et al., Caspase activation by granzyme B is indirect, and caspase autoprocessing requires the release of proapoptotic mitochondrial factors. Immunity, 2003. 18(3): p. 319-29.

4. Andrade, F., et al., Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition. EMBO J, 2007. 26(8): p. 2148-57.

5. Metkar, S.S., et al., Human and mouse granzyme A induce a proinflammatory cytokine response. Immunity, 2008. 29(5): p. 720-33.

6. Buzza, M.S. and P.I. Bird, Extracellular granzymes: current perspectives. Biol Chem, 2006. 387(7): p. 827-37.

7. Korkmaz, B., et al., Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol Rev, 2010. 62(4): p. 726-59.

8. Hendel, A., et al., Granzymes in age-related cardiovascular and pulmonary diseases. Cell Death Differ, 2010. 17(4): p. 596-606.

9. Granville, D.J., Granzymes in disease: bench to bedside. Cell Death Differ, 2010. 17(4): p. 565-6.

10. Shen, Y., et al., Topical small molecule granzyme B inhibitor improves remodeling in a murine model of impaired burn wound healing. Exp Mol Med, 2018. 50(5): p. 68.

62

OP-17

Efficient short splice-switching morpholino antisense oligonucleotides for the treatment of Duchene Muscular Dystrophy

Ugur Akpulata,b,c , Haicui Wanga,b, Kerstin Beckera,b,James Novakd, Terence

Partridged, Sebahattin Ciraka, b

aCenter for Molecular Medicine Cologne (CMMC), University of Cologne, German; bUniversity Hospital Cologne, Department of Pediatrics, Cologne, Germany; cDept. of Medical Biology, Faculty of Medicine, Kastamonu University, Turkey; dChildren’s National Health System, Children’s Research Institute, Center for Genetic

Medicine Research, Washington DC, USA (Times New Roman, 11-Font-italic) E-mail: uakpulat@gmail.com, sebahattin.cirak@uk-koeln.de

Duchenne muscular dystrophy (DMD) is an X-linked recessive lethal muscle-wasting disease affecting 1:3500-5000 newborn males and caused by out-of-frame mutations in DMD. Milder phenotype of the allelic disease, Becker muscular dystrophy (BMD) caused by in-frame mutations has served as model for exon skipping strategy as a promising therapeutic attempt for DMD patients. Morpholino antisense oligonucleotides (PMO) have been employed to exclude disruptive exons from the mutant DMD transcript has elicited the restoration of truncated dystrophin, but partly functional protein in analogy to BMD, in dystrophic muscle. Recently, the FDA conditionally approved Eteplirsen, a 30-nucleotide PMO, for exon 51 skipping that is amenable for 13% of all DMD patients. However its clinical beneficial effect for restoring dystrophin protein in DMD patients remains debatable. Systemic delivery of Eteplirsen to DMD patients has shown a dose dependant response, better for higher doses but with high variability and patchy dystrophin expression (Cirak et al., 2011). To identify the factors leading to this variability, we investigated the influence of myofiber regeneration on exon skipping by treating dystrophin-null mdx mice with a high-dose of morpholino together with timed pulses of BrdU. Robust PMO uptake and efficient elicitation of dystrophin expression was localized to lesions regenerating during the 3 days prior to, or coincident with, PMO administration. PMO accumulation was exclusive to inflamed regions where it entered inflammatory cells, differentiating myoblasts and newly forming myotubes. We conclude much of the variability in PMO-induced dystrophin expression reflects the limitations on delivery imposed by the need for two concomitant events: first, release of PMO from the vascular bed into the interstitium by inflammatory exudation associated with myopathic lesions; second, fusion of PMO-loaded differentiated myoblasts into the newly repairing segments of muscle fibres (Novak et al., 2017). Clinical trials proofed that the PMO backbone safe for systemic delivery in men but higher doses of Eteplirsen could not be administered due to excessive economic costs (about $300,000/patient annually). Sufficient levels of dystrophin restoration require high dose PMO administration because PMO uptake is mainly localised to regenerating sites of skeletal muscle. Reducing the length of the PMO would make their production more economic and allow higher dosing. We designed shorter 25-mer PMOs targeting same region as Eteplirsen on the pre-mRNA of DMD to compare their efficacies on dystrophin-null exon 52 deleted mouse myoblast cell line in-vitro and mdx52 mice in-vivo. All of three shorter PMOs and Eteplirsen has shown dose dependent response, while skipped-dystrophin induction was similar in amount at higher doses only one of them was comparable with Eteplirsen at lower doses.

63

Our findings are of importance to improve PMO delivery and reduce variability for exon skipping mediated therapies, especially for DMD and other inherited primary muscle diseases. Shorter PMO antisense oligonucleotides would allow higher doses at the same economic cost and lead to higher levels of dystrophin restoration in DMD muscle and ultimately increase the clinical efficacy. Keywords: Exon Skipping, Duchenne Muscular Dystrophy, Eteplirsen, PMO, Splice Switching Oligonucleotides References:

1. Cirak et al., 2011, The Lancet 2. Novak et al., 2017, Nature Communication

64

OP-18

BSA binding and in silico studies of synthesized azinane based 1,3,4-oxadiazole derivatives: Pharmacological Evaluation

Javed Iqbala, Aziz-ur-Rehmana, Hira Khalidb

a Department of Chemistry, Government College University Lahore, Pakistan-1,2; b Department of Chemistry, Government College University Lahore, Pakistan-3

E-mail: dr.hknajmee@gmail.com: hirakhalid@fccollege.edu.pk A series of compounds have been synthesized bearing pharmacologically important 1,3,4-oxadiazole and piperidine moieties. The spectral data analysis by 1H-NMR, 13C-NMR, IR and EI-MS elucidated the structures of synthesized molecules. Docking studies justified the different types of interaction of the presented compounds with amino acids while bovine serum albumin (BSA) binding interactions verified their pharmacological effectiveness. The antibacterial screening of these compounds demonstrated excellent to moderate activity against S. typhi and B. subtilis but moderate to weak activity against the remaining three bacterial strains taken into account. Seven compounds were the most active members as acetyl cholinesterase inhibitors. The outstanding action of the all the presented compounds was received against the urease inhibition. Compounds 7l, 7m, 7n, 7o, 7p, 7r, 7u, 7v, 7x and 7v were highly active ones showing extreme level of activity against urease enzyme with IC50

values of 2.14±0.003, 0.63±0.001, 2.17±0.006, 1.13±0.003, 1.21±0.005, 6.28±0.003, 2.39±0.005, 2.15±0.002, 2.26±0.003 and 2.14±0.002 μM as compared to the thiourea used as reference standard with IC50 value of 21.25±0.15 μM and would be able to replace the existing drugs after their comprehensive in vivo studies.

Keywords: BSA-Binding, Piperidine, Oxadiazole

Figure 1. Docked conformation of AChE inhibitors 7x (Cyan), 7x (Magenta) super-positioned over experimental conformation of donepezil (Magenta) in AChE main binding cavity. References 1. H. Khalid & A. Rehman, et al, Synthesis, Biological Evaluation And Molecular Docking Of N\'- (Aryl/alkylsulfonyl)-1-(Phenylsulfonyl) Piperidine-4- Carbohydrazide Derivatives. (2014) Turk J Chem.; 38, 189-201 2. H. Khalid, et al, Synthesis, Spectral Analysis And Anti-Bacterial Study Of N-Substituted Derivatives Of 2-(5-(1(Phenylsulfonyl)Piperidin-4- Yl)-1,3,4-Oxadiazol2-Ylthio)Acetamide. (2016) J Saudi Chem Soc.; 20, S615-S623

65

OP-19

Integrative Analysis of Transcriptome Data and Genome-Scale Metabolic Networks to Identify Candidate Drug Targets and

Drugs for Parkinson’s Disease

Ali Kaynara, Işıl Aksan-Kurnazb, Tunahan Çakıra

aGebze Technical University, Department of Bioengineering, Gebze, Kocaeli bGebze Technical University, Department of Molecular Biology and Genetics, Gebze, Kocaeli

E-Mail: akaynar@gtu.edu.tr Background: Integrative analysis of genome-wide data with molecular interaction networks has high potential to reveal molecular mechanisms of diseases and predict candidate biomarkers, drug targets and drugs. Methods: In this work, we use transcriptome datasets from Parkinson's Disease patients, obtained from public database NCBI Gene Expression Omnibus, to investigate predictive nature of the brain-specific genome-scale metabolic network, iMS570. A modified and compartmented version of the model called iBrain575 that incorporates alpha-synuclein accumulation was used with molecular crowding constraint to predict highly-probable biomarkers, drug targets and drugs. The computational method called Gene Inactivity Moderated by Metabolism and Expression (GIMME) is first used to find inactive reactions in the model by mapping the transcriptome data on the metabolic network. Using this method, a context-specific set of reactions are identified, which is further used for the prediction of metabolic flux distribution specific to Parkinson's Disease. The approach called Minimization of Metabolic Adjustment (MOMA) is used to predict disease-state fluxes such that they are as close as possible to the healthy-state metabolic fluxes. Results: The simulation results predict a decrease in glucose and oxygen uptake rates, a significant increase in lactate secretion, a decrease in ATP production and a significantly lower flux through the Krebs cycle, in agreement with the major metabolic mechanisms of the disease. Additional simulations were performed to identify drug targets whose inhibition can bring the metabolism of disease case cells closer to the healthy metabolism. Available drugs for the identified drug targets were identified from drug databases. Conclusions: Use of molecular crowding constraint together with transcriptome data is a successful strategy to calculate metabolic fluxes specific for Parkinson’s Disease and to understand the mechanism of the disease and to identify potential drug targets and drugs. This research was financially supported by TUBITAK (Grant Number:315S302) and by The Turkish Academy of Sciences - Outstanding Young Scientists Award Program (TUBA-GEBIP 2015). Keywords: Parkinson’s disease, molecular crowding, genome-scale metabolic networks, transcriptome data, constraint-based metabolic modelling

66

OP-20

Anticancer Activity of Quinoline and Dihydroquinoline Derivatives

Meltem SELVİTOPUa, İsmail Furkan TURANa , Seçil ERDEN TAYHANa, Esra KOÇb

aDepartment of Genetics and Bioengineering, Faculty of Natural Science and Engineering, Gaziosmanpasa University, Tokat, Turkey

bDepartment of Chemistry, Faculty of Arts and Sciences, Gaziosmanpasa University, Tokat, Turkey E-mail: meltem.selvi.5@gmail.com

Background: Cancer is a constantly increasing disease in the population. Recent scientific studies have investigated new compounds targeting cancer cells. Quinoline is a heterocyclic aromatic organic compound having the chemical formula C₉H₇N. Methods: In the first step of this work, the synthesis of the original therapeutic products quinoline and dihydroquinoline derivatives was carried out. After the synthesis process, antiproliferative effects of quinoline and dihydroquinoline derivatives were tested in vitro on breast cancer (MCF7), prostate cancer (PC3) and colon cancer (HT29) cell lines. DMSO-soluble substances were added to the cultured cells at a specific concentration range (0.4 mM - 200 mM) and incubated for 48 hours. In the mentioned analysis, the antiproliferative effect was determined by the MTT assay. From the results obtained from the test, IC50 values of the related compounds were calculated by GraphPad Prism 6.0. software. Results: As a result, it was found that methoxy dihydroquinoline and bromine dihydroquinoline were effective on the PC3, MCF7 and HT29 cell lines and decreased cell proliferation. These compounds were effective on all cell lines at 200 mM, whereas all administration doses (0.4 mM - 200 Mm) reduced only PC3 cell line proliferation. Conclusions: In conclusion, methoxy dihydroquinoline and bromine dihydroquinoline may be potentially useful as novel chemotherapeutic raw material candidates. Keywords: Quinoline derivatives, dihydroquinoline derivatives, anticancer activity. References: 1. Wang M, Lee RJ, Bi Y, Li L, Yan G, Lu J, Meng Q, Teng L, Xie J. Transferrin-conjugated liposomes loaded with novel dihydroquinoline derivatives as potential anticancer agents. (2017) PLoS ONE; 12(10):e0186821.

67

OP-21

Nanoparticles as Potential Nanocarriers for pH-responsive Investigation of the Stabilization of Topotecan Anticancer Drug

Bilsen Turala, Erdal Ertaş a, Servet Tural a

aDepartment of Chemistry, Faculty of Education, Dicle University, Diyarbakır, Turkey E-mail: bilsentural@gmail.com

Background: Cancer is one of the leading causes of death worldwide. To date, cancer treatment remains a challenge because isolating cancer cells from healthy cells is difficult, and most effective treatments, namely, chemotherapy and radiotherapy, cause inadvertent damage to healthy cells1,2,3. A large variety of biocompatible magnetic ferrofluid nanoparticles (MFFNPs) have established useful applications in biomedical fields, such as hyperthermia, magnetic resonance imaging (MRI), and drug delivery inside the body4. The pH-responsive drug delivery systems are the most investigated models since the pH values, notably vary in different tissues and cellular compartments as well as tumor microenvironments5. Topotecan (TPT) was selected as an anti-cancer drug. TPT can easily hydrolyze at physiological conditions (pH=7.4). To keep anticancer drug in the lactone form, it was efficiently loaded into phosphate-starch modified magnetic ferrofluid nanoparticles, and the stability of TPT in the carriers was fully examined with HPLC6. It was found that the drug was highly stable in active form in the prepared nanocarriers. Mechanical studies on the stabilization of TPT anticancer drug with phosphate-starch modified superparamagnetic iron oxide (SPIO) nanoparticles were carried out using HPLC, ATR-FTIR and SEM. Methods: In this work, we characterized phosphate-starch modified magnetic ferrofluid nanoparticles and drug loading and pH-responsive release conditions and drug loading efficiency were studied with phosphate-starch modified magnetic ferrofluid nanoparticles for the anti-cancer drug topotecan. Results: Biocompatible and biodegradable magnetic ferrofluid nanoparticles (MFFNPs) was characterized with different analytical devices such as Scanning Electron Microscope (SEM), Vibrating Sample Magnetometer (VSM), Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) and Energy Dispersive X-Ray Analysis (EDX. Drug loading and release studies were followed by High-Performance Liquid Chromatography (HPLC). Conclusions: One of the major shortcomings of topotecan is the hydrolyzation of the lactone ring moiety of the drug under physiological conditions i.e. at pH 7 or above, revealing low biologic activity and high toxicity7. Thus, lactone ring was a crucial structural neccessity for both passive diffusion of TPT into cancer cells as well as desired TPT interaction with the pharmacological target DNA topoisomerase 1. In the present work, we developed an efficient and simple strategy for maintaining lactone moiety of topotecan via biodegradable biocompatible phosphate-starch modified superparamagnetic iron oxide (SPIO) nanoparticles. Keywords: Drug release; Magnetic ferrofluid; Magnetic targeting; Topotecan

68

References: 1. Misra R, Acharya S, Sahoo SK. Cancer nanotechnology: application of nanotechnology in cancer therapy. (2010) Drug Discov Today; 15 (19–20): 842–850 2. Anand, P., Kunnumakara, A. B., Sundaram, C., Harikumar, K. B., Tharakan, S. T., Lai, O. S., ... & Aggarwal, B. B. Cancer is a preventable disease that requires major lifestyle changes. (2008) Pharmaceutical research; 25(9), 2097-2116. 3. Trichopoulos, D., Li, F. P., & Hunter, D. J. What causes cancer? (1996) Scientific American; 275(3), 80-87. 4. Mehta RV, Desai R, Bhatt P, et al. Synthesis and characterization of certain nonmagnetic particles coated with citrate and dextran molecules. (2006) Indian J Pure Appl Phys; 44: 537–542. 5. Murugan, C., Rayappan, K., Thangam, R., Bhanumathi, R., Shanthi, K., Vivek, R., ... & Kannan, S. Combinatorial nanocarrier based drug delivery approach for amalgamation of anti-tumor agents in breast cancer cells: an improved nanomedicine strategy. (2016) Scientific reports; 6, 34053. 6. Kizişar, Dilay, et al. Investigation of The Stabilization of Camptothecin Anticancer Drug via PSA-PEG Polymeric Particles. (2016) Anadolu University Journal of Science and Technology–A Applied Sciences and Engineering, 17.1: 221-231. 7. Mert O, Esendaglı G, Dogan A L, Demir A S. Injectable biodegradable polymeric system for preserving the active form and delayed-release of camptothecin anticancer drugs. (2012) RSC Adv; 2: 176-185.

69

OP-22

Searching New MAO-B Hit Inhibitors with Integrated Computational Drug Design Methods

Yusuf Serhat Isa,b,d, Serdar Durdagia,c,*, Busecan Aksoydana,c, Mine Yurtseverb,* aComputational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of

Medicine, Bahcesehir University, Istanbul 34353, Turkey bDepartment of Chemistry, Istanbul Technical University, Istanbul 34469, Turkey

cNeuroscience Program, Graduate School of Health Sciences, Bahcesehir University, Istanbul 34349, Turkey dVocational High School, Department of Chemical Technology, Istanbul Gedik University,

Istanbul 34876, Turkey

E-mails: serdar.durdagi@med.bau.edu.tr, mine@itu.edu.tr

Monoamine oxidase (MAO) enzymes, MAO-A and MAO-B have an important role in the metabolism of monoamine neurotransmitters.1,2 Their main role is to catalyze the oxidative deamination of monoamine neurotransmitters such as dopamine and serotonin.3 MAO inhibitors are prescribed in the treatment of several neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). In this study study, approximately 2.5x105 molecules from Otava Green Chemical Collection were virtually screened for their binding affinities against MAO-B enzyme using firstly rigid docking (HTVS) protocol.3 The %90 of the molecules were eliminated due to their low docking scores. The remaining molecules were evaluated for their therapeutic activity potentials, pharmacokinetic and toxicity properties by using topology based binary quantitative structure−activity relationship (QSAR) models implemented in MetaCore/MetaDrug platform. The filtered ligands having acceptable pharmacokinetic and therapeutic properties were further screened by induced fit docking (IFD) method. At the end, two molecules as most potent hit MAO-B inhibitors were identified. The structural and dynamic profiles of these molecules in the binding site of MAO-B were followed by the MD simulations. We observed that the hit molecules perform better as MAO-B inhibitors than the marketed drug selegiline and they have higher affinity against MAO-B than MAO-A as desired. Moreover, common fingerprints of known inhibitors as well as identified hits were used as input in the text mining studies for the identification of therapeutically active selective novel MAO-B inhibitors. Keywords: Virtual Screening, MAO-B inhibitors, QSAR, MD simulations, text mining, docking References: [1] Kalgutkar, A. S., Dalvie, D. K., Castagnoli, N., Jr., et al. (2001) Interactions of nitrogen-containing xenobiotics with monoamine oxidase (MAO) isozymes A and B: SAR studies on MAO substrates and inhibitors. Chem. Res. Toxicol. 14, 1139−1162. [2] Shih, J. C., Chen, K., and Ridd, M. J. (1999) Monoamine oxidase: from genes to behavior. Annu. Rev. Neurosci. 22, 197−217. [3] Yusuf Serhat Is, Serdar Durdagi, Busecan Aksoydan, Mine Yurtsever (2018) Proposing Novel MAO-B Hit Inhibitors Using Multidimensional Molecular Modeling Approaches and Application of Binary QSAR Models for Prediction of Their Therapeutic Activity, Pharmacokinetic and Toxicity Properties. ACS Chem. Neurosci. 9, 1768−1782.

70

OP-23

Carbonic anhydrase II: The effect of single nucleotide polymorphisms on enzyme structure and function

T. Allan Sanyangaa, Bilal Nizamia,b and Özlem Tastan Bishopa

aResearch Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, Faculty of Science, Rhodes University, Grahamstown, 6139, South Africa

bInstitute of Materials and Environmental Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Magyar tudósok körútja 2, 1117 Budapest, Hungary

E-mail: O.TastanBishop@ru.ac.za

Background: Carbonic anhydrase 2 (CA-II) is a metalloenzyme responsible for the reversible hydration of CO2. The active-site contains a Zinc metal ion present in a tetrahedral geometry, which is coordinated by three histidine residues and one water molecule. Histidine 64 functions as a proton shuttle within the active-site by alternating between “out” and “in” conformations [1]. Within CA-II, non-synonymous single nucleotide polymorphisms (nsSNPs) have been associated with the phenotypes osteopetrosis and renal tubular acidosis, because of the SNP associated enzyme function and stability reduction [2, 3]. As previous research into CA-II has focused on inhibition, this study investigates the effect of six nsSNPs on the structure and function of CA-II, and sets a foundation for drug discovery centered on enzyme stability and functional rescue. Methods: Six validated nsSNPs were identified and modelled, with and without CO2 bound as a ligand. The Variant Analysis Portal (VAPOR) [4], was used to predict potential effect of SNPs on enzyme stability and function. Quantum mechanical calculations were performed for metal parametrization using Gaussian 09, and the Ambertools17 MSPB (metal site parameter builder) [5] scripts used to calculate bond length and angles, and dihedral angles of the Zn ion and coordinating atoms. Using the AMBER ff14SB forcefield, topologies were generated and molecular dynamics analysis performed using Gromacs over 200 ns. Dynamic residue network analysis was performed using the MD-TASK [6] suite to observe changes occurring at residue level. Results: The nsSNPs H107Y, K18E, K18Q, N252D, P236H and P236R were identified. VAPOR predicted that all SNPs are associated with a reduction in stability within CA-II. In the presence of CO2, H107Y and K18E distort rotation of His64 resulting in the formation of π-stacks with Tyr7. His64 distortion is indicated in Figure 1. Figure 1A shows that His64 in the wild-type and H107Y face in different directions with the SNP rotation closer to Tyr7; this explains the interactions. In H107Y Tyr7 also hydrogen bonds with the ND1 atom of His64. Trp5 hydrogen bonds to atom HE2 of His64 in both K18E and N252D. Figure 1B compares His64 distortion in the wild-type and K18E. Though both histidines are facing the same direction, the orientation differs as evidenced by the direction of ND1 faces. In the absence of CO2, Trp5 π-stacks with His64 in P236H and P236R and hydrogen bonds with the HE2 atom. Betweenness centrality revealed that Glu117 is the most important residue for communication in all proteins. Conclusions: Research into the effects of nsSNPs on CA-II was successful. SNP effect occurs away from the SNP location and is centered around the active-site of the CA-II. In the wild-type, His64 is capable of free rotation between the “in” and “out” conformations and the rotation time is not rate limiting on enzyme activity [7]. The coupling of π-stacks and hydrogen bonds in the SNPs however, means that higher energy is required by His64 to break bonds and facilitate alternation between conformations. This could potentially render the change in conformation of His64 as a rate limiting step. Keywords: nsSNPs, carbonic anhydrase II, VAPOR, MD-TASK

71

Figure 1: Distortion of the rotation of His64 within CA-II due to the presence of SNPs. The green color indicates the wild-type protein and the blue color indicates the SNP. The blue surface represents Tyr7 and red represents the ND1 atom. A: variant H107Y; B: variant K18E References 1. Silverman, D.N. and R. McKenna, Solvent-mediated proton transfer in catalysis by carbonic anhydrase. Accounts of chemical research, 2007. 40(8): p. 669-675. 2. Nicoletta, J.A. and G.J. Schwartz, Distal renal tubular acidosis. Current opinion in pediatrics, 2004. 16(2): p. 194-198. 3. Wilson, C.J. and A. Vellodi, Autosomal recessive osteopetrosis: diagnosis, management, and outcome. Archives of disease in childhood, 2000. 83(5): p. 449-452. 4. Brown, D.K. and Ö. Tastan Bishop, HUMA: A platform for the analysis of genetic variation in humans. Human mutation, 2018. 39(1): p. 40-51. 5. Li, P. and K.M. Merz, MCPB.py: A Python Based Metal Center Parameter Builder. Journal of Chemical Information and Modeling, 2016. 56(4): p. 599-604. 6. Brown, D.K., D.L. Penkler, O. Sheik Amamuddy, C. Ross, A.R. Atilgan, C. Atilgan, and Ö. Tastan Bishop, MD-TASK: a software suite for analyzing molecular dynamics trajectories. Bioinformatics, 2017. 33(17): p. 2768-2771. 7. Fisher, Z., J.A. Hernandez Prada, C. Tu, D. Duda, C. Yoshioka, H. An, L. Govindasamy, D.N. Silverman, and R. McKenna, Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II. Biochemistry, 2005. 44(4): p. 1097-1105.

72

OP-24

Computational investigation on the escape mechanism and fitness cost of noroviruses upon mutations

Abdulkadir KOCAK Gebze Technical University, Department of Chemistry

E-mail: kocak@gtu.edu.tr Background: Human Noroviruses (HuNoVs) infect host cell via binding to histo-blood group antigens (HBGAs) through its interaction between VP1 envelope protein and HGBA cell surface antigens. This interaction is required for virus particle to enter inside the host cell. This attracts great attention to develop therapeutics that will block the interaction between VP1 and HBGAs as a cure for virus infection. Efforts in developing therapeutics against virus infection mostly fail due to emergence of drug resistance that is consequence of presence of high mutation rates in virus genome during virus' life cycle. Here, we computationally analyzed the affinity of a drug target, wild type VP1 envelope protein and its three variants to a therapeutic antibody FAB5I2 and to carbohydrate core structures of A- and H-type HGBA antigens. Methods: In the first part, the starting structures of the VP1-FAB5I2 complex variants (PDB id=5KW9, 2ZL5, 4P12 and 4RDJ) were retrieved from the Protein Data Bank. Since the mutant proteins were lack of FAB5I2, hypothetical mutant VP1-FAB5I2 complexes were generated by copying coordinates of FAB5I2 of the wild type after superimposing and aligning the protein structures. The molecular dynamics simulations were carried out using Gromacs 5.1 software package with Amber ff99SB-ILDN force field. For each complex three repetitive MD simulations of ~80 ns at the NPT ensemble was carried out using Langevin Dynamics. The stepwise procedure of the MD simulations were described elsewhere [1-2]. In the second part, VP1-sugar (of A- and H-type antigens) complexes (PDB id=2ZL6, 2ZL7, 4P1V, 4P26, 3ASQ and 3ASP were retrieved from protein databank. The ligands were extracted and partially optimized using Gaussian 09 software package at B3LYP/6-311++G(d,p) by freezing the heavy atoms. The ligands were parametrized using Antechamber at Generalized Amber Force Field (GAFF) and RESP charges. Original sugar structures were replaced with optimized ones. The same procedure mentioned in the first part was used for the rest of the protein preparation steps. Free energy studies were done by means of MMGBSA, MMPBSA and BAR perturbation methods. Results: In the VP1-FAB5I2 interaction part of the project, MD analysis along with free energy calculations by means of MMPBSA clearly showed that the binding affinity of VP1 protein to FAB5I2 drastically dropped upon mutations. In the VP1-HBGA interaction part of the project, the calculations are still ongoing. Conclusions: MD simulation data suggested that all the mutations develop escape mechanism toward the FAB5I2 antibody upon mutations. In addition, the location of the mutation seems more important than the number of mutations. Free energy calculations also show the affinity loss upon mutation. Fitness cost studies will be complementary to these studies and we are currently working on that.

73

Keywords: Norovirus, therapeutic antibody, HBGA blockage, molecular dynamics, drug resistance References

1. Kocak A, Erol I, Yildiz M, and Can H Computational insights into the protonation states of catalytic dyad in BACE1–acyl guanidine based inhibitor complex. (2016) Journal of Molecular Graphics and Modelling, 70:226-235.

2. Kocak, A. and Yildiz M, Docking, molecular dynamics and free energy studies on aspartoacylase mutations involved in Canavan disease. (2017) Journal of Molecular Graphics and Modelling,74:44-53.

74

OP-25 Diastereoselectivity in Benzylation Reaction of 5-Methyl-Derivatives of

Thiobarbituric Acids

Semin Funda Oğuza, Burcu Başa, Sevgi Sarıgülb, İlknur Doğanb

aDepartment of Chemical Engineering, Yeditepe University, İstanbul, Turkey b aDepartment of Chemistry, Bogazici University, İstanbul, Turkey

E-mail: funda.oguz@yeditepe.edu.tr Background: Barbituric and thiobarbituric acid derivatives are very well-known heterocyclic compounds for their different uses in medicine and industry.1,2 When aromatic and alkyl groups are bonded to C-5 of the heterocyclic ring of barbituric acids, their biological activity increases significantly 2,3. Asymmetric synthesis of thiobarbituric acid derivatives have high synthetic and medicinal value. Methods: The benzylation reactions were conducted by reacting 5-methyl-1-(o-aryl)- and 5-methyl-1-phenyl-2-thiobarbituric acids with benzylbromide in the presence of LDA or cinchonine, which is known as an environmentally friendly natural chiral organocatalyst, in THF at low temperatures as shown in Figure 1. Results: When LDA was used in benzylation reaction of 5-methyl derivatives of thiobarbituric acids as a base a partial diastereoselectivity was observed, whereas when cinchonine was used a complete diastereoselectivity was achieved (Figure 1).

Figure 1. Diastereoselectivity in Benzylation Reaction of 5-methyl derivatives of Thiobarbituric Acids Conclusion: An important conclusion about this study is that benzylation reactions of thiobarbituric acid derivatives can be done diastereoselectively using (+)-cinchonine as a chiral basic catalyst. Keywords: organocatalyst, cinchonine, thiobarbituric acids, diastereoselectvit, chirality

75

References 1. Rauf, A.; Shahzad, S.; Bajda, M.; Yar, M.: Ahmed, F.; Hussain, N.; Akhtar, M. N.; Khan, A.; Jon´czyk Peddibhotla

S. Design and synthesis of new barbituric- and thiobarbituric acid derivatives as potent urease inhibitors: Structure activity relationship and molecular modeling studies. (2015), Bioorganic & Medicinal Chemistry; 23: 6049-6058.

2. Yan, Q.; Cao, R.; Yi, W.; Chen, Z.; Wen, H.; Ma, L.; SongH. Inhibitory effects of 5-benzylidine barbiturate derivatives on mushroom tyrosinase and their antibacterial activities (2009), European J. of Medicinal Chemistry; 44: 4235-4243.

3. Faidallah, H. M.; Khan, K. A. (2012), Synthesis and biological evaluation of new barbituric and thiobarbituric acid fluro analogs of benzenesulfonamides as antidiabetic and antibacterial agents. J. Fluorine Chem; 142: 96-104.

76

OP-26

Clay mineral particles as imaging and drug delivery agents

Maide Gökçe Bekaroğlua, Fuad Nurilib, Sevim İşçia* aIstanbul Technical University, Faculty of Science and Letters, Dept. of Physics, Maslak 34469,

Istanbul, Turkey; bDepartment of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

E-mail: iscisev@itu.edu.tr Background: Clay minerals have always been widely used in production of pharmaceutical formulations due to their many favorable properties such as their high adsorption capacity, high specific surface area, ion exchange capacity, colloidal structure, low toxicity etc. Although clay minerals are widely used as raw pharmaceutical materials they have not yet been investigated as targeted drug delivery agents for tumors at specific areas. [1-4]. Montmorillonite (Mt), a clay mineral of the smectite group can be used as a medical imaging and drug delivery agent to be used in transcatheter arterial embolization (TAE) applications. In this embolization technique, the arterial vessels around tumor site are blocked by drug loaded micro sized Mt particles so tumor is deprived of oxygen and nutrition while loaded drugs are released by the particles into the tumor site. Both devascularization and drug release around tumor site can progressively shrink the targeted tumor size. Certain properties of Mt such as size, adsorption capability, and biocompatibility indicate that Mt particles are highly applicable and unique for TAE and imaging applications. Methods: In order to obtain drug releasing clay mineral particles that are suitable for TAE that can also enable medical imaging, interactions of purified montmorillonite (PMt) with a computed tomography (CT) contrast material and an antitumor drug were investigated. Initially, In-vitro cytotoxicity assays using normal cells were carried out in order to assess PMt biocompatibility. Then, swelling and adsorption behaviour of PMt in the presence of the CT contrast material or antitumor drug were investigated using DLS, XRD and FTIR in order to determine proper concentration where particle sizes are suitable for successful TAE applications. Particles suitable for TAE procedure were further investigated using In-vitro and In-vivo tests. Results: Results showed both CT contrast material and antitumor drug could penetrate interlayer spaces and were adsorbed by the surfaces of the PMt particles. Particle sizes required to successfully obtain embolization (at least 40 μm) were achieved as the size of the PMt increased due to both the adsorption and coagulation. Preliminary embolization and CT imaging tests of particles in a rabbit renal model, in-vitro drug release profiles and in-vitro drug activity on MCF-7 (human breast adenocarcinoma) cells were also satisfactory.

77

Embolization was successfully performed in rabbits without any complications related to the procedures. No embolic material reflux was observed. The pre- and post-embolization comparison was shown at the first rabbit’s renal artery and its branches. The renal artery and its branches were clearly identifiable in the image before the embolization procedure.

In vitro anticancer activity of pure DOX and DOX loaded clay particles (PMt+800DOX) against the MCF-7 cell line was investigated in order to assess PMt+800DOX particles’ anticancer drug potential. The amount of pure DOX was adjusted to be equal as the amount of loaded DOX onto PMt+800DOX particles. DOX and PMt+800DOX exhibit similar toxicity to MCF-7 cells. Conclusions: This study demonstrated that embolic particles that can also be used as drug releasing and imaging agents can be obtained by using PMt particles. This novel and economic approach for synthesis of embolic particles showed promising results that synthesized particles would be promising for embolization therapies and drug delivery applications. Keywords: Montmorillonite; drug delivery; imaging agent; cancer therapy; arterial embolization References: 1. Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: Possibilities and limitations. Applied Clay Science 36, 22-36 2. Jayrajsinh, S., Shankar, G., Agrawal, Y.K., Bakre, L., 2017. Montmorillonite nanoclay as a multifaceted drugdelivery carrier: A review. J Drug Deliv Sci Tec 39, 200-209 3. Viseras, C., Cerezo, P., Sanchez, R., Salcedo, I., Aguzzi, C., 2010. Current challenges in clay minerals for drug delivery. Applied Clay Science 48, 291-295. 4. Yang, J.H., Lee, J.H., Ryu, H.J., Elzatahry, A.A., Alothman, Z.A., Choy, J.H., 2016. Drug-clay nanohybrids as sustained delivery systems. Applied Clay Science 130, 20-32

78

OP-27 Influence of Biphalin on Human Immortalized Corneal Epithelial Cell Cultures

O. Melike GEDARa, Afsun SAHINb, Adriano MOLLICAc, Erdost YILDIZd, Kerem KABADAYIe

aAssistant Professor, Bahçeşehir University, Faculty of Medicine, Department of Ophthalmology, Istanbul, TURKEY

bAssociate Professor, Koc University, Faculty of Medicine, Department of Ophthalmology, Istanbul, TURKEY cAssociate Professor, University ”G. d’ Annunzio” of Chieti-Pescara, Department of Pharmacy, Chieti, ITALY

dKoc University, Faculty of Medicine, Istanbul, TURKEY eMedical Student, Bahçeşehir University, Faculty of Medicine, Istanbul, TURKEY

E-mail: melikegedar@gmail.com Background: Topical analgesic drugs (eg. non-selective opioid agonists) are not used to reduce pain following physical/chemical or surgical trauma due to reduce corneal wound healing. However, biphalin, a selective mu and delta receptor agonist, has shown to have analgesic action in the cornea of the rodents and at the same time does not impair wound healing in the corneal epithelium as opposed to other non-selective opioid receptor agonists. As far as our knowledge there is no study about biphalin has the same effect on wound healing in the human corneal epithelium. In this experimental study, we aimed to investigate the effects of biphalin, a selective opioid receptor agonist, on human corneal epithelium wound healing. Methods: Immortalized human corneal epithelial cells (HCEC) were cultured in serum-free keratinocyte medium and then exposed to biphalin at increasing concentration and time to yield 3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium-bromide (MTT) and in vitro toxicity levels of biphalin were determined. After detecting the highest non-toxic biphalin dose, the relative wound area (RWA) was calculated in cell culture by using the “in vitro scratch assay” method and the effect of biphalin on corneal wound healing was measured. Afterwards, the effect of biphalin on corneal epithelial cell migration was assessed by transwell migration assay and the effect on proliferation was assessed by calculating Ki67 proliferation index. In all experiments, the effect of biphalin was inhibited by using non-selective opioid receptor competitive agonist, naloxone, and the results were compared. Results: As a result of MTT assay, biphalin did not show any toxicity for up to 21 days at concentrations below 100 μM. In the “in vitro scratch assay”, 10 μM, 50 μM and 100 μM biphalin significantly accelerated corneal wound healing compared to the control group after 6 hours (p <0.001). Biphalin was found to accelerate corneal epithelial cell migration and proliferation compared with the control group. Conclusions: The selective opioid receptor agonist biphalin accelerates wound healing of the corneal epithelium. Corneal epithelial healing may not be affected by the analgesic effect of biphalin in the treatment of pain following posttraumatic or collagen cross-linking. In the next step, the experimental animal model will be used to investigate the effects of biphalin in vivo corneal wound healing. Keywords: biphalin, human corneal epithelial cell (HCEC), wound healing, opioid agonists

79

OP-28

Evaluation of carbapenem resistance using phenotypic and genotypic techniques in Enterobacteriaceae isolates

Kazım Sahina , Ayse Tekinb, Sule Ozdasc, Demet Akind, Hande Yapislare,

Aziz Ramazan Dileka and Emine Sonmeza aDepartment of Microbiology, Medical Faculty, Recep Tayyip Erdogan University, Rize, Turkey�

bDepartment of Infectious Diseases, Cengiz Gökcek Women and Obstetrics Hospital, Gaziantep, Turkey. cDepartment of Molecular Biology and Genetics, Medical Faculty, Istanbul Bilim University,

Istanbul, Turkey. dDepartment of Pharmacology, Medical Faculty, Bahcesehir University, Istanbul, Turkey.

eDepartment of Physiology, Medical Faculty, Acıbadem Mehmet Ali Aydınlar University, Istanbul, Turkey. E-mail: demet.akin@med.bau.edu.tr

Background: Bacterial resistance to antibiotics is increasing worldwide. Antibiotic-resistant strains can lead to serious problems regarding treatment of infection. Carbapenem antibiotics are the final treatment option for infections caused by serious and life-threatening multidrug-resistant gram-negative bacteria (1,2,3). Therefore, an understanding of carbapenem resistance is important for infection control. In the study described herein, the phenotypic and genotypic features of carbapenem-resistant Enterobacteriaceae strains isolated in our hospital were evaluated. Methods: In total, 43 carbapenem-resistant strains were included in this study. Sensitivity to antibiotics was determined using the VITEK 2 system. The modified Hodge test (MHT) and metallo-β-lactamase (MBL) antimicrobial gradient test were performed for phenotypic identification. Resistance genes IMP, VIM, KPC, NDM-1, and OXA-48 were amplified by multiplex PCR. Results: The OXA-48 gene was detected in seven strains, and the NDM-1 gene in one strain. No resistance genes were detected in the remainder of strains. A signi cant correlation was observed between the MHT test and OXA-48 positivity, and between the MBL antimicrobial gradient test and positivity for resistance genes (p < 0.05). Conclusion: The finding of one NDM-1-positive isolate in this study indicates that carbapenem resistance is spreading in Turkey. Carbapenem resistance spreads rapidly and causes challenges in treatment, and results in high mortality/morbidity rates. Therefore, is necessary to determine carbapenem resistance in Enterobacteriaceae isolates and to take essential infection control precautions to avoid spread of this resistance. Keywords: Enterobacteriaceae, Carbapenem resistance, OXA-48, NDM-1, Modified Hodge test, Metallo-β-lactamase antimicrobial gradient test

80

Figure 1: Relationship between MHT and MBL E-test results with OXA-48 and NDM-1 genes positivity

References:

1. Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. (2011) Emerg Infect Dis; 17:1791–8. �

2. Patel JB, Rasheed JK, Kitchel B. Carbapenemases in Enterobacteriaceae: activity, epidemiology, and laboratory detection (2009 ) Clin Microbiol Newsl. 31:55–62. �

3. Shah PM. Parenteral carbapenems. (2008) Clin Microbiol Infect. 14(1):175–80. �

Page 4 of 6Sahin et al. Ann Clin Microbiol Antimicrob (2015) 14:44

in-patients were significantly higher compared to patients infected with carbapenem-sensitive strains (48 and 20  %, respectively; p =  0.001). In this study, it was noted that receiving a transplant was an independent fac-tor in carbapenem-resistant K. pneumoniae infection. In another study, the mortality rate was 60  % in a hetero-geneous patient population that included carbapenem-resistant K. Pneumoniae-infected transplant recipients [17]. In addition, staying in the ICU, surgical procedures, using catheter, length of hospitalization and using of cephalosporins and aminoglycosides are risk factors for carbapenem-resistant K. pneumoniae infections [18, 19].

In our study, sensitivity was determined using the auto-mated VITEK®2 Compact system and the antimicrobial gradient test. Of the 43 strains, all were ertapenem-resist-ant, 95.3  % were meropenem-resistant and 83.7  % were imipenem-resistant. A resistance rate of 97.7 % to ertape-nem was detected using antimicrobial gradient test tech-niques, whereas the resistance rate was 100 % according to the VITEK®2 automated system. Additionally, mero-penem resistance was calculated using the antimicrobial gradient test (93 %) and the automated system (90.7 %).

The MHT is one of the simplest techniques used to indicate carbapenemase activity. In this test, carbapenem inhibition zone diameter and MIC values are considered sufficient [4] and valuable for the determination of KPC, OXA and MBL enzymes in laboratories in which dilution techniques cannot be performed [2, 20]. Herein, a cor-relation was found between MHT technique results and OXA-48 gene positivity (p = 0.004) (Table 3). Raghuna-than et  al. conducted studies in which they determined the carbapenem resistance of Enterobacteriaceae strains by comparing PCR and MHT techniques. The authors revealed a 96 % correlation between PCR positivity and MHT positivity in terms of KPC genes (causing carbap-enem resistance) [21]. MHT is not sufficient to detect OXA-48 or other carbapenemase producing isolates.

Combining carbapenemase inhibitor test, using phenylb-oronic acid, with MHT is a good indicator fort the detec-tion of carbapenemase producing isolates [22].

In the present study, the MBL antimicrobial gradient test was used to detect class B metallo-β-lactamases. Of the 43 isolates, MBL positivity was found in two. Of these two isolates, only one was NDM-1 positive according to PCR results. A correlation was observed between MBL antimicrobial gradient test results and NDM-1 positiv-ity/negativity by multiplex PCR (Table 3). NDM-1 resist-ance has been described in several case reports in recent years [14, 23]. However, class D OXA carbapenemase-mediated carbapenem resistance has become prevalent, especially among Enterobacteriaceae strains causing nosocomial infections. In our country, an OXA-48 type β-lactamase belonging to class D carbapenemases was detected for the first time at the Istanbul University Fac-ulty of Medicine Hospital. This carbapenemase enzyme was isolated from K. pneumoniae strains obtained from a urine culture of an in-patient [24]. The OXA-48 car-bapenemase enzyme has mostly been identified in K. pneumoniae strains in Turkey, Lebanon and Belgium [14, 25, 26]. Two studies evaluated carbapenem resistance in ESBL-producing carbapenem-resistant K. pneumoniae strains using disc diffusion and antimicrobial gradient test techniques [27, 28]. Of the 14 strains examined, the OXA-1 gene was detected in all, the OXA-48 gene in two, and the NDM-1 gene in two [27]. In addition, the IS1999 location was shown in strains expressing OXA-48, indicating that ERT resistance was higher in OXA-48 positive strains compared to other carbapenems [29]. According to studies performed in Greece and Spain, all carbapenem-resistant K. pneumoniae strains were posi-tive for the OXA-48 gene. The OXA-48 gene was associ-ated with the IS1999 element [30, 31]. In Turkey, the first NDM-1 resistance gene was identified in 2011 by Poirel et al. [14]. This gene was isolated from an allogeneic stem cell-transplanted leukemia patient from Iraq. From this isolated case, NDM-1 was identified as the first resist-ance gene detected from an international origin [14]. Yanik et al. [12] conducted a study in Samsun-Turkey in which the carbapenemase enzyme was detected using the MHT technique in gram-negative strains resistant to at least one of the antibiotics (imipenem, meropenem or doripenem). PCR was performed to detect NDM-1, but no NDM-1 gene resistance was found in any of the 210 clinical isolates. These results indicate that NDM-1 is not yet common in Turkey. The NDM-1 resistance gene with no history of foreign contacts was firstly determined by Alp et al. [23], in which 137 carbapenem-resistant K. pneumoniae strains were examined for resistance genes. Of 127 strains, OXA-48 was detected in 91.5 %, IMP in 3.2  %, NDM-1 in 4.3  %, and both OXA-48 and NDM-1

Table 3 Relationship between MHT and MBL E-test results with OXA-48 and NDM-1 genes positivity

MHT modifiye Hodge test, MBL E-test metallo-beta-lactamase E-test

Test Positive Negative p

n % n %

OXA-48

MHT

Positive 3 43 32 88.9 0.004

Negative 4 57 4 11.1

NDM-1

MBL E-test

Positive 1 100 1 2.4 0.000

Negative 0 0 41 97.6

81

OP-29

May the Thiol-Disulfide Level be an Indicator for Diabetic Retinopathy

İsa Yuvacıa a Sakarya Üniversitesi Tıp Fakültesi Eğitim Araştırma Hastanesi Göz Kliniği

E-mail: mdisay@hotmail.com

Background: Comparison of serum thiol-disulfide balance in patients with type 2 diabetes who has retinopathy or not. Methods: This study was planned and conducted as a prospective controlled study on diabetic patients who were regularly followed up and a voluntary control group who is similar to them demographically in our clinic. The patients with diabetes and non-retinopathy (DM) constituted one group and patients with diabetic retinopathy (DRP) constituted the other one, while the healthy volunteers with similar demographic data constituted the control group (C). Following a routine ophthalmoscopic examination, blood samples were drawn from the patients on whom it was worked study and from volunteers at least 8 hours after fasting, and samples centrifuged and stored at -80 C till biochemical analysis made. The data belonging to the groups were assessed by using the SPSS program. Results: While mean age of patients was 54.2 years in the C group, it was 56.59 years in the DM group and 58.8 years in the DRP group. Mean spherical equivalents of participants was detected as 0.53, 0.1 and 0.15 D (in the same order). While mean intraocular pressure values were found as 15.60, 17.31 and 16.57 mmHg, mean front anterior eye lengths were 22.93, 23.16 and 23.43 mm. Their mean fasting blood glucose levels were detected as 97.35, 177.59 and 245.80 mg / dl. The mean glycosylated hemoglobin levels were 5.44, 8.92 and 10.17. Mean diabetes durations of patients were 0, 7.18 and 11.09 years. Thiol levels in group C patients were found as follows, respectively; the total thiol is 375.18, native thiol 345.98, disulfide 14.60, disulfide native thiol ratio (DNTO) 4.30, disulfide total thiol ratio (DTTO) 3.94, and the native thiol total thiol ratios (NTTO) 92.10. (Rates were evaluated as %). These values were found in the DM group as 402.97, 361.38, 20.79, 5.99, 5.32 and 89.34, while in the DRP group, they were found as 370.29, 329.09, 20.60, 6.46, 5.70 and 88.59. When the findings were analyzed, it was not found any statistically significant difference between the native and total thiol levels between the groups. Disulfide values were found to be significantly higher in the DM and DRP groups than the control group. However, these values were not found as a significantly difference between DM and DRP groups. While DNTR, DTTO and NTTO values were significantly different between the group K and the DM and DRP groups, it was not found any significant difference between DM and DRP groups. Conclusions: Inadequacy occurs in the antioxidant systems of the body in the course of time, in the case of diabetes. This leads to changes in balance of thiol and disulfide in the body. The thiol-disulfide values were found to be high in the DM and DRP groups in our study namely, the thiol and disulfide balance were found to be impaired, as noted in various previous studies. It was not found any statistically significant difference in terms of DM and DRP groups. Keywords: Diabetes, retinopathy, thiol, disulfide

82

OP-30

Oncogenic mutations on Rac1 affect global intrinsic dynamics underlying GTP and PAK1 binding

Saliha Ece Acuner-Ozbabacana,b, Fidan Sumbula,c, Hamdi Torund, Turkan Haliloglua aDepartment of Chemical Engineering and Polymer Research Center, Bogazici University, Istanbul, Turkey; bPresent address: Department of Bioengineering, Istanbul Medeniyet University, Istanbul, Turkey; cPresent

address: U1067 INSERM & Aix Marseille Universite 163 avenue de Luminy, 13009 Marseille, France; dDepartment of Mathematics, Physics and Electrical Engineering, Northumbria University, Newcastle, UK.

E-mail: Turkan Haliloglu (halilogt@boun.edu.tr) and Hamdi Torun (hamdi.torun@northumbria.ac.uk) Background: Rac1 (Ras-related C3 botulinum toxin substrate 1) is an important GTPase, found in all eukaryotic organisms, regulating cell responses such as cell adhesion, cytoskeleton rearrangement, lamellipodia and membrane ruffle formation, NADPH oxidase activation for antibacterial defense and induction of gene expression, through binding of numerous effector proteins in its active state [1, 2]. Rac1 is a small member of Ras superfamily and Rho family of GTPases, associated with the plasma membrane, acting as a binary molecular switch between the GTP-bound active state (ON) and the GDP-bound inactive state (OFF) [3]. Its activation-inactivation cycle is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (Figure 1). One of the most important downstream effectors of Rac1 is a serine/threonine kinase, p21-activated kinase 1 (PAK1). Overexpression or mutational activation of PAK isoforms (mostly PAK1) by upstream elements (e.g. Rac1) have oncogenic signaling effects in cell proliferation, survival, invasion and metastasis [4]. Methods: We explore mutation-dependent dynamic regulation of intrinsic binding states and interactions of Rac1 in a framework by Single Molecule Force Spectroscopy (SMFS) experiments using Atomic Force Microscopy (AFM) corroborated with elastic network model (ENM) analysis, Molecular Dynamic (MD) simulations and structural modeling of protein-protein complexes. Results: The unbinding free energy landscape (FEL) of Rac1-PAK1 dissociation reaction for the GTP loaded wild-type (wt) Rac1-PAK1 shows two pathways (so-called high- and low-strength binding states), whereas the number of pathways is reduced to one for constitutively active Q61L and oncogenic Y72C mutations with unbinding rates shifted towards the high-strength binding state of the wt Rac1. On the other hand, MD simulations show that Q61L and Y72C, and two more oncogenic mutants P29S and Q61R, attenuates the intrinsic dynamic event underlying binding of the wt Rac1 with GTP as well as the upstream and downstream partners. While the cooperativity of GTP-binding sites around p-loop and Switch I -including also some of the PAK1 binding sites in the vicinity- decreases mostly for the constitutively active Q61L, the cooperativity of some other PAK1-binding residues (around C terminus) increases. Together with the predicted binding energy calculations of model complex structures of Rac1-PAK1, this dynamic event could plausibly be ascribed to a more favorable PAK1 interaction. To this end, the association of these mutations also with the hinge sites of the global modes of motion rationalizes this behavior as a perturbation to Rac1's intrinsic functional dynamics. Conclusions: Although the positions of the case study mutations (as well as other oncogenic mutations) appear sporadically, they have commonality of aligning with the global hinges that coordinate the cooperative dynamics and global motions. Hence, when the changes in the intrinsic dynamics of GEF, GAP and PAK1 binding regions of Rac1 are considered; as a result of

83

an allosterically regulated interplay between important functional regions on Rac1, both upstream and downstream interactions are affected leading to the activation of important cell signaling networks in cancer. Keywords: Protein Dynamics, Allostery, Molecular Simulations, Gaussian Network Model, AFM

Figure 1: Rac1 activation/deactivation cycle. References: 1. Jaffe AB, Hall A: Rho GTPases: biochemistry and biology. (2005) Annual review of cell and developmental

biology; 21:247-269. 2. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A: The small GTP-binding protein rac regulates growth

factor-induced membrane ruffling. (1992) Cell; 70(3):401-410. 3. Vetter IR, Wittinghofer A: The guanine nucleotide-binding switch in three dimensions. (2001) Science;

294(5545):1299-1304. 4. Radu M, Semenova G, Kosoff R, Chernoff J: PAK signalling during the development and progression of

cancer. (2014) Nature reviews Cancer; 14(1):13-25.

84

OP-31

Structural, Vibrational and Kinetic Analysis of a Series of Ibuprofen Derivatives by Using Ab-Initio Quantum Mechanical Calculations

Nil E. Binbaya , Veysel Binbayb aDicle University, Vocational School, Department of Electronics 21280, Diyarbakır, Turkey

bDicle University, Institute of Natural Science, Department of Physics, 21280, Diyarbakır Turkey

E-mail: nbinbay@gmail.com ; veysel.binbay@gmail.com Background: The Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) has been widely being used for treating inflammation. And also, they possess high incidence of gastrointestinal (GI) side effects, which mainly caused by their non-selective profiles about binding to both of Cyclooxygenase enzymes (COX-1 and COX-2)1. To date, it has been representing a major challenge to find COX-2 selective inhibitors as novel drug candidates. One of the promising strategies of achieving this goal has been the modification of known NSAIDs, such as ibuprofen. In such an effort, a series of derivatives of ibuprofen (6-substituted thiazolo[3,2-b]-1,2,4-triazole-5(6H)-one) were synthesized at Hacettepe University, Turkey, in an earlier study2. Here we report a detailed structural and vibrational frequency analysis of these novel derivatives based on purely ab-initio quantum mechanical calculations. Moreover we report a comparison between theoretical calculations and experimental data. Some kinetic and thermodynamic parameters, such as chemical hardness, electronegativity and electrophilicity of these novel derivatives were also calculated and reported. Methods: Theoretical calculations have been carried out by using a general ab initio quantum chemistry package GAMESS (the General Atomic and Molecular Electronic Structure System)3,4. DFT (Density Functional Theory) studies were carried out by hybrid Becke5,6, three-parameter, Lee–Yang–Parr (B3LYP) exchange-correlation functional. Results: It is shown that, theoretical calculations and experimental results are highly correlated. Conclusions: It is concluded that, some of these novel agents has promising parameters among the others as novel anti-inflammatory drug candidates. Keywords: Ibuprofen, DFT, GAMESS, References: 1. Vane J R, Botting R M, Anti-inflammatory drugs and their mechanism of action.(1998) Inflammation Res.; 47 (Suppl. 2):

S78−S87. 2. Uzgören-Baran A, Tel B C, Sarıgöl D, Öztürk E İ, Kazkayası İ, Okay G, Ertan M, Tozkoparan B Thiazolo[3,2-b]-1,2,4-triazole-

5(6H)-one substituted with ibuprofen: Novel non-steroidal anti-inflammatory agents with favorable gastrointestinal tolerance. (2012) European Journal of Medicinal Chemistry ; 57: 398-406.

3. Gordon M S, Schmidt M W in Theory and Applications of Computational Chemistry, the first forty years. (2005) pp 1167-1189 C.E.Dykstra, G.Frenking, K.S.Kim, G.E.Scuseria (editors), Elsevier, Amsterdam.

4. Schmidt M W, Baldridge K K, Boatz J A, Elbert S T, Gordon M S, Jensen J J, Koseki S, Matsunaga N, Nguyen K A, Su S, Windus, T L, Dupuis M, Montgomery J A, (1993) J.Comput.Chem.; 14: 1347-1363

5. Becke A D, Semiempirical hybrid functional with improved performance in an extensive chemical assessment. (1993) J Chem Phys.; 98: 5648

6. Becke A D, Density-functional thermochemistry. IV A new dynamical correlation functional and implications for exactexchange mixing. (1996) J Chem Phys.;104:1040–1046

85

OP-32

In silico and in vitro Attenuation of Pseudomonas aeruginosa Quorum Sensing by Psoromic Acid

Barış GÖKALSINa, Ayla YILDIZ a, Arhun Ali BALKANa, Serdar DURDAĞI b, Cenk SESALc,

aMarmara University, Institute of Pure and Applied Sciences, Biology Department, İstanbul, Turkey, bBahcesehir University, Department of Biophysics, School of Medicine, İstanbul, Turkey, cMarmara

University, Faculty of Arts and Sciences, Biology Department, İstanbul, Turkey E-mail: bgokalsin@gmail.com

Background: Cystic Fibrosis (CF) is a genetic disease that affects respitory and digestive systems. Pseudomonas aeruginosa infections are presented to be the main cause for high mortality rates in CF patients. P. aeruginosa populations can regulate their virulence gene expressions via the bacterial communication system: quorum sensing (QS). The novel approach to reduce virulence of these bacteria without risking antimicrobial resistance is to inhibit QS and block bacterial communication by employing signal blocking antagonists. Therefore, discovering QS inhibitors (QSIs) from natural compounds bears significance. Lichens produce unique secondary metabolites that have potential antivirulence properties. Psoromic acid is a natural compound that can be found in lichens. This study investigates the antivirulence properties of psoromic acid on P. aeruginosa by inhibiting its QS systems. Methods: Cultures of P. aeruginosa lasB-gfp, rhlA-gfp and pqsA-gfp monitor strains were utilized in QS inhibition screens. Bacteria were treated with serial dilutions of psoromic acid. Growth and green fluorescent protein (GFP) expressions were monitored using multimode microplate reader (Biotek-Cytation 3) for 16 hours, measuring absorbance and fluorescence every 15 minutes. Fluorescence of GFP expression was measured at 485 nm excitation and 535 nm emission wavelengths. Molecular docking methods were also performed for crucial QS proteins of P. aeruginosa. The 3D crystal structures of LasI, LasR and PqsR proteins were obtained from Protein Data Bank with PDB codes 1RO5, 3IX3 and 4JVC, respectively. Azithromycin, an antibiotic and QSI for CF treatment available in market, and psoromic acid structures were downloaded from ZINC Database. Protein-ligand preparation and docking processes were performed using Glide module of Maestro/Schrödinger software. Results: In vitro QSI screens have shown that psoromic acid inhibits expressions of QS regulated lasB-gfp, rhlA-gfp and pqsA-gfp fusions at certain concentrations. Psoromic acid treated samples with concentrations between 312 µM and 78 µM resulted in lower fluorescence levels compared to control. Results indicate that psoromic acid is capable of inhibiting P. aeruginosa las, rhl and pqs systems approximately 80.2%, 57.8% and 68.4% respectively at 312 µM. Molecular docking results are evaluated considering docking scoring function (kcal/mol). Conclusions: According to results, psoromic acid is capable of inhibiting QS systems and thus reduce virulence of P. aeruginosa considerably. QSIs can keep virulence and pathogenicity in control and leave bacteria vulnerable. Screening natural compounds such as psoromic acid can provide opportunities for future QSI drugs. Further studies should be considered to inhibit QS signal receptors using competitive natural or synthetic molecules and their homologue structures. Acknowledgement: This study was funded by the project: TUBITAK 216S885. Keywords: Pseudomonas aeruginosa, quorum sensing, psoromic acid

86

POSTER PRESENTATIONS

87

P-1

Cross-talk between autophagy and endoplasmic reticulum stress in atiprimod-induced apoptotic cell death in MDA-MB-468 and MDA-MB-231

breast cancer cells

Ajda Coker-Gurkana, Esin Can*a, Semanur Sahin*a, Elif-Damla Arisana, Pinar Obakan-Yerlikayaa

*equal contribution

aIstanbul Kultur University, Dept. of Molecular Biology and Genetics, 34156, Bakirkoy-Istanbul E-mail: a.coker@iku.edu.tr

Background: Constitutive activation of STAT3 through receptor tyrosine kinases triggered breast cancer cell growth, invasion-metastasis. Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine), a cationic amphiphilic agent, has anti-proliferative, anti-carcinogenic effects on in vitro and in vivo in hepatocellular carcinoma, myeloid leukemia and breast cancer. Autophagy is a vesicule-mediated clearance mechansim and one of the handicap against drug-induced apoptotic cell death. Our aim in this study is to modulate the endoplasmic reticulum stress and autophagy regulation during atiprimod induced apoptotic cell dearh in MDA-MB-231 and MDA-MB-468 breast cancer cells. Methods: Dose- and time-dependent effect of atiprimod was determined by MTT cell viability, trypan blue and hanging drop assays. Annexin-V/PI staining and PI FACS flow analysis was performed to demonstrate the effect of atiprimod on cell cycle arrest and apoptotic cell death. ER stress, autophagy and apoptotic key markers expression profiles were observed by immunoblotting. CHOP activation and LC3-II cleavage was determined by fluorescence tagged-LC3 gene inserted plasmid-mediated FACS flow analysis in MDA-MB-468 and MDA-MB-231 breast cancer cells. Results: Dose- and time-dependent atiprimod treatment inhibits cell viability, colony formation in MDA-MB-468 and MDA-MB-231 breast cancer cells. Atiprimod (2 �M) triggered PERK, BiP, CHOP upregulation and nuclear locazation of CHOP and ATF6α proteins in breast cancer cells. Concominantly, atiprimod induced autophagy vacuole formation, upregulation of Atg-5, Atg-12 proteins in MDA-MB-468 breast cancer cells. Moreover, atiprimod triggered caspase-dependent apoptotic cell death through modulating Bcl-2 family members in MDA-MB-231 and MDA-MB-468 breast cancer cells. Conclusions: Atiprimod induced apoptotic cell death via activating ER stress and autophagy in MDA-MB-231 and MDA-MB468 breast cancer. Keywords: Breast cancer, Atiprimod, Endoplasmic reticulum stress, Autophagy, Apoptosis References: 1. Amit-Vazina M, Shishodia S, Harris D, Van Q, Wang M, Weber D, Alexanian R, Talpaz M, Aggarwal BB, Estrov

Z. Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells. (2005) Br J Cancer. ; 11;93(1):70-80.

2. Sulaiman NB, Mohan CD, Basappa S, Pandey V, Rangappa S, Bharathkumar H, Kumar AP, Lobie PE, Rangappa KS. An azaspirane derivative suppresses growth and induces apoptosis of ER-positive and ER-negative breast cancer cells through the modulation of JAK2/STAT3 signaling pathway. Int J Oncol.; 2016 49(3):1221-9.

88

P-2

Preparation and characterization of Ceranib-2 anticancer drug delivery system based on Rosin Ester/PEG nanostructure

Ali Ben Taleb* , Selcan Karakus, Ezgi Tan, Merve Ilgar , Ayben Kilislioglu

Istanbul University Cerrahpasa, Istanbul, Turkey. *E-mail: alimbentaleb66@gmail.com

Background: The design of effective drug delivery system for poorly soluble potent anticancer drugs with highly cellular bioavailability, and good colloidal stability is an essential challenge in modern pharmaceutical formulation. Polymer encapsulation drug delivery system nowadays become an attractive approach to overcome many pharmaceutical issues associated with anticancer formulation [1]. Rosin gum is a promising natural abundant polymer obtained from pin tree. It has been used in many applications due to its properties such as biodegradability, biocompatibility, easy of processing, inexpensive, good drug release profile, and good film forming capability [2]. However, the drug delivery system of rosin ester nanostructure is not yet studied in detail. In this study, the drug delivery system using an amphoteric nanostructure based on PEGylated rosin ester for Ceranib-2 is synthesized and characterized as novel ceramidase inhibitors (CDs). The nanostructure is conjugated with a certain cell ligand like folic acid in order to overcome bioavailability and pharmacokinetic properties of ceramidase inhibitors drugs. Method: Stable homogeneous nanocapsules (RE / PEG/ Ceranib-2) were prepared by using sonication and evaporation techniques. The nanostructure was characterized using DLS, SEM. Results: Intensity particle size distribution analysis of rosin ester nanostructure was obtained. The DLS results showed homogeneous distribution of the system (Fig 1.0) and the average particle size was obtained around 350 nm. However, PDI was 0.426 which indicates well polydispersity of the system. According to the SEM micrographs the average diameters (270 nm) were in agreement with hydrodynamic diameters obtained from DLS. Discussion: RE / PEG/ Ceranib-2 nanocapsules were formed successfully with the aid of the electrostatic interaction of Rosin ester /PEG. Conclusion: Based on the results obtained one could expect that Rosin ester /PEG nanocapsules can be used successfully as a drug delivery nanosystem. Keywords: Rosin ester, PEG, Ceranib-2, nanoencapsulation, sonication. References: 1. Hughes G A, Nanostructure-mediated drug delivery. (2005) Nanomedicine: Nanotechnology, Biology and Medicine; 1(1):22–30. 2. Baveja SR, Ranga RKV, Arora J, Examination of natural gums and mucilages as sustaining materials in tablet dosage forms, (1989) J Pharm Sci, 51(4), 115- 118.

89

P-3

Homology Modeling, Molecular Dynamics Simulation and Docking studies of Allatostatin Receptor type C of

Thaumetopoea pityocampa

Aida Shahrakia, Necla Birgul-Iyisona, Serdar Durdagib a Department of Molecular Biology and Genetics, Bogazici University, 34342 Bebek-Istanbul, Turkey

b Department of Biophysics, School of medicine, Bahcesehir University, Goztepe, Istanbul, Turkey Background: Allatostatins (ASTs) are neuropeptides with critical role in regulating metamorphosis, food intake and many other important physiological functions in insects. They exert their function through binding to their cognate receptors which are G-protein Coupled Receptor (GPCRs) [1]. Activation of the receptor inhibits the Juvenile Hormone (JH) secretion which manages most of the insects’ developmental activities [2]. Thus, molecules capable of activation of these receptors are providing another mode of action for the next-generation pesticides. In this project, the Allatostatin type C receptor (AstR-C) of Thaumetopoea pityocampa (T.pit), a pest residing in the Mediterranean regions of the world [3] was targeted with final aim to design agonist molecules for the receptor. Methods: Receptor and its native ligand sequences were derived from the Whole Genome Sequencing [4] data. SWISS-MODEL server was used for the homology modeling of the receptor. Each model was applied to 25ns molecular dynamics (MD) simulations to check the structural integrity. The ligand structure was constructed and then refined. The possible binding sites of the receptor were predicted, and grid was generated, accordingly. The docking was performed. Results: Mouse opioid receptors (PDB ID: 6DDE) showed to be a promising template and the constructed model showed acceptable stability during the simulation according to the RMSD and RMSF plots. In order to keep the model in its active state during the simulation, G-protein was also added to the system and the MD simulations were repeated. The backbone showed acceptable stability. Docking studies were done using this template. Conclusions: Using homology modeling method, a reliable model of AstR-C of T.pit was constructed. The stability and reliability of the model was tested by molecular dynamics simulation studies. The docking studies were performed to find the ligand binding pocket of the receptor. Obtained information would be used in the next steps which would be designing agonist molecules for AstR-C with final goal of proposing next generation pesticides having less side effects on the ecosystem. Keyword: GPCR, Alatostatin receptor, Homology modeling, MD Simulation, Docking References: 1. Schoofs, Liliane, Arnold De Loof, and Matthias Boris Van Hiel. Neuropeptides as regulators of behavior in insects:(2017)Annual review of entomology 62 : 35-52. 2. Ivanovic, Jelisaveta. Hormones and metabolism in insect stress:(2018) CRC Press. 3. Williams, David T., and Gillian Jonusas. The influence of tree species and edge effects on pheromone trap catches of oak processionary moth Thaumetopoea processionea (L.) in the UK.:(2018). Agricultural and Forest Entomology 4. Ekblom, Robert, and Jochen BW Wolf. A field guide to whole-genome sequencing, assembly and annotation: (2014) Evolutionary applications 7.9:1026-1042.

90

P-4

Novel Imatinib Analogues Featuring (Thio) urea Motifs: Design, Molecular Modeling and Assessment of Cytotoxic and Apoptotic Potential in Human

Leukemia and Lymphoma

Aslı Türea, Özlem Bingöl Özakpınarb , İlkay Küçükgüzela a Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

Marmara University, 34668; b Department of Biohemistry, Faculty of Pharmacy, Marmara University, 34668.

E-mail: ikucukguzel@marmara.edu.tr

Background: Imatinib is the first protein kinase inhibitor that has been developed with rational drug design approach and has been used in clinic for years. It is effective against a range of leukemias (e.g., chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic myelomonocytic leukemia, chronic eosinophilic leukemia), as well as gastrointestinal stromal cancer [1]. Imatinib owes its clinical success in CML therapy to the inhibition ability of Abl kinase protein [2]. It is known that overexpression of Abl by the induction of v-Abl or Bcr-Abl causes CML [3]. Methods: By the help of molecular docking implementations with Abl kinase we designed imatinib analogues and evaluated their anticancer activity through this research. Molecular modelling studies were run with DFG-out inactive states of Abl kinase. Possible binding conformations of each ligand were evaluated. Interactions with hinge region and interactions with DFG motif of the kinase proteins which are both crucial for kinase inhibition were visualized after docking processes. With respect to anticancer activity studies, cytotoxicity of thirty-two different compounds and imatinib as experimental control were tested initially on Raji (Burkitt’s lymphoma), K562 (CML) and NIH3T3 (fibroblast) cells. Percent inhibition and IC50 values were determined subsequently. Compounds which exhibited better cytotoxicity results towards K562 and Raji cells were selected for further studies. Apoptosis studies including Annexin V staining and measurement of caspase activation were performed and detection of the loss of mitochondrial membrane potential (MMP) was screened. Results: Docking conformations showed favorable superposition of phenylaminopyrimidine cores of designed (thio)urea analogues and imatinib. In addition, these analogues revealed out significant anticancer activity in comparison with imatinib. The most active derivative (compound 31) had an IC50 value of 16.95 µM on K562 cells which was comparable to that of imatinib (IC50=11.22 µM). Conclusions: A series of novel imatinib analogues were designed, synthesized and evaluated as potential anticancer agents. Further in silico and in vitro studies will be discussed in detail. Keywords: Analog design, imatinib, Bcr-Abl, cancer, ureas / thioureas.

91

References:

1. Levitzki A. Tyrosine Kinase Inhibitors: Views of Selectivity, Sensitivity, and Clinical Performance. (2013) Annu Rev Pharmacol Toxicol.; 53:161-185.

2. Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J. Structural Mechanism for STI-571 Inhibition of Abelson Tyrosine Kinase. (2000) Science; 289(5486): 1938-1942.

3. Nowell PC. Discovery of the Philadelphia chromosome : a personal perspective. (2007) J Clin Invest.; 17(8): 2033-2035.

92

P-5

Biofilm and pqs System Inhibition Properties of Norlichexanthone against Pseudomonas aeruginosa

Ayla YILDIZa, Barış GÖKALSINa , Arhun Ali Balkana, Hiroko KAWAKAMIb, Kojiro HARAb,

Serdar DURDAĞIc, N. Cenk SESALd aMarmara University, Institute of Pure and Applied Sciences, Biology Department, İstanbul, Turkey,

bAkita Prefectural University, Department of Biological Production, Akita, Japan, cBahcesehir University, Department of Biophysics, School of Medicine, İstanbul, Turkey, dMarmara University,

Faculty of Arts and Sciences, Biology Department, İstanbul, Turkey E-mail: yildizzayla@gmail.com

Background: Pseudomonas aeruginosa, opportunistic and drug resistant pathogen, is associated with high morbidity and mortality rates. Quorum Sensing (QS) system is a bacterial communication system to sense their population density in their surrounding milieu via signal molecules. Several virulence factors and related bacterial behaviors including biofilm formation are regulated by QS. P. aeruginosa utilizes four hierarchically related QS pathways. These are LasI/LasR, RhlI/RhlR, PqsR-controlled quinolone system and iqs system. pqs signaling system has great importance in the pathogenesis of infections via controlling virulence factors such as pyocyanin, elastase etc. Moreover, P. aeruginosa forms biofilm by secreting extracellular matrix for surface attaching. It has been reported that biofilm forms are more resistant up to 1000-3000 times to antibiotics compared to their planktonic forms. Most researchers have focused on QS inhibition as an alternative strategy to overcome drug resistance. Natural and synthetic QS inhibitors have been reported up to date. The natural compounds synthesized from lichens are important resources in the development of pharmaceutical raw materials since lichens have various pharmacological properties such as antimicrobial, antioxidant, antitumor etc. Methods: Nolichexanthone, isolated from lichen-forming fungi (LFF) cultures of Lecanora sp. was tested against P. aeruginosa for evaluation of inhibitory effects on pqs system and biofilm formation at a certain dosage of 150, 75 and 37,5 μg/ml. pqsA-gfp biosensor strains of P. aeruginosa were used to monitor QS inhibition and also anti-biofilm activity was tested on PAO1 wild type strain. QS and biofilm inhibition tests were performed in 96-well microplates. Fluorescence of GFP expression was measured at 485 nm excitation and 535 nm emmission for every 15 mins for 24 hr to detect QS activity. Biofilms were stained with 0.1% crystal violet and measured at OD: 590 nm in microplate reader (Cytation 3-BioTek). Molecular docking studies were performed by the Glide module of Maestro/Schrodinger’s molecular modeling package and the Glide/SP (Standard Precision) protocol was used. Norlichexanthone was docked with PqsR protein (PDB code: 4JVC). Results: The inhibition rates for pqs system was approximetely 72.5%, 49.8% and 40.6%at concentrations of 150, 75 and 37,5 μg/ml of nolichexanthone. Also, we observed that biofilm formation was inhibited by nolichexanthone at same concentrations for 22.9%, 12.6% and 15.2%, respectively. According to in silico experiments, docking score of 4JVC protein was determined as 7.025 kcal/mol,

93

Conclusions: Our results demonstrate that QSI potential of norlichexanthone was considerably high against pqs system, especially for the highest dose. On the other hand, the same inhibitory potential was not detected in biofilm formation. In this way, we may suggest that biofilm formation is not only related to pqs system. Also, high scores were obtained for molecular docking results of 4JVC protein belonging to pqs system, indicating that norlichexanthone may have potential to inhibit PqsR protein. Consequently, in silico and in vitro results were found to be consistent and norlichexanthone may be utilized in drug discovery by further research. Acknowledgement: This study was funded by the project: TUBITAK 216S885. Keywords: Pseudomonas aeruginosa; quorum sensing; biofilm; molecular docking; lichen secondary metabolites

94

P-6

Docking Simulations of Cyclic and Linear Peptides to Discover Novel αvβ3 Integrin Inhibitors

Ayşe Berçin BARLASa, İsmail Hakkı AKGÜNa aEge University, Faculty of Engineering, Bioengineering Department, Bornova, İzmir, TURKEY

E-mail: aysebercinb@gmail.com, ismail.hakki.akgun@ege.edu.tr Background: Integrins are membrane receptor (cell adhesion) proteins which provide cell-cell and cell-surface interaction. They play an important role, especially αvβ3 sub-class, for tumor growth and angiogenesis (1). Consequently, integrins are important therapeutic target proteins for cancer therapy. Up to date, many molecules have been studied that contain the Arginine-Glycine-Aspartic Acid (RGD) peptide sequence or mimetics of this motif as integrin inhibitors. In addition, studies on integrin inhibitors that do not contain RGD have increased recently (2, 3). Success of peptidomimetic (eptifibatide) studies (4) and possibility to generate high number of both linear and cyclic peptides with different sequence length encourages us to work on to generate novel integrin inhibitors. Methods: Integrin αvβ3 crystal structure was obtained from RCSB Protein Data Bank (PDB ID: 1L5G) (5). Both linear and cyclic peptide structures were created by using Bioware web server’s CycloPs (6) tool. Ligand and receptor preparations were performed using PyMol (7). In ligand preparation process hydrogen were added to the structures and saved in sdf data format. In receptor preparation process all heteroatoms and molecules were deleted from receptor file except Mn2+ ions in binding pocket. Docking experiments were performed using Rosetta Online Server (ROSIE) ligand docking tool (8). In each docking simulation we have set to server generate 200 structures. Results: Docking score of RGD tripeptide structure was calculated as -11.131. At the same time docking score of the inhibitor molecule Cilengitide in the PDB structure of 1L5G was calculated as -10.282. On the other hand, some of the other cyclic peptide derivatives have shown close docking score to both RGD and Cilengitide. For instance docking score of the cyclic pentapeptide c(ARGDD) and c(CRGDC) were calculated as -9.693, -9.359 respectively. On the contrary binding scores of the linear forms of these cyclic peptides were not as decent as cyclic peptides (Table 1). Conclusions: Our docking simulations results are showing that cyclic peptides are more favourable over linear ones. This enables us to generate novel structures to be used as integrin inhibitors. Keywords: Integrins, inhibitor, cyclic peptide, computational design, docking

95

References: 1. Hatley R J, Macdonald S J, Slack R J, Le J, Ludbrook S B, Lukey P T. An αv-RGD Integrin

Inhibitor Toolbox: Drug Discovery Insight, Challenges and Opportunities. (2018) Angewandte Chemie International Edition; 57(13), 3298-3321.

2. Höltke C. isoDGR-Peptides for Integrin Targeting: Is the Time Up for RGD? (2018) J. Med. Chem.; 61(17): 7471–7473.

3. Francesca N, Paissoni C, Quilici G, Gori A, Traversari C, Valentinis B, Sacchi A, Corti A, Curnis F, Ghitti M, Musco G. Succinimide-Based Conjugates Improve IsoDGR Cyclopeptide Affinity to αvβ3 without Promoting Integrin Allosteric Activation. (2018) J. Med. Chem.; 61(17):7474–7485.

4. Phillips, D. R., & Scarborough, R. M., Clinical pharmacology of eptifibatide. (1997) Am J Cardiol, 80(4), 11B-20B.

5. Xiong J P, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman S L, Arnaout M A. Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. (2002) Science; 296: 151-155.

6. Duffy, F. J., Verniere, M., Devocelle, M., Bernard, E., Shields, D. C., & Chubb, A. J., CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids. (2011) J. Chem. Inform. Model., 51(4), 829-836.

7. Schrodinger LLC (2010) The PyMOL Molecular Graphics System, Version 1.7.x 8. Deluca, S., Khar, K., Meiler, J., Fully Flexible Docking of Medium Sized Ligand Libraries with

RosettaLigand., (2015) PLoS ONE 10(7): e0132508. doi:10.1371/journal.pone.0132508.

Table 1. Docking scores of some linear and cyclic peptides on ROSIE server.

Ligand Docking

Score Ligand

Docking

Score Ligand

Docking

Score

RGD -11.131 c(ARGDD) -9.693 c(CRGDC) -9.359

Cilengitide -10.282 ARGDD -5.607 CRGDC -5.862

96

Figure 1: Docking structures of a) Cilengitide b) RGD c) c(ARGDD) d) ARGDD e) c(CRGDC) f)

CRGDC.

a) b)

f) e) d) c)

97

P-7

Design, Synthesis and Cytotoxic Activity Studies of 5-Aminouracil Derived Novel Organic Molecules

Gamze Koza, Ayşe Haliça, Cenk Serhan Özverelb, Ömer Koza

aDepartment of Chemistry, Faculty of Engineering and Natural Sciences, Bursa Technical University, 16310, Bursa, Turkey;

bDepartment of Bioengineering, Faculty of Engineering, Ege University, 35100, İzmir, Turkey E-mail: aysehalic@gmail.com

Background: DNA is the material of inheritance and it controls the structure and function of cells. Recently, there has been an emerging interest in studies related to the interaction of small molecules with nucleic acids because of their relevance in the development of new reagents for biotechnology and medicine.1 5-Substituted uracils stand out among the bioactive derivatives of uracil and their activity ranges from antiviral, anticancer, anti-inflammatory, antitumor and antibacterial.2 On the other hand, imine-DNA binding studies are very recent and the results are promising.3

Methods: Here we have synthesized and characterized a series of novel 5-arylideneaminouracil molecule. Two model molecules were used for molecular docking studies using DFT method. All samples were tested for their in vitro cytotoxicity against PC-3, A549, SHSY-5Y and HEK293 cell lines.

Results: The computational studies related to 5-aminouracil imine derivatives and DNA interaction revealed that these small molecules can act as groove binders. We have successfully obtained 13 samples and tested their cytotoxic activities.

Conclusions: 5-arylideneaminouracils were evaluated as DNA binding agents for the first time. The interaction strength with DNA through hydrogen bonding is encouraging for a small organic ligand compared to the well-known groove binders.

Keywords: aminouracil, DNA binding, molecular docking, synthesis References: 1. Khare D, Pande R. (2012) Der Pharma Chem.; 4:66-75. 2. Baraldi P. G, Romagnoli R, Guadix A. E, Pineda de las Infantas M. J. P, Gallo M. A, Espinosa A, Martinez A,

Bingham J. P, Hartley J. A. J. (2002) Med. Chem.; 45:3630-3638. 3. Kirubavathy S. J, Velmurugan R, Karvembu R, Bhuvanesh N. S. P, Enoch I. V. M. V, Selvakumar P. M,

Premnath D, Chitra S. (2017) Journal of Molecular Structure; 1127:345-354.

98

P-8

How Does PtdIns(3,5)P2 Affect Mitotic Exit and Cytokinesis

Baris Bekdas. Ayse Koca Caydasi Department of Molecular Biology and Genetics, Koc University, Istanbul

E-mail:bbekdas18@ku.edu.tr.

Background: Phosphoinositides are evolutionary conserved phospholipid derivatives found in cellular membrane. Each phosphoinositide interacts with specific proteins to perform cellular functions such as membrane trafficking, nuclear events, signal transduction and cytoskeletal organization (1). Phosphotidylinositol (3,5) Biphosphate (PtdIns(3,5)P2) is the least abundant phosphoinositide derivative in a cell. Some neurological diseases such as Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth Disease are associated with PtdIns(3,5)P2 deficiency. Although PtdIns(3,5)P2 plays roles in membrane trafficking, stress response, vacuole/endolysosome structure and function - and transcriptional regulation-, no mitotic function has been attributed to PtdIns(3,5)P2. Previous studies demonstrated that PtdIns(3,5)P2 physically interacts with microtubule regulators, centrosome and some components of exit from mitosis (2). Therefore, we think that PtdIns(3,5)P2 might have a function in exit from mitosis. Methods: To test that PtdIns(3,5)P2 plays a role in exit from mitosis, we have analysed metaphase-anaphase transition, microtubule dynamics, contraction properties of actomyosin ring in cytokinesis by live cell fluorescence microscopy and compared those between wild type and PAS component - deleted strains. In addition, we performed genetic analysis to reveal whether PtdIns(3,5)P2 synthesis have epistatic interactions with known pathways for exit from mitosis Therefore, the genes responsible for synthesis of PtdIns(3,5)P2 were deleted in MEN mutant strains to determine any synthetic lethality. Results: According to our results, PAS components show synthetic interactions with many mutant strains which are defective in mitotic exit. Conclusions: Our genetic analysis demonstrated that PtdIns(3,5)P2 synthesis promotes MEN activity and contributes mitotic exit in yeast through yet an unknown mechanism. Keywords: PtdIns(3,5)P2, PAS component, Mitotic Exit Pathway References:

1. 1 Rautio Mathews, Chrisotphe K.; van Holde, K.E.; Ahern, Kevin G. (2005). Biochemistry Third Edition 2. 2 J, Kumpulainen H, Heimbach T, Oliyai R, Oh D, Järvinen T, Savolainen J. Prodrugs: Design and clinical

app Jones DR, González-García A, Díez E, Martinez-A C, Carrera AC, Meŕida I. The identification of phosphatidylinositol 3,5-bisphosphate in T-lymphocytes and its regulation by interleukin-2. J Biol Chem. 1999 Jun 25;274(26):18407-13. PMID 10373447

99

P-9

Norlichexantone against Pseudomonas aeruginosa las and rhl QS Systems

Barış GÖKALSINa, Ayla YILDIZa, Arhun Ali BALKANa, Hiroko KAWAKAMIb, Kojiro HARAb, Serdar DURDAĞIc, N. Cenk SESALd

aMarmara University, Institute of Pure and Applied Sciences, Biology Department, İstanbul, Turkey, bAkita Prefectural University, Department of Biological Production, Akita, Japan, cBahcesehir

University, Department of Biophysics, School of Medicine, İstanbul, Turkey, dMarmara University, Faculty of Arts and Sciences, Biology Department, İstanbul, Turkey

E-mail: bgokalsin@gmail.com

Background: Pseudomonas aeruginosa is an opportunistic and drug resistant pathogen. It is associated with high morbidity and mortality rates especially in immunocompromised patients. Quorum Sensing (QS) system is a bacterial communication system to sense their population density in their surrounding milieu via signal molecules. Several virulence factors and related bacterial behaviors including biofilm formation are regulated by QS. Most researchers have focused on QS inhibition as an alternative strategy to overcome drug resistance. Natural and synthetic QS inhibitors (QSIs) have been reported up to date. The natural compounds synthesized from lichens are unique and important resources in the development of pharmaceutical raw materials. This study investigates the antivirulence properties of norlichexanthone, fractionationed from LFF extracts of Lecanora sp. against P. aeruginosa by inhibiting its QS systems. Methods: Cultures of P. aeruginosa lasB-gfp and rhlA-gfp monitor strains were utilized in QS inhibition screens. Bacteria were treated with serial dilutions of norlichexanthone. Growth and green fluorescent protein (GFP) expressions were monitored using multimode microplate reader (Biotek-Cytation 3) for 16 hours, measuring absorbance and fluorescence every 15 minutes. Fluorescence of GFP expression was measured at 485 nm excitation and 535 nm emission wavelengths. Molecular docking methods were also performed for crucial QS proteins of P. aeruginosa. The 3D crystal structures of LasI and LasR proteins were obtained from Protein Data Bank with PDB codes 1RO5 and 3IX3. Azithromycin, an antibiotic and QSI for CF treatment available in market, and norlichexanthone structures were downloaded from ZINC Database. Protein-ligand preparation and docking processes were performed using Glide module of Maestro/Schrödinger software. Results: In vitro results indicate that norlichexanthone is capable of inhibiting las and rhl QS systems by approximately 82.7% and 52.1% respectively for 150 µg/ml. Molecular docking results are evaluated by docking scoring function (kcal/mol). Docking scores were determined as -7.851 for LasR and -4.765 for LasI. Conclusions: Results show that in vitro inhibition rates and in silico docking results have shown a consistency and lichen secondary metabolite norlichexanthone can be employed as a QSI. Screening unique natural compounds such as norlichexanthone can provide opportunities for future QSI drugs. Keywords: Pseudomonas aeruginosa; quorum sensing; lichen secondary metabolites

100

P-10

Determination of Potential CK2 Inhibitors Using Fragment Based Drug Discovery

Doğa Özşena, Beril Ateşa, Hande Abeşa,, Sezen Alsancaka, Yeşim Çamlısoya, Nihan Çelebi Ölçüma,*

aDepartment of Chemical Engineering, Yeditepe University, İstanbul, 34755 *Corresponding author: nihan.olcum@yeditepe.edu.tr

Abstract The serine/threonine protein kinase, CK2 (casein kinase 2) which is found in many cells in human body and it is classified as a messenger-independent protein. It has been detected that, CK2 shows a significant role in increasing cell growth for both normal and cancer cell and it acts as a suppressor of apoptosis so it is associated with cancer. [1][2][3] CX-4945, also known as Silmitasertib, (5-(3 chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid), is the only potential inhibitor for CK2 that has been passed to phase 2 in clinical trials as a drug. CX-4945 is an orally administered, highly specific, ATP-competitive inhibitor of CK2 and it shows cytotoxiticiy and apoptosis. A number of molecules similar to the CX-4945 complex, which is used as a CK2 inhibitor in human clinical trials, are being developed.

Fragment based drug design, which is used for reducing attrition and providing leads for previously intractable biological targets[4] , was performed for CX-4945 molecule to investigate new potential molecules as CK2 inhibitors. Also druglikeness and structural properties of these molecules were investigated computationally and compared with properties of CX-4945. Moreover, pharmacophore search was performed to determine similarity with CX-4945 and docking was applied to reveal protein-ligand interaction.From the knowledge of this work, insight for indentification of dericatives with higher biological activity will be provided so that it will guide the synthesis studies. Key words: Fragment based drug design , CK2 intibitors, CX-4945, Potential drug molecules References

1. Chon, H., J., Bae, J., K., Lee, Y., Kim, J., “The Casein Kinase 2 Inhibitor, CX-4945, as an Anti-cancer Drug in Treatment of Human Hematological Malignancies”, Frontiers in Pharmacology, Vol 6, No 70, 1-2, 2015.

2. David W. LITCHFIELD1,“Protein kinase CK2: structure, regulation and role in cellular decisions of life and death”, 2003

3. J. H. Trembleya, G. Wanga, G. Ungerc, J. Slatond, K. Ahmeda, “CK2: A key player in cancer biology”, Cellular and Molecular Life Sciences, Birkhuser Verlag, Basel, 2009

4. Christopher W. Murray & David C. Rees, “The rise of fragment-based drug discovery”, Nature Chemistry, Vol 1, pages187–192 (2009)

101

P-11

Drug repositioning for the treatment of cervical cancer through host-pathogen protein-protein interactions

Beste Turanlia,b,*, Medi Korib,* , Kazim Yalcin Argab

aDepartment of Bioengineering, Istanbul Medeniyet University, Istanbul, Turkey; bDepartment of Bioengineering, Marmara University, Istanbul, Turkey

*Equal contribution E-mail: beste.turanli@medeniyet.edu.tr

Background: Cervical cancer (CESC) is a malignant neoplasm which is the second most common cancer among women worldwide. Development of cervical cancer is triggered by oncogenic Human Papillomavirus (HPV) infection. Although HPV has hundreds of different sub-types with oncogenic potential, HPV-16 and -18 are the responsible for up to almost 80% of cervical cancer cases [1]. Determination of new drugs or drug combinations for CESC were discussed in the concept of drug repositioning for gynecologic tumors [2]. In this study, we aimed to investigate highly interacted host-pathogen protein-protein interactions (PPIs) in HPV-16 and -18, separately. Afterward, hub host proteins in the PPIs and their interacting drug associates were applied to repurpose drugs for sub-type specific disease treatment. Methods: HPV-16 and HPV-18 host-pathogen PPIs were acquired by PHISTO [3]. Degree and Betweenness as network parameters were calculated for each interaction. Additionally, differentially expressed genes were calculated by using two independent transcriptome datasets (GSE7803 and GSE9750) for each type as mentioned in our previous study [1]. Host protein evidence has been supported by proteome data acquired from Human Protein Atlas [4]. Afterward, the statistical significance of drug-protein interactions was acquired by geneXpharma [5]. All information was integrated to calculate scores for drug-protein interaction pairs. Top 5% of all drug-gene interactions were investigated in both sub-types, separately. Results: Results showed 84 interactions among 63 drugs and 17 drug targets in HPV-16 cases whereas 40 interactions among 39 drugs and 15 drug targets in HPV-18. 20 of the drugs including docetaxel, celecoxib, metformin, etc. were common. Our analysis determined not only anti-neoplastic agents which are already used for CESC therapy, but also different drug groups treating various diseases such as cardiovascular, psychiatric or rheumatologic. Conclusions: The identification of sub-type specific drug and its targets may improve the specificity and sensitivity of the treatment by using efficient drug repositioning strategies as presented. As soon as the results are tested in vitro and in vivo studies, the launching of the drug to the market can be faster. Keywords: cervical cancer, drug repositioning, host-pathogen interaction, drug target

102

References:

1. Kori M, Arga KY. Potential biomarkers and therapeutic targets in cervical cancer: Insights from the meta-analysis of transcriptomics data within network biomedicine perspective. (2018) PLoS One

2. Banno K, Iida M, Yanokura M, Irie H, Masuda K, Kobayashi Y, Tominaga E, Aoki D. Drug repositioning for gynecologic tumors: a new therapeutic strategy for cancer. (2015) ScientificWorldJournal.

3. Durmuş Tekir S, Çakir T, Ardiç E, Sayilirbaş AS, Konuk G, Konuk M, Sariyer H, Uǧurlu A, Karadeniz I, Özgür A, Sevilgen FE, Ülgen KÖ. PHISTO: Pathogen-host interaction search tool. (2013) Bioinformatics; 29 (10), 1357-1358.

4. Uhlen M, Fagerberg L, Hallstrom, B. M., Lindskog, C., Oksvold, P., Mardinoglu, A., … Ponten, F. Tissue-based map of the human proteome. (2015) Science; 23;347(6220):1260419.

5. Turanli B, Gulfidan G, Arga KY. Transcriptomic-Guided Drug Repositioning Supported by a New Bioinformatics Search Tool: geneXpharma. (2017) Omi. A J. Integr. Biol.; 21: 584–591.

103

P-12

Characterization of a Novel Function of Cohesin in Mitotic Control

Betül Sarı, Ayşe Koca Çaydaşı Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey

E-mail: bsari17@ku.edu.tr Background: Mitotic exit requires cyclin dependent kinase (Cdk) inactivation. In budding yeast, Cdc14 phosphatase is responsible for reverting Cdk phosphorylation, via dephosphorylating Cdk targets directly and leading proteasomal degradation of cyclin subunits indirectly (1). To achieve mitotic exit, Cdc14 should be released into cytoplasm where it associates with its targets (1). “Mitotic Exit Network” is the main network that is responsible for mitotic exit in budding yeast. Mitotic Exit Network (MEN) is under the control of a special mechanism so called Spindle Position Checkpoint (SPOC). If the mitotic spindle is misaligned in mother cell or cytoplasmic microtubule-cortex interaction is not achieved due to microtubule defects, it is sensed by the SPOC which then blocks mitotic exit (2). In our preliminary study, surprisingly, we observed that partial inactivation of cohesin complex allowed mitotic exit of budding yeast cells otherwise arrested in late mitosis due to the SPOC activity. Our data indicates that the decrease in functionality of cohesin complex helps cells arrested in late mitosis to exit from mitosis. Importantly, suggested role of cohesin applies at anaphase/telophase/ cytokinesis phases of cell cycle so beyond its role in sister chromatid cohesion in metaphase. Here, I will present data that will help understanding the novel function of cohesin in molecular detail. Mutations in cohesin cause several disorders such as cohesinopathies and are associated with several cancers (3). Importantly, it is thought that these disorders arise from functions of cohesin other than its role in sister chromatid binding. Functions of cohesin from which cohesinopathies and cohesin-related cancers originate remain unknown. Novel cellular functions of cohesin and elucidation of its molecular mechanisms are important for understanding of these cohesin-related diseases which might be the first step in pinpointing the cure with effective drugs in the near future. Methods: To find proteins which genetically interact with cohesin in the proposed pathway, a dosage-suppression genetic screen was performed using 2-micron yeast genomic library. Additionally, to examine the effects of the identified genes in cell cycle, these genes will be deleted from wild type genome and the outcomes in terms of cell cycle kinetics will be analyzed by microscopy and Western blot studies. Results: We have selected 370 colonies that were able to revert the growth rescue phenotype of cohesin mutants. Plasmids from these colonies were isolated. We are currently back transforming these plasmids to the cohesin mutant strains in order to identify the true plasmids that bear genes responsible from the phenotype. Conclusions: Cohesin has an additional function in cell cycle regulation other than its known role in sister chromatid cohesion. Keywords: Mitotic control, cohesin, genetic screen

104

References:

1. Stegmeier F1, Amon A. Closing Mitosis: The Functions of the Cdc14 Phosphatase and Its Regulation. (2004) Annu Rev Genet.;38:203-32

2. Pereira G, Schiebel E. Kin4 Kinase Delays Mitotic Exit in Response to Spindle Alignment Defects. (2005) Molecular Cell; 19(2):209–221.

3. Michaelis C, Ciosk R, Nasmyth K. Cohesins: Chromosomal Proteins that Prevent Premature Separation of Sister Chromatids. (1997) Cell.; Oct 3;91(1):35-45

105

P-13

Synthesis and Characterization of Novel Branched Magnetic Chitosan Nanocomposite for Anti-Cancer Drug Loading and

Release System

Bilsen Turala, Tuba Tarhanb, Servet Tural a aDepartment of Chemistry, Faculty of Education, Dicle University, Diyarbakır, Turkey

bVocational Higher School of Health Services, Mardin Artuklu University, Mardin, Turkey E-mail: bilsentural@gmail.com

Background: Chemotherapy is used one of the best methods in cancer treatment. One of the most important problems of cancer chemotherapy nowadays is used that the anti-cancer drugs do not have the ability to recognize cancer cells and show toxic effects on healthy cells 1,2,3. In addition, administration of high doses of the drug is given to the body to provide a therapeutic dose concentration, this situation is causes severe side effects and systemic toxicity, and if not controlled it can result in death. Therefore, delivering the drug to target cell within the body and developing of drug delivery system providing controlled release are of great importance. In recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. It is possible to eliminate most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. Methods: In this study, we synthesized and characterized the novel magnetic branched carboxymethyl chitosan nanocomposite with NαNα-Bis (Carboxymethyl)-L-Lysine Hydrate (NTA)4,5,6 and drug loading and release conditions and drug loading efficiency were studied with the novel branched magnetic chitosan for the anti-cancer drug topotecan. Results: the novel biocompatible and biodegradable branched polymer was successfully synthesized and characterized with different analytical devices such as Transmission Electron Microscopy (TEM), Scanning Electron Microscope (SEM), Vibrating Sample Magnetometer (VSM), Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-Ray Analysis (EDX), and X-Ray Photoelectron Spectroscopy (XPS). Drug loading and release studies were followed by High-Performance Liquid Chromatography (HPLC). Conclusions: The drug loading yield was found to 54.1% for the novel branched polymer nanocomposite. Percentage of drug released was found to 2.68 % during 90 minute and it was observed that the release came to balance after 40 minute. On the other hand, it was found to the novel branched polymer has good drug loading capacity. Keywords: Drug release; Magnetic nanocomposite; Magnetic targeting; Novel branched chitosan Acknowledgments This project is funded by the Scientific and Technological Research Council of Turkey (TUBITAK, 116Z778).

106

References 1. Aydemir A.T. Loading of Superparamagnetic PMMA (SP-PMMA) Latexes by Chemotherapic Drugs and Determınation of The Cytotoxic Effects on Human Hepatoma Cell Lines (Hep3B). Balikesir University, Institute of Science, Department of Biology (2011) Balıkesir. 70. 2. Sinha R, Kim GJ, Nie S, and Shin DM. Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery. (2006) Mol. Cancer. Ther.; 5:1909-1917. 3. Wang AZ, Gu F, Zhang L, Chan J M, Radovic-Moreno A, Shaikh M R, Langer RS, and Farokhzad OC. Biofunctionalized Targeted Nanoparticles for Therapeutic Applications. (2008) Expert opinion on biological therapy; 8:1063-1070. 4. Park J-H, Im K-H, Lee S-H, Kim D-H, Lee D-Y, Lee Y-K, Kim K-M, Kim K-N. Preparation and characterization of magnetic chitosan particles for hyperthermia application. (2005) Journal of Magnetism and Magnetic Materials; 293(1): 328-333. 5. Schweiger C, Pietzonka C, Heverhagen J, Kissel T. Novel magnetic iron oxide nanoparticles coated with poly (ethylene imine)-g-poly (ethylene glycol) for potential biomedical application: synthesis, stability, cytotoxicity and MR imaging. (2011) International Journal of Pharmaceutics; 408(1): 130-137. 6. Zhou H, Tao K, Ding J, Zhang Z, Sun K, Shi W. A general approach for providing nanoparticles water-dispersibility by grinding with poly (ethylene glycol). (2011) Colloids and Surfaces A: Physicochemical and Engineering Aspects; 389(1): 18-26.

107

P-14

In silico Thermostability Studies for Muscular Dystrophy Causing Mutation in the hISPD Protein

Busecan Aksoydana,b*, Haicui Wangc,*, Hülya-Sevcan Daimagülerc, Rosanne Sprutec,

Sebahattin Cirakc,1, Serdar Durdagia,*,1 aComputational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of

Medicine, Bahcesehir University, Istanbul, Turkey; bGraduate School of Health Sciences, Neuroscience Programme, Bahcesehir University, Istanbul, Turkey cCenter for Molecular Medicine (CMMC) and

Department of Pediatrics, University of Cologne, Germany

e-mail: sebahattin.cirak@uk-koeln.de and serdar.durdagi@med.bau.edu.tr

Background: Alpha-dystroglycan is an O-mannose-linked glycoprotein mainly responsible for the binding of extracellular ligands. Aberrant glycosylation of α-dystroglycan causes a group of muscular dystrophies with variable brain and eye involvement collectively termed dystroglycanopathies. Human isoprenoid synthase domain (hISPD) containing protein is a cytidyltransferase homodimer protein, which synthetizes a nucleotide sugar for the α-dystroglycan O-mannosylation in muscle and brain. The deficiency and mutations in the ISPD protein is known to be one of the common causes for dystroglycanopathies. While many patients with ISPD mutation have a severe phenotype due to loss-of-function mutations, other patients have a milder phenotype with amino acid substitutions. It had been postulated that the mutations may not only alter catalytic activity but also ligand binding, stability and homodimer formation [1-3]. We hypothesized whether these mutations may also affect the thermostability of the protein. To this end, we developed complementary in silico and experimental approaches. There are several objectives of this study including the examination of the effects of certain mutations to ligand binding, and the effects of temperature increment on the hISPD protein structure via different in silico approaches.

Methods: The crystal structure of hISPD (PDB ID, 4CVH [2]) was used for further simulations. 100 ns and 1μs of molecular dynamics (MD) simulations were performed via Desmond [4] and GROMACS 5.1.4 [5] in physiological conditions. In addition, simulated annealing simulations are also performed, which facilitates heating the systems from 310 K to 348 K. Results & Conclusions: According to our results obtained from in silico approaches, the effects of selected mutations result with increased or decreased thermostability could be discriminated in simulated annealing studies, and each protomer are found to effect ligand binding in a positive or negative manner as well as the mutations. Keywords: dystroglycanopathies, hISPD, molecular dynamics Acknowledgements: The numerical calculations reported in this paper were partially performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources). This work was supported by the German Research Foundation Emmy Noether Grant (DFG) to S.C.

108

References:

1. Cirak, Sebahattin, et al. "ISPD gene mutations are a common cause of congenital and limb-girdle muscular dystrophies." Brain 136.1 (2013): 269-281. 2. Riemersma, Moniek, et al. "Human ISPD is a cytidyltransferase required for dystroglycan O-mannosylation." Chemistry & biology 22.12 (2015): 1643-1652. 3. Gerin, Isabelle, et al. "ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto α-dystroglycan." Nature communications 7 (2016): 11534. 4. Banks, Jay L., et al. "Integrated modeling program, applied chemical theory (IMPACT)." Journal of computational chemistry 26.16 (2005): 1752-1780. 5. Abraham, Mark James, et al. "GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers." SoftwareX 1 (2015): 19-25.

109

P-15

In-silico identification of Toxoplasma gondii 3-Oxoacyl-[Acyl-Carrier-Protein] Reductase Inhibitors by Virtual High-throughput Screening

Can Aygün1, Özal Mutlu2

1Marmara University, Institute of Pure and Applied Sciences, Department of Biology, Istanbul, Turkey

2Marmara University, Faculty of Arts and Sciences, Department of Biology, Istanbul, Turkey

E-mail: canaygun@marun.edu.tr

Background: Toxoplasma gondii, is an intracellular protozoan parasite which is a member of the Apicomplexa phylum and ever-present worldwide in every country amounting to approximately 30% infected globally and potentially lethal in congenital form1,2. Apicomplexans have an exclusive fatty acid synthesis pathway, subcellularly localized to the apicoplast organelle unique to them. FAS-II pathway has a plant-like nature of a prokaryotic origin absent in the host thus is a suitable drug target with potentially much less severe and fewer side effects upon treatment3, 4. 3-Oxoacyl-[Acyl-Carrier-Protein] Reductase enzyme (FabG) (EC:1.1.1.100) of FAS II is essential for the production of host-derived vital long and very long chain fatty acids via carbon chain elongation4,5,6. The present study was conducted to search for possible suitable target binders to TgFabG from compound libraries, upon whose discovery further assessment and experimentation may find an eligible inhibitory drug compound. Methods: A three-tier high-throughput virtual screening protocol was followed. After an initial high-throughput virtual screening of a custom-made rich compound library, comprised of PubChem/Coumarin and ZINC chemical compound databases, the top 20% of the compounds were first SP docked and the top 10% of those compounds were then XP docked using Schrödinger Glide. The 15 best ligands were selected for future analyses based on Glide XP scores. The three best ligands were also visually displayed and further elucidated upon in their respective protein-ligand complexes. These were then taken to a further Molecular Dynamics analysis. Results: Three ligands: L1 (Pubchem ID: 101720177), L2 (Pubchem ID: 101024380) and, L3 (Pubchem ID: 85706708) were discovered with extra precise binding energies; -13.748, -11.432, -11.382 kcal/mol respectively with L2 H-bonding directly to one active site residue Lys156. Conclusions: Substances inspected here with sufficiently high default binding affinities to the TgFabG active site pocket may be available for further possible drug design experiments as lead compounds. Keywords: FabG, Virtual Screening, Molecular Docking, Toxoplasma gondii, Drug Design

110

References:

1. Pappas G, Roussos N, Falagas ME. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. (2009) Int J Parasitol.; 39:1385–94

2. Dubey J. P. The History of Toxoplasma gondii—The First 100 Years. (2008) J. Eukaryot. Microbiol.; 55(6):467-475

3. Maier T, Leibundgut M, Ban N. The Crystal Structure of a Mammalian Fatty Acid Synthase. (2008) SCIENCE.;321:1315-1321

4. Mazumdar J, Wilson EH, Masek K, Hunter CA, Striepen B. Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. (2006) PNAS.;103(35):13192-13197

5. Martins-Duarte ES, Carias M, Vommaro R, Surolia N, de Souza W. Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii. (2016) J. Cell Sci.;129:3320-3331

6. Tymoshenko S, Oppenheim RD, Agren R, Nielsen J, Soldati-Favre D, Hatzimanikatis V. Metabolic Needs and Capabilities of Toxoplasma gondii through Combined Computational and Experimental Analysis. (2015) PLOS Comp. Sci.;11(5):1-28

111

P-16

In Silico Prediction Studies of Polypeptidic Immune Epitopes of Toxoplasma gondii FabG Protein for Potential Vaccine Development

Can Aygün1, Özal Mutlu2

1Marmara University, Institute of Pure and Applied Sciences, Department of Biology, Istanbul, Turkey 2Marmara University, Faculty of Arts and Sciences, Department of Biology, Istanbul, Turkey

E-mail: can-_-aygun@hotmail.com Background: TgFabG (EC: 1.1.1.100) in Toxoplasma gondii is an enzyme of the type II fatty acid synthesis pathway found in apicomplexan pathogens, subcellularly located to a unique and exclusive plastid organelle, the apicoplast. Its function is essential to the integrity of the parasitic protozoan therefore substantial hindrance of its processes can and will be lethal to the apicomplexan. The prevalence of infection and severity of congenital toxoplasmosis outcomes such as severe pre/post-natal neurodegeneration or even death, and the fact that there is no vaccine for humans currently available, it is of dire importance that synthetic polypeptides which act as therapeutic agents be discovered1, 2, 3, 4, 5. Methods: The TgFabG sequence was obtained from NCBI Refseq (Accession: XP_002371341.1) and the whole sequence including the N-terminal 129 residue long unstructured signal peptide was submitted to IEDB MHCI and II prediction servers and Bepipred 2.0 for an HLA epitope search6. The 3D homology model of the protein consisting of the structured domain with no signal peptide was provided to the IEDB Ellipro and Discotope 2.0 servers for linear and discontinuous B-cell epitope predictions. For MHC I predictions the ANN-Align method was used and for MHC II predictions NN-Align was selected. Alleles were: HLA-A*01:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-B*07:02, HLA-B*08:01, HLA-B*40:01, HLA-B*44:02, HLA-B*53:01 for MHC I and HLA-DRB1*0101, HLA-DRB1*0301, HLA-DRB1*0401,05; HLA-DRB1*0701; HLA-DRB1*0802; HLA-DRB1*0901; HLA-DRB1*1201; HLA-DRB1*1302; HLA-DRB1*1501; HLA-DRB3*0101,0202; HLA-DRB1*1101; HLA-DRB4*0101; HLA-DRB5*0101 for MHC II epitope predictions. Predicted epitopes’ physiochemical parameters were computed on the ProtParam server. Results: Epitopic polypeptides including 191GDGPAVAALCEELLQQYP208 (Bepipred), 220TRDNLFIRMNEQEWNDVI237 (Linear B-Cell), HLA-DRB1*07:01 - 275NPGQANYAASKAGMI289 (NN-Align), HLA-DRB1*03:01 - 216NAGITRDNLFIRMNE240 (NetMHCII 2.3), A*01:01 - 238NTNLNSAFY246 (ANN-Align & NetMHC4.0) have been selected as the most optimal epitopes based on their predicted IC50 and, their instability index, aliphatic index, grand average of hydropathicity (GRAVY) values computed by ProtParam in addition to proximity to active and/or catalytic site residues. Conclusions: At least one epitopic polypeptide region was found per prediction method with desirable binding affinities (<100nM IC50), stability, hydrophilicity, aliphaticity and proximity to significant regions. Keywords: Vaccine Development, Toxoplasma gondii, Immune Epitope Research, FabG

112

References: 1. Torgerson PR, Mastroiacovo P. The global burden of congenital toxoplasmosis: a systematic review. (2013) Bull. World. Health. Organ.;91:501-508 2. Dubey JP. The History of Toxoplasma gondii—The First 100 Years. (2008) J. Eukaryot. Microbiol.;55(6):367-475 3. Mazumdar J, Wilson EH, Masek K, Hunter CA, Striepen B. Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. (2006) PNAS.;103(35):13192-13197 4. Robert-Gangneaux F, Darde M-L. Epidemiology of and Diagnostic Strategies for Toxoplasmosis. (2012) Clin. Microbiol. Rev.;25(2):264-296 5. Martins-Duarte ES, Carias M, Vommaro R, Surolia N, de Souza W. Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii. (2016) J. Cell Sci.;129:3320-3331 6.Kovjazin R, Volovitz I, Daon Y, Vider-Shalit T, Azran R, Tsaban L, Carmon L, Louzoun Y. Signal Peptides and Trans-membrane Regions are Broadly Immunogenic and have High CD8+ T Cell Epitope Densities:Implications for Vaccine Development. (2011) Mol. Immunol.;48(8):1009-1018

113

P-17

SAR of 2,4-dianilinopyrimidine containing methanesulfonamide (TRE-069) as potent and selective EGFR T790M/C797S mutant inhibitor for

anticancer treatment

Chong Hak CHAEa, Kwangho LEEb, Gildon CHOIb aChemical Platform Technology Division, Korea Research Institute of Chemical Technology, Daejeon

34114, Korea. *E-mail: chchae@krict.re.kr; bBio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea.

*E-mail: kwangho@krict.re.kr;

Background: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related death globally, with epidermal growth factor receptor (EGFR) activating mutations such as EGFR L858R and EGFR ex19 deletions being the main target in East Asian and European descendants. Methods: In this study, the 2,4-dianilinopyrimidine containing ortho-methanesulfonamide (TRE-069) was identified as a potent EGFR T790M/C797S inhibitor and its binding mode was explained on molecular model study. Results: Analysis of interactions between the compounds and EGFR residues reveals information valuable for the assessment of binding affinity, including the hydrogen bonding and hydrophobic effects. Conclusions: With favorable selective EGFR mutant activity profile, further lead optimization is on-going and will be reported in due course. Keywords: Epidermal growth factor receptor T790M/C797S, Kinases, Nonsmall cell lung cancer, Diaminopyrimidines

114

Figure 1: Proposed binding mode of TRE-069 (thick yellow sticks) and brigatinib (thin magenta lines) in the EGFR. EGFR is drawn by ribbons along with the interacting residues represented as sticks, and hydrogen-bonding interactions are marked with dashed red lines. References: 1. Choe H, Jeon B, Jung M, Jeon M, Shin I, Cho B, Choi G, Chae C, Lee K. Structure–Activity Relationship Study of 2,4-Dianilinopyrimidine Containing Methanesulfonamide (TRE-069) as Potent and Selective Epidermal Growth Factor Receptor T790M/C797S Mutant Inhibitor for Anticancer Treatment. (2017) Bull. Korean Chem. Soc.; 38:1353–1357

115

P-18

Preparation of Radiolabeled Methotrexate Loaded Silica Nanoparticles for Diagnostic Purposes

Çiğdem İçhedefa, Burcu Aydına, E. İlker Medinea, Serap Teksöza aEge University, Institute of Nuclear Sciences, 35100, Bornova-İzmir

E-mail: cigdem_ch@yahoo.com

Background: Recently, the possibilities of providing nanoparticles (NP) in the transport of therapeutic and target-specific drugs have received increasing interest from scientists and the pharmaceutical industry. Anticancer drugs have some adverse effects, such as healthy tissue toxicity, low stability, and resistance to tumor cells. Many different drug delivery systems such as liposomes, polymers, mycelia, and antibodies have been investigated in order to overcome these adverse effects. Among these carrier systems, mesoporous silica nanoparticles have promising properties. Methods: In this study, methotrexate (MTX), an anticancer drug used in breast cancer treatment, loaded mesoporous silica nanoparticles (MSN) was synthesized. Optimum synthesize conditions was determined by examining parameters such as particle size, morphology, drug trapping capacity of synthesized nanoparticles. The characterization of nanoparticles was performed by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM). Then the synthesized MTX loaded mesoporous silica nanoparticles were radiolabeled with Tc-99m in order to evaluate the breast cancer diagnosis potential of the drug delivery system. Results:The synthesized MTX loaded silica nanoparticles have the particles size of 100-250 nm and the SEM analyses proved that they have homogenous spherical size. The radiolabeling yield of the silica nanoparticles were determined by thin layer chromatography with an efficient yield. Conclusions: The optimized drug delivery system to be obtained at the end of the project can be evaluated for further studies for use in carrier formulations of anticancer drugs. Keywords: Silica nanoparticles, Radiolabeling, Diagnosis Acknowlegement: This project was funded by Ege University Scientific Research Project numbered 2017NBE006.

116

References:

1. Lu J, Liong M, Li Z, Zink JI, Tamanoi F. Biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals. (2010) Small; 6(16):1794-1805.

2. Kim J, Kim HS, Lee N, Kim T, Kim H, Yu T, Song IC, Moon WK, Hyeon T. Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a mesoporous silica shell for magnetic resonance and fluorescence imaging and for drug delivery.(2008) Angew. Chem. Int. Ed; 47(44):8438-8441.

3. De Barrosa ALB, Ferraza KSdeO, Dantasa TCS, Andradea GF, Cardosob VN, de Sousa EMB. Synthesis, characterization, and biodistribution studies of 99mTc-labeledSBA-16 mesoporous silica nanoparticles. (2015) Materials Sci and Eng. C.; 181-188.

117

P-19

Novel insights into Spindle Position Checkpoint of budding yeast

Dilara Kocakaplan, Ayse Koca Caydasi Koç University, Department of Molecular Biology and Genetics, 34450, Sariyer,

Istanbul/Turkey aykoca@ku.edu.tr

Background: The accurate positioning of the mitotic spindle in the mother to daughter cell polarity axis is monitored by a surveillance mechanism Spindle Position Checkpoint (SPOC) in S. cerevisiae.1 Upon detection of the mispositioned spindle, SPOC inhibits the Mitotic Exit Network (MEN) signal transduction pathway to ensure that mitotic exit and cytokinesis are delayed until the cell corrects the spindle positioning. When the spindle is correctly aligned, Bub2–Bfa1 GTPase-activating protein (GAP) is inactivated by its regulator Cdc5 polo-like kinase, driving MEN initiation that causes full activation of the Cdc14 phosphatase, resulting mitotic exit. Kin4 kinase is another Bfa1 regulator that plays a key role in SPOC activation by phosphorylation and by changing the binding dynamics of SPOC components to Spindle Pole Body (SPB).2 Identification of novel mitotic exit inhibitors are currently one the main interest in the field. New players in the SPOC and MEN pathways will help understand not only control of mitotic exit but also production of daughter cells with different fates. Here, I will present the results of a genetic screen designed to discover novel mitotic exit inhibitors with a focus on a particular hit of the screen. Methods: Synthetic Genetic Array (SGA) by yeast knock out library, Yeast-Two-Hybrid (Y2H), SPOC assay, epistasis analysis, cassette deletion, live cell imaging, point mutation Results: Alongside the expected genes (KIN4, BFA1 and BUB2), we found 26 genes that are potential mitotic exit inhibitors. Only one out of the 26 genes identified in the screen caused SPOC deficiency when deleted. The Y2H data showed that this protein possibly interacts with polarity related proteins. Conclusions: Revealing the novel components of SPOC constitutes a crucial point in terms of establishing the spatial and temporal regulation of the spindle positioning in asymmetrically dividing cells. The efforts in forming a complete Mitotic Exit Network ultimately contributes to the understanding of proper cell division that promises the generation of the new drug targets of molecules in pathways possessing the defective elements in the cell cycle. Keywords: S. cerevisiae, asymmetric cell division, SPOC, MEN References: 1. Caydasi AK, Pereira G: Spindle alignment regulates the dynamic association of checkpoint proteins with yeast spindle pole bodies. (2009) Dev Cell. ; (1):146-56 2. Caydasi AK, Lohel M, Grünert G, Dittrich P, Pereira G, Ibrahim B: A dynamical model of the spindle position checkpoint. (2012) Mol Syst Biol. ; 8:582.

118

P-20

COMPARATIVE ANALYSIS OF TWO INHIBITOR BINDING SITES ON THE BmrA EFFLUX PUMP

Duygu Senturk Karagöza, Onur Sercinogluaa, Pemra Ozbek Saricaa, Cedric Orelleb, Jean-Michel Jaultb and Berna Sariyar Akbuluta

aDepartment of Bioengineering, Marmara University, Kadiköy, 34722, Istanbul, Turkey bUniv Lyon, CNRS, UMR5086 “Molecular Microbiology and Structural Biochemistry”, 7

passage du Vercors, 69367, Lyon Cedex 7, France E-mail: duygu.senturk@marun.edu.tr

Background: Antimicrobial antibiotic resistance causing drug efflux has constituted a worldwide health problem. The necessity of bringing the antibacterial susceptibility back and returning the existing antibiotics to the clinic have generated the center of attention of these efflux pumps' inhibition1. The current work focalized on novel inhibitors of the ATP binding cassette drug efflux pump BmrA (from Bacillus subtilis), which is a homodimer is highly similar to the eukaryotic pump P-glycoprotein (P-gp)2. The current inhibitors of P-gp are targeting two major sites; the nucleotide binding domain (NBD) and the groove between the two halves of the protein that constitute the pump. Taking reference P-gp as the starting point, the chemical library was screened in order to search new inhibitors which are the best binders targeting the NBD and cavity sites of BmrA. Methods: The initial work contained the homology modeling studies of BmrA pump using Modeller 9.19 program, taking Escherichia coli pump EcMsbA as the template and Staphylococcus aureus SAV1866 (pdb code: 2HYD) for the secondary structure information. The obtained best model was evaluated with ProCheck, ProSa and Verify3D for suitability. Virtual screening of the library was then carried using AutoDock Vina 1.1.23 and the binding energies were calculated accordingly. Results: Our preliminary results have indicated that there exist ligands showing high binding energies for both NBDs and cavity. According to binding energies comparison, new potential inhibitors are found different for NBD and cavity sites of BmrA. Conclusions: There are common ligands which are considering potential inhibitors. However, the results indicate that cavity site is more preferable due higher binders energies were obtained at cavity site docking studies. Keywords: Multi drug resistance efflux pumps inhibitors, Virtual Screening

119

Figure 1: BmrA model was constituted with Modeller 9.19. References: 1. Mahmood, A.; Prüfert, F.; Efiana, N.A. et al. Cell-penetrating self- nanoemulsifying drug

delivery systems (SNEDDS) for oral gene delivery. Expert Opin Drug Deliv, 2016, 13, 1503–1512.

2. Steinfels, E.; Orelle, C.; Fantino, J.R.; Dalmas, O.; Rigaud, J.L.; Denizot, F.; Di Pietro, A.; Jault, J.M. Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis. Biochemistry. 2004, 43, 7491–7502.

3. Trott, O.; Olson A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comput Chem, 2010, 31, 455–461.

120

P-21

The identification of protein expression profile of PC3 prostate cancer and PNT1A prostate epithelial cells following anti-obesity

drug Orlistat treatment regardless of AMPK signaling

Elif Damla Arısan, Esra Nebiler, Ajda Çoker Gürkan, Pınar Obakan Yerlikaya Istanbul Kültür University, Department of Molecular Biology and Genetics,

Atakoy Campus 34156 Istanbul-Turkey E-mail: d.arisan@iku.edu.tr

Background: Prostate cancer is the second leading cancer type after lung cancer incidence in male population worldwide. Obesity is one of the leading factors that triggers aggressive and metastatic prostate cancer cases. Recent findings indicated that the identification of therapeutic potential of these diseases related specific molecular targets might provide a new therapy model or design of combinational drug studies. It was well-established that increased fatty acid synthesis and cholesterol metabolism triggers poor prognosis in prostate cancer cells. For this reason, a lipase inhibitor Orlistat (Xenical, Roche) becomes critical in the treatment of cancer cells as an anti-carcinogenic agent. Similar to lipid deregulation, problems at androgen receptor (AR) signaling cascade and metabolic sensor protein adenosine mono phosphate kinase (AMPK) becomes apparent targets in aggressive forms of prostate cancer. Therefore, silencing of AMPK in the androgen irresponsive prostate cancer cells is a promising strategy to suppress excess lipogenesis pathways. In this study, we aimed to clarify the molecular targets of Orlistat regardless of AMPK in aggressive PC3 prostate cancer cells and as well as PNT1A prostate epithelial cells. Methods: Following the generation of AMPK knock-out PC3 and PNT1A cells, the effect of orlistat was determined via MTT cell viability, wound healing, colony formation and screening of protein targets of orlistat regardless of AMPK expression profile. All results were compared to parental PC3 and PNT1A cells. Results: Orlistat induced apoptosis through alteration of AMPK related fatty acid synthase and lipid oxidation signaling cascades in PC3 cells, but PNT1A prostate epithelial cells did not exert similar effect. Conclusions: Orlistat as a fatty acid synthase inhibitor is an effective therapeutic agent in the treatment of aggressive forms of prostate cancer through modulatin lipid biosynthesis and oxidation pathways differently in the presence or absence of AMPK (Supported by TUBITAK 116Z804). Keywords: Orlistat, prostate cancer, FASN, AMPK, apoptosis

121

P-22

Cytotoxic Effect of Novel 1-Ethyl and 2-Methyl Indole Derivatives on Breast Cancer Cells: ER and Aromatase as Their Potential Therapeutic Targets

Elif Incea, Alev Tascioglu Aliyeva, Atilla Akdemirb, Kubra Durgunc, Sibel Suzenc, Hande Gurer-Orhana

aDepartment of Pharm. Toxicology, Faculty of Pharmacy, Ege University, Izmir, Turkey; bDepartment of Pharmacology, Faculty of Pharmacy, BezmiAlem University, Istanbul, Turkey cDepartment of

Pharm. Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey E-mail: hande.gurer.orhan@ege.edu.tr

Background: According to IARC data, breast cancer is the most frequent cancer among women and the first cause of death by cancer in women from undeveloped regions. Preventive strategies and more effective therapies is an expanding field in recent years. An indolic hormon, melatonin (MLT), regulates circadian rhytm and has antiproliferative effects on various breast cancer types via hormon related or unrelated mechanisms. Although MLT has low toxicity its short half life is an important limitation in its therapeutic use. In the present study in vitro short and long term cytotoxicity of novel indol based melatonin analogues were evaluated both in ER+ (MCF-7) and triple negative (MDA-MB 231) breast cancer cells. Active compounds on ER+ but not in triple negative breast cancer cells were further investigated for their potential effects on selected hormon-related targets; aromatase (responsible for local estrogen synthesis) and estrogen receptor (ER). Methods: Molecular docking studies of the compounds against aromatase and ER were performed by using the Gold Suite software package. Short and long term cytotoxicity of the compounds were evaluated by MTT and clonogenic assay, respectively. The in vitro inhibitory effect of MLT analogues on aromatase was evaluated by using a fluorescent substrate, 7-methoxy-4-trifluoromethyl. Potential ER antagonistic effects were evaluated by two-stepped MCF-7 proliferation assay. Results: In total 37, 1-ethyl indole and 2-methyl indole derivatives were screened for their cytotoxic potential both in MCF-7 and MDA-MB-231 cells with MTT assay. Three compounds showed dose-dependent inhibition curves, with IC50 values ranging from triple digit nanomolar to single-digit micromolar range in MCF-7 cells without having a significant cytotoxic effect on MDA-MB 231 cells. They also decreased colony formation dramatically in MCF-7 cells. Out of those 3 compounds, two of them were found to have both aromatase inhibiting and ER antagonistic effects. Conclusions: Two of the tested compounds are found to be promising candidates for further studies on ER+ breast cancer therapy by their antiestrogenic effects via two different mechanisms; by inhibiting aromatase enzyme and interfering with ER-dependent cell proliferation. Keywords: Breast cancer cells, indole-based melatonin analogues, aromatase inhibitor, estrogen receptor antagonist

122

Figure 1: IC50 values of the active compounds in MCF-7 cells via MTT assay after 72 hr incubation. References: 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence

and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer.

2. Risk C. Melatonin: A Molecule for Reducing Breast Cancer Risk. Molecules. 2018;23(2):336.

-9 -8 -7 -6 -5 -40

20

40

60

80KD1M6KD3

IC50KD1

9.073e-006M6

4.120e-006KD3

5.986e-007

LogC (M)

%vi

abili

ty

123

P-23

Design and Synthesis of Novel Heterobivalent Molecules having Therapeutic Potential for Treatment of Parkinson’s Disease

Elif Keskina, Essam Hamied Ahmed Hanashalshahabya, Ozge Sensoyb, Mustafa Guzela

aInternational School of Medicine, Istanbul Medipol University, Kavacık, Istanbul, Turkey; bSchool of Engineering and Natural Sciences, Istanbul Medipol University, Kavacık,

Istanbul, Turkey keskinelifedi@gmail.com, alshahabyessam@yahoo.com

Background: Parkinson’s disease (PD), which is the second main neurodegenerative disorder, is caused by degeneration of dopamine producing neurons, which are located in the striatum. Once this problem occurs, it brings along some crucial symptoms as well such as tremor, slowness, stiffness, and balance problems. It has been shown that simultaneous binding of Adenosine 2A receptor (A2AR) agonist and antagonist to the tetramer, which is the dominant oligomer formed by A2AR and D2R in the striatum, can increase D2R signaling more than that is exerted by antagonist alone. These findings suggest that hetero-bivalent ligands might be used for this purpose, and they can provide opportunity of using lower dose of L-dopa than that of currently being used for the therapy of the disease. Therefore, the risk of onset of motor complications such as motor fluctuations and dyskinesia might be alleviated. This study has been aimed to design and synthesize novel hetero-bivalent ligands that target tetramer to prevent antagonistic effect of A2AR on D2R, thus treating Parkinson’s disease. Method: The pharmacophore groups of the heterobivalent ligands have been developed by combining certain parts of A2AR antagonist (istradefylline) and A2AR agonist (CGS-21680). Synthesis procedure for one of the heterobivalent ligands developed is given in Figure 1. Istradefylline and CGS-21680 have been synthesized according to the literature reports2,3. Results: Synthesis Strategy of linked Istradefylline-Ligands has been investigated which is considered one of the main moieties to get our target molecules shown in Scheme 1. Keywords: Anti-Parkinson, A2AR-D2R heterotetramer, heterobivalent ligands, in silico modeling

124

Figure 1: Design of linked heterobivalent ligands

Scheme 1: linked Istradefylline-Ligands Synthesis Strategy

References:

1. Bonaventura J, Navarro G, Casado-Anguera V, Azdad K, Rea W, Moreno E, Cortes A Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer. (2015) Proceedings of the National Academy of Sciences; 112(27):3609-3618.

2. Jörg, M., Shonberg, J., Mak, F. S., Miller, N. D., Yuriev E., Scammells P. J., Capuano B. Novel adenosine A2A receptor ligands: A synthetic, functional and computational investigation of selected literature adenosine A2A receptor antagonists for extending into extracellular space.(2013) Bioorganic & Medicinal Chemistry Letters; 23:3427–3433.

3. Hutchison A. J, Williams M, De Jesus R, Yokoyama R, Oei H. H, Ghai G. R, Jarvis M. F. 2-(Arylalkylamino)adenosin-5’-uronamides: a new class of highly selective adenosine A2 receptor ligands. (1990) Journal of Medicinal Chemistry; 33(7):1919-1924.

125

P-24

Dynamical characterization of TCRpMHC complex: A comparative molecular dynamics study

Elif Naz Bingöl, Onur Serçinoğlu, Pemra Ozbek Department of Bioengineering, Marmara University, Goztepe, Istanbul, Turkey

E-mail: enazbingol@gmail.com Background: T-cell mediated immunity works against microbial pathogens as a major defense system in higher organisms. The fractions of proteins and peptides from pathogens are presented with peptide-loaded major histocompatibility complex (pMHC) to T-cell receptors (TCR) to trigger immune response. Understanding the interaction of MHC and TCR is crucial in the treatment of diseases such as cancer and autoimmune diseases. The low affinity in interaction of TCR and pMHC is the main characteristic property of this interaction. In earlier studies, dynamical changes of MHC were observed during the interaction with TCR and the existence of similar effects were suggested for TCR as well, along with the consideration of a possible allosteric effect1–5. Methods: To this end, we employed molecular dynamics (MD) simulations on TCR, pMHC and TCRpMHC complexes to understand the changes in bound and unbound states of the TCR. Molecular dynamics (MD) simulations were performed using GROMACS simulation package6 and OPLS-AA / L all-atom force field7 for 100 ns after 1000 step energy minimization. The simulations are conducted in the NPT ensemble with pressure of 1 bar and 310 K and 2 fs time step is used. Results: The bound and unbound states of the TCR displays highly different RMSD distributions along the trajectory. Also, the difference in the cross-correlations of these three systems highlights the important regions in the interaction of TCR and pMHC. The changes mostly center around the interaction sites. Conclusions: The analysis of the simulations indicate that the interaction of pMHC and TCR alters the dynamics of TCR molecule more than pMHC. Besides, the activation of the TCR by pMHC molecule is necessary for immune response. Therefore, the notable dynamical change in TCR can be an indication of a response which is induced by the binding of pMHC molecule. To conclude, one can also say that this change can be an allosteric effect of the pMHC binding. Keywords: molecular dynamics, major histocompatibility complex, immune response

126

References: 1.Baker, B. M., Scott, D. R., Blevins, S. J. & Hawse, W. F. Structural and dynamic control of T-cell receptor specificity, cross-reactivity, and binding mechanism. Immunol. Rev. 250, 10–31 (2012). 2.Knapp, B., Dunbar, J., Alcala, M. & Deane, C. M. Variable Regions of Antibodies and T-Cell Receptors May Not Be Sufficient in Molecular Simulations Investigating Binding. J. Chem. Theory Comput. 13, 3097–3105 (2017). 3.Knapp, B. & Deane, C. M. T-Cell Receptor Binding Affects the Dynamics of the Peptide/MHC-I Complex. J. Chem. Inf. Model. 56, 46–53 (2016). 4.Natarajan, K. et al. ARTICLE An allosteric site in the T-cell receptor Cb domain plays a critical signalling role. Nat. Commun. 8, (2017). 5.Hawse, W. F. et al. Cutting Edge: Evidence for a Dynamically Driven T Cell Signaling Mechanism. J. Immunol. 188, 5819–5823 (2012). 6.Pronk, S. et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845–854 (2013). 7.Kaminski, G. A., Friesner, R. A., Tirado-Rives, J. & Jorgensen, W. L. Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides †. J. Phys. Chem. B 105, 6474–6487 (2001).

127

P-25

Profiling the mutational spectrum of hereditary breast and colorectal cancer patients in Turkey using multigene panel testing

Elifnaz Çelika,d, Tuğba Kızılboğaa,d, Gizem Alkurta,d, İzzet Akçaya,d, Jale Yıldıza,d, Fikret Ezbercib, Levent Doğanayc,d, Gizem Dinler Doğanaya,d

aIstanbul Technical University, Department of Molecular Biology and Genetics, Maslak, Istanbul, Turkey; bUniversity of Health Sciences, Umraniye Teaching and Research Hospital, Department of

General Surgery, Umraniye, Istanbul, Turkey; cUniversity of Health Sciences, Umraniye Teaching and Research Hospital, Department of Gastroenterology and Hepatology, Umraniye, Istanbul, Turkey;

dGLAB (Genomic Laboratory), Istanbul Association of Northern Anatolian Public Hospitals, Umraniye and Maslak, Istanbul, Turkey

E-mail: elfnz.celik@yahoo.com

Background: Breast and colorectal cancers are the most common types in Turkey and leading cause of death in affected individuals. Although the majority of cancer arises from mutations sustained sporadic over a lifelong, still 5 to 10% of all the cancer cases are caused by genetic alterations that are transmitted through generations and appear in hereditary cancer category [1]. The individuals with hereditary risk-carriers tend to develop cancer at an earlier age than average age and have more than one cancer type. It is important to conduct genetic testing to take preventive actions for these carrier patients and their relatives. In this study, we sequenced gene coding, 3’ and 5’ regions of 20 cancer susceptibility genes and filtered rare variants. We selected allele frequency equal or smaller than 1% as well as no record in 1000 Genome population database to characterize clinical features and variants and to reveal mutational spectrum of those patients. Methods: The patients were classified based on their risk status by using NCCN guidelines. 276 breast patients, 125 colorectal patients and 345 controls were sequenced with three different panels using Illumina MiSeq and NextSeq500 instruments. The variant calling process were done using both Sophia DDM software and GATK Best Practices pipeline. The classification of variants, which are resulted from variant calling process, was done according to the guideline prepared by ACMG using numerous source, such as in silico tools (SIFT, PolyPhen-2, MutationTaster, HSF), population (1000G, ExAC, ESP6500) and clinical (ClinVar, OMIM, ORPHANET) databases. The variant frequencies in the patient groups were compared to the controls. Results: The total number of variants are 950 in breast cancer patients, 522 in colorectal cancer patients and 1444 in controls. 23 (n=22, 7.97%), 13 (n=13, 10.4%) and 13 (n=13, 3.71%) variants identified as pathogenic or likely pathogenic were found in breast cancer, colorectal cancer and control groups, respectively. Besides, the numbers of variants classified as unknown significance are 88 (n=69, 25%) for breast cancer, 44 (n=34, 27.2%) for colorectal cancer and 110 (n=93, 26.96%) for control groups. In addition, 62 (n=51, 18.61%), 48 (n=41, 32.8%) and 93 (n=80, 23.19%) novel variants were identified in breast, colorectal and control groups, respectively.

128

Conclusions: The association between novel mutations as well as VUS found in our study group and diseases may provide opportunity for development of candidate drugs specific to the breast as well as colorectal cancers. Keywords: Hereditary cancer, next generation sequencing, mutational spectrum, breast cancer, colorectal cancer

129

P-26

Synthesis of Biologically Active Novel Sulfonamide Derivatives

Elmas Begüm Çakmaka, Özge Özmenb, Dilay Kahvecioğluc, Belma Zengin Kurtd, Fatih Sönmeza,e

aSakarya University, Faculty of Arts and Science, Department of Chemistry, Sakarya, Turkey aBezmialem Vakif University, Faculty of Pharmacy, Istanbul, Turkey

cMarmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey

dBezmialem Vakif University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey

eSakarya Uygulamalı Bilimler University, Pamukova Vocational High Scholl, Sakarya, Turkey E-mail: elmasbegumcakmak@gmail.com

Background: Carbonic anhydrase (CA) is a metal-based enzyme that is commonly found in all organisms and contains Zn +2 ion in its active site. The carbonic anhydrase enzyme has 16 different isoenzymes, of which CA IX and CA XII are mostly present in tumor cells. Since CA IX is seen in solid tumors that are membrane-bound and cell-surface, it is expressed especially in renal cell carcinomas, cervical, ovarian, colon, head and neck carcinomas. In recent studies, the necrosis around the tumor is explained by the overexpression of the increased CA IX enzyme in this region and the pH control being dependent on this enzyme. Therefore, it is thought that carbonic anhydrase inhibitors will play an important role in the synthesis of new generation anticancer drugs. The most commonly used compounds as a carbonic anhydrase inhibitor are the sulfonamide group. Such compounds interact with the zinc atom at the center of the enzyme to form a trigonal-bipramidal structure. They also interact with glutamate and threonine in the active site. Methods: Aldehyde derivatives are solved in DMF at room temperature with propargyl bromide and K2CO3. The 4-aminobenzenesulfonamide was reacted with NaNO2 and NaN3 to obtain the 4-azidobenzenesulfonamide. 4-azidobenzenesulfonamide and propargyllated aldehyde derivatives were reacted with CuSO4.5H2O / Natrium ascorbate in THF:H2O at room temperature. Results: New sulfonamid derivatives were synthesized at four steps in high yield. All the new compounds were characterized by 1H NMR, 13C NMR, IR, and elemental analysis. Conclusions: The biological activity studies of the synthesized compounds are continuing. Keywords: Sulfonamid, carbonic anhydrase,

130

Figure 1: Synthesis of new sulfonamid derivatives. References:

1. Pastoreka J, Pastorekova S, Hypoxia-induced carbonic anhydrase IX as a target for cancer therapy: From biology to clinical use (2015) Seminars in Cancer Biology, 31, 52–64.

2. Duda D, Govindasamy L, Agbandje-McKenna M, Silverman CTDN, McKenna R, The refined atomic structure of carbonic anhydrase II at 1.05 Å resolution: implications of chemical rescue of proton transfer, (2003) Acta Cryst. 59, 93-104.

131

P-27

A Structure-Based Drug Design Study; Interactions of Bos taurus Enolase and

Coumarin Derivatives Through Molecular Docking and

Molecular Dynamic Simulation

Emrah SARIYERa,b, Sinem YAKARSONMEZa, Ozkan Danisc, Dilek TURGUT-BALIKa aYıldız Technical University, Faculty of Chemical and Metallurgical Engineering,

Department of Bioengineering, 34210, Istanbul, Turkey bGumushane University, Faculty of Engineering and Natural Sciences,

Department of Genetics and Bioengineering, 29100, Gumushane, Turkey cMarmara University, Faculty of Arts and Sciences, Department of Chemistry,

34722, Istanbul, Turkey E-mail: esaryer@yahoo.com

Background: Tropical theileriosis is thick-borne disease caused by Theileria annulata and causes huge economic loss among domestic animals in wide geographic area such as North Africa, the Mediterranean coastal area, South Europe, Asia. Theileriosis has high morbidity, mortality and Theileria annulata infects wild and domestic animals especially Bos taurus (cattle) and Bos indicus species [1,2]. Parvaquone and Buparvaquone have been used in the treatment of theileriosis since 1980. However, drug resistance has been reported against the parasite in a study published in 2010 [3-4]. The need for the identification of new drug candidates for theileriosis caused high financial losses has arisen. Methods: In this present study, compounds that potentially inhibits one of the parasite’s metabolic enzyme called enolase has been tested against its counterpart in the host Bos taurus. The envisaged drug candidates K8, K9 and K21 coumarin derivatives (benzo-alpha-pyrone compounds) were analyzed in the interactions with Bos taurus enolase in silico. In previous studies, the unknown 3-D structure of the enzyme was determined by homology modeling method and simulated by molecular dynamic methods until the molecule becomes thermodynamically stabilized. Firstly, the coumarin derivatives have been docked to Bos taurus enolase using AutoDock by molecular docking methods [5]. Two different positions with the lowest binding energies and the highest binding affinities were selected as the initial conformation. Bos taurus enolase-coumarin complexes were minimized during 10000 cycles steepest descent algorithm and 90000 cycles conjuge gradient algorithm. System was heat up 300 K control with Langevin Thermostat and simulated for 100 ns using Berendsen thermostat [6]. The RMSD (Root-mean-square deviation of atomic positions) and RMSF (Root mean square fluctuation of atomic fluctuation) plots were drawn using CPPTRAJ from AMBER trajectory files and the final 3-D structure was determined [7].

132

Results: Binding energies of all of the compounds are close to each other when the calculated binding energies based on molecular docking are compared. The 2-phosphoglyserate (2PG) substrate were docked to the active site of the enolase after coumarin derivatives were docked to the enolase and the binding energies were ranked as K8-1 <K9-1 <.K8-2 <K21-1 <K9-2 <K21-2. During the simulation, the K9 has moved away from the system. K21 remains stable in both positions, but the K8 displaced away from the cavity during the simulation. Besides, the interaction between the substrate and enolase during the simulation was analyzed and found to interact with the catalytic residues of the enzyme. Conclusions: The interactions of enolase-coumarin and enolase-substrate were analyzed in this study. The findings is to be a preliminary and supportive study for in vitro inhibition studies. Keywords: Structure-Based Drug Design, Molecular Docking, Molecular Dynamic Simulation This research has been supported by Yıldız Technical University Scientific Research Projects Coordination Department. Project Number: 2015-07-04-KAP07 The numerical calculations reported in this paper were fully/partially performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources)

References: 1.Dolan, T.T. Recent Developments in the Research and Control of Theileria Annulata: Proceedings of a Workshop Held at ILRAD, Nairobi, Kenya, 17-19 September 1990, ILRI (aka ILCA and ILRAD), 1992. 2. Glass, E.J., Preston, P.M., Springbett, A., Craigmile, S., Kirvar, E., Wilkie, G., et al. Bos taurus and Bos indicus (Sahiwal) calves respond differently to infection with Theileria annulata and produce markedly different levels of acute phase proteins. International journal for parasitology. 2005, 35, 337-47. 3. Mhadhbi, M., Naouach, A., Boumiza, A., Chaabani, M.F., BenAbderazzak, S., Darghouth, M.A. In vivo evidence for the resistance of Theileria annulata to buparvaquone. Veterinary parasitology. 2010, 169, 241-7 4. Mhadhbi, M. Chaouch, M. Ajroud, K. Darghouth, M.A. ve BenAbderrazak, S., (2015). "Sequence polymorphism of cytochrome b gene in Theileria annulata Tunisian isolates and its association with buparvaquone treatment failure", PloS one, 10: e0129678. 5. Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S. and Olson, A. J. (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexiblity. J. Computational Chemistry 2009, 16: 2785-91. 6. D.A. Case, D.S. Cerutti, T.E. Cheatham, III, T.A. Darden, R.E. Duke, T.J. Giese, H. Gohlke, A.W. Goetz, D. Greene, N. Homeyer, S. Izadi, A. Kovalenko, T.S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, D. Mermelstein, K.M. Merz, G. Monard, H. Nguyen, I. Omelyan, A. Onufriev, F. Pan, R. Qi, D.R. Roe, A. Roitberg, C. Sagui, C.L. Simmerling, W.M. Botello-Smith, J. Swails, R.C. Walker, J. Wang, R.M. Wolf, X. Wu, L. Xiao, D.M. York and P.A. Kollman (2017), AMBER 2017, University of California, San Francisco. 7. Daniel R. Roe and Thomas E. Cheatham, III, "PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data". J. Chem. Theory Comput., 2013, 9 (7), pp 3084-3095

133

P-28

Comparative Modelling and Molecular Dynamic Simulation of Bacteriodes fragillis Enolase

Erennur UGURELa, Sinem YAKARSONMEZa, Emrah SARIYERa,b, Ozal MUTLUc, Osman Mutluhan UGURELa , Dilek TURGUT-BALIKa

aYıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, 34210, Istanbul, Turkey

bGumushane University, Faculty of Engineering and Natural Sciences, Department of Genetics and Bioengineering, 29100, Gumushane, Turkey

cMarmara University, Faculty of Arts and Sciences, Department of Biology, 34722, Istanbul, Turkey E-mail: esaryer@yahoo.com

Background: Bacteriodes fragilis is an anaerobic, non–spore-forming, gram negative bacteria that found in the human intestinal flora and also has pathogenic features. It is known that the bacteria causes some human intestinal infections and additionally promotes to progress of colon cancer[1,2]. In recent years, increase of B. fragilis resistance against many common antibiotics has been reported [3,4]. Therefore, identification a new target would be necessary to develop alternative drugs. Enolase from B. fragilis (BfENO) was selected as a drug target in this study, because it is a key enzyme that is responsible for interconversion of 2- phosphoglyceric acid to phosphoenolpyruvate in the glycolytic pathway. In addition, enolase is found on cell surface of several bacteria and also mediates pathogen invasion with fibronectin and plasminogen binding functions [5,6]. Methods: Template structures in homology modelling analyses were identified by PSI-Blast using protein sequence of B. fragilis enolase (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Open and closed conformations of BfENO were modelled by MODELLER 9.15 [8]. BfENO homology models were minized with 10000 cycles steepest descent algorithm and 90000 cycles conjuge gradient algorithm using AMBER software package [15]. Open and closed conformations of BfENO were simulated for 50 ns by molecular dynamic methods. All these models were validated after the simulation by using ERRAT, RAMPAGE, ProSA, ProQ, QMEAN, VERIFY3D servers [9,10,11,12,13,14]. Results: The structure of group A Streptococcal enolase (PDB ID: 3ZLH) and the structure of Staphylococcus aureus enolase in complex with PEP (PDB ID: 5BOE) were used as template structures to build open and closed conformation models of BfENO, respectively. The built homology models have been minimized to remove bad contacts and energies and was simulated for 50 ns to thermodynamically reach the equilibrium of the system. The system reached equilibrium after approximately 20 ns and the average RMSD value of open and closed conformations of BfENO were calculated as 1.768 and 1.721, respectively. After the simulation, it was confirmed that open and closed conformation models of BfENO were modelled with high accuracy according to validation results. Conclusions: Homology model of unknown 3-dimensional structure of Bacteroides fragilis enolase was built in this study. The generated 3-D structures were simulated using molecular dynamic methods to thermodynamically stabilize and the accuracy of the structures were validated using web based applications. Overall, the reliable structure would lead to structure based drug design studies. Keywords: Colon cancer, Bacteroides fragilis, Enolase, Homology modelling, MD simulation,

134

This research has been supported by Yıldız Technical University Scientific Research Projects Coordination Department. Project Number: FBA-2018-3339 The numerical calculations reported in this paper were fully/partially performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources)

References:

1) Toprak, U. N. Yagci, A. Gulluoglu, B. Akin, M. Demirkalem, P. Celenk, T. and Soyletir, G., "A Possible Role of Bacteroides fragilis Enterotoxin in the Aetiology of Colorectal Cancer", (2006), Clinical Microbiology and Infection, 12: 782-786.

2) Wexler, H.M., "Bacteroides: The Good, The Bad, And The Nitty-Gritty", (2007), Clinical Microbiology Reviews, 20: 593-621.

3) Snydman, D. R., Jacobus, N. V., McDermott, L. A., Golan, Y., Goldstein, E. J. C., Harrell, L, Jenkins S, Newton D, Pierson C, Rosenblatt J, Venezia R, Gorbach SL, Queenan AM, Hecht DW. “Update on resistance of Bacteroides fragilis group and related species with special attention to carbapenems 2006–2009”. (2011), Anaerobe, 17(4), 147-151.

4) Sherwood, J. E., Fraser, S., Citron, D. M., Wexler, H., Blakely, G., Jobling, K., and Patrick, S. “Multi-drug resistant Bacteroides fragilis recovered from blood and severe leg wounds caused by an improvised explosive device (IED) in Afghanistan”. (2011), Anaerobe, 17(4), 152-155.

5) Díaz-Ramos, À., Roig-Borrellas, A., García-Melero, A., and López-Alemany, R. “α-Enolase, a multifunctional protein: its role on pathophysiological situations”. (2012), BioMed Research International, 2012.

6) Bergmann, S., Wild, D., Diekmann, O., Frank, R., Bracht, D., Chhatwal, G. S., and Hammerschmidt, S. “Identification of a novel plasmin (ogen)-binding motif in surface displayed α-enolase of Streptococcus pneumonia”. (2003), Molecular microbiology, 49(2), 411-423.

7) Esgleas, M., Li, Y., Hancock, M. A., Harel, J., Dubreuil, J. D., and Gottschalk, M. “Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis”. (2008), Microbiology, 154(9), 2668-2679.

8) B. Webb, A. Sali, Protein structure modeling with MODELLER, Protein Structure Prediction (2014) 1-15. 9) Congreve, M., Andrews, S. Perspective in Medicinal Chemistry: Structure-Based Drug Design. Current

Topics in Medicinal Chemistry. 2017, 17, 93-4. Morris, A.L. MacArthur, M.W. Hutchinson, E.G. ve Thornton, J.M., (1992). "Stereochemical quality of protein structure coordinates", Proteins: Structure, Function, and Bioinformatics, 12: 345-364.

10) Colovos, C. ve Yeates, T.O., (1993). "Verification of protein structures: patterns of nonbonded atomic interactions", Protein Science, 2: 1511-1519.

11) Wiederstein, M. ve Sippl, M.J., (2007). "ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins", Nucleic acids research, 35: W407-W410.

12) N. Eswar, B. Webb, M.A. Marti-Renom, M. Madhusudhan, D. Eramian, M.y. Shen, U. Pieper, A. Sali, Comparative protein structure modeling using Modeller, Current protocols in bioinformatics 15(1) (2006) 5.6. 1-5.6. 30.

13) Benkert, P., Tosatto, S.C.E. and Schomburg, D. (2008). "QMEAN: A comprehensive scoring function for model quality assessment." Proteins: Structure, Function, and Bioinformatics, 71(1):261-277.

14) D. Eisenberg, R. Lüthy, J.U. Bowie, VERIFY3D: assessment of protein models with three-dimensional profiles, Methods in enzymology, Elsevier1997, pp. 396-404.

15) D.A. Case, D.S. Cerutti, T.E. Cheatham, III, T.A. Darden, R.E. Duke, T.J. Giese, H. Gohlke, A.W. Goetz, D. Greene, N. Homeyer, S. Izadi, A. Kovalenko, T.S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, D. Mermelstein, K.M. Merz, G. Monard, H. Nguyen, I. Omelyan, A. Onufriev, F. Pan, R. Qi, D.R. Roe, A. Roitberg, C. Sagui, C.L. Simmerling, W.M. Botello-Smith, J. Swails, R.C. Walker, J. Wang, R.M. Wolf, X. Wu, L. Xiao, D.M. York and P.A. Kollman (2017), AMBER 2017, University of California, San Francisco.

135

P-29

Isozyme Selective QSAR Models of Carbonic Anhydrases with Non-classical Inhibitors, Coumarins and Sulfocoumarins

Erol Eroğlua

aAkdeniz University, Education Faculty, Mathematics and Sciences Education Department, 07058, Antalya, Turkey eeroglu@akdeniz.edu.tr

Background: First time, about a decade ago, scaffold was identified as a inhibitor of CAs [1]. Since then, quite large number of derivatives of coumarin and sulfacoumarin were synthesized and their inhibition activities towards different isozymes of CA measured and published. Some of these derivatives exhibit great inhibition selectivity for the different isozymes of CA. Here, we present some QSAR models which may help to mechanistically uncover the rationale of isozyme selectivity of these compounds. Methods: Most of the molecular descriptors were calculated using DFT method together with B3LYP functional and 6-31G+(d,p) basis set. Best Multi Linear Regression (BMLR) algorithm of Codessa 3 software was used for the feature selection and model construction. Leverage approach was used to determine domain of application (AD) of the obtained models. Results:

Isozyme selectivity N (training)

R2 Q2 S2 F N (test) R2 SE

Ki (CAII-CAI) 25 0.78 0.62 0.18 13.80 3 0.99 0,10 ΔpKi= -340.48 + 309.52(MBL_O) + 16.36(FNSASA-3) -54.20(LUMO) + 0.09(NCCBo)+ 0.89(Q_Pol) Ki (CAIX-CAII) 25 0.78 0.63 0.82 13.66 5 0.57 0.65 ΔpKi= 18.21 - 14.82 (FMMNC) + 124.35(LUMO) + 46.77(XYZ_Box) – 34.02(XY_Rec) + 0 01(PSASA-2) Ki (CAXII-CAII) 21 0.80 0.69 0.68 23.67 5 0.88 0.35 ΔpKi= -41.51 + 0.07(MCQMEL) – 101.99(HOMO-1) + 10.76(MBL_CO_Bo)

Conclusions: Frontier orbital such as LUMO and HOMO-1, charge distribution and geometry related descriptors play a critical role in the selectivity inhibition of different isozymes of CAs by coumarins and sulfocoumarins. Keywords: QSAR, DFT, Coumarin, Carbonic Anhydrase Reference: [1] Maresca, A., Temperini, C., Vu, H., Pham, N. B., Poulsen, S. A., Scozzafava, A., ... & Supuran, C. T. (2009). Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors. Journal of the American Chemical Society, 131(8), 3057-3062.

136

P-30

New Imidazo[2,1-b]Thiazol Derivatives: Synthesis, Anticancer Activity And Molecular Docking

Başoğlu, F.a, Ece, A.b, Cimok, S.c, Ulusoy Güzeldemirci, N.c

aCyprus Health and Social Sciences University, Faculty of Dentistry, Department of Basic Medical Science, Mersin 10, Turkey;

bBiruni University, Faculty of Pharmacy, Department of Pharmacetical Chemistry, Istanbul, Turkey; cIstanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey

E-mail: faika.basoglu1990@gmail.com Background: In this study the synthesis of new compounds bearing an imidazo[2,1-b]thiazole moiety, 3-alkyl/aryl-2-[[[6-(4-bromophenyl)imidazo[2,1-b]thiazol-3-yl]acetyl]hydrazono]-5-methyl-4-thiazolidinones (5-13) were synthezised and their antcancer activities were evaluated. Additionally, the ligand interactions with a potential target were studied using molecular docking technique [1,2] Methods: In this study, new imidazo[2,1-b]thiazole derivatives were investigated by using both experimental and Schrödinger program packages. Both all of the synthesized molecules and X-ray the structure of cyclin-dependent kinase 2 (CDK2) (PDB ID: 1FVT) in complex with an oxindole inhibitor [3-5] were prepared by using related modules of Schrödinger. The docking calculations were performed by using the Glide SP (standard precision) module of Schrödinger Suite. Results: Molecular docking studies were performed to see possible binding modes of the synthesized compounds and also docking scores of the compounds 5–13 together with the native ligand, are obtained. Conclusions: In this study, the results obtained from experimental and in silico shed light on the synthesis and discovery of potential anticancer drug candidates. Keywords: Imidazo[2,1-b]thiazole, anticancer, molecular modelling

137

Figure 1: 2D ligand interaction diagram for inactive copound 5 (left), active compound 11 (middle), and native ligand (right) References: 1. Ulusoy N. Synthesis and antituberculosis activity of cycloalkylidenehydrazide and 4-zza-1-thiaspiro[4.5]decan-3-one derivatives of imidazo [2,1-b]thiazole (2002) Arzneim-Forsch/ Drug Res; 52(7): 565-571. 2. Andreani A, Bonazzi D, Rambaldi M. Potential Antitumor Agents, VII. 5-Substituted 6-phenylimidazo[2,1-b]thiazoles (1982). Arch Pharm; 315: 451-456. 3. Ibrahim HS, Abou-Seri SM, Abdel-Aziz HA. 3-Hydrazinoindolin-2-one derivatives: Chemical classification and investigation of their targets as anticancer agents (2016). Eur J Med Chem; 122: 366-381. 4. Hongo F, Takaha N, Oishi M, Ueda T, Nakamura T. CDK1 and CDK2 activity is a strong predictor of renal cell carcinoma recurrence (2014). Urologic Oncology, 32: 1240-1246. 5. Zaman MS, Shahryari V, Deng G, Thamminana S, Saini S et al. Up-regulation of microRNA-21 correlates with lower kidney cancer survival (2012). Plos One; 7(4): e31060.

138

P-31

AN INVESTIGATION ON THE PHARMACOLOGICAL COMPONENTS OF HERBAL TEAS THAT HAVE BEEN USED FOR MILK-INCREASING ACTIVITIES

BY GC / MS / MS

Fidan Pesen Özdoğan1, Seyfullah Oktay Arslan1, Enis Macit2 1Ankara Yıldırım Beyazıt Üniversitesi, Tıp Fakültesi, Tıbbi Farmakoloji Anabilim Dalı, Bilkent, ANKARA

2Sağlık Bilimleri Üniversitesi, Tıp Gülhane Tıp Fakültesi, Toksikoloji Bilim Dalı, Etlik, ANKARA ABSTRACT The present study was aimed to detect the phytochemical components of herbal teas that are used to improve mother’s milk production by gas chromatography/mass spectrometry/mass spectrometry (GC/MS/MS). The methanolic extracts were extracted by maceration method and extraction using rotary evaporator. The volatile oils were obtained by Clevenger method. The GC/MS/MS analysis of the volatile oils or the methanol extracts of the mix of herbal teas or each one of them include Foeniculum vulgare, Pimpinella anisum, Trigonella foenum graceum, Urtica dioica, Nigella sativa listed as several compounds in this presentation. In the future researches, pharmacological active components of these herbal teas need to be taken into account. Some of phytoestrogenic compounds are basically trans-anethole, estragole, diosgenin, etc. Keywords: Foeniculum vulgare, Pimpenella anisum, Trigonella foenum graceum, Urtica diotica Nigella Sativa, GC/MS/MS

139

P-32

A synergistic apoptotic effect of combined treatment usnic acid with tamoxifen in breast cancer

Gamze Guney Eskilera, Isıl Ezgi Eryılmazb, Beste Yurdacanc, Unal Egelib,

Gulsah Cecenerb, Berrin Tuncab

aDepartment of Medical Biology, Faculty of Medicine, Sakarya University, Sakarya, Turkey bDepartment of Medical Biology, Faculty of Medicine, Uludag University, Bursa, Turkey

cDepartment of Pharmacology, Faculty of Pharmacy, Ege University, Izmir, Turkey E-mail: gamzeguney@sakarya.edu.tr

Background: Tamoxifen (Tam) is extensively used as an anti-estrogen for premenopausal women with breast cancer. However, Tam has been associated with increased risk of developing endometrial cancers and high dose of Tam induces toxic side effects. Additionally, acquired resistance to Tam is a major limitation for the successful treatment of breast cancer (1-3). In the previous studies, usnic acid (UA) has exhibited a variety of biological activities including anti-tumor, anti-oxidant and anti-inflammatory properties (4,5). For this purpose, we investigated the potential synergistic effects of Tam and UA on breast cancer in the current study. Methods: To evaluate the potential of the Tam and UA combined effect, MCF7 breast cancer cells were treated with Tam (100-1000 nM) and UA (12.5-150 μM) for 24, 48 and 72 h. The in vitro inhibition of UA and Tam was analyzed by WST-1 analysis. The effect of combination treatment on cell apoptosis was determined by Annexin V and cell cycle analysis. Besides, apoptotic morphology was observed by acridine orange-ethidium bromide (AO/EB) fluorescence staining. Results: The viability of MCF7 cells reduced to 35.0% and 37.5%, when Tam (100 nM) combined with UA at 12.5 and 25 µM for 24 h, respectively (p<0.01). However, after incubation with 100 nM of Tam and 12.5 µM of UA alone, the viability of MCF7 cells decreased to 86.9% and 89.7%, respectively (p<0.01). The percentage of total-apoptotic cells was nearly 77.0% and 71.6% at 12.5 µM UA+100 nM Tam and 25 µM UA+100 nM Tam for 24 h, respectively. After the MCF7 cells were incubated with 12.5 µM UA+100 nM Tam and 25 µM UA+100 nM Tam, the percentage of cells in the G0/G1 phase considerably increased from 51.0% to 70.8% and 73.2%, respectively (p<0.01). Additionally, we observed chromatin condensation, holes and cell shrinkage in MCF7 cells treated with Tam and UA alone. Similar anti-proliferative and apoptotic effects were observed in MCF7 cells treated with in combination Tam with UA. Thus, the combination of Tam and UA led to a remarkable decrease in cell viability and synergistically induced apoptosis and G0/G1 arrest in MCF7 cells. Conclusions: These findings suggested that the combination of Tam with UA had potential synergistic effects, in vitro. Furthermore, UA might be a potential candidate for enhancing efficacy and reducing toxicity of Tam in the treatment of breast cancer. Keywords: Breast cancer, Usnic acid, Tamoxifen, Synergistic effect, Combined treatment

140

References: 1. Lim E, Metzger-Filho OWE. The natural history of hormone receptor–positive breast cancer. (2012). Oncol (Willist Park); 26(8):688-694. 2. Jiang Q, Zheng S, Wang G. Development of new estrogen receptor targeting therapeutic agents for tamoxifen-resistant breast cancer. (2013). Future Med Chem.;5(9):1023–1035. 3. García-Becerra R, Santos N, Díaz L, Camacho J. Mechanisms of resistance to endocrine therapy in breast cancer: focus on signaling pathways, miRNAs and genetically based resistance. (2012) Int J Mol Sci.;14(1):108-145. 4. Zuo S, Wang L, Zhang Y, Zhao DN, Li QS, Shao D, Fang XD. Usnic acid induces apoptosis via an ROS-dependent mitochondrial pathway in human breast cancer cells in vitro and in vivo. (2015) RSC Adv.; 5: 153-162. 5. Einarsdottir E, Groeneweg J, Bjornsdottir GG, Harðardottir G, Omarsdóttir S, Ingólfsdóttir K, Ögmundsdóttir HM. Cellular mechanisms of the anticancer effects of the lichen compound usnic acid. (2010) Planta Medica; 76: 969-974.

141

P-33

Structure-Function Relationship in Anti-Apoptotic Bcl-xL

Gamze Tanriver,a,b Saron Catak,a and Gerald Monardb

a Department of Chemistry, Bogazici University, 34342 Bebek, Istanbul, TURKEY; bUniversité de Lorraine, UMR 7565 SRSMC, Vandoeuvre-les-Nancy, FRANCE

E-mail: gamze.tanriver@boun.edu.tr

Background: B-cell lymphoma-2 (Bcl-2) family proteins regulate mitochondrial pathway of apoptosis and maintain balance between life and death of cells.(1, 2) In 1980s the first member of this family, Bcl-2, was identified.(3) The Bcl-2 family is primarily divided into three sub-families; namely, anti-apoptotic, pro-apoptotic and BH3-only proteins. Bcl-xL is an anti-apoptotic protein, also known as “pro-survival protein” and is located in both cytosol and mitochondrial outer membrane (MOM). Basically Bcl-xL binds and inhibits BH3-only proteins leading to activation of pro-apoptotic proteins and apoptosis. Structurally, Bcl-xL consists of 8 helices, a C-tail known as the trans membrane domain (TMD) and a flexible loop between α1 and α2.(4) However, limited structural information about full-length Bcl-xL are available in the literature. Recently, importance of the C-tail and loop section have been reported.(5, 6) Results: MD studies of full-length Bcl-xL have been performed. Outcomes will be shown. Conclusions: In this study, we have modelled full-length Bcl-xL alone and in complex with BH3-only peptide to elucidate contributions of the loop and the C-tail to anti-apoptotic function of Bcl-xL. The structure and dynamics of Bcl-xL were investigated through micro second long MD simulations to understand the outcome of the structural changes on the Bcl-xL’s function. Keywords: Bcl-2 family, Bcl-xL, Molecular Dynamic Simulation. References: 1. Campbell, K. J., and Tait, S. W. G. Targeting BCL-2 regulated apoptosis in cancer. (2018) Open Biol.;

8:1–11. 2. Cory, S., and Adams, J. M. The bcl2 family: regulators of the cellular life-or-death switch. (2002) Nat.

Rev. Cancer; 2:647–656. 3. Tsujimoto, Y., Yunis, J., Onorato-Showe, L., Erikson, J., Nowell, P. C., and Croce, C. M. Molecular

cloning of the chromosomal breakpoint of B-Cell lymphomas and leukemias with the t(11;14) chromosome translocation. (1984) Science; 224:1403–1406.

4. Muchmore, S. W., Sattler, M., Liang, H., Meadows, R. P., Harlan, J. E., Yoon, H. S., Nettesheim, D., Chang, B. S., Thompson, C. B., Wong, S.-L., Ng, S.-C., and Fesik, S. W. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. (1996) Nature; 381:335–341.

5. Maity, A., Sinha, S., Ganguly, D., and Ghosh Dastidar, S. C-terminal tail insertion of Bcl-xL in membrane occurs via partial unfolding and refolding cycle associating microsolvation. (2016) Phys Chem Chem Phys.; 18:24095–24105.

6. Yao, Y., Fujimoto, L. M., Hirshman, N., Bobkov, A. A., Antignani, A., Youle, R. J., and Marassi, F. M. Conformation of BCL-XL upon membrane integration. (2015) J. Mol. Biol.; 427:2262–2270.

142

P-34

The investigation of the antitumor agent toxicity on the electron transport chain enzymes, catalase activities and lipid peroxidation levels in lung, heart

and brain tissues of rats

Gizem Kursunluoglua,b, Dilek Taskirand, *Hulya Ayar Kayalia,c aIzmir Biomedicine and Genome Center (IBG), 35340 İzmir, TURKEY

bDepartment of Chemistry, The Graduate School of Natural and Applied Sciences, Dokuz Eylul University, 35160 İzmir, TURKEY

cDepartment of Chemistry, Division of Biochemistry, Faculty of Science, Dokuz Eylul University, 35160 İzmir, TURKEY

d Department of Physiology, Ege University School of Medicine, 35100 İzmir, TURKEY *E-mail: hulya.kayali@ibg.edu.tr

Background: Cisplatin is one of the most active cytotoxic agent in the cancer treatment but its clinical use is involved in toxicity. Mitochondria which is the major target of cisplatin, therefore ETC was affected from cisplatin toxicity by inducing ROS generation, electron leaks, mitochondrial impairment. It has shown that cisplatin may have important direct interactions with mitochondria. To address the interaction, it was hypothesis that mitochondrial electron transport chain (ETC) enzymes; succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) enzyme activities, nucleotide levels as well as catalase (CAT) enzyme and membrane lipid peroxidation (LPO) level can be effected by cisplatin. Methods: Male Sprague Dawley adult rats were treated Cisplatin and Capsaicin. In this study, cisplatin levels, isolation of mitochondria, Succinate Dehydrogenase Activity Assay (SDH), Cytochrome c Oxidase Activity Assay (COX), The cytosolic preparation for CAT activity, Lipid Peroxidation, Nucleotide levels and Protein Determination has been carried out in the lung, heart and brain tissues. Results: In the lung, heart and brain tissues after cisplatin exposure at 1st day the significantly decreased SDH (Figure 1) and COX (Figure 2) activities besides ATP and ADP levels compared to control group could be due to cisplatin mediated mitochondrial dysfunction. The higher LPO level in the studied tissues after cisplatin exposure at 1st and 4th day compared to control may be inferred highly electron leakage involved in ETC and ROS can cause extensive tissue damage such as membrane lipid destruction. It was also studied capsaicin possible protective effect on impaired ETC by cisplatin. SDH and COX activities in cisplatin+capsaicin exposed heart and brain were higher to cisplatin groups while LPO levels were lower. Conclusions: In the current study, cisplatin transport carried out to all studied tissues and decreases in cisplatin levels at 4th day were low in brain compared to the lung and heart tissues. It suggested that blood-brain barrier to protect by certain molecules. The observations suggest that cisplatin inhibited SDH, COX as well as ATP synthase. In the lung, heart and brain tissues after cisplatin exposure at 1st day the significantly decreased SDH activity compared to control group could be due to cisplatin mediated mitochondrial dysfunction and the SDH enzyme was inhibited (1). However, the SDH activities in all studied tissues increased and nearly reached their controls during 14 days following cisplatin exposure. The COX activities

143

in lung, heart and brain tissues were found to be markedly decreased in cisplatin exposed rats compared to control as like in SDH activity. These observations strongly suggest that cisplatin may induce alterations in the activity of electron transport chain enzymes. In conclusion, the present study indicates that cisplatin lead to toxicity because of the impairment of the balance between ETC and antioxidant system. Therefore, rats were treated with also capsaicin besides cisplatin to determine whether capsaicin have antioxidant effect on these systems. The results of cisplatin+capsaicin groups suggest that capsaicin have antioxidant capacity to scavenge ROS to prevent membrane damage. Keywords: Cisplatin, capsaicin, ETC, CAT, LPO

Figure 1. Variations in SDH activity in lung (a), heart (b) and brain (c) tissues depending on the days. Lung (¾¾¾), heart (¾¾¾) tissues, brain (¾¾¾), controls (¾¾¾). The values are the mean ±S.E.M. (n=6).

Figure 2. Variations in COX activity in lung (a), heart (b) and brain (c) tissues depending on the days. Lung (¾¾¾), heart (¾¾¾) tissues, brain (¾¾¾), controls (¾¾¾). The values are the mean ±S.E.M. (n=6).

References:

1. Kruidering M, De Water B.V, De Heer E, Mulder, G.J.J, Nagelkerke F. Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. (1996) J Pharmacol Exp Ther.; 280(2):638-49.

b c a

a b c

144

P-35

Pharmacophore Generation and Molecular Dynamics Studies of Novel Glycogen Synthase Kinase 3 Beta Enzyme Inhibitors

Gozde Yalcina, Ilkay Yildizb a Biotechnology Institute, Ankara University, Ankara 06110 Turkey; b Department of Pharmaceutical

Chemistry, Faculty of Pharmacy, Ankara University, Ankara 06110 Turkey E-mail: gozdeyalcin@ankara.edu.tr

Background: Glycogen Synthase Kinase 3 Beta (GSK3β) is a serine-threonine kinase and it has been associated with Parkinson disease and Alzheimer Diseases (AD). GSK3β mediates the hyper-phosphorylation of tau, the increased production of Aβ from APP (via β and γ secretase-mediated cleavage) and causes to impairments in learning and memory in AD [1]. Methods: Several Acetylcholinesterase inhibitors bearing IC50 values from BindingDB [2] were chosen as a training set. Molecular Docking studies for determination of the predictive positions of ligands on binding site were implemented by LigandFit protocol of Discovery Studio Client 3.5 (DSC) [3]. 3D-QSAR Pharmacophore Generation module of DSC was used to generate new pharmacophore hypothesis. Novel compounds were obtained by Library Analysis module of DSC. Molecular Dynamics Simulations of these compounds were implemented by AMBER14 Package [4]. Results: 3D structure of GSK3β was retrieved from RSCB protein data bank. PDB ID: 1Q41 [5] was chosen due to its resolution (2.1 Å) and the overlap between 3D sequence and Fasta sequence of enzyme (98.82%). Best LigandFit docking results of ligands and GSK3β were given in Figure 1. Conclusions: Acetylcholinesterase inhibitors were used with the intent of developing multi-target directed AD drugs. Known GSK3β inhibitor (indirubin) showed a worse binding profile than Acetylcholinesterase inhibitors. Keywords: Glycogen Synthase Kinase, Alzheimer Diseases, 3D-QSAR Pharmacophore Generation, LigandFit Protocol, multi-target directed drug design

Figure 1: LigandFit results of Acetylcholinesterase inhibitors.

145

References: 1. Hooper C, Killick R, Lovestone S. The GSK3 hypothesis of Alzheimer’s disease. (2008) J

Neurochem.;104(6):1433-1439 2. Chen X, Gilson ML and MK. BindingDB: A Web-Accessible Molecular Recognition

Database. (2001) Comb Chem High Throughput Screen.;4(8):719-725 3. Accelrys Software Inc. Discovery Studio 3.5 Client. 2012� 4. D.A. Case, J.T. Berryman, R.M. Betz, D.S. Cerutti, T.E. Cheatham, III, T.A. Darden, R.E.

Duke, T.J. Giese, H. Gohlke, A.W. Goetz, N. Homeyer, S. Izadi, P. Janowski, J. Kaus, A. Kovalenko, T.S. Lee, S. LeGrand, P. Li, T. Luchko, R. Luo, B. Madej, K.M. Merz DMY and PAK. AMBER2014. (2014)

5. Bertrand JA, Thieffine S, Vulpetti A, et al. Structural characterization of the GSK-3β active site using selective and non-selective ATP-mimetic inhibitors. (2003) J Mol Biol.;333(2):393-407.

146

P-36

Investigation of Molecular Interactions Between KRAS G12C and Known Anticancer Agents Influential in Lung Cancer

Gozde Yenice, Serap Yilmaz

Trakya University Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Edirne, Turkey E-mail: gozdeyenice@trakya.edu.tr

Background: RAS proteins have critical roles in the maintenance of cell homeostasis by regulating the basic functions of proliferation, survival, growth, migration, death etc. On the contrary, the uncontrolled and impaired RAS functions can evoke undesirable circumstances such as cancers and the other disorders (1). RAS genes (HRAS, NRAS and CRAS) are the predominantly mutated oncogenes in especially lung, colorectal and pancreatic cancer cases, among which KRAS is the most frequently occurred isoform (2). Various mutations were identified on this protein. One of them is that the mutation in 12 glycine positions is usually replaced by cysteine (mutation G12C), providing an electrophilic targeting of the mutant form, but not the wild type form that can be effective option for drug design studies. The inhibitors developed can irreversibly and selectively bind the mutant G12C RAS and stabilize the protein in inactive GDP form (3). Methods: Known KRAS (G12C) inhibitors were prepared and tested to several KRAS (G12C) proteins which were taken from PDB, to define the active site and crucial amino acids of protein using Discovery Studio 3.5 (4). The interactions between known inhibitors and KRAS (G12C) protein were investigated, after that anticancer agents which are used in treatment of lung cancer, were tested to the active site of KRAS protein. Results: When the interactions of known inhibitors were examined, it was found that Gly15, Lys16, Cys12, Pro34, Asp33, Tyr32, Val29, Asp30, Gly13, Thr35 and Gln61 amino acids in the active site played an important role for inhibition of protein and similar interactions were found in the tested anticancer agents. Conclusions: Tested anticancer agents are known to exhibit anticancer activity through different mechanisms, but they may also have ability to inhibit KRAS G12C protein. For the future directions, it is suggested to investigate the KRAS G12C protein inhibitor activities of these agents. Keywords: KRAS G(12C), lung cancer, RAS, RAS-effector interaction References:

1. Takashima A, Faller DV. (2013) Expert Opin Ther Targets. 17(5):507–531. 2. Waters A, Der C. Cold Spring Harb Perspect Med 2018;8:a031435. 3. Marín-Ramos NI, Ortega-Gutiérrez S, et al. (2018). Seminars in Cancer Biology.

Article in press. 4. Accelrys, Discovery Studio v3.5. (2012).

147

P-37

Identification of Novel Hit Molecules Against B-cell leukemia/lymphoma-2 (BCL2)

Gurbet Tutumlua, Ismail Erola,b, and Serdar Durdagia

aComputational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey

bDepartment of Chemistry, Gebze Technical University, Kocaeli, Turkey B-cell leukemia/lymphoma-2 (BCL2) family proteins are one of the important oncogenic targets used in anti-cancer drug designing studies. In this study, in order to identify new hits against BCL2, both ligand-based and structure-based techniques were used. Initially 212520 small molecules were retrieved from Specs database and binary QSAR based models from MetaCore/MetaDrug platform were used to screen against a defined therapeutic activity, “cancer”. The therapeutic activity value ranges from 0 to 1 and a value of more than 0.5 indicates potential of small molecule against selected disease model. In this study, we used a higher threshold value (0.8) for filtering the bioactive molecules. The molecules that have higher therapeutic activity values than 0.8 were used in the 26 different toxicity QSAR models. Combined disease and toxicity QSAR models resulted about 250 hit molecules that have high therapeutic activity (>0.8) and no toxicity in 26 toxicity models. These models were then screened against BCL2 targets using docking simulations and their docking scores were compared with known inhibitors (i.e., positive controls). Selected hit molecules which are the molecules that have higher docking scores than positive controls and they construct known crucial were used in molecular dynamics (MD) simulations. Structural and dynamical properties of ligand-protein complex is investigated for better understanding of hit molecules at the binding pocket of the target.

148

P-38

Design and characterization of montelukast sodium loaded Kollidon® SR nanoparticles

Gülsel Yurtdaş Kırımlıoğlua, Şennur Görgülüb a Department of Pharmaceutical Technology, Faculty of Pharmacy, Anadolu University,

Eskişehir, Turkey

bPlant, Drug and Scientific Researches Center, Anadolu University (AUBİBAM), Eskişehir, Turkey

E-mail: gyurtdas@anadolu.edu.tr

Background: Montelukast sodium (MS), a selective leukotriene receptor antagonist is considered as a beneficial common therapy to treat both bronchial asthma and allergic rhinitis [1]. The most important drawbacks of conventional MS formulation is that undergoes hepatic first pass metabolism and experiences side effects including headache, drowsiness, gastrointestinal disturbances, sleep disorders, hypersensitivity reactions [2]. It shows biological half-life of 2.5-5.5 h, thereby decreasing bioavailability up to 64%. Thus, these characteristics make MS a promising candidate for formulation of a sustained release dosage form.

Methods: Polymeric nanoparticles were prepared using Buchi B-190 mini spray dryer (BUCHI, Switzerland). Briefly, accurately weighed Kollidon SR (KSR) (1g) was dissolved in methanol (100 mL). MS was added to the solution under mild agitation (250 rpm). Final transparent solution was then spray-dried using the spray dryer with an inlet temperature of 120°C and an outlet temperature of 65°C. White dry powders were collected and kept in tightly-closed and colored vials at room temperature until being analysed. Compositions of formulations prepared are summarized in Table 1. During the characterization studies particle size, polydispersity index, morphology, zeta potential and thermal analyses were performed.

Table 1: Compositions of formulations prepared. Code KSR (g) MS (g) Methanol (mL)

F1 1 0.05 100

F2 1 0.10 100

F3 1 0.20 100

PLCB 1 - 100

Results: Particle size and zeta potential measurements were shown in Table 2. Particle sizes of all formulations were found to be in the nanoscale range. Morphological studies demonstrated spherical shape of all formulations. Zeta potential values measured displayed negatively charged characteristic. Possible MS polymeric matrix interaction was shown with DSC analyses.

149

Table 2: Mean particle size, PI, zeta potential of formulations prepared (SE: Standard Error) (n=3).

Code Particle size (nm) ± SE PI ± SE Zeta Potential (mV) ± SE

F1 421.87 ± 12.01 0.22 ± 0.16 -13.77 ± 0.60

F2 436.73 ± 32.51 0.48 ± 0.03 -17.33 ± 0.47

F3 438.00 ± 48.90 0.42 ± 0.13 -20.83 ± 0.92

PLCB 403.13 ± 26.82 0.49 ± 0.07 -20.14 ± 0.23

Conclusions: As indicated with this study, polymeric nanoparticles prepared with single spray drying procedure is able to enhance physicochemical characteristics of drug. Possible MS matrix interaction were determined to be a promising approach for extended delivery of MS. Keywords: Montelukast sodium, Kollidon® SR, nanoparticle, spray drying, characterization References:

1. Ranjan OP, Nayak, UY, Reddy MS, Dengale SJ, Musmade PB, Udupa N. Osmotically controlled pulsatile release capsule of montelukast sodium for chronotherapy: Statistical optimization, in vitro and in vivo evaluation. (2014) Drug Deliv.; 21(7):509-518.

2. Priyanka K, Sathali AA, Preparation and evaluation of montelukast sodium loaded solid lipid nanoparticles. (2012) J. Young Pharm.; 4(3):129-137.

150

P-39

Identification of Novel MDM2-p53 Protein-Protein Interaction Inhibitors as New Anticancer Hits

Gulsah Aydina,b,c , Elif Sedef Develid , Selvi Durmus Erimd ,

Serdar Durdagic and Mine Yurtseverb

aCoordinationship of Specialization in Environmental and Health Technologies, Duzce University, Duzce, Turkey;

bDepartment of Chemistry, İstanbul Technical University, İstanbul, Turkey; cComputational Biology and Molecular Simulations Laboratory, Department of Biophysics,

School of Medicine, Bahcesehir University, Istanbul, Turkey; dMedical Biochemistry Department, Istinye University, Istanbul, Turkey

E-mail: gulsahaydinn@gmail.com

Background: The tumor suppressor protein p53 is a powerful anti-tumoral molecule which is an important role in the regulation of cell cycle, DNA repair, and apoptosis. [1–4] The majority of human cancers are caused by mutations or deletions of alleles in p53 [1–6]. p53 activity is effectively inhibited by its negative inhibitor Murine Double Minute 2 (MDM2) oncogenic protein. The presence of excessive MDM2 in cells has been determined to cause in ∼11% of all tumors and observed at higher levels in certain tumor types such as hepatocellular carcinoma (44%), soft tissue sarcomas (31%) and osteosarcomas (20%) [3,7]. Therefore, development of inhibitors of the MDM2 is very important in anticancer drug design studies. Methods: MDM2 (PDB:4HBM) were prepared for experimentation using protein preparation module of Schrodinger’s Maestro Molecular Modeling Suit. [8] Hydrogen atoms were added; missing side chains and loops were fixed using Prime module of Maestro. Protonation states of amino acid residues were determined by PROPKA at pH 7.0. [9,10] Water molecules around the catalytic side were kept during protein preparation and rest of the water molecules were removed. Optimization were performed by QPLS_2005 force field. Drug-like ligands were downloaded from Databases for molecular docking simulation studies. Prior to the docking, all ligands were prepared with the ligand preparation LigPrep module of the Maestro. The physiological medium and ionization states were determined for the all ligands at pH 7.4. Docking simulation studies were performed with virtually screening using Glide algoritms in Schrodinger’s Maestro Molecular Modeling Suit. Then a small number of selected ligands were docked using GOLD, IFD and QPLD docking methods and ADME, MetaCore and MetaDrug studies were performed for this selected ligand. İn vitro studies were carried out with several ligands predicted to give hope when all results were evaluated together. Results: As a result of our virtual screening studies, we observed that ligands that give top docking score interactions with critical amino acids, and when we examine ADME, MetaCore and MetaDrug properties of these ligands, we observed that there are no obstacles in the determination of anticancer drug candidates. We performed in vitro trials of several of these ligands and found promising results.

151

Conclusions: The results of suggested molecules were compared with standard inhibitors and promising results have been observed. Keywords: p53-MDM2 interaction inhibitors, molecular docking simulations, anticancer hits References: 1. Vogelstein B, Lane D, Levine A, Surfing the p53 network, (2000) Nature 408: 307–310. 2. Almerico A, Tutone M, Pantano L, Lauria A, (2012) Biochemical and Biophysical Research Communications 424: 341–347 3. Toledo F, Wahl G, Regulating the p53 pathway: in vitro hypotheses, in vivo veritas, (2006) Nat. Rev. Cancer 6: 909–923. 4. Vousden K, Lane D, P53 in health and disease, (2006) Nat. Rev. Mol. Cell Biol. 8:275–283. 5. Hollstein M, Sidransky D, Volgelstein B, Harris C, P53 mutations in human cancers, (1990) Science 253: 49–53. 6. Joerger A, Fersht A, Structural biology of the tumor suppressor p53, (2008) Annu. Rev. Biochem. 77:557-582. 7. Oliner J, Kinzler K, Meltzer P, George D, Vogelstein B, Amplification of a gene encoding a p53-associated protein in human sarcomas, (1992) Nature 358: 80–83. 8. Madhavi G, Adzhigirey M, Day T, Annabhimoju R, Sherman W, Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments, (2013) J. Comput. Aided. Mol. Des. 27: 221–234. 9. Bas D, Rogers D, Jensen J, Very fast prediction and rationalization of pKa values for protein-ligand complexes, (2008) Proteins Struct. Funct. Genet. 73:765–783. 10. Li H, Robertson A, Jensen J, Very fast empirical prediction and rationalization of protein pK a values, (2005) Proteins Struct. Funct. Genet. 61:704–721.

152

P-40

Drugs as Potent Catalyst for the Reaction of Oxindole and Nitrosobenzene

Hande Abeşa, Sezen Alsancaka, Yeşim Çamlısoya, Nihan Çelebi-Ölçüma aDepartment of Chemical Engineering, Yeditepe University, İstanbul, Turkey

E-mail: sezen.alsancak@yeditepe.edu.tr Background:

Quantum mechanical calculations have successfully demonstrated numerous organocatalytic reaction mechanisms and revealed the observed reaction results, however, most predictive organocatalyst design strategies are faced with synthetic feasibility and are not practical. The object of this study is to facilitate the examination of potential small organocatalyst candidates for the target reaction with the new computational approach. Methods:

New computational approach1 which combines quantum mechanical calculations with drug design tools were proposed. For the implementation of this approach, reactions of 3-substituted 2-oxindoles with nitrosobenzene, which are of interest in the product range, have been selected. From this reaction it is possible to obtain two different products with different bioactivity (Figure 1). The aminoxylation products are found in many natural and pharmaceutical active products.2 On the other hand, hydroxyamination products are found in the structures of potential drug candidates in the treatment of malaria and stress-related disorders.3 Due to different bioactivities of these products, their selective synthesis plays a crucial role in pharmaceutical industry. Catalytic active site models were constructed using quantum mechanical calculations to understand the structures responsible for the selectivity of the catalytic mechanism and the target reaction. The cinchona alkaloid-based organocatalyst functional groups that catalyze the target reaction were identified as pharmacophore groups. Results:

Catalytic group maps were created using arrangement of these pharmacophore groups in the active site models. The catalytic activity and selectivity of determined lead molecule will be examined using quantum mechanical calculations. Conclusions:

The resulting catalytic group maps were screened against conformational library of active pharmaceutical ingredients on the markets. Keywords: active pharmaceutical ingredients, quantum mechanic, oxindole, nitrosobenzene

Figure 1: Hydroxyamination and Aminoxylation Reactions of 2-oxindoles

NPG

O

R

PhNO

Cinchona Alkaloids NPG

O

RNOH

Ph

NPG

O

ROHNPh

+

hydroxyamination aminoxylation

153

References: 1. Tübitak 1001, “Determination of Active Organocatalysts Using Computational

Methods”, 114Z791. 2. S. Hibino, T. Choshi, Natural Product Reports, 2001, 18, 66-87.

3. S. Peddibhotla, Current Bioactive Compounds, 2009, 5, 20-38.

154

P-41

Should HOMA INDEKS be given directly from in biochemistry laboratory? Hayrullah Yazara, Omer Emre Oza

aSakarya University Faculty of Medicine, Department of Medical Biochemistry, Sakarya, Turkey

E-mail: drhyazar@hotmail.com Background: HOMA Index get used by doctors to glysemıc ındex tracking in recent years. How many doctors use and know HOMA Index ? Our purpose; create awareness about HOMA Index and stop diabetic process before it starte. Methods: FBG (Fasting Blood Glucose) and insulin be ordered every patient’s HOMA calculations directly by automation in our Hospital. (SUEAH) The Docs who wants it can use HOMA to treatment planning . Our reserch work has been investigated how often HOMA is used and how much is known by doctors to treatment planning. Results: There are different formulas in calculations of HOMA but common formula is; FBG(mg/dl) x Fasting Insulin ((μU/lt)/405) . It is very important patients give blood when they are fasting to method’s reliability. Doctors who order FBG (Fast Blood Glucose) and insulin in automation, they can also check patient’s HOMA. 100 physicians working in the Sakarya Education Research Hospital provided our participation. 60 of our doctors are assistants. 25 experts. 15 'is academician. Conclusions: So HOMA Index is too important to early diagnosis and treatment methods about increasingly diseases in socieyt (Type II DM, Obesity, Metabolic Syndrome, Dyslipidemia et al.) According to our opinion HOMA is going to be routine biochemical parameter. Keywords: HOMA Index, Insulin, FBG Figure 1: Use of HOMA Index. Of the 100 doctors, 23 reported that the Homa Index test was working. Seven of these doctors are assistants, 10 are specialists, and 6 are researchers. Two of our specialist doctors have actively reported that it works in some cases, even if they do not use the homa index parameter. References: 1.Hill NR, Levy JC, Matthews DR (2013). "Expansion of the homeostasis model assessment of β-cell function and insulin resistance to enable clinical trial outcome modeling through the interactive adjustment of physiology and treatment effects. Diabetes Care 36 (8): 2324–30. doi:10.2337/dc12-0607. PMID 23564921.

155

P-42

PREPARATION OF BIOCOMPATIBLE CARRIER IMPLANT IN COLCHICINE

CONTROLLED RELEASE SYSTEM

Hesna URAL KAYALIK, a Sema ÇETİNb

a. Ufuk Üniversitesi SHMYO, b. Kırıkkale Üniversitesi Fen Bilimleri Enstitüsü Biyoloji ABD E-mail: drhesnaural@gmail.com

Background: In this study, it is designed to create a continual system that obtained slow basal colchicine release. Methods: Poly(2-hydroxyethyl-methacrylate-methyl methacrylate) (p(HEMA-MMA)) copolymer, swellable with water has been prepared with UV-starter fotopolimerizasyon method in a cylindrical form for colchicine controlled release and effective hydrogel implant formulation. p(HEMA-MMA) composition with different monomer ratio was prepared and scanning electron micrograph (SEM) for the surface structure of prepared carrier implant material and differential scanning calorimetry (DSC) for thermal stability were performed. It was investigated the swelling behaviors with the transfer of solvent molecules to the hydrogels (1,2). The kinetic of colchicine oscillation in continuous flow oscillation system containing physiological phosphate buffer was evaluated with loaded three different colchicine doses (0.5, 1 and 2 mg/ml). The oscillated amount of colchicine were determined by using the spectrophotometric method. Power Law, zeroth order and Higuchi model equations were used in order to assess the mechanism of colchicine oscillation. The most appropriate results in drug loading and release studies were obtained in a 1:1 (V/V) ratio of HEMA:MMA monomer composition. It was seen that the smooth surface in the SEM image of the carrier hydrogel implant. According to the DSC result; it was found to decrease thermal stability with the participation of the MMA comonomer to the structure of pHEMA hydrogel (3,4). It was observed that equilibrium water content in physiologic phosphate buffer of p(HEMA-MMA) copolymer hydrogel is lower when compared with pHEMA. Results: It was concluded that these formulations generated in the study can be successfully applied for basal colchicine level three weeks. Keywords: Colchicine, pHEMA, hydrogel, controlled release. References 1. Poplack DG, Morgolin. Management of common cancers of childhood. In: Poplack DC, editors. Principles and Practice of Pediactric Oncology I. Philedelphia: Saunders, 1997:409-504. 2. Lanzkowsy P. Editors. Leukemias. San Diego: Academic pres, 1999:359-411. 3. C.J., van Oss, R.J., Good, M.K., Additive and nonadditive surface tension components and the interpretation of contact angles. Chaudury Langmiur 4, 884(1988).

156

P-43

Characterization and analysis of anti-tumor properties of newly synthesized half-sandwich organoruthenium (II) complexes [(η6-p-cym)RuCl2(η1-S-TSC) in

colorectal cancer cell lines

Elif Subasi a, Esra Bulut Atalayb, Duygu Erdogan b, Betül Sen c, Bilge Pakyapan d, Hülya Ayar Kayalıa,e

a Department of Chemistry, Faculty of Science, Dokuz Eylül University, 35160 Izmir, Turkey b Izmir International Biomedicine and Genome Institute, Dokuz Eylül University,

35340 Izmir, Turkey c Department of Physics, Faculty of Science, Dokuz Eylül University, 35160 Izmir, Turkey

d Institute of Science and Technology, Ege University, 35100 Izmir, Turkey e Izmir Biomedicine and Genome Center, Dokuz Eylül University, 35340 Izmir, Turkey

E-mail: esrablt-2011@hotmail.com Background: Some metal-based drugs, especially platinum-based drugs, have been used in various cancer treatments despite of having high toxicity and self-defense systems of cancer cells [1,2]. In recent years, the medicinal properties of other metal-based drugs such as ruthenium (Ru) have been investigated to be an alternative to widely used platinum-based drugs [3]. Methods: New organoruthenium(II) complexes [(η6-p-cym)RuCl2(η1-S-TSC)], (TSC: TSC1, I; TSC2, II; TSC3, III; TSC4, IV) have been prepared from the reaction of [{(η6-p-cym)RuCl}2(µ-Cl)2] with the corresponding new thiosemicarbazones: TSC1, (2-acetyl-5-chloro-thiophene-4-phenyl-3-thiosemicarbazone); TSC2 (2-acetyl-5-methyl-4-phenyl-3-thiosemicarbazone); TSC3, (2-acetyl-5-chloro-thiophene-4-methyl-3-thiosemicarbazone) and TSC4, (2-acetyl-5-methyl-thiophene-4-methyl-3-thiosemicarbazone) in methanol and the complexes have been identified by elemental analysis, FT-IR and 1H NMR spectroscopy, and XRD analysis. Then all the ligands and ruthenium complexes assessed for anticancer properties in vitro against three different colorectal cancer stage’s cell lines (Caco-2, DLD-1, and SW620) and a noncancerous cell (CCD18Co). Results: Results of MTT assay for all complexes showed higher cytotoxicity with IC50 values as 0.1-0.33 µM in colorectal cell lines expect SW620 with IC50 values as 47.4-84.2 µM than in normal cells. The morphological and fluorescence imaging revealed that the drug candidates could induce an apoptosis and easily be taken into the cells and accumulate in the nucleus. As a result of the DNA and BSA binding experiments, the Ru-complexes showed high binding affinity with CT-DNA whereas there was weak interaction with protein (BSA). It was also observed that the molecules cause the cell cycle arrest by affecting the G0/G1 and/or S phase. However, it was not understood from Western blotting that the complexes prefer the apoptotic pathway or the autophagic pathway. Conclusions: The study clearly demonstrated they regard as highly promising alternative to platinum counterparts in cancer treatment due to high biological properties of newly synthesized Ru complexes. Keywords: Half-sandwich organoruthenium (II) complexes, colorectal cancer, cytotoxicity, anticancer property

157

Figure 1: The overview of the study. References:

1. Liu W, Gust R. Metal N-heterocyclic carbene complexes as potential antitumor metallodrugs. (2013) Chem Soc Rev.; 42:755-773.

2. Starobova H, Vetter I. Pathophysiology of chemotherapy-induced peripheral neuropathy. (2017) Front Mol Neurosci.; 10:174.

3. Tavsan Z, Yaman P. K, Subasi E, Kayali H. A. Screening organometallic thiophene containing thiosemicarbazone ruthenium (II/III) complexes as potential anti-tumour agents, (2018) J Biol Inor Chem., 23:425-435.

158

P-44

Extraction, Isolation, Characterization and Oral Bioavailability of Curcumin for the Medicinal Purposes

İbrahim Demirtaş

Department of Chemistry, Faculty of Science, Çankırı Karatekin University, Cankırı-Turkey; Mada Grup Aş., Technopark Ankara, Ankara-Turkey

E-mail: ibdemirtas@gmail.com Background: The use of complementary and traditional medicine (CTM) by patients with cancer may result in potentially negative effects that can impact the efficacy and safety of conventional anticancer treatments. Greater than 85% of patients have reportedly admitted they would discontinue the use of herbal agents if a potentially harmful herb-drug interaction was suspected (1). The study methodology reflects an important and objective approach for describing a general trend of curcumin as a herbal medicine use by patients with cancer. Methods: Curcuma longa L. (turmeric) consists of a complex combination of natural chemicals as well as curcumin. The constituents of turmeric have been characterized as three curcuminoids: curcumin (diferuloylmethane; the primary constituent and the one responsible for its vibrant yellow color), demethoxycurcumin, and bisdemethoxycurcumin, as well as volatile oils (tumerone, atlantone, and zingiberone), sugars, proteins, and resins. A simple method has been designed for their efficient extraction and separation. Their structures have been confirmed by 1H NMR spectroscopy and unique mass fragmentation pattern. Results: While numerous pharmacological activities, including antioxidant and antimicrobial properties, have been attributed to curcumin, this study focuses on curcumin's scale up purification and characterization for a herbal medicine purposes. Curcumin's effect on cancer (from an anti-inflammatory perspective) will also be discussed; however, an exhaustive review of its many anticancer mechanisms is outside the scope of this presentation. Conclusions: A number of different curcumin compounds and analogs were isolated and purified that may be more effective and better absorbed. Curcumin's diverse array of molecular targets affords it great potential as a therapeutic agent for a variety of inflammatory conditions and cancer types. Keywords: Curcuma longa, curcumin, scale up preparation, characterization References: 1. McCune JS, Hatfield AJ, Blackburn AA, Leith PO, Livingston RB, Ellis GK. Potential of hemotherapy-herb interactions in adult cancer patients. (2004) Support Care Cancer; 12:454-462.

159

P-45

Dimerization Interfaces of AT1 Receptor

Ismail Erola,b, Bunyemin Cosuta and Serdar Durdagib aDepartment of Chemistry, Gebze Technical University, Kocaeli, Turkey; bComputational Biology and

Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey

E-mail: i.erol@gtu.edu.tr Background: G protein-coupled receptors (GPCRs) are the largest membrane protein family, targeted by 35% FDA approved drugs [1]. This make them one of the most largest drug targets in human diseases. Angiotensin II type 1 receptor (AT1R) belongs to Class A GPCR family, binds Angiotensin II (A-II), which is a hormone responsible from ion/fluid balance and causes vasoconstriction and an increase in blood pressure [2]. GPCR oligomerization is a long debated topic and recent advances in both structural and computational biology provide an opportunity to shed light into oligomerization interfaces and also consequences of this phenomena, in terms of G Protein or β-arrestin coupling. Methods: In this study, since only inactive state of AT1R is available (PDB ID, 4YAY), first we modeled the active structure of AT1R [3], and based on the literature we used crystal GPCR dimer interfaces to construct the AT1R dimer sets (i.e., five interfaces: TM1,2,8; TM4; TM4,5; TM5 and TM6,7). First set consist of two inactive AT1R, second set: one inactive and one active AT1R, and the last set consist of two active AT1R. Obtained dimers were then inserted into POPC membrane and subjected to 200ns and 500ns (two independent runs) classical molecular dynamic simulation (cMD) using Gromacs [4]. Results: Since homooligomerization of GPCRs have different pharmacological outcomes, this study may shed light into dimerization and its consequences in AT1R. Conclusions: Thus, it may be pharmacologically relevant to use dimer forms of AT1R in target-driven based ligand design challenges. Keywords: Molecular Dynamics Simulation, Dimerization, G-Protein-Coupled Receptors, Angiotensin II Type 1 Receptor Acknowledgments This study has been financially funded by Scientific and Technological Research Council of Turkey (TUBITAK). Project Number: TUBITAK 214Z122. The numerical calculations reported in this abstract were fully performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources). References: 1. Hauser et al., Nature Reviews Drug Discovery, 16, 829–842 (2017). 2. Durdagi et al., European Journal of Medicinal Chemistry, 145, 273-290 (2018). 3. Pándy-Szekeres et al., Nucleic Acids Research, 46, D440-D446 (2017). 4. Abraham et al., SoftwareX, 1-2, 19-25 (2015).

160

P-46

A Molecular Modelling Study and Effect of Nitroimidazole Compounds on Proliferation and COX-2 Expression in Prostate Cancer Cells

Necla Kulabaşa, Sevil Şenkardeşa, Özge Çevikb, İlkay Küçükgüzela, Ş. Güniz Küçükgüzela

aMarmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpaşa, İstanbul, TURKEY; bDepartment of Biochemistry, School of Medicine, Adnan Menderes University,

Aydın, TURKEY. E-mail: necla.kulabas@marmara.edu.tr

Background: Prostate cancer is now one of the most common cancer among men worldwide. COX-2 plays an important role in prostate inflammation and carcinogenesis and are associated with increased angiogenesis, tumor invasion, and resistance to apoptosis (1). Similarly, overexpression of COX- 2 has been shown to induce cancer formation in transgenic mice (2). Docking studies with synthesized compounds (3a-g) against COX-2 receptor indicated that they exhibit specific interactions with key residues located in the site of the COX-2 structure. The analysis of the docking results of 1-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-(4-nitrophenoxy)propan-2-ol (3b) and methyl 4-[2-hydroxy-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propoxy]benzoate (3d) indicated high binding energy (comparable with celecoxib) and the best docking score. The present study aims to investigate the effect of these two nitroimidazole derivatives (3b and 3d) on COX-2 expression and proliferation in prostate cancer cell lines PC-3 and DU145. Methods: Synthetic methods and analysis of compounds were performed as previously described (3). Crystal structure of COX-2 (pdb:3LN1, resolution 2.4 Å, co-crystallized with the inhibitor celecoxib) was downloaded from the Protein Data Bank (www.rcsb.org) and the molecular docking studies were performed using Autodock version 4.0. Evaluation of the antiproliferative activity was performed using tetrazolium salt MTT assay as reported (4). The pCOX-2-Luc reporter plasmid encoding firefly luciferase under the control of the COX-2 promoter was transfected in PC-3 and DU145 cells and 24-h post-transfection cell lysates were subjected to luciferase activity assay (5). Luciferase activity assay was performed employing the Dual-Glo Luciferase and measured using a luminometer for three different concentrations (2.5, 10 and 50 �M). Results: According to in silico studies, the binding energy of the docked compounds 3b and 3d COX-2 was found -8.4 and -7.8 kcal/mol, respectively. Compound 3b exhibited antiproliferative activity with an IC50 value 89.01 �M against the PC-3 cell line. Compound 3d exhibited good cytotoxicity on DU145 and PC-3 cell line with IC50 value 59.81 �M and 31.00 �M, respectively. According to luciferase assay, 3b and 3d reduced the promoter activity of COX-2 in prostate cancer cells in a dose-dependent manner. By the in vitro and in silico studies, selected compounds not only showed high affinity for COX-2 binding site but also, suppressed the COX-2 gene expression. Conclusions: Variation in the COX-2 gene could alter COX-2 enzyme expression and function. It has been reported that, pharmacological and genetic inactivation of COX-2 led to suppression of cell growth and might have a general anticancer effect. Thus, 3b and 3d might undergo further studies as potential COX-2 inhibitors with antiproliferative properties. Keywords: COX-2, luciferase, nitroimidazole, prostate cancer, molecular docking,

161

Figure 1: Binding orientation of compounds 3b (magenta) and 3d (blue) with celecoxib (yellow) as a reference ligand in the active site of COX-2 References:

1. Tsujii M, Kawano S, Tsuji S, Saeaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. (1998) Cell; 93:705-716.

2. Liu CH, Chang SH, Narko K. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. (2001) J Biol Chem.; 276: 18563-18569.

3. Şenkardeş S, Kulabaş N, Kalaycı S, Şahin F, Küçükgüzel İ, Küçükgüzel ŞG. Synthesis and antimicrobial activity of novel ether-linked derivatives of ornidazole. (2018) 12th International Symposium on Pharmaceutical Sciences; P-487, p:404-405, Ankara, Turkey.

4. Çoruh I, Çevik Ö, Yelekçi K, Djikic T, Küçükgüzel ŞG Synthesis, anticancer activity, and molecular modeling of etodolac-thioether derivatives as potent methionine aminopeptidase (type II) inhibitors. (2018) Arch Pharm.; 351(3-4): 1-16.

5. Manvar D, Pelliccia S, La Regina G, Famiglini V, Coluccia A, et al. New 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides inhibit hepatitis C virus replication via suppression of cyclooxygenase-2. (2015) Eur J Med Chem.; 90:497-506.

162

P-47

Docking Simulations of Novel N-Acetyl-Tryptophan and Biphenyl-3-ol Derivatives on Acetylcholinesterase Enzyme

İsmail Hakkı AKGÜNa, Turgut Mesut YILMAZa

aEge University, Faculty of Engineering, Bioengineering Department, Bornova, İzmir, TURKEY E-mail: ismail.hakki.akgun@ege.edu.tr, turgutmesutyilmaz@gmail.com

Background: Our efforts on generating new lead molecules revealed some remarkable results. For example, docking simulation studies carried out by using Autodock Vina scored binding energy of molecule 1 (M1) (Figure 1) with -14.2 kcal/mol against acetylcholinesterase enzyme (RSCB code 1eve (1, 2)) while crystal pose of donepezil was scored as -11.2 kcal/mol (Figure 2). According to our notes M1 was generated virtually using two fragments molecule 2 (M2) (N-acetyl-tryptophan) and molecule 3 (M3) (biphenyl-3-ol derivative) (Figure 1). In this work we have focused our attentions to simulate binding energies of other molecules that constituted with using M2 and M3, against acetylcholinesterase enzyme with further filters. Methods: Search on our local database revealed that there are totally 1138 molecules generated by using M2 and 1958 with using M3. Before docking these molecules against acetylcholinesterase we have applied brain penetration and filter using SwissADME web server (3) to increase success of finding reliable hit for further in-vitro and in-vivo screenings. After this filter total number of the molecules generated using M2 decreased to 158 (Group 1). Meanwhile the number of the molecules which have passed brain penetration filter was 95 (Group 2) for the ones generated with M3. The molecules that have passed filter were docked using Autodock Vina (4) with the pdb structure (1eve) mentioned above. Results: Lowest binding energy for the molecules in group 1 was – 10.3 kcal/mol (M4) while highest energy was – 7.6 kcal/mol (M5) (Figure 3). None of the molecules in group 1 have shown better binding energy then donepezil. However, in group 2 lowest binding energy was determinated as -12.1 kcal/mol (M6) and highest was -9.8 kcal/mol (M7) (Figure 4). Nine of the molecules in group 2 has shown better binding energy than donepezil molecule. Conclusions: Our results are indicating that 9 of the total 253 molecules have advantage over donepezil based on their binding energies and these compounds can be used as lead molecule in the studies to generate novel acetylcholinesterase inhibitors. Keywords: Docking, Virtual Screening, Acetylcholinesterase, Novel Molecules

163

References:

1. Kryger, G., Silman, I., & Sussman, J. L.: Structure of acetylcholinesterase complexed with E2020 (Aricept®): implications for the design of new anti-Alzheimer drugs. (1999) Structure; 7(3), 297-307. 2. Huang, N., Shoichet, B. K., & Irwin, J. J.: Benchmarking sets for molecular docking. (2006) J. Med. Chem.; 49(23), 6789-6801. 3. Daina, A., Michielin, O., & Zoete, V.: SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. (2017) Sci. Rep.; 7, 42717. 4. Trott, O., & Olson, A. J.: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. (2010) J. Comput. Chem., 31(2), 455-461.

Molecule 1 (M1)

Molecule 2 (M2) Molecule 3 (M3)

Figure 1. Structures of molecule 1 (M1) and fragments molecule 2 (M2) and molecule 3 (M3)

Figure 2. Simulated docking poses of molecule 1 (M1) and crystal structure of donepezil.

164

M4 ( - 10.3 kcal/mol) M5 ( - 7.6 kcal/mol)

Figure 3. Docking pose and 2D structures of M4 and M5.

M6 ( - 12.1 kcal/mol) M7 ( - 9.8 kcal/mol) Figure 4. Docking pose and 2D structures of M6 and M7.

165

P-48

The Cerebral Vasospasm; How can we unreval this problem with the vasodilator, Hydrogen Sulfide?

İsmail Kayaa, İlker Deniz Cingözb, Hasan Emre Aydına, Soner Sahinc aKütahya Medical Sciences University, Department of Neurosurgery, Kütahya

bİzmir Alsancak State Hospital, Department of Neurosurgery, İzmir cBahcesehir University, Department of Neurosurgery, Istanbul

E-mail: dr.ikaya@gmail.com

Background: Cerebral vasospasm following subarachnoid hemorrhage (SAH) is the most important complication which effects mortality and morbidity of patients with intracranial aneurysm although effective treatment. Today, the mechanisms of vasospasm is not understood in spite of experimetal and clinical researches. In the past due to the toxic properties of known hydrogen sulfide after discovery of vasodilatation in cardiovascular system, its effects on other systems started to be examined. Methods: In this study, 40 adult Sprague Dawley female rats, weighing 200 to 250 grams were used and was divided two groups. All the rats were maintained in cages at 22 °C and fed with the same amount of standard animal feed throughout the study. The group which treated with Hydrogen Sulfide compared with Nimodipine. Results: Our first findings suggest that hydrogen sulfide was effective on vasospasm through to the Ca channel receptors like Nimodipine. Conclusions: As a conclusion, our study compared hydrogen sulfide with nimodipine, which known to has vasodilator effects. Hydrogen sulfide, which a natural active ingredient and has considered to be beneficial on vasospasm. Keywords: hydrogen sulfide, vasospasm, subarachnoid hemorrhage References:

1. Rautio J, Kumpulainen H, Heimbach T, Oliyai R, Oh D, Järvinen T, Savolainen J. Prodrugs: Design and clinical applications. (2008) Nat Rev Drug Discov.; 7(3):255-270

166

P-49

Synthesis and Molecular Modeling of New (S)-Naproxen Thioethers as Possible Breast Cancer Agents

Kaan Birgüla,e, Abdullahi Ibrahim Ubab, Ozan Çuhadarc, Sevgi Koçyiğit Sevinçc, Pınar Mega

Tiberc, Oya Orunc, Özgür Yılmazd, Kemal Yelekçib, Ş. Güniz Küçükgüzela aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, 34668, Haydarpaşa,

Istanbul, Turkey; b Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, Kadir Has University, 34083, Istanbul, Turkey; c Department of Biophysics, School of Medicine, Marmara

University, 34854 Maltepe, Istanbul, Turkey; d TUBITAK Marmara Research Center, 41470, Gebze, Kocaeli, Turkey; e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Altınbaş University, 34144, Bakirkoy,

Istanbul, Turkey. E-mail: kaanbirgul93@gmail.com

Background: Methionine aminopeptidase2 (MetAP2) is a bifunctional protein that plays a critical role in the growth of different types of tumours (1). Naproxen ((S)-2-(6-methoxy-2-naphthyl)propanoic acid) is a non-steroidal anti-inflammatory drug (NSAID), also known to have anticancer activities (2). Besides, thiosemicarbazides, 1,2,4-triazole-3-thiones and thioethers have been reported to have anticancer activity on different cancer cell lines (3-5). On the light of the foregoing, new naproxen derivatives were synthesized. The molecular binding of the compounds on MetAP-2 was performed and their anticancer effects on MCF-7 human breast adenocarcinoma cancer cell line was studied. Methods: The naproxen ester (methyl (S)-2-(6-methoxynaphthalen-2-yl)propanoate) [1] and naproxen hydrazide ((S)-2-(6-methoxynaphthalen-2-yl)propanoic acid hydrazide) [2] were prepared according to the reported method (6). Naproxen hydrazide and substituted isothiocyanates were refluxed to give new (S)-2-(6-methoxynaphthalen-2-yl)propanoyl-N-substituedthiosemicarbazide [3]. The naproxen thiosemicarbazides were treated with 4N NaOH solution and (S)-5-(1-(6-methoxynaphthalen2-yl)ethyl)-4-substitued-1,2,4-triazole-3-thiol was obtained [4]. Lastly, the naproxen triazoles were treated with substituted benzyl chloride with the presence of potassium carbonate in ethanolic solution to give the corresponding thioeters. Results: The purpose of this study is to synthesize new naproxen derivatives, investigate molecular binding on MetAP2 active side and studied anticancer activity on MCF-7 cancer cell line. The structure of the compounds were elucidated by FT-IR, 1H-NMR and their purity were proven by elemental analysis and TLC. Our compounds showed good inhibition against MCF-7 cell lines and the docking results were correlated with the anticancer activity. Conclusions: In this study, we designed and synthesized new naproxen derivatives which could have possible anticancer activity on breast cancer. To understand the mechanism of action, we also performed the docking studies on MetAP2 enzyme. The results were satisfying and promising in terms of anticancer effects of the novel naproxen compounds. Acknowledgement: The synthesis, structure elucidation and molecular docking of novel compounds were supported by a grant of TUBITAK (Project number: 215S009) Keywords: Naproxen, Anticancer, MetAP2, molecular docking, MCF-7.

167

References: 1. Selvakumar P., Lakshmikuttyamma A., Dimmock J. R., Sharma R. K. (2006). Methionine aminopeptidase 2 and cancer. Biochimica et Biophysica Acta 1765;148-154 2. Motawi T.M., Bustanji Y., El-Maraghy S., Taha M.O., Al-Ghussein M.A. (2014). Evaluation of naproxen and cromolyn activities against cancer cells viability, proliferation, apoptosis, p53 and gene expression of survivin and caspase-3. J Enzyme Inhib Med Chem, 29(2):153-161. 3. Küçükgüzel Ş.G., Coşkun G.P. (2016). Macromolecular drug targets in cancer treatment and thiosemicarbazides as anticancer agents. Anticancer Agents Med Chem, 16(10):1288-1300 4. Küçükgüzel Ş.G., Süzgün P.Ç. (2015). Recent advances bioactive 1,2,4-triazole-3-thiones. Eur J Med Chem, 97:830-870 5. Çoruh I., Çevik Ö., Yelekçi K., Djikic T., Küçükgüzel Ş.G. (2018). Synthesis, anticancer activity and molecular modeling of etodolac-thioether derivatives as potent methionine aminopeptidase (type II) inhibitors. Arch Pharm Chem Life Sci. 2018;351:e1700195 6. Amir M., Kumar H., Javed S. A. (2007). Non-carboxylic Analogues of Naproxen: Design, Synthesis, and Pharmacological Evaluation of some 1,3,4-Oxadiazole/Thiadiazole and 1,2,4-Triazole Derivatives. Arch. Pharm. Chem. Life Sci. 2007;340,577-585.

168

P-50

Computational insight into the phthalocyanine-DNA binding via docking and molecular dynamic simulations

Lalehan Ozalpa Safiye Sağ Erdema, Başak Yüce-Dursuna, Özal Mutlub, Mehmet Özbilc

aChemistry Department, Faculty of Arts and Sciences, Marmara University, Goztepe, Istanbul, Turkey.

bBiology Department, Faculty of Arts and Sciences, Marmara University, Goztepe, Istanbul, Turkey. cDepartment of Molecular Biology and Genetics, Faculty of Arts and Sciences, Istanbul Arel University,

Buyukcekmece, Istanbul, Turkey. E-mail: lalehanozalp@marun.edu.tr

Background: Today, nucleic acids are of great importance as drug targets and developing novel drugs that are able to interact with those targets is the basic and crucial step for drug design. It has been demonstrated that metalloporphyrins easily bind to nucleic acids, leading to fracture in DNA strands. Cationic porphyrins are known as functional compounds that attach to DNA and photodynamically modify the intended site of a DNA molecule by a mechanism similar to that of anti-tumor drugs. Phthalocyanines (Pcs) show slight differences than porphyrins since they possess isoindole rings providing them a larger π system. In spite of a number of experimental studies that aim to enlighten the binding mechanism of Pcs to DNA, there is a gap in the literature regarding the molecular modelling studies on this subject. The main goal of the present study is to contribute to fill this gap, to investigate the interactions between DNA and Pc derivatives possessing varying metals (Zn, Ni, Cu, Mg, Ca and Fe) in search of the effect of substituent and central metal, and to direct future experimental studies. Methods: Quaternized metallophthalocyanines (Q-MPcs) were optimized by quantum mechanical DFT/wB97x/6-31 G (d,p) calculations and docked into a B-form DNA segment (PDB ID: 2DND) employing Autodock Vina and Molegro software. Docked complexes were then subjected to a 20 ns MD simulation using YASARA program Structure Suit. Results: Autodock Vina results reveal that each Q-MPc derivative binds to DNA strongly with similar binding energies and almost identical binding modes. Although changing the metal has no significant effect on binding, presence of quaternary amine substituents increases the binding constant Kb by about 2-fold comparing to the core Pc (ZnPc). Contrary to Autodock Vina, the calculated Molegro Virtual Docker binding scores have been more diverse indicating that the scoring function of Molegro is better in differentiating these metals. Additional calculations employing molecular dynamics (MD) simulations verified the stability of Q-MPc-DNA complexes which remained in contact after 20 ns via attractive interactions mainly between DNA backbone and the Pc metal center. Conclusions: According to output of two docking programs and MD simulations; Q-FePc was found to be the common strong DNA binder. Considering iron’s being a vital constituent for vertebrates, Q-FePc can be seen as the best candidate for medical use. Q-CuPc and QZnPc are the next potential candidates1. Keywords: Molecular modelling, Autodock Vina, Molegro Virtual Docker, molecular dynamics, drug design

169

Figure 1: Snapshots of Q-FePcs obtained from a 20 ns MD simulation

References: 1. Ozalp, L., Sağ Erdem, S., Yüce-Dursun, B., Mutlu, Ö. & Özbil, M. Computational insight into the phthalocyanine-DNA binding via docking and molecular dynamics simulations. Comput. Biol. Chem. 77, 87–96 (2018).

170

P-51

Clay mineral particles as imaging and drug delivery agents

Maide Gökçe Bekaroğlua, Fuad Nurilib, Sevim İşçia*

a Istanbul Technical University, Faculty of Science and Letters, Dept. of Physics, Maslak 34469, Istanbul, Turkey;

b Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. E-mail: iscisev@itu.edu.tr

Background: Clay minerals have always been widely used in production of pharmaceutical formulations due to their many favorable properties such as their high adsorption capacity, high specific surface area, ion exchange capacity, colloidal structure, low toxicity etc. Although clay minerals are widely used as raw pharmaceutical materials they have not yet been investigated as targeted drug delivery agents for tumors at specific areas. [1-4]. Montmorillonite (Mt), a clay mineral of the smectite group can be used as a medical imaging and drug delivery agent to be used in transcatheter arterial embolization (TAE) applications. In this embolization technique, the arterial vessels around tumor site are blocked by drug loaded micro sized Mt particles so tumor is deprived of oxygen and nutrition while loaded drugs are released by the particles into the tumor site. Both devascularization and drug release around tumor site can progressively shrink the targeted tumor size. Certain properties of Mt such as size, adsorption capability, and biocompatibility indicate that Mt particles are highly applicable and unique for TAE and imaging applications. Methods: In order to obtain drug releasing clay mineral particles that are suitable for TAE that can also enable medical imaging, interactions of purified montmorillonite (PMt) with a computed tomography (CT) contrast material and an antitumor drug were investigated. Initially, In-vitro cytotoxicity assays using normal cells were carried out in order to assess PMt biocompatibility. Then, swelling and adsorption behaviour of PMt in the presence of the CT contrast material or antitumor drug were investigated using DLS, XRD and FTIR in order to determine proper concentration where particle sizes are suitable for successful TAE applications. Particles suitable for TAE procedure were further investigated using In-vitro and In-vivo tests. Results: Results showed both CT contrast material and antitumor drug could penetrate interlayer spaces and were adsorbed by the surfaces of the PMt particles. Particle sizes required to successfully obtain embolization (at least 40 μm) were achieved as the size of the PMt increased due to both the adsorption and coagulation. Preliminary embolization and CT imaging tests of particles in a rabbit renal model, in-vitro drug release profiles and in-vitro drug activity on MCF-7 (human breast adenocarcinoma) cells were also satisfactory. Embolization was successfully performed in rabbits without any complications related to the procedures. No embolic material reflux was observed. The pre- and post-embolization comparison was shown at the first rabbit’s renal artery and its branches. The renal artery and its branches were clearly identifiable in the image before the embolization procedure. In vitro anticancer activity of pure DOX and DOX loaded clay particles (PMt+800DOX) against the MCF-7 cell line was investigated in order to assess PMt+800DOX particles’ anticancer drug potential. The amount of pure DOX was adjusted to be equal as the amount of loaded DOX onto PMt+800DOX particles. DOX and PMt+800DOX exhibit similar toxicity to MCF-7 cells.

171

Conclusions: This study demonstrated that embolic particles that can also be used as drug releasing and imaging agents can be obtained by using PMt particles. This novel and economic approach for synthesis of embolic particles showed promising results that synthesized particles would be promising for embolization therapies and drug delivery applications.

Keywords: Montmorillonite; drug delivery; imaging agent; cancer therapy; arterial embolization References: 1. Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: Possibilities and limitations. Applied Clay Science 36, 22-36 2. Jayrajsinh, S., Shankar, G., Agrawal, Y.K., Bakre, L., 2017. Montmorillonite nanoclay as a multifaceted drugdelivery carrier: A review. J Drug Deliv Sci Tec 39, 200-209 3. Viseras, C., Cerezo, P., Sanchez, R., Salcedo, I., Aguzzi, C., 2010. Current challenges in clay minerals for drug delivery. Applied Clay Science 48, 291-295. 4. Yang, J.H., Lee, J.H., Ryu, H.J., Elzatahry, A.A., Alothman, Z.A., Choy,

172

P-52

Molecular Dynamics Studies of Functionalized Pyrene Adsorption on Single-

Walled Carbon Nanotubes with Structural Defects

Mehdi Merana, Seniha Günera, Özge Kürkçüoğlu aDepartment of Chemical Engineering, Istanbul Technical University, Istanbul, Turkey

E-mail: mmeran@itu.edu.tr Background: Single walled carbon nanotubes (SWNTs) have aroused a great deal of interest in the areas of nano-science and nano-devices since their discovery. Enormous progress has been made over the last decade in characterizing carbon nanotubes and developing different approaches to increase their biocompatibility for biomedical applications, especially for drug delivery. However, point defects on SWNTs represent a rich and mostly untapped system. In fact, defects can dominate physical properties of SWNTs, therefore, defect control is very critical for particular applications. Methods: In this study, we performed all-atom molecular dynamics (MD) simulations of SWNTs with structure defects and functionalized pyrene molecules with hydrophilic moieties, used to coat the hydrophobic SWNT to increase its solubility in water (or body fluids). We generated different SWNTs with 5-7-7-5 Stone-Wales defects. We monitored the self-assembly of SWNT−Pyr-COOH, SWNT−Pyr-OH, and SWNT−Pyr-(OH)2 complexes to reveal atomic-scale insights into the adsorption process and their dynamical behaviors. 5 ns long MD simulations in NVT ensemble at 298 K were performed using the COMPASS force field in Materials Studio. Results: MD results showed that the adsorption of functionalized pyrenes on SWNT is significantly affected by the defects, which can reduce the biocompatibility of the nanotubes. Locations of defects define the number of Pyr-COOH, Pyr-OH, and Pyr-(OH)2 adsorbed on the SWNT sidewall. Conclusions: We noted concave/convex distortions of the SWNT sidewall due to one or more pentagons or heptagons defects, which can affect the drug delivery capabilities of these nano-vectors. Keywords: MD Simulation, Single walled carbon nanotubes, Vacancy defect References: 1. He, H., Pan, B. Studies on Structural Defects in Carbon Nanotubes. (2009) Front. Phys. China 4 (3), 297–306. 2. Charlier, J. C. Defects in Carbon Nanotubes (2002). Acc. Chem. Res.

173

P-53

New and Potent eEF2K Inhibitors for the treatment of TNBC

Ferah Comert Ondera,b, Bulent Ozpolatb, Serdar Durdagic, Mehmet Aya aCanakkale Onsekiz Mart University, Department of Chemistry, Faculty of Science and Arts, Natural

Products and Drug Research Laboratory bThe University of Texas MD Anderson Cancer Center, Department of Experimental Therapeutics, TX

77030, USA

cBahcesehir University, School of Medicine, Department of Biophysics, Computational Biology and Molecular Simulations Laboratory

E-mail: mehmetay06@comu.edu.tr Background: Triple-negative breast cancer (TNBC) which constitutes 15-20% of all breast cancers, is a subtype and aggressive type of breast cancer that is a heterogeneous disease that can be classified into estrogen receptor α-positive (Era +) and HER2+ tumors as well as triple-negative (TN) tumors [1]. The activity of eEF-2 kinase is increased in many malignant cell lines and certain cancer tissues [2]. Eukaryotic elongation factor 2 kinase (eEF2K) is indicated as a potential molecular driver in several cancers such as pancreatic, brain and breast cancers [3,4]. For this purpose, we performed docking studies of Etodolac (known drug) which is a pyranocarboxylic acid and non-steroidal anti-inflammatory drug (NSAID) with antipyretic and analgesic activities, and its derivatives, by using computational techniques as eEF2K inhibitors and then in vitro studies. Methods: Computational studies were performed by using Schrödinger 2015 programme to investigate of in vitro activity of the compounds for the treatment of TNBC and also, the potent activity of the compounds were determined by using clonogenic survival assay and western blot analysis. Further studies are still ongoing. Results: There are significant inhibitions at the different concentrations (2.5, 5 μM and etc.) after 2 hours treatments with MDA-MB-231 cell. Conclusions: The results show that etodolac and its derivatives may be potent eEF2K inhibitors. Keywords: TNBC, in silico techniques, eEF2K inhibitor, Etodolac References: 1. Prat A, Perou CM. Deconstructing the molecular portraits of breast cancer. (2011) Mol

Oncol 5: 5 – 23. 2. Hait WN, Wu H, Jin S, Yang JM (2006) Elongation factor-2 kinase: its role in protein synthesis

and autophagy. Autophagy. 2(4):294-6. 3. Tekedereli I, Alpay SN, et al. Targeted silencing of elongation factor 2 kinase suppresses

growth and sensitizes tumors to doxorubicin in an orthotopic model of breast cancer. (2012) PloS one. 7:e41171.

4. Bayraktar R, Pichler M, et al. MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase. (2017) Oncotarget 8(7): 11641-11658.

174

P-54

Evaluation of SUEAH laboratory productivity commission Mehmet Ozdina, Hayrullah Yazarb

aSakarya University Education and Research Hospital, Sakarya, Turkey; bSakarya University Faculty of Medicine, Department of Medical Biochemistry, Sakarya, Turkey. E-mail: drmozdin33@gmail.com Background: Our aim in this research is to evaluate the laboratory productivity commission (lpc) studies of Sakarya University Training and Research Hospital (SUEAH) in terms of laboratory cost analysis. Methods: Our method for laboratory productivity analysis is to evaluate the commission studies statistically in terms of cost analysis by retrospectively scanning the data six months before and six months after the establishment of the LPC (laboratory productivity commission) through automation. The tests to be examined in the study are; FOLAT, estradiol, ferritin, FSH, Progesterone, TOTAL PSA, T3, T4, Ca2 +, AFP, Anti-Thyroglobulin, Anti TPO, Beta hCG, c-peptide, CA 125, CA 15-3, Ca 19-9, CEA, DHEA-sulphate , TSH, SHBG, Vit B1. Results: All of the tests that were run were found to decrease in the number of requests. However, the decrease in some tests was noteworthy. The highest number of requests from these tests is the T3 test with a decreasing 42.19%. If the minimum number of requests is decreasing, the C-peptide is at a rate of 3.22%. Conclusions: Laboratory productivity shows the first data from commission studies; there is a very serious amount of unnecessary and testable requests in hospitals. This situation can be corrected by the laboratory productivity commissions to be established. Keywords: laboratory productivity commission, productivity analysis, hospital information management system References: 1. Erkol Umit, Ağırbaş İsmail. Hastanelerde Maliyet Analizi ve Faaliyet Tabanlı Maliyetleme Yöntemine dayalı Bir Uygulama. (2011) Ankara Üniversitesi Tıp Fakültesi Mecmuası, 64(2).

Graphic 1. Comparison before and after lpc in working tests

175

Tests N Savings Provided Decrease rate Number Before the meeting After the meeting Materiality 1 AFP 7.035 4.711 2.324 33,03 *** 2 Anti

Tiroglobulin 4.190 2.624 1.566 37,37 ***

3 Anti TPO 4.427 2.999 1.428 32,26 *** 4 Beta hCG 8.065 7.688 377 4,67 OD 5 c-peptid 2.643 2.558 85 3,22 OD 6 CA-125 6.188 4.967 1.221 19,73 ** 7 CA- 15-3 6.462 4.984 1.478 22,87 ** 8 CA- 19-9 8.282 6.181 2.101 25,37 *** 9 CEA 8.303 5.683 2.620 31,55 *** 10 dhea sulfat 1.052 984 68 6,46 OD 11 Folat 21.992 17.022 4.970 22,60 ** 12 Estradiol 3.921 3.293 628 16,02 * 13 Ferritin 23.722 19.195 4.527 19,08 ** 14 Fsh 4.434 3.960 474 10,69 OD 15 Progesteron 1.121 1.014 107 9,55 OD 16 Prolaktin 4.681 4.213 468 10,00 OD 17 PSA total 4.145 3.343 802 19,35 ** 18 F-t3 31.708 18.330 13.378 42,19 *** 19 F-t4 40.439 29.890 10.549 26,09 *** 20 TSH 54.520 42.805 11.715 21,49 *** 21 Shbg 273 209 64 23,44 *** 22 Vit b12 32.427 29.349 3.078 9,49 OD TOTAL 280.030 216.002 64.028 22,13 **

Table 1. Level of Significance of Productivity obtained by in-service training seminars given at the hospital. The test with the most savings is the T3 test with 42.19%. The least-saved test is c-peptid with a rate of 3.22%.

176

P-55

Synthesis and identification of iodized antiseptic drugs

MEKAHLIA Leilaa, MERAH Abdelalia a Laboratory of Pharmaceutical Mineral Chemistry, Department of Pharmacy, faculty of Medicine,

Badji Mokhtar University, Annaba, Algeria. E-mail: mekahlialeila@hotmail.fr

The antiseptic is a drug for external use that enables to protect and clean the skin by acting on the microorganism level during the antisepsis process. Therefore, these drugs are very important in combatting the infectious disease. As a result, it is very important for us to make this research which objective is to make a physicochemical synthesis and identification for the antiseptic dugs which are made of the iodine. The active ingredient in these drugs is the iodine which is present in their formulas in different quantities. Qualitative and Quantitative identification of these different antiseptic preparations already synthesized in the previous part as well as that of Betadine 10% have been carried out. The qualitative identification is the set of parameters (physical state, color, odor, boiling point, melting point and solubility in water) that make it possible to identify the nature of substances tested. For the quantitative identification of iodine, we have used two methods of dosage: a direct volumetric dosage and a direct spectrophotometric dosage. For the solution of the lugol, Alcoholic iodine and the betadine 10%, a volumetric dosage using the idometry is used concentrating on the oxydoreductric properties of the I2/2Ishowing that the iodine concentration changes from one drug to another. The concentration of the iodine found in these drugs corresponds the norms with a relative gap of 3% for the betadine. A direct spectrophotometric dosage is proposed to determine the concentration of the diiodine in the dyeing of the officinal iodine. The concentration found also corresponds the norms with a relative gap of 3, 2 %. All these preparations show that the concentration of the diiodine is satisfactory to ensure a good antiseptic activity respecting the conservation and using conditions.

Keywords: infectious disease, antiseptic drug, iodine, synthesis, identification.

References:

1. European pharmacopoeia, « Identity reactions of ions and functional groups», TOME 1, 8th edition, CEDEX, FRANCE: Council of Europe, (2013), p128.

177

P-56

Investigation of cell migration effects of tekamen and methotrexate on

MDA-MB 231 breast cancer cell line

Melike BUGULb, Esin GUVENIR CELIKb, Hacer KAYAb, Onur EROGLUa

aBilecik Şeyh Edebali University, Biotechnology Research Center, Bilecik/Turkeyb

bBilecik Şeyh Edebali University Department of Molecular Biology and Genetics, Faculty of Science and Letters, Bilecik/Turkey

E-mail: onur.eroglu@bilecik.edu.tr

Background:

Combination therapy, a treatment modality that combines two or more therapeutic agents, is a cornerstone of cancer therapy. Tekamen has topoisomerase I activity by stabilizing the cleavable complex between topoisomerase I and DNA, resulting in DNA breaks that inhibit DNA replication and trigger apoptotic cell death. Methotrexate binds to and inhibits the enzyme dihydrofolate reductase, resulting in inhibition of purine nucleotide and thymidylate synthesis and, subsequently, inhibition of DNA and RNA syntheses. Combined treatment can provide benefit and reducing side effects of drugs. The aim of this study is to understand effects of Tekamen and Methotrexate on cell migration on MDA-MB-231 breast cancer cell line. Methods:

Cell viabilty was measured by MTT assay100 μM Tekamen for wound healing, the MDA-MB-231 cell line was cultured being 105 cells in 100 μL in a 6-well culture plate. After cells have 90% confluency, cells treated with 100 μM Tekamen, 60 μM Methotrexate and 75 μM Tekamen+ 60 μM Methotrexate. Widths of scar were observed at 0, 24, 48 and 72 hours by inverted microscope. Results:

We compared effect of monotherapy and polytherapy of drugs on cell migration and wound size drug-applied breast cancer cell line to control group. We observered amount of cell viabilties after drug treatment are 54,3%, for 100µM tekamen,40,89% for 60µM Methotrexate and 59,47% for 75 μM Tekamen+ 60 μM Methotrexate combine treatment. We observed to inhibit cell migration with combine treatment at for 72 hours. These results show that combine therapy is more effective than monotherapy because cancer cells are less to have resistance to multiple drugs treatment and this approach increases the chance of effective treatment.

178

Conclusions:

Although combination therapy can be toxic if one of the agents used is chemotherapeutic, the toxicity is significantly less because different pathways will be targeted. These results suggest that combine therapy may use for cancer theraphy because of decreasing the resistance of cancer cells to drugs. We found a decrease of migration activity of cells with Tekamen+Methotrexate. Using combination Tekamen and Methotrexate may be an candidate for breast cancer theraphy in the future.

Keywords: Tekamen, Methotrexate, cell migration, breast cancer

References:

1. Irinotecan Hydrochloride, National Center for Biotechnology Information, U.S. National Library of Medicine, Modify Date: 2018-08-26; Create Date: 2005-08-08

2. Methotrexate, National Center for Biotechnology Information, U.S. National Library of Medicine,Modify Date: 2018-08-26; Create Date: 2005-06-24

179

P-57

Anticancer Activity of Quinoline and Dihydroquinoline Derivatives

Meltem SELVİTOPUa, İsmail Furkan TURANa, Seçil ERDEN TAYHANa, Esra KOÇb aDepartment of Genetics and Bioengineering, Faculty of Natural Science and Engineering,

Gaziosmanpasa University, Tokat, Turkey bDepartment of Chemistry, Faculty of Arts and Sciences, Gaziosmanpasa University,

Tokat, Turkey E-mail: meltem.selvi.5@gmail.com

Background: Cancer is a constantly increasing disease in the population. Recent scientific studies have investigated new compounds targeting cancer cells. Quinoline is a heterocyclic aromatic organic compound having the chemical formula C₉H₇N. Methods: In the first step of this work, the synthesis of the original therapeutic products quinoline and dihydroquinoline derivatives was carried out. After the synthesis process, antiproliferative effects of quinoline and dihydroquinoline derivatives were tested in vitro on breast cancer (MCF7), prostate cancer (PC3) and colon cancer (HT29) cell lines. DMSO-soluble substances were added to the cultured cells at a specific concentration range (0.4 mM - 200 mM) and incubated for 48 hours. In the mentioned analysis, the antiproliferative effect was determined by the MTT assay. From the results obtained from the test, IC50 values of the related compounds were calculated by GraphPad Prism 6.0. software. Results: As a result, it was found that methoxy dihydroquinoline and bromine dihydroquinoline were effective on the PC3, MCF7 and HT29 cell lines and decreased cell proliferation. These compounds were effective on all cell lines at 200 mM, whereas all administration doses (0.4 mM - 200 Mm) reduced only PC3 cell line proliferation. Conclusions: In conclusion, methoxy dihydroquinoline and bromine dihydroquinoline may be potentially useful as novel chemotherapeutic raw material candidates. Keywords: Quinoline derivatives, dihydroquinoline derivatives, anticancer activity. References: 1. Wang M, Lee RJ, Bi Y, Li L, Yan G, Lu J, Meng Q, Teng L, Xie J. Transferrin-conjugated liposomes loaded with novel dihydroquinoline derivatives as potential anticancer agents. (2017) PLoS ONE; 12(10):e0186821.

180

P-58

DETECTION OF THE INTERACTION SURFACE OF BAG-1S WITH RAD23B

Mert GÖLCÜKa, Umut GERLEVİKb,c, Gizem DİNLER DOĞANAYa aDepartment of Molecular Biology-Genetics & Biotechnology, Istanbul Technical University,

Istanbul, Turkey, bDepartment of Molecular Biology and Genetics, Istanbul Technical University,

Istanbul, Turkey cDepartment of Biostatistics and Medical Informatics, School of Medicine,

Acıbadem Mehmet Ali Aydınlar University, İstanbul, Turkey E-mail: golcuk@itu.edu.tr

Background: Bcl-2 associated athanogene-1 (BAG-1) is a protein can act as an adapter protein and has various roles in cell, including transcription regulation, hormonal activities, proteasomal degradation, apoptosis, cell proliferation, oncogenesis, cell motility and activation of multiple signaling pathways[1, 2]. UV excision repair protein RAD23 homolog B (RAD23B) is a protein in mainly proteostasis mechanism by acting as a shuttle between nucleotide excision repair and proteasomal degradation pathways responsible of carrying the ubiquitinated proteins to proteasome complex[3]. RAD23B is a multi-ubiquitin receptor, and BAG-1 has a ubiquitin-like (UBL) domain. Earlier experimental pulldown studies from our lab revealed that BAG-1 and RAD23B could interact with cellular stress in MCF-7 breast cancer cells. Interaction between these two proteins was observed in breast cancer cells whereas it was not in non-tumorigenic breast cells, indicating the interaction between BAG-1 and RAD23B could be crucial in breast cancer tumorigenesis. Therefore, it will be important to study in detail the interaction surface of these proteins to reveal possible sites for therapeutic intervention. Methods: Crystal structure of BAG-1S and RAD23B complex is not found in Protein Data Bank (PDB), so we used BAG-1S (PDB ID: 1wxv) and RAD23B (PDB ID: 1uel) crystal structures separately and studied the interaction surface using PRISM webserver[4, 5]. After continuing with the lowest energy scored model of PRISM, the crucial residues were detected by analyzing the interaction surface. Molecular Dynamics (MD) simulations with CHARMM36[6] forcefield were repeatedly applied to the model from PRISM webserver, called wild type (WT), and mutant (I82K) structure. All MD simulations were applied on NAMD[7] software, under NPT conditions while system preparations and all analyses were performed via VMD[8] software. Results: After all trajectories were observed by animating and analyzing by some common patterns in literature such as RMSD, RMSF, salt bridges and binding energies, WT kept its form stable through the trajectories whereas I82K mutant showed disruption in the interaction surface of BAG-1S and RAD23B as moving away from each other. Thus, Ile82 residue is probably vital. Conclusions: In this study, crucial residues in the interaction surface between RAD23B and BAG-1S were revealed to identify a putative site for therapeutic intervention. Knowledge about this interaction surface could allow a target when developing a drug against breast cancer.

181

Keywords: BAG-1, RAD23B, Molecular Dynamics, Breast Cancer, Interaction Surface References: 1. Chen, J., et al., Distinct BAG-1 isoforms have different anti-apoptotic functions in BAG-

1-transfected C33A human cervical carcinoma cell line. Oncogene, 2002. 21: p. 7050. 2. Cutress, R.I., et al., BAG-1 expression and function in human cancer. British Journal Of

Cancer, 2002. 87: p. 834. 3. Ahn, J.-M., et al., Proteogenomic Analysis of Human Chromosome 9-Encoded Genes

from Human Samples and Lung Cancer Tissues. Journal of Proteome Research, 2014. 13(1): p. 137-146.

4. Baspinar, A., et al., PRISM: a web server and repository for prediction of protein–protein interactions and modeling their 3D complexes. Nucleic Acids Research, 2014. 42(W1): p. W285-W289.

5. Tuncbag, N., et al., Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM. Nature Protocols, 2011. 6: p. 1341.

6. Huang, J. and A.D. MacKerell, CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data. Journal of Computational Chemistry, 2013. 34(25): p. 2135-2145.

7. Phillips, J.C., et al., Scalable molecular dynamics with NAMD. Journal of Computational Chemistry, 2005. 26(16): p. 1781-1802.

8. Humphrey, W., A. Dalke, and K. Schulten, VMD: Visual molecular dynamics. Journal of Molecular Graphics, 1996. 14(1): p. 33-38.

182

P-59

Synthesis, Characterization and Drug Delivery Applications of Photoresponsive Coumarin Functional Amphiphilic Copolymers

Merve Keklika, Binnur Aydogan Temela,b aDepartment of Biotechnology, Institute of Health Sciences, Bezmialem Vakif University, Fatih,

Istanbul 34093, Turkey; bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, Fatih, Istanbul 34093, Turkey

E-mail: merve.keklikk@gmail.com Background: Drug delivery systems are constantly evolving due to the need to minimize the side effects while increasing therapeutic activity. One of the most interesting drug delivery systems in recent times has been the self-assembly of stimuli-responsive amphiphilic block copolymers in aqueous solutions. Among them, the light-sensitive ones are of particular interest. These structures, which are capable of photo-reversible dimerization and are widely used in polymeric systems, generally proceed with a [4π + 4π] - or [2π + 2π] - cyclochemical mechanism which can be reversed by the application of light of a suitable wavelength.1-3 In this work, RAFT polymerisation technique and copper-catalyzed "click" reaction were used for the synthesis of photoresponsive coumarin functional amphiphilic block copolymers. Method: Poly(glycidyl methacrylate) polymer was synthesized by RAFT polymerization and it was then functionalized with coumarin groups via simple ring opening reaction and copper-catalyzed azide-alkyne cycloaddition reaction. Resulting coumarin functional polymer was used as a macro RAFT agent for the polymerization of poly(ethylene glycol) methacrylate. Final amphiphilic copolymers were used for preparation of polymeric micelles which were further loaded with DOX in order to examine their potential as drug carriers. All polymers were characterized by 1H NMR, FT-IR, DSC, UV-Vis and Fluorescence spectroscopy. Particle sizes of polymeric micelles were characterized using DLS and TEM measurements. Results: Coumarin functional amphiphilic copolymers were synthesized and their micelles were formed in water. 1H NMR, FT-IR, UV-Vis and fluorescence data clearly revealed the successful synthesis of all structures. A hydrophobic drug DOX was used to study encapsulation of guest molecules inside the hydrophobic core of (PGMA-g-Cum)-b-P(PEGMA) copolymer micelle. Photodimerization characteristics of coumarin pendants in polymer micelles were investigated by UV-Vis spectroscopy. Polymer micelles were irradiated at 365 nm and obtained UV spectra showed a clear decrease with time. Conclusions: (PGMA-g-Cum)-b-P(PEGMA) is a novel photoresponsive block copolymer that self-assembles into polymeric micelles in water. The micelles showed high loading capacity for DOX and drug release study was clearly demonstrated. The micelles were also efficiently crosslinked at 365 nm by dimerization of coumarin functional groups in the micellar core. Keywords: RAFT polymerization, click chemistry, amphiphlic copolymers, coumarin, drug delivery

183

Figure 1: Schematic illustration of the amphiphilic copolymer with coumarin pendants and formation of polymeric micelles. References:

1. Liu Y, Chang H, Jiang J, Yan X, Liu Z, Liu Z. The photodimerization characteristics of anthracene pendants within amphiphilic polymer micelles in aqueous solution. (2014) RSC Adv.; 4: 25912-25915.

2. Shi Y, Cardoso RM, van Nostrum CF, Hennink WE. Anthracene functionalized thermosensitive and UV-crosslinkable polymeric micelles. (2015) Polym. Chem.; 6: 2048-2053.

3. Jin Q, Maji S, Agarwal S. Novel amphiphilic, biodegradable, biocompatible, cross-linkable copolymers: synthesis, characterization and drug delivery applications. (2012) Polym. Chem.; 2, 2785-2793.

HO

OS

CN

SCH2(CH2)10CH3

Sn

O OOH

N3

OOOH

NNN

O

OO

m

OO

OH

k

kSelf-assembly

184

P-60

Synthesis of Biologically Active Novel Naproxen Derivatives Containing Urea

Özlem Beyazyüza, Merve Nur Tokgöza, Belma Zengin Kurtb, Fatih Sönmeza,c

aBezmialem Vakif University, Faculty of Pharmacy, Istanbul, Turkey bBezmialem Vakif University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,

Istanbul, Turkey cSakarya Uygulamalı Bilimler University, Pamukova Vocational High Scholl, Sakarya, Turkey

E-mail: mervenurtkgz@gmail.com

Background: Inflammation is a key factor in tumor growth and progress. In recent studies, it has been observed that cyclooxygenase-2 (COX-2) expression is strongly associated with different types of cancer [1]. A potent non-steroidal anti-inflammatory (NSAID) drug, commonly used in the treatment of acute and chronic inflammation, propanoic acid derivative Naproxen is involved in the production of prostaglandins that produce musculoskeletal disorders that cause pain, swelling, and inflammation. Studies have shown that specific COX-2 inhibitors inhibit colon cancer formation and growth of tumor tissue in many studies [2]. In this study, it is aimed to synthesize more active derivatives of naproxen which are propanoic acid derivative COX inhibitors. The resulting compounds are expected to exhibit anticancer properties by providing COX inhibition. Methods: Naproxen was mixed with phosphoroxychloride and thiosemicarbazides at 75 °C for 30min. The mixture was cooled and water was added to it. It was stirred at 100 °C for 4 hours. The mixture was cooled and neutralized with 50% NaOH. The precipitated product was filtered from the crucible and dried in a vacuum oven. It was crystallized in methanol. The obtained naproxen thiadiazole amine was reacted with different isocyanate derivatives to synthesize new urea derivatives of non-steroidal anti-inflammatory drugs containing propanoic acid. Results: New naproxen derivatives containing urea group were synthesized at two steps in high yield. All the new compounds were characterized by 1H NMR, 13C NMR, IR, and elemental analysis. Conclusions: The biological activity studies of the synthesized compounds are continuing. Keywords: Naproxen, urea, thiadiazole ring, NSAID.

185

Figure 1: Synthesis of naproxen urea derivatives.

References: 1. De Cicco P, Panza E, Ercolano G, Armogida C, Sessa G, Pirozzi G, Cirino G, Wallace JL,

Ianaro A., ATB-346, a novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo. (2016) Pharmacol. Research. 114, 67-73.

2. Reddy BS1, Hirose Y, Lubet R, Steele V, Kelloff G, Paulson S, Seibert K, Rao CV, Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. (2000) Cancer Research. 60(2), 293-297.

186

P-61

Comparison of allosteric pathway identification of CREB-binding protein using

computational methods

Metin Yazara,b, Pemra Ozbek Sarıcab aDepartment of Bioengineering, Marmara University, Goztepe, Istanbul, Turkey; b Department of

Genetics and Bioengineering, Istanbul Okan University, Tuzla, Istanbul, Turkey E-mail: metinyazar88@yahoo.com.tr

Background:

One of the key processes for biological molecules is allostery, which is transmission of a stimulus from one site to a physically distinct site.1 Detecting changes by allosteric signals can be complex as fluctuations in the protein can occur regularly, while signaling can shift this ensemble complicating the detection of allosteric mechanism from static snapshots2-3. Molecular dynamics (MD) simulations are valuable tools for the study of allostery4-5. CREB-binding protein (CBP) is a transcriptional co-activator and play crucial roles in some biological processes such as cellular differentiation, development and growth control6. CBP is a transcriptional scaffold which interacts with many transcription factors, so many protein binding domains are available in CBP that allosteric regulation happens. One such modular binding domain in CBP is the KIX domain, which is a small single-domain with bundle of three R helices and two short 310 helices7. The activation domain of mixed lineage leukemia (MLL) can bind to one site of KIX domain and binding can result positive allostery for another transcriptional factor that can also bind to KIX8-9. WISP is a program which is useful for detection of suboptimal pathways via protein structure network (PSN) using cross correlation matrix10. gRINN is a tool which is developed for the analysis of pairwise residue interaction energy data from protein MD simulation trajectories via protein energy network (PEN) using interaction energy matrix and could be an example application for investigation of allosteric pathway11. To compare existing allosteric pathways in literature which can be studied with experimentally, we aimed to employ both gRINN and WISP software to be able to find allosteric pathways in CBP when KIX is bound. Methods:

To investigate possible allosteric pathways of CBP, representative structure is downloaded from Protein Data Bank (PDB) 12 (PDB ID:2LXS)13. 100 ns parallel molecular dynamics simulations with/without MLL protein in the structure were carried out with NAMD 2.12 software14 with CHARMM36 force field15 at 300 K. In the next step, pairwise amino acid non-bonded interaction energies and possible shortest path identifications for allostery were employed via gRINN. Similarly, possible shortest paths were also identified with WISP.

187

Results:

Possible shortest paths that we found using gRINN and WISP software have similarity and agree with the allosteric pathways suggested experimentally in the literature13,16,17. Also, amino acid count and frequency in shortest path in both WISP and gRINN have significant similarities between key residues in allosteric pathways in the literature. Conclusions: Our study showed that possible allosteric paths can be identified via computational tools such as gRINN and WISP. Keywords: Allostery, allosteric pathway, molecular dynamics, CREB binding protein, protein structure network, protein energy network References: 1. Hertig S, Latorraca NR, Dror RO. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations. PLoS Comput Biol.; (2016):12(6). 2. Cui Q, Karplus M. Allostery and cooperativity revisited. Protein Science (2008).; 17:1295–307. 3. Tsai CJ, Nussinov R. A unified view of "how allostery works". PLoS Comput Biol. (2014); 10. 4. Swain JF, Gierasch LM. The changing landscape of protein allostery. Current Opinion in Structural Biology. (2006); 16:102–8. 5. Karplus M, Kuriyan J. Molecular dynamics and protein function. Proceedings of the National Academy of Sciences. (2005); 102:6679–85. 6. Goodman RH, Smolik S. CBP/p300 in cell growth, transformation, and development. Genes&Development. 2000. 14: 1553-1577. 7. De Guzman RN, Goto NK, Dyson HJ, Wright PEJ. Structural basis for cooperative transcription factor binding to the CBP coactivator. Mol.Biol. 2006, 355, 1005–1013. 8. Ernst P, Wang J, Huang M, Goodman RH. Korsmeyer, S. MLL and CREB Bind Cooperatively to the Nuclear Coactivator CREB-Binding Protein. J.Mol. Cell. Biol. 2001, 21, 2249–2258. 9. Parker D, Rivera M, Zor T, Henrion-Caude A, Radhakrishnan I, Kumar A, Shapiro LH, Wright PE, Montminy M, Brindle PK. Role of Secondary Structure in Discrimination between Constitutive and Inducible Activators. Mol. Cell. Biol. 1999, 19, 5601–5607. 10. Van Wart AT, Durrant J, Votapka L, Amaro RE. Weighted Implementation of Suboptimal Paths (WISP): An Optimized Algorithm and Tool for Dynamical Network Analysis. J. Chem. Theory Comput. (2014), 10, 511−517 11. Serçinoğlu O, Ozbek P. gRINN: a tool for calculation of residue interaction energies and protein energy network analysis of molecular dynamics simulations. Nucleic Acids Res. (2018).. 12. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. (2000) The Protein Data Bank. Nucleic Acids Research, 28: 235-242. 13. Brüschweiler S, Konrat R, Tollinger M. Allosteric communication in the KIX domain proceeds through dynamic repacking of the hydrophobic core. ACS Chem Biol. 2013 Jul 19;8(7):1600-10. 14. Phillips JC, Braun R, Wang W, et al. Scalable Molecular Dynamics with NAMD. Journal of computational chemistry. (2005);26(16):1781-1802. 15. Huang J, MacKerell AD. CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data. Journal of computational chemistry. (2013);34(25):2135-2145. 16. Goto NK, Zor T, Martinez-Yamout M, Dyson HJ, Wright PE. Cooperativity in transcription factor binding to the coactivator CREB-binding protein (CBP). The mixed lineage leukemia protein (MLL) activation domain binds to an allosteric site on the KIX domain. J Biol Chem. 2002 Nov 8;277(45):43168-74. 17. Palazzesi F, Barducci A, Tollinger M, Parrinello M. The allosteric communication pathways in KIX domain of CBP. Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):14237-42.

188

P-62

Comparative Assesment of Structural and Dynamical Properties of Transforming (H-RasG12D), Non-transforming (H-RasG12P) and Wild Type HRas Protein

Metehan ILTER a, Ozge Sensoya aIstanbul Medipol University, The School of Engineering and Natural Sciences, 34810, Istanbul, Turkey

E-mail:milter@st.medipol.edu.tr Background: Ras proteins are responsible for various cytoplasmic processes such as cell proliferation, differentiation, growth and survival1-5. Hence, mutants in this protein family result in the onset of cancer. However, G12P mutant preserves the intrinsic GTPase activity, thus not leading to any transformation in the cell, whereas G12D mutation, for instance, causes to disappear of the intrinsic GTPase activity6,7. Results obtained from this study might help locate novel regions on the protein surface which may be crucial for modulating transforming property of the RAS protein. Methods: In this study, atomistic molecular dynamics simulations were performed to investigate structural and dynamic properties of GDP/GTP-bound H-RasG12P, GDP/GTP-bound H-RasWT and GTP-bound H-RasG12D. Root-mean-square Fluctuation (RMSF) profiles were calculated to examine any differences in the dynamics of crucial residues, namely T35, G60, Q61 and Y32, among the systems studied. In addition to RMSF calculations, we also measured various distances between certain residue pairs to have an idea on the stability of the nucleotide binding pocket. Moreover, to have an insight more on the global dynamical properties of the systems Dynamic Cross Correlation Analysis (DCCA) and Principle Component Analysis (PCA) were made. Results: RMSF profiles reveal that Switch I (residues 30-38) and II (residues 59-76) regions are more flexible in H-RasG12D than in H-RasG12P and H-RasWT. However, Y32 fluctuates more in the H-RasG12P mutant. Distance distribution plots have shown that the nucleotide binding pocket is found in the closed conformation in H-RasG12P whereas it is found in a mixture of open and closed conformations in H-RasG12D and H-RasWT. Dynamic Cross Correlation Analyses have revealed that Switch I and Switch II pair together with Switch II and allosteric switch display anti-correlation with respect to each other’s motion in HRASG12D, whereas these two domain pairs display correlation in HRASG12P. Moreover, exchange of GDP with GTP reduces anti-correlation in all the systems studied. Lastly, PCA has shown that dominant contribution to the overall motion has come from Switch I and II in H-RasG12D than other two systems. Interestingly, in HRASG12P, Y32, which is responsible for intrinsic GTP-ase activity of the protein, has been also shown to dominantly contribute to the overall motion in HRASG12P 10. Conclusions: To the best of our knowledge, this is the first study where a comparative analysis of dynamic and structural properties of transforming, non-transforming mutant and wild type protein has been made. According to that, non-transforming mutant seems to represent a more catalytically competent state than the wild-type protein. Therefore, mimicking properties of non-transforming mutant might increase the success rate of therapeutic molecules which are used to modulate the transforming property of RAS proteins. Acknowledgement: This study was supported by TUBITAK 2209-A Undergraduate Research Support Program, project number: 1919B011701434. The numerical calculations were

189

partially performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources). Keywords: H-Ras, Non-transforming and transforming mutants, Molecular Dynamics Simulations, Essential Dynamics

References

1. Bryant, K. L., Mancias, J. D., Kimmelman, A. C., & Der, C. J. (2014). KRAS: Feeding pancreatic cancer proliferation. Trends in Biochemical Sciences. https://doi.org/10.1016/j.tibs.2013.12.004 2. Cox, A. D., & Der, C. J. (2010). Ras history. Small GTPases, 1(1), 2–27. https://doi.org/10.4161/sgtp.1.1.12178 3. Drosten, M., Dhawahir, A., Sum, E. Y. M., Urosevic, J., Lechuga, C. G., Esteban, L. M., … Barbacid, M. (2010). Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival. The EMBO Journal, 29(6), 1091–1104. https://doi.org/10.1038/emboj.2010.7 4. Pylayeva-Gupta, Y., Grabocka, E., & Bar-Sagi, D. (2011). RAS oncogenes: weaving a tumorigenic web. Nature Reviews Cancer, 11(11), 761–774. https://doi.org/10.1038/nrc3106 5. Wiesmüller, L., & Wittinghofer, F. (1994). Signal transduction pathways involving RAS. Cellular Signalling, 6(3), 247–267. https://doi.org/10.1016/0898-6568(94)90030-2 6. Franken, S. M., Scheidig, A. J., Krengel, U., Rensland, H., Lautwein, A., Geyer, M., … Wittinghofer, A. (1993). Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21H-ras. Biochemistry, 32, 8411–8420. https://doi.org/10.2210/PDB1AGP/PDB 7. Mishra, A. K., & Lambright, D. G. (2016). Invited review: Small GTPases and their GAPs. Biopolymers, 105(8), 431–448. https://doi.org/10.1002/bip.22833 8. Ahmadian, M. R.; Zor, T.; Vogt, D.; Kabsch, W.; Selinger, Z.; Wittinghofer, A.; Scheffzek, K. Proc Natl Acad Sci USA 1999, 96, 7065–7070

9. Gibbs, J. B., Schaber, M. S., Allard, W. J., Sigal, I. S., & Scolnick, E. E. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5026-5030. 10. Bunda, S., Heir, P., Srikumar, T., Cook, J. D., Burrell, K., Kano, Y., … Ohh, M. (2014). Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation. Proceedings of the National Academy of Sciences.

190

P-63

Synthesizing Graphene Oxide at Large-Scale via Modified Hummers' Method

as Drug Carrier Systems

Rumeysa Hilal Çelik1, Murat Kazanci 2*

1 Nanoscience and Nanoengineering Program, Natural Sciences Institute, Istanbul Medeniyet University, 34700 Istanbul Turkey

2 Biomedical Engineering Department, Faculty of Engineering and Natural Sciences, Istanbul Medeniyet University, 34700 Istanbul Turkey

E-mail:muratkazanci.tr@gmail.com

Background:

There are many studies aimed at the use of targeted drug or gene delivery systems, especially in cancer treatment studies [1]. In the last decade, one of the most popular of these structures have been graphene and their derivatives due to their extraordinary properties [2, 3]. Graphene, can be defined as a single layered molecule formed from sp2 hybridized carbon atoms in the 2D honeycomb lattice. The sp2 benzene annular structure provide to graphene and its derivatives elasticity, conductivity, high mechanical strength, high surface area and biocompatibility [4, 5]. Graphene oxide (GO) is the oxidized form of graphene and its derivatives are the most commonly used form among graphene types in biomedical applications due to the high degree of alcohol, carboxylic acid, and epoxide groups. These functional groups provide higher biocompatibility, water solubility, capability of biomolecules absorptions (such as proteins and genes) and functionalization by various molecules (such as polymers or markers). As a result of many studies which carried out using graphene and its derivatives, it has been found that this material is suitable for use in drug or gene carrier systems [1-3].

The aim of this study is synthesizing graphene oxide at large-scale via modified Hummers' method as drug carrier systems. Methods: GO was synthesized from a commercial synthetic graphite powder by using a modified Hummers and Offeman method [6, 7]. The ice bath was used throughout the experiment to prevent explosion due to temperature increase. Briefly, 1 gr graphite powder and 0.5 gr NaNO3 was placed in a flask at a 2:1 ratio. Under continuous stirring, concentrated sulphuric acid (H2SO4, 18 M, 23 mL) was slowly added to the flask. After stirring for 30 minutes, the potassium permanganate (KMnO4, 3 g) was added to the mixture in small portions to avoid a rapid increase in temperature (10 °C). The reaction temperature was raised to 35 ºC and the mixture was stirred for 30 minutes. After completion of the reaction, distilled water (46 mL) was slowly added to the solution. The mixture was kept at 98 °C for 15 minutes without boiling. The process was ended by adding 140 mL of distilled water and hydrogen peroxide (H2O2, 30%, 10 mL) to the suspension. The yellow-brown GO solution thus obtained was centrifuged at 10000 rpm for 15 minutes to remove the supernatant. The pellet was washed three times with diluted HCl (5%, 450 mL) and acetone before dried in vacuum.

191

Results: After vacuum drying, the sample was characterized by FT-IR. The obtained peaks in the FT-IR measurements display the graphene oxide fingerprints. The band approximately at 3600 cm-1 refers to the stretching of the hydroxyl group (-OH) from the C-OH vibration. The band at 3210 cm-1 refers to the hydroxyl group (-OH) stretching of the COOH group. The band at 1720 cm-1 refers to the C=O stretching vibration of the aldehyde groups. The band at 1622 cm-1

refers non-oxidized sp2 hybridization from C = C bonds of aromatic rings. The band at 1223cm-

1 refers to the C-O-C stretch of the epoxy groups [8].

Figure 1: Characteristic GO peaks obtained as a result of FT-IR analysis. Conclusions: The GO can be functionalized by linking several molecules to its molecular structure via π bonds through the alcohol, carboxylic acid and epoxy groups. For this reason, GO molecules have a great potential to use in targeted drug delivery systems. Many studies in the literature support this view. As a result, we concluded that the synthesis of GO by the modified Hummers' method is very suitable for industrialization and large-scale production. Keywords: Graphene oxide, drug delivery, targeting treatment, Modified Hummers' Method References:

1. Feng, L., L. Wu, and X. Qu, New horizons for diagnostics and therapeutic applications of graphene and graphene oxide. Adv Mater, 2013. 25(2): p. 168-86.

2. Nurunnabi, M., et al., Bioapplication of graphene oxide derivatives: drug/gene delivery, imaging, polymeric modification, toxicology, therapeutics and challenges. Rsc Advances, 2015. 5(52): p. 42141-42161.

3. Goenka, S., V. Sant, and S. Sant, Graphene-based nanomaterials for drug delivery and tissue engineering. J Control Release, 2014. 173: p. 75-88.

4. Novoselov, K.S., et al., Electric field effect in atomically thin carbon films. science, 2004. 306(5696): p. 666-669. 5. Geim, A.K. and K.S. Novoselov, The rise of graphene. Nature materials, 2007. 6(3): p. 183. 6. Hummers Jr, W.S. and R.E. Offeman, Preparation of graphitic oxide. Journal of the American Chemical Society, 1958.

80(6): p. 1339-1339. 7. Marcano, D.C., et al., Improved synthesis of graphene oxide. ACS nano, 2010. 4(8): p. 4806-4814. 8. Ţucureanu, V., A. Matei, and A.M. Avram, FTIR spectroscopy for carbon family study. Critical Reviews in Analytical

Chemistry, 2016. 46(6): p. 502-520.

192

P-64

The Fine Tuning of the Hedgehog and Autophagy Pathways to Combat Cholangiocarcinoma

Nihan Aktas, Mona El Khatib

E-mail: monaalkhatib.00@gmail.com Background: The American Cancer Society has estimated that cases in liver cancer have increased more than three times since 1980. Cholangiocarcinoma (CCA) is one of the most common liver cancer types with a 5-year survival rates ranging between 15% and 30% for cholangiocarcioma patients [1]. Aberrant signaling pathways could be a main driver in CCA pathogenesis. Hedgehog (Hh) Pathway is one of these pathways, which has role in tissue differentiation and stem cell maintenance [2, 3]. Aberrant activation of this pathway has been observed in several carcinomas including cholangiocarcinoma [3, 4]. Hh pathway is known to crosstalk and regulate autophagy, which is a lysosomal degradation process involved in the regulation of metabolic processes, organelle turnover and cellular homeostasis [5-9]. Based on this crosstalk between Hh pathway and autophagy and aberrant activation of Hh pathway observed in cholangiocarcinoma, we are planning to understand the effects of combination therapy using Hh and autophagy inhibition on cholangiocarcioma progression. Methods: Accordingly, we performed MTT cell proliferation assay on treated EGI-1 and TFK-1 cholangiocarcinoma cell lines with chloroquine, ammonium chloride, hydroxychloroquine and GANT61 in order to check the effects of autophagy and Hh inhibiton on cholangiocarcinoma proliferation. Using IC50 and IC20 values, we treated TFK-1 cells with chloroquine, hydroxychloroquine and ammonium chloride, EGI-1 cells with chloroquine and checked percentage of apoptotic cells/control. Results: Proliferation of TFK-1 and EGI-1 cell lines decreased after 24h, 48h and 72h treatment with autophagy inhibitors ammonium chloride, chloroquine, hydroxychlorquine and Hh inhibitor, GANT61. We observed a 30% decrease in the proliferation of TFK-1 cells after hydroxychloroquine, chloroquine and ammonium chloride treatment and a 90% decrease in the proliferation of GANT61 treated TFK-1. Hydroxychloroquine and chloroquine treatment decreased cell proliferation to 10% and we observed 70% and more than 90% dead cells after ammonium chloride and GANT61 treatment, respectively. Percentage of apoptotic cells/control increased after 48h chloroquine, hydroxychloroquine and ammonium chloride treatment. We observed a 1.5X, 2.5X and 3.2X fold-increase in the apoptotic cell percentage compared to control after chloroquine, hydroxychloroquine and ammonium chloride, respectively and almost 420% of apoptotic cells/control in chloroquine treated EGI-1 cells. Conclusion: Although Hh pathway has been known to be deregulated in CCA patients, targeting Hh pathway alone might not be effective to combat CCA because of the complexity of the disease. Considering the crosstalk between Hh and autophagy pathways, we are planning to continue with combination therapy using our obtained data to reveal the effects of autophagy activation upon Hh inhibition on progression of CCA cells.

193

Keywords: cholangiocarcinoma, autophagy, Hedgehog pathway, combination therapy References:

1. www.cancer.org 2. Cilloni, D., Saglio, G. 2012. “Molecular Pathways: BCR-ABL”, Clinical Cancer Research,

18, 930-7. 3. Zuo, M., Rashid, A., Churi, C., Vauthey J., N., Chang, P., Li, Y., Hung, M., C., Li, D., Javle,

M. 2015. “Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer”. British Journal of Cancer, 112, 1042–1051.

4. El Khatib, M., Kalnytska, A., Palagani, V., Kossatz, U., Manns, M., P., Malek, N., P., Wilkens, L,. Plentz, R., R. 2103. “Inhibition of Hedgehog Signaling Attenuates Carcinogenesis InVitro and Increases Necrosis of Cholangiocellular Carcinoma”, Hepatology. 57, 1035-1045.

5. Xu, Y., ANnY., Wang, X., Zha, W., Li, X. 2014. “Inhibition of the Hedgehog pathway induces autophagy in pancreatic ductal adenocarcinoma cells”, Oncology Reports, 31, 707-712.

6. Jimenez-Sanchez, M., Menzies, F., M., Chang, Y., Y., Simecek, N., Neufeld, T., P.,. Rubinsztein, D., C. 2012. “The Hedgehog signalling pathway regulates autophagy”, Nature Communications, 3,1200.

7. Evangelisti, C., Evangelisti, C., Chiarini, F., Lonetti, A., Buontempo, F., Neri, L.M., McCubrey, J.A., Martelli, A.M. 2015. “Autophagy in acute leukemias: A double-edged sword with important therapeutic implications”, Biochimica et Biophysica Acta, 1853, 14-26.

8. Altman, J., K., Szilard, A., Goussetis, D., J., Sassano, A., Colamonici, M, Gounaris, E., Frankfurt, O., Giles, F., J., Eklund, E., A., Beauchamp E., M., Platanias, L., C. 2014. “Autophagy Is a Survival Mechanism of Acute Myelogenous Leukemia Precursors during Dual mTORC2/mTORC1 Targeting”, Clinical Cancer Research, 20, 2400-9.

9. Martelli, A., M., Chiarini, F., Evangelisti, C., Cappellini, A., Buontempo, F., Bressanin, D., Fini, M., McCubrey, J., A. 2012. “Two hits are better than one: targeting both phosphatidylinositol 3-kinase and mammalian target of rapamycin as a therapeutic strategy for acute leukemia treatment”, Oncotarget, 3, 371-94.

194

P-65

Anti-apoptotic Bag-1 Isoforms are involved in Endoplasmic Reticulum Associated Degradation

Nisan Denizce Cana, Tuğba Kızılboğa Akgüna, Baran Dingiloğlua, Serena Muratçıoğlub, Efe Elbeylib, Özlem Keskinb, Gizem Dinler Doğanaya

aIstanbul Technical University; bKoç University E-mail: nisandc@gmail.com

Background: Bag-1 is an anti-apoptotic adaptor protein, which involves in the regulation of transcription, cell proliferation, tumorigenesis, apoptosis, and several cellular signaling pathways via interacting with heat shock proteins, E3 ligases, nuclear hormone receptors, Raf-1 and Bcl-2. Bag-1 enhances cell survival and has high expression levels in many types of cancer, especially in breast cancer. In human cells, Bag-1 has three major isoforms as Bag-1S, Bag-1M and Bag-1L, which are derived from alternative translation initiation of a single mRNA transcript. Bag-1 isoforms have different cellular localizations and may also have different interacting partners. In this study, we aimed to detect interacting partners of each isoform to determine their role in breast cancer. Methods: Bag-1 isoforms with an N-terminal TAP-tag were cloned in an expression vector and further used for the transfection of MCF-7 breast cancer cells. After successful overexpression of Bag-1 isoforms with their interacting proteins, each isoform was purified as complexes from the cells. Purified interactomes were digested to their peptides and then peptide mapping analyses were performed using LC-MS/MS (Waters Synapt G2-Si HDMS). Further identified proteins in the complexes were confirmed by immunoblotting and PRISM, a protein-protein interaction prediction tool was used to map the interaction surfaces of proteins in complexes. Results: Interacting partners of Bag-1S, Bag-1M and Bag-1L were identified by LC-MS/MS analyses. Among all identified proteins, some heat shock proteins, endoplasmic reticulum resident chaperones, lectins, protein disulfide isomerases, ubiquitin ligases, and transitional endoplasmic reticulum ATPase found as common interacting partners for all Bag-1 isoforms and those mainly participate in endoplasmic reticulum-associated protein processing. Immunoblotting assays confirmed those interactions for all isoforms and protein-protein interaction predictions showed the possibility of existence of a large complex near the endoplasmic reticulum that formed by Bag-1 isoforms via BAG domain and UbL (ubiquitin-like) domain. Conclusions: Considering the mechanisms that Bag-1 is involved, known partners and newly identified interacting proteins whose functions are well known in protein homeostasis pointed out the role of Bag-1 in endoplasmic reticulum associated degradation (ERAD). Prediction of interaction surfaces suggested that Bag-1 is capable of interacting with identified proteins simultaneously and forms a large complex that functions in the degradation of unfolded proteins and locates between endoplasmic reticulum and proteasome.

195

Keywords: Bag-1, breast cancer, protein-protein interaction, mass spectrometry, endoplasmic reticulum This work is supported through TUBITAK 115Z169 grant and ITU internal funds.

Figure 1: Working model of the complex formed by Bag-1 in ERAD mechanism.

References:

Takayama, S., & Reed, J. C. Molecular chaperone targeting and regulation by BAG family proteins. (2001) Nature cell biology; 3(10), E237-E241. Kabbage, M., &Dickman, M. B. The BAG proteins: a ubiquitous family of chaperone regulators. (2008) Cellular and Molecular Life Sciences; 65(9), 1390-1402. Lüders, J., Demand, J., & Höhfeld, J. The ubiquitin-related BAG-1 provides a link between the molecular chaperones Hsc70/Hsp70 and the proteasome. (2001) Journal of Biological Chemistry; 275(7), 4613-4617.

196

P-66

Targeting long non-coding RNAs (lncRNAs) to overcome tamoxifen resistance in Estrogen Receptor alpha (ERα) positive breast cancer

Oguzhan Tarmana, Nevin Beldera, Hilal Bala, Yasser Riazalhosseinib, Ozgur Sahina, c

a Bilkent University, Department of Molecular Biology and Genetics, Ankara, TURKEY; b McGill University, Department of Human Genetics, Montreal, CANADA; c University of South Carolina,

Department of Drug Discovery and Biomedical Sciences, Columbia, SC, USA. E-mail: oguzhan.tarman@bilkent.edu.tr

Estrogen receptor alpha (ERα)-positive breast cancer (BC) is the most common BC subtype treated by hormone therapy agents including tamoxifen. Although tamoxifen has significantly improved patient survival, more than 40% of patients are de novo resistant or develop acquired resistance leading to recurrence and mortality [1]. Tamoxifen resistance has been associated with activation of several receptor tyrosine kinases, such as EGFR, HER2, FGFR1, and IGF-1R [2, 3]. Recently, we have shown that targeting Phosphodiesterase 4D (PDE4D) overcomes tamoxifen resistance by inducing cAMP/ER stress/JNK/p38/apoptosis axis [4]. Furthermore, deregulation of a class of non-coding RNAs, microRNAs, e.g., miR-375, miR-519 and miR-221, were shown to be important modulators of tamoxifen resistance by us and others [5-7]. Long noncoding RNAs (lncRNAs), another class of non-coding RNAs, play important roles in cancer pathophysiology via their involvement in cellular mechanisms, such as sequence-specific chromosome modifications, gene expression regulation and signaling pathway modulations; however, little is known about their functions in tamoxifen resistance. Therefore, we aimed at identifying lncRNAs involved in tamoxifen resistance and their underlying molecular mechanisms. In this line, we have developed and published two different, clinically relevant isogenic lines of tamoxifen resistance models using MCF7 and T47D human BC cell lines (referred TamR). To identify differentially expressed lncRNAs and mRNAs between tamoxifen resistant and sensitive cells, we performed transcriptome analysis using RNA-Seq. In accordance with RNA-Seq results, upregulation of our candidate lincRNA (c-linc) was validated in tamoxifen resistant cells and xenografts via qRT-PCR. We observed an increase in tamoxifen sensitivity in resistant cells upon siRNA, shRNA or CRISPR-Cas9-mediated knockdown of c-linc. Moreover, we found that high c-linc expression predicted worse relapse-free and disease-free survival in tamoxifen-treated ERα-positive breast cancer patients. Importantly, localization of lncRNAs dictates their mechanism of action, therefore we examined the localization of c-linc and found it to be mostly localized to cytoplasm. Cytoplasmic lincRNAs are known to interact with proteins or acts as miRNA sponges. To identify interacting proteins and miRNA partners, we are currently performing lincRNA pull-down followed by mass spectrometry and RNA-Seq/miR-Seq on c-linc knockdown samples, respectively. Sensitizer role of c-linc will further be tested using nanoparticles with antisense oligonucleotides (ASOs) against c-linc in combination with tamoxifen on ER+, tamoxifen resistant patient-derived xenografts (PDXs). Prognostic potential of c-linc will be validated using ISH of lincRNA on FFPE tissues of ERα positive BC patients. In conclusion, we believe that targeting c-linc is a promising alternative strategy for tamoxifen sensitization, and c-linc could be an independent prognostic factor for the outcome of tamoxifen-treated, ERα positive BC patients. Considering that RNA-based first therapy is

197

approved by FDA recently, targeting functionally deregulated lincRNAs might be a useful strategy for cancer treatment in future. Keywords: ERα-positive breast cancer, long non-coding RNAs (lncRNAs), tamoxifen resistance, prognostic biomarker, nanoparticles Acknowledgement: This project was supported by TUBITAK grant number 215Z357. References: 1. Davies, C., et al., Long-term effects of continuing adjuvant tamoxifen to 10 years versus stopping at 5 years after diagnosis of oestrogen receptor-positive breast cancer: ATLAS, a randomised trial. Lancet, 2013. 381(9869): p. 805-16. 2. Gutierrez, M.C., et al., Molecular changes in tamoxifen-resistant breast cancer: relationship between estrogen receptor, HER-2, and p38 mitogen-activated protein kinase. J Clin Oncol, 2005. 23(11): p. 2469-76. 3. Kirkegaard, T., et al., AKT activation predicts outcome in breast cancer patients treated with tamoxifen. J Pathol, 2005. 207(2): p. 139-46. 4. Mishra, R.R., et al., Reactivation of cAMP Pathway by PDE4D Inhibition Represents a Novel Druggable Axis for Overcoming Tamoxifen Resistance in ER-positive Breast Cancer. Clin Cancer Res, 2018. 24(8): p. 1987-2001. 5. Ward, A., et al., Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer. Oncogene, 2013. 32(9): p. 1173-82. 6. Ward, A., et al., MicroRNA-519a is a novel oncomir conferring tamoxifen resistance by targeting a network of tumour-suppressor genes in ER+ breast cancer. J Pathol, 2014. 233(4): p. 368-79. 7. Miller, T.E., et al., MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1. J Biol Chem, 2008. 283(44): p. 29897-903.

198

P-67

Biophysical Links Between Polymorphism, Stability and Peptide- Binding in the Major Histocompatibility Complex

Onur Serçinoğlu, Pemra Ozbek Department of Bioengineering, Marmara University, Goztepe, Istanbul, Turkey

E-mail: onursercin@gmail.com Background: Evolutionary rates in protein structures vary from site to site. They are influenced by structural and functional constraints: residues that are less important for maintaining the fold or function of the protein tend to evolve faster than more important ones1. In this context, the human peptide-loaded Major Histocompatibility Complex (pMHC) is an interesting system. The complex is a trimer, consisting of a Human Leukocyte Antigen (HLA) chain, b-2-m protein and a peptide. It presents peptides from pathogens to cytotoxic T-cells, hence it evolves to maintain a wide array of peptide repertoire, resulting in a high number of alleles. On the other hand, experimentally elucidated structures of different alleles with different peptide ligands display an “ultra-conserved” structural fold2. Moreover, it is known that peptide ligands dramatically influence complex stability as well. Here, we aim to identify the biophysical basis of this conserved structural fold as well as peptide-induced stability using computational molecular biophysics and bioinformatics methods. Methods: We quantified the conservation of each position in the peptide binding groove using Evolutionary Trace3 (ET) analysis on 9869 HLA allele sequences from the IMGT/HLA database. Then, we modeled the structures of 1111 alleles that have the whole HLA chain sequence identified previously in complex with 10 high affinity peptide ligands as predicted by netMHCpan 3.0. Finally, we analysed local frustration in the structure using the frustratometer24 algorithm on produced homology models. Results: Minimally frustrated positions in MHC binding groove are predominantly conserved whereas non-conserved binding pocket positions show neutral frustration (Figure 1). This finding establishes a direct link between polymorphism and structural stability. Moreover, peptide ligands stabilize the F-pocket in the binding groove, which has been reported recently in MD simulations5. Conclusions: Our findings clearly indicate that the human MHC maintains its observed fold by avoiding high levels of polymorphism at minimally frustrated positions (structural constraint) while allowing for variation to occur at other positions. Keywords: major histocompatibility complex, human leukocyte antigens, protein biophysics, protein evolution, peptide-protein interaction

199

Figure 1: (A) real-value Evolutionary Trace (rvET) versus (B) Single Residue Frustration Index (SRFI) on the peptide binding groove. References: 1. Echave J, Spielman SJ, Wilke CO. Causes of evolutionary rate variation among protein sites. (2016) Nat Rev Genet. ;17(2):109-121.. 2. Wieczorek M, Abualrous ET, Sticht J, et al. Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation. (2017) Front Immunol.;8:292. doi:10.3389/fimmu.2017.00292. 3. Wilkins A, Erdin S, Lua R, Lichtarge O. Evolutionary trace for prediction and redesign of protein functional sites. (2012) Methods Mol Biol. ;819:29-42. 4. Parra RG, Schafer NP, Radusky LG, et al. Protein Frustratometer 2: a tool to localize energetic frustration in protein molecules, now with electrostatics. (2016) Nucleic Acids Res. 2016;44(W1):W356-W360. 5. Fisette O, Wingbermühle S, Schäfer L V. Partial Dissociation of Truncated Peptides Influences the Structural Dynamics of the MHCI Binding Groove. (2017) Front Immunol. 2017;8:408.

200

P-68

Anti-proliferation Activity of Novel Heterocyclic Compounds in Tumor Cell Lines and Their Flow Cytometric Analysis

Ömer Tahir Günkaraa, Nüket Öcala, Fulya Günayb, Yuk Yin (Peter) Ngb aChemistry Department, Faculty of Science and Arts, Yildiz Technical University,

Davutpasa Campus, Istanbul, Turkey; bIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Department of Genetics and

Bioengineering, Santralistanbul Campus, 34060, Eyüp, Istanbul, Turkey E-mail: omerrrgunkara@hotmail.com

Background:

Cancer treatment has been a major endeavor of research and development in academia and pharmaceutical industry for the last many years as it is one of the leading causes of death. Many of the available anticancer agents exhibit undesirable side effects such as reduced bioavailability, toxicity and drug-resistance. Therefore, the search for novel and selective anticancer agents is urgently required due to problems associated with currently available anticancer drugs. Because of their biological activities, five- and six-membered heterocyclic compounds such as piperidine, pyridine, piperazine, oxazole, thiazolidine and pyrrole have become an important subject for pharmaceutical chemists from the beginning of the 20th century. Methods:

In this study, it was aimed to detect the anti-proliferation activities of novel Tandospirone analogues in various tumor cell lines by the MTT cell proliferation assay. HeLa, PANC1, REH, Nalm6, Jurkat, K562 and Molt4 cells were plated in 96-wells plate with various concentration of Tandospirone analogues. After 24h, MTT were added and incubated for 4 h. Finally, DMSO were added and the absorbance at 540 nm was determined. Triplicate wells were assayed for each condition. Growth Inhibition (GI) value was calculated from the average of the obtained results according to the formula: %GI: [OD(control)-OD(sample)]/ OD(control)*100 IC50 value was calculated and the dose was used for the apoptotic activity assay. The cells were exposed to the appropriate compound overnight. After that, the cells were harvested and stained with AnnexinV/PI according to the manufacturer’s protocol and analyzed by flow cytometry. Results:

We demonstrated that several novel compounds of the tandospirone analogues were able to inhibit the growth in various cancer cell lines. Among them, compound 4 and 6 showed high growth inhibitory activity, 60.3% and 82.5% respectively, while compound 15 showed high growth inhibition in Nalm6 (58.6%) and K562 (66.0%) cell lines. We observed a high percentage of AnnexinV/PI positive in all cell lines after incubation with the drugs. In the K562

201

and Molt-4 cells, high apoptosis was seen after the incubation with all compound 4 and 6. These results suggest that these compounds can induce apoptosis in cancer cells. Conclusions: To our knowledge, this is the first in vitro study describing the anti-cancer activity of anti-depressive reagent analogues in cancer cell lines. We conclude that the novel tandospirone analogues may represent a new group of anti-proliferative agents for the treatment of cancer. Keywords: Anti-cancer agent, Flow cytometry test, MTT test, Tandospirone analogues.

Figure 1: Some of the synthesized heterocyclic compounds as an anti-cancer agent.

References:

1. Kossakowski J, Jarocka M. Synthesis of new N-substituted cyclic imides with an expected anxiolytic activity. XVII. Derivatives of 1-ethoxybicyclo[2.2.2]-oct-5-one-2,3-dicarboximide. (2001) IL Farmaco; 56: 785-789.

202

P-69

Investigating the molecular mechanisms of paclitaxel resistance through

PI3K/Akt ve EMT signaling pathways in MCF-7 breast cancer

Özlem Sönmez, Pelin Özfiliz Kılbaş, Elif Damla Arısan, Ajda Çoker Gürkan, Pınar Obakan Yerlikaya, Narçin Palavan Unsal

Istanbul Kültür University, Dept of Molecular Biology and Genetics, Atakoy Campus 34156 Istanbul E-mail: sonmez_ozlem_@hotmail.com

Background:

Chemotherapy is widely used for cancer treatment, but common drug resistance phenomenon renders potential therapeutic success. Paclitaxel (PTX) resistance represents a challenge in breast cancer treatment. Therefore, the investigation of the resistance mechanism developed against chemotherapeutic drugs, which are frequently used in breast cancer, is the basic step for understanding the mechanisms of drug resistance and developing new treatment methods. In this context, phosphatidylinositol-3-kinase (PI3K)/Akt and epithelial mesenchymal transition (EMT) signaling pathways, which trigger metastasis and drug resistance mechanism through downstream effectors (1). Methods:

To generate PTX-resistant cells, parental MCF-7 breast cancer cells were exposed to PTX in dose (5-100 nM) and time (1-75 days)-dependent manner. 100 nM PTX concentration was used for further drug-resistance experiments. To investigate the viability and growth potential of cells, growth assay, colony forming assay were performed. Morphological changes were observed by light and fluorescent microscopy. The effect of PTX-resistance on PI3K/Akt and EMT pathways, immunoblotting assay were performed.

203

Figure 1. Generating PTX-resistant MCF-7 breast cancer cells. a) Schema for development of PTX-resistant cells in parental MCF-7 cells in dose-dependent manner (5-100 nM). b) Morphological changes of untreated wild type and PTX-resistant MCF-7 cells against 100 nM at day 75 sub-culturing. Results:

The Multidrug Resistance Protein (MDR) expression was upregulated in MCF-7 breast cancer cells, which were exposed to PTX in dose-dependent manner (5-100 nM) for 75 days compared to parental cells. PTX-resistance caused the transition of cells from epithelial to mesenchymal characteristic morphologically (Figure 1). Also, PTX-resistant cells showed higher colony formation potential compared to parental MCF-7 breast cancer cells and we confirmed the increased survival ratio with upregulation of phospho- Akt (Ser 473) protein levels. As one of the EMT markers, we investigated the downregulation of E-cadherin level in PTX-resistant MCF-7 cells which is associated with the mesenchymal transition of cells. Conclusions:

Our preliminary findings suggested that changes in the expression levels of mesenchymal biomarkers lead to understand the potential role of PTX resistance in breast cancer survival and progression (This project is supported by TUBİTAK 2209A-2017/2-1919B011703621). Keywords: breast cancer, paclitaxel-resistance, PI3K/Akt, EMT singnaling pathways References:

1. Wang, Y. and B.P. Zhou, Epithelial-mesenchymal Transition.

204

P-70

Investigation the relationship between cholesterol metabolism and cancer through miR-33 in estrogen receptor targeting therapies in breast cancer cells Pelin Özfiliz Kılbaş, Özlem Sönmez, Elif Damla Arısan, Ajda Çoker Gürkan, Pınar Obakan Yerlikaya, Narçin Palavan Unsal Istanbul Kültür University, Dept of Molecular Biology and Genetics, Atakoy Campus 34156 Istanbul e-mail: p.ozfiliz@iku.edu.tr Background: The development of effective diagnostic methods gains importance in aggressive breast cancer patients to perform appropriate treatment strategies. Obesity linked lipid and cholesterol irregular metabolism takes part among the risk factors that increase the metastatic properties and aggressiveness of estrogen receptor (ER) positive breast cancer. Therefore, elucidating the network of lipid metabolism, cholesterol level and breast cancer may significantly contribute the clinical and therapeutic applications of breast cancer outcome. In order to clarify of these issues, designed biomarkers are used to enlighten the role of breast cancer risk factors as the potential targets in the diagnosis of breast cancer. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that have been used as biomarkers in the diagnosis of cancer. Alternations of miRNA levels, depending on the tissue specify and the target genes, are primary detectable points on the diagnosis of cancer. Previous studies showed that, miR-33 is a significant molecule in targeting cholesterol and lipid metabolism in cancer. miR-33 is downregulated in metastatic breast cancer patients and knockdown of this microRNA impairs the development of breast cancer (1). Methods: To investigate the network between cholesterol metabolism and cancer through targeting miR-33 in ER+ MCF-7 and ER- MDA-MB-231 breast cancer cells, 17-�-Estradiol (activation of estrogen) and Fulvestrant (inhibition of estrogen) treatments were performed in dose and time-dependent manner. The viability, colony forming and growth potentials of cells against 17-�-Estradiol and Fulvestrant were investigated by MTT, colony formation assay and growth assay. The expression level of miR-33 was analyzed by quantitative PCR assay in the treatment of 17-�-Estradiol and Fulvestrant in both MCF-7 and MDA-MB-231 breast cancer cells. Results: The exact role of miR-33 on cell survival, growth, proliferation and colony formation potentials in MCF-7 cells was determined according to the estrogen levels. MCF-7 and MDAMB-231 cells, which differs in estrogen receptor status, showed different cell viability, growth and colony forming potentials in the treatment of 17-�-Estradiol and Fulvestrant. The expression level of miR-33 significantly upregulated in MCF-7 cells against Fulvestrant treatment (100 nM), however MDA-MB-231 cells showed a slight upregulation in miR-33 expression level. We also investigated the expression levels of Fatty Acid Synthase and Acetly-CoA Carboxylase, fatty acid and lipid metabolism signaling pathway markers, and found that MCF-7 and MDA-MB-231 cells showed different expression patterns of these important markers associated with different estrogen levels related with miR-33 expression. Conclusions: miR-33 might be a potential diagnostic and therapeutic marker to contribute the link between cholesterol metabolism and enhanced ER+ breast cancer development

205

considering the mechanistic effect on breast cancer cell survival and cell death decision provided from this project (This project is supported by SIE Scholarships Grants 2017 -2018). Keywords: Breast cancer, cholesterol metabolism, obesity, adipogenesis, miRNA References: 1. Gerin, I., et al., Expression of miR-33 from an SREBP2 intron inhibits cholesterol export and fatty acid oxidation. J Biol Chem, 2010. 285(44): p. 33652-61.

206

P-71

Epibrassinolide induces apoptosis in MIA PaCa-2 and PANC-1 pancratic cancer

cells via activating endoplasmic reticulum stress

Pınar Obakan-Yerlikaya, Sedef Çevikli*, Şevval Özağar*, Elif Damla Arısan, Ajda Coker-Gurkan

*equal contribution Istanbul Kultur University, Dept. of Molecular Biology and Genetics, 34156, Bakirkoy-Istanbul

E-mail: p.obakan@iku.edu.tr Background:

Epibrassinolide (EBR) with a similar polyhydroxysteroid structure of steroid hormones in vertebrates, is a member of brassinosteroids (BR) from plant growth regulators. Our recent studies indicated that EBR induces apoptotic cell death in a p53-independent way in cancer cells, but not in normal epithelial cells such as fetal human colon (FHC) or mouse embryonic fibroblasts (MEF). Our previous study on the proteomic alterions upon EBR treatment revealed that EBR acts on one of the ER lumen-resident proteins, calreticulin (CALR), a chaperone localized in endoplasmic reticulum lumen which has role in protein folding. The incidence and mortality rates of pancreatic cancer are close due to its aggressive type. It is difficult to diagnose and usually resistant to chemotherapeutic agents. In the development of pancreatic cancer, mutations in tumor suppressor genes such as K-Ras, CDKN2A / p16, p53 and SMAD4 are usually the causative factors. We aimed to understand the EBR-induced apoptosis by evaluating ER stress and UPR molecular signatures in different pancreas cancer cell lines. Methods:

The effect of EBR on tumor formation was determined with colony formation and scratching assays. The apoptotic effect of EBR was determined by FACS flow following annexin V-PI staining, while the apoptotic protein and ER stress marker expressions were shown with immunoblotting. Results:

EBR induced apoptosis in 1 and 10 μM concentrations and diminished tumor formation. The wound healing was decreased following exposure of EBR to both MIA PaCa-2 and PANC-1 cells. EBR treatment was also able to trigger ER stress which was shown with CALR and ER stress biomarkers alterations such as IRE1α, PERK and ATF6. Conclusions:

EBR is a potential apoptotic agent for pancreatic cell lines via ER stress induction. Keywords: Pancreatic cancer, epibrassinolide, apoptosis, calreticulin, endoplasmic reticulum stress

207

P-72

Synthesis, Structure, DNA Interaction Studies and Cytotoxic Activities of

Nickel(II) Complexes Containing Schiff Base Ligands

Ramazan Güpa, Cansu Topkayab, Esin Sakallı Çetinc aDepartment of Chemistry, Muğla Sıtkı Koçman University, Muğla, Turkey; bCenter of Research

Laboratories, Muğla Sıtkı Koçman University, Muğla, Turkey; cDepartment of Medical Biology, Muğla Sıtkı Koçman University, Muğla, Turkey

E-mail: rgup@mu.edu.tr

Background:

Metal complexes, in particular transition metal complexes, offer potential for unique biochemistry character based on their accessible redox states as well as their wide range of coordination numbers and geometries [1]. In recent years, interactions of transition metal complexes with biological molecules such as DNA have been intensively studied [2]. Many studies have justified that DNA is the primary intracellular target of anticancer drugs owing to the interaction between small molecules and DNA, which can cause damage in cancer cells, blocking the division and resulting in cell death [3]. Methods: In the present study, two new Ni(II) complexes of Schiff base ligand of p-subtitue isonitrosoacetophenone (-H, -CI) and 1,3-diaminopropan were synthesized and characterized by structural, analytical, and spectral methods such as 1H-NMR, 13C-NMR, FT-IR and elemental analysis. DNA interactions of Ni(II) complexes have been investigated using CT-DNA and pBR322 with UV-titration and gel electrophoresis methods, respectively. DNA cleavage abilities of the compounds were analyzed by monitoring the conversion of the supercoiled form to nicked circular form and linear form of pBR322. The binding ability of compounds to CT-DNA is performed by using UV-titration method. Briefly, a compound binding with DNA through intercalation usually results in hypochromism and bathochromism of the absorption band and the absorption intensity of the ligand is increased (hyperchromism) upon increasing the concentration of CT-DNA due to a damage of the CT-DNA structure. In vitro cytotoxicities of the complexes were studied using standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) bioassay in human breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative). Results: In vitro assay results show that Ni (II) complexes of Schiff base ligands synthesized in this study have considerable DNA binding and cleaving activities. Further, the in vitro cytotoxic effect of the complexes examined on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that both two complexes exhibited substantial cytotoxic activity.

208

Conclusions: The observed data revealed that these compounds could be used as potent antitumor drugs after further analysis and screening.

Keywords: Anticancer, DNA cleavage, DNA binding, Cytotoxicity, MTT * This study is supported by TUBITAK (The Scientific and Technological Research Council of Turkey) with the project number 215Z236

References:

1. Deng J, Su G, Chen P, Du Y, Gou Y, Liu Y. Evaluation of DNA binding and DNA cleavage of nickel (II) complexes with tridentate α-N-heterocyclic thiosemicarbazones ligands. (2018) Inorg Chim Acta.: 471:194-202.

2. Barone G, Terenzi A, Lauria A, Almerico AM, Leal JM, Busto N, Garcia B. DNA-binding of nickel(II), copper(II) and zinc(II) complexes: Structure–affinity relationships. (2013) Coordin Chem Rev.; 257(19-20):2848-2862.

3. Zuber G, Quada Jr JC, Hecht SM. Sequence Selective Cleavage of a DNA Octanucleotide by Chlorinated Bithiazoles and Bleomycins. (1998) J Am Chem Soc.: 120:9368-9369.

209

P-73

Protein Networks: Development of a web tool to analyze protein

structural topology

Rasim Murat Aydinkal, Onur Sercinoglu, Pemra Ozbek Sarica Department of Bioengineering, Marmara University, Goztepe, Istanbul, Turkey

E-mail: a.rasimmurat@gmail.com

Background:

To identify the functions and dynamics of biological macromolecules, the visualization of topological features of protein networks on 3D structure is crucial. Studies on investigation of protein networks have become important to analyse structural features of proteins. Furthermore, there is still a need for useful tools that can visualize and analyze the network under the modern data analysis environments. Features of current applications are very narrow with fundamental analysis options. The main objective of this study is to develop a web tool to visualize and analyze the protein structure networks where the edge weights are defined by custom network metrics. Methods: Protein structure data is the starting point of network construction. This tool supports output of a model structure obtained from any molecular modelling program in PDB format, or the required data can be downloaded from the Protein Data Bank (1). Nodes are added with their 3D coordinates of the representative atoms in each network type using the information in the PDB file, to analyse the 3D topology of protein structure. This tool is able to analyze multi-chain proteins. Any time during the session, user can select chain(s), network type, threshold for network construction and residue separation along the backbone chain (Fig. 1). Results: Shortest path between selected two nodes can be calculated based upon the optional algorithms such as Dijsktra (2), Floyd-Warshall (3), Bellman-Ford (4). When shortest paths are defined, they are visualized both on the interaction energy network graph and 3-dimensional structure of the protein. Conclusions: This study represents a flexible and efficient tool for assessing the significance of network properties through a biologically meaningful data. Thus, we believe this tool is useful for the initial analysis of reconstructed networks, as well as subsequent network-based research. Keywords: network analysis, shortest path, web tool

210

Figure 1: Screenshots of the web application. (A) visualization of protein structure using WebGL API; (B) protein structure network graph; (C) shortest path calculation panel; (D-E) degree centrality plots of a sample data.

References:

1. H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne, "The Protein Data Bank," Nucleic Acids Research, vol. 28, pp. 235–242, 2000.

2. E. W. Dijkstra, "A note on two problems in connexion with graphs," Numerische mathematik, vol. 1, no. 1, pp. 269–271, 1959.

3. R. W. Floyd, "Algorithm 97: shortest path," Communications of the ACM, vol. 5, no. 6, p. 345, 1962.

4. R. Bellman, "On a routing problem," Quarterly of Applied Mathematics, vol. 16, no. 1, 87–90, 1958.

211

P-74

Understanding Allosteric Interaction Network within A2AR/A2AR/Gαs-D2R/D2R-Gαi complex bound to Heterobivalent Ligand Samman Mansoor a , Gulru Kayik b , Serdar Durdagi b , Ozge Sensoy a a The School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul, Turkey; b Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey E-mail: smansoor@st.medipol.edu.tr Background: Parkinson’s disease (PD), which is the second most common neurodegenerative disease, is resulted from disruption of cells which are responsible for providing dopamine to the striatum. Most commonly used drugs for the treatment of the disease, such as L-DOPA, only target D 2 R receptor. On the other hand, it has been shown that D 2 R form oligomer with A 2 AR in the striatum, which has an antagonistic effect on D 2 R. Recently, the stoichiometry of the oligomer sub-unit has been discovered to be tetramer, which is pre-coupled to G-protein as well (1). Moreover, it has been shown that simultaneous occupation of both A 2 AR protomers in the tetramer by an agonist and an antagonist efficiently prevents the antagonistic effect of A 2 AR on the D 2 R (2). Here, we hypothesize that simultaneous occupation of the protomers can be efficiently provided by heterobivalent ligands and so effective D 2 R signaling can be maintained. In this project, we aim to test this hypothesis at the molecular level by means of computational tools. Methods: As a first step in the project, the tetramer is modelled. Afterwards, heterobivalent ligands are developed based on the 3D model of the tetramer. The stability of the heterobivalent ligands is investigated by means of classical molecular dynamics simulations. One of the stable ligand is selected for further studying the allosteric interaction network within the tetramer. Subsequently, the dynamics of D2R dimer in the tetramer is investigated by means of accelerated molecular dynamics. Results: Our preliminary results show that hydrocarbon-based linker groups provide more stability to the heterobivalent ligands than PEG-based groups do. In addition, involvement of PRO-GLY repeat in the linker group further increases the stability. Accelerated molecular dynamics simulation results show that in the presence of the heterobivalent ligand D 2 R attain more active-like state. Keywords: (Parkinson's Disease, Heterobivalent ligands, A 2 AR-D 2 R Heterotetramer, Accelerated Molecular Dynamics simulation, allosteric interaction)

212

Figure 1 : The proposed model for tetramer-G protein comlex References: 1. Navarro, G., Cordomí, A., Casadó-Anguera, V., Moreno, E., Cai, N. S., Cortés, A., ... & Lluís, C. (2018). Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase. Nature communications , 9 (1), 1242. 2. Bo naventura,J.; Navarro,G.; Casado-Anguera,V.; Azdad,K.; Rea,W.; Moreno,E.; Cortes,A. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer. Proceedings of the National Academy of Sciences, 2015, 112(27), 3609-3618.

213

P-75

Radiolabeling of Gemcitabine Loaded Multifunctional drug delivery system

(SPION-PLGA-GEM)

Selin Yılmaza, Burcu Aydına, Çiğdem İçhedefa, Serap Teksöza aEge University, Institute of Nuclear Sciences, Department of Nuclear Applications,

Bornova-İzmir E-mail: selin9076@hotmail.com

In recent years, a large number of nanoparticle drug delivery systems have been developed to raise efficacy of anticancer drugs. In this work, we present an encapsulation of gemcitabine by superparamagnetic iron oxide nanoparticles (SPIONs) and polylactic-co-glycolic acid (PLGA). It is well-known that magnetic nanoparticles have a strong magnetic field effect, biocompatibility and superparamagnetic properties. For this reason, it can be used in biomedical applications such as drug delivery systems. Also, the use of biodegradable copolymers of poly lactic-co-glycolic acid (PLGA) for delivery of anticancer drugs has widely increased in treatment of cancer types, too. Gemcitabine (2ʹ, 2ʹ-diflurodeoxycytidine), is used to treat several types of cancers including lung, ovarian, pancreatic, bladder, breast and colon cancers. The aim of current study was to develop a novel multifunctional nanoparticle, which encapsulates SPION and Gemcitabine in PLGA to form multifunctional drug delivery system. The SPIONs were synthesized by coprecipitation method and coated with PLGA. After that, gemcitabine (GEM) was loaded to SPIO - PLGA nanoparticles to form multifunctional drug delivery systems. SPIO-PLGA-GEM nanoparticles were synthesized with double emulsion technique (w/ o / w). Drug loaded nanoparticles was radiolabeled with technetium-99m tricarbonyl core and the quality control was performed using thin layer radiochromatography (TLRC) method using various mobile phase solutions. Studies have been performed on MCF7 cancer line to determine the biological behavior of gemcitabine loaded SPIO-PLGA nanoparticles in vitro. Keywords: Magnetic nanoparticles, drug delivery, gemcitabine, in vitro References:

1. Alberto R, Schibli R, Egli A, Schubiger AP. A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules: Synthesis of [99mTc(OH2)3(CO)3]+ from [99mTcO4]- in Aqueous Solution and Its Reaction with a Bifunctional Ligand. (1998) Journal of the American Chemical

Society; 120(31): 7987-7988. 2. Vandana M, Sahoo SK. Long circulation and cytotoxicity of PEGylated gemcitabine and its

potential for the treatment of pancreatic cancer.(2010) Biomaterials; 31: 9340-9356. 3. McBain, S. C., Yiu, H. H. P., & Dobson, J. Magnetic nanoparticles for gene and drug delivery.

(2008) International Journal of Nanomedicine; 3(2), 169- 80. 4. Wilczewska AZ, Niemirowicz K, Markiewicz K. H, Car H. Nanoparticles as drug delivery

systems.(2012) Pharmacological Reports; 64: 1020-1037.

214

P-76

Warfarin dosing according to CYP2C19 and VKORC1 genotyping

DUZENLI Selmaa, Zeynep Gunes OZUNALb aBolu Abant Izzet baysal University, Medical Faculty, Dept. of Medical Genetics, Bolu

bMaltepe University, Medical Faculty, Dept. of Medical Pharmacology, Istanbul E-mail: duzenlis@gmail.com

Background: Metabolisation differs in individuals due to their genotypes for drug metabolizing enzymes like the cytochrome P450 enzyme system. A large number of cytochrome P450 genes are known to be subject to functionally significant polymorphisms. CYP3A, CYPSD6, CYP2C9 and CYP2C19 account for the majority of P450-mediated drug metabolism. Warfarin, the most prescribed anticoagulant, that has a narrow therapeutic range is metabolised differently in patients. CYP2C9 (cytochrome P450) and the VKORC1 (vitamin K epoxide reductase complex subunit 1) polymorphisms occur frequently in patients who are warfarin sensitive and require lower doses, whereas patients with VKORC1 missense mutations are warfarin resistant and require higher doses. To compare the CYP2C9 and VKORC1 alleles and genotype frequencies among 118 Turkish individuals, we genotyped seven CYP2C9 and eight VKORC1 alleles.

Methods: Patient DNA was isolated from peripheral whole blood, extracted DNA was performed by microarray analysis for CYP2C19 and VKORC1 SNPs. The genotyping panel was as follows: CYP2C9 *1/*2/*3/*4/*5/*6/*11, VKR 3673G/A, 5808T/G, 6009C/T, 6484C/T, 6853G/C, 7566C/T, 8773C/T, 9041G/A. 118 patient DNAs were genotyped for CYP2C9 and VKORC1 genes.

Results: On the basis of their genotypes, the assigned CYP2C9 metabolic phenotypes were distributed as extensive, intermediate, and poor metabolizers. After genotyping, the results are used at the warfarindosing database to mainatin an acurate anticoagulant dose. Table 1:

CYP2C9 and VKORC1 combined genotype warfarin sensitivity and dosing outcome CYP2C9 VKORC1a n=patient (%) total estimated

metabolization rate

estimated maintenance warfarin daily dose

*1/*1 extensive metabolizer allele

slow metabolizer allele/s

20 (23.6) intermediate metabolizer

3-4 mg

intermediate metabolizer allele/s

27 (31.86) bintermediate metabolizer

5-7 mg

extensive metabolizer allele/s

1 (1,18) extensive metabolizer

5-7 mg

n(%)=48 (56.64) 48 (56.64)

215

*1/*2 or *1/*3 intermediate metabolizer alleles

slow metabolizer allele/s

22 (25.96) bintermediate metabolizer

3-7 mg

intermediate metabolizer allele/s

34 (40.12) intermediate metabolizer

3-4 mg

extensive metabolizer allele/s

3 (3.54) bextensive metabolizer

5-7 mg

n (%)=59 (69.62) 59 (69.62) *2/*2, *2/*3 or *3/*3 slow metabolizer alleles

slow metabolizer allele/s

3 (3.54) slow metabolizer 0.5-2 mg

intermediate metabolizer alleles

6 (7.08) bintermediate metabolizer

2-4 mg

extensive metabolizer allele/s

2 (2.36) intermediate metabolizer

3-4 mg

n (%)=11 (12.98) 11 (12.98) total patient 118 (100)

a VKORC1 metabolizing effect changes due to SNP, only the metabolizing effect of appropriate SNP is given in table bproceed extra carefully with dosing, outcome is debatable Conclusions: The polymorphisms of CYP2C9 and VKORC1 alleles indicate that clinical testing of these genes is essential for initiating individual warfarin therapy. Genotype information ensures to select the optimal dose for the individual patient at the start of warfarin therapy. In the era of pharmacogenomics the combined effects of genes and epigenetic transformation factors such as life style on a particular phenotype should be investigated to give the individual a satisfactory individualized medical advice. Keywords: pharmacogenomics, pharmacogenetics, drug,warfarin, resistance Web resources: http://warfarindosing.org http://www.pharmgkb.org References: 1. Anke-Hilse Maitland-van der Zee and Ann K. Daly. Pharmacogenetics and Individualized Therapy, Wiley,

2012. ISBN 978-0-470-43354-6 (cloth). 2. Stuart A. Scott, Lisa Edelmann, Ruth Kornreich, Robert J. Desnick. Warfarin

pharmacogenetics: CYP2C9 and VKORC1 Genotypes Predict Different Sensitivity and Resistance Frequencies in the Ashkenazi and Sephardi Jewish Populations.Am J Hum Genet. 2008 Feb 8; 82(2): 495–500.

3. Ryan P. Owen, Li Gong, Hersh Sagreiya, Teri E. Klein, Russ B. Altman. VKORC1 Pharmacogenomics Summary Pharmacogenet Genomics. Author manuscript; available in PMC 2011 Oct 1. Pharmacogenet Genomics. 2010 Oct; 20(10): 642–644.

4. Scott S.A., Sangkuhl K., Stein C.M., Hulot J.S., et al. Clinical Pharmacogenetics Implementation Consortium guidelines for CYP2C19 genotype and clopidogrel therapy: 2013 update. Clinical pharmacology and therapeutics. 2013;94(3):317-23.

216

P-77

Synthesis of new organoruthenium complexes and investigation of anticancer

properties in ovarian cell lines

Seminay Gulera, Gizem Kursunluoglua, Egemen Orkun Sadanb, Ozkan Ipekb, Elif Subasib,

Hulya Ayar Kayalia, aIzmir International Biomedicine and Genome Institute, (IBG), 35340 İzmir, TURKEY

bDepartment of Chemistry, Division of Inorganic Chemistry, Faculty of Science, Dokuz Eylul University, 35160 İzmir, TURKEY

cDepartment of Chemistry, Division of Biochemistry, Faculty of Science, Dokuz Eylul University, 35160 İzmir, TURKEY

E-mail: elif.subasi@deu.edu.tr

Background: Ovarian cancer is one the most common gynecological cancer type leading cause of death due to the lack of early diagnosis. The standard therapy for ovarian cancer involves the use of platinum-based chemotherapy drugs. But, their efficacy, safety and toxicity are still under debate (1). Ruthenium complexes have come in view as powerful candidate to replace chemotherapy drugs. Recently, ruthenium, which is found to have anticancer activity, has much higher biocompatibility and bioselectivity than platinum-based drugs commonly used in cancer treatment. These important unique features have made ruthenium complexes known as promising anticancer drugs candidates (2). However, the mechanism of anticancer properties of Ruthenium complexes still remains unclear. Thus, in this study we aim to investigate the influence of newly synthesized Ruthenium complexes on ovarian cancer cells. Methods:

Ruthenium complexes were synthesized. Two different human ovarian cancer cell lines, A2780 (primary human ovarian cancer cell line) and Ovcar-3 (metastatic human ovarian cancer cell line), were used. Viability of cells was determined with MTT assay after different concentration of Ruthenium complexes were applied to ovarian cancer cells. Following MTT assay, the intracellular distribution of Ruthenium complexes was searched by DNA and BSA binding assays. DNA thermal denaturation method was used to demonstrate the stability of the resulting Ruthenium complex bound DNA and after this experiment, groove binding mode was investigated. DNA fragmentation analysis were performed to determine anticancer effect that occurs as a result of apoptosis. Finally, transepithelial resistance measurements were carried out to examine the change in junctional properties that hold cells together. Results:

[Ru(p-cymene)(N,S-TSC)Cl]Cl complexes were synthesized and identified by using FT-IR, 1H NMR spectroscopy. Biological activity of these newly synthesized Ru-complexes were investigated. It was found that ruthenium complexes had cytotoxic effect on both primary and metastatic ovarian cancer cells. Ruthenium complexes have been shown to aid in the elimination of cancer cells by demonstrating effective cytotoxicity.

217

Conlusion: Today, platinum-based drugs are among the anticancer compounds which are not intended to be used not only because they lead to drug resistance but also because they have important side effects in patients. Therefore, the interest in new Ruthenium complexes has been increasing every day. This study has shown that newly synthesized ruthenium complexes are effective on ovarian cancer. Keywords: Ovarian cancer, Ruthenium complexes, cytotoxic effect, anticancer property

References:

1. Vargas-Hernández VM, Moreno-Eutimio MA, Acosta-Altamirano G, Vargas-Aguilar VM. Management of recurrent epithelial ovarian cancer. (2013) Gland Surg; 3(3): 198–202.

2. Popolin CP, Reis JBP, Beccener AB, Graminha AE, Almeida MAP, Corrêa RS, Colina-Vegas LA, EllenA J, Batista AA, Cominetti MR. Cytotoxicity and anti-tumor effects of new ruthenium complexes on triple negative breast cancer cells. (2017) Plos One; 12(9):e0183275

218

P-78

Investigation of the Lipidic Nano Carriers Containing Olaparib for the

Assessment of Human Pancreatic Cell Cytotoxicity

Şennur Görgülü aAnadolu University, Yunus Emre Campus, Plant, Drug and Scientific Researches Center,

26470-Eskişehir, Turkey E-mail: sennur@gmail.com

Background:

Pancreatic cancer is a type of cancer in which the diagnosis is very difficult and only symptomatic data can be obtained when it spreads to the entire pancreas, so only a few of the patients have the chance to have surgery. For this reason, it is important to develop new treatments. The aim of the study was to investigate the cytotoxic effects of a novel lipidic nano carrier system to increase the cytotoxic effects of the formulation on pancreatic cell lines through comparison to the anticancer agent. Methods: Within the scope of this study, Olaparib, an oral poly (adenosine diphosphate [ADP] -ribose) polymerase (PARP) inhibitor, has been obtained solid lipid nanoparticle formulations with two different lipid as a new generation anticancer drug. The cytotoxic and apoptotic effects of the obtained formulations on human normal pancreatic cells (hTERT-HPNE) and human pancreatic cancer cells (PANC-1 and Capan-1) were investigated in vitro. xCELLigence Real Time Cell Analysis System were used to determine the IC50 values of the formulations on cell viability over 72 hours. Apoptotic cell measurements were performed with flow cytometry device via Annexin V-FITC/PI and Caspase-3 analysis kits. Results: Based on the results of the cytotoxicity studies, a higher anticancer effect was observed with Olaparib loaded lipidic nano carriers, with IC50 values being approximately three-fold to six fold higher than that seen with the Olaparib on pancreatic cancer cell lines, respectively. Conclusions:

As a result, it has been shown that nanoparticle size formulations are developed that are more effective than the single active agent at lower concentrations in cancer cells and less harmful in normal cells. Keywords: Olaparib, PARP, SLN, Pancreatic Cancer, Apoptosis References: 1. Parhi R, Suresh P. Preparation and characterization of solid lipid nanoparticles-a review. (2012)

Curr. Drug. Discov. Technol. 9(1):2-16. 2. Vincent A, Herman J, Schulick R, Hruban R.H, Goggins M. Pancreatic cancer. (2011) Lancet. 378

(9791): 607-620. 3. Chen, A. PARP inhibitors: its role in treatment of cancer. (2011) Chinese Journal of Cancer. 30(7),

463–471.

219

P-79

Cell Cycle Arrest Properties and DAPI Staining of Benzimidazole/p-cymene

Ruthenium Complexes to PC-3 Prostate Cancer Cells

Serap Şahin-Bölükbaşıa,b, Neslihan Şahinc, İsmail Özdemird, Brian S Cummingsb, aCumhuriyet University, Faculty of Pharmacy, Biochemistry Department, Sivas, Turkey

bUniversity of Georgia, Faculty of Pharmacy, Department of Pharmaceutical and Biomedical Sciences, Athens, GA, USA

cCumhuriyet University, Faculty of Education, Department of Basic Education, Sivas, Turkey dInönü University, Faculty of Science and Art, Department of Chemistry, Malatya, Turkey

Email: wserap@yahoo.com

Background:

Cancer is one of the serious health problems and the major causes of death in all over the world. Prostate cancer is a serious health problem and a major cause of death in men after lung cancer. Although contemporary treatment for cancer consists of various drugs, there is a growing tendency to search for new anticancer drugs because of the major side effects of these drugs [1]. A lot of coordination compounds have been studied for biological activity. Increasing interest has focused on organometallic compounds, specifically on ruthenium(II)-benzimidazole compounds, in last years [2-3]. Differentiation of substitutents around the benzimidazole nucleus have provided a wide spectrum of biological activities [4-5]. Herein, we report the cell cycle arrest properties and DAPI staining of four Ru-Benzimidazole-p-cymene compounds include different ligands (naphthalenomethyl, 3,4,5-trimethoxybenzyl, diizoprophylethly and morpholinoethyl) against PC-3 human prostate cancer cells. Methods:

The cell lines PC-3 (CRL-1435, human prostate adenocarcinoma) were grown in F-12 media, containing 10% (v/v) FBS, and 100 Units/ml penicillin and 100 μg/ml streptomycin in a 37°C humidified incubator with 5% CO2. All four ligands and complexes synthesized according to the literature and all compounds were characterized using elemental analysis, FT-IR, 1H NMR and 13C NMR spectroscopy [6]. The compounds were dissolved in DMSO; stock solutions were then diluted to the desired final concentrations with sterile complete medium immediately before each experiment. The final DMSO concentration never exceeded 0.5%, which was not toxic to the cells under the drug exposure conditions used in this study. DAPI (4'-6-diamidino-2-phenylindole) staining and cell cycle arrest were made according to the literatures [7-8]. Results:

Chromatin condensation was analyzed by fluorescent microscopy using the DNA binding fluorescent dye DAPI. DAPI staining revealed that these complexes induced chromatin condensation and nuclear fragmentation, suggesting the induction of apoptosis. Inhibition of cancer cell proliferation by cytotoxic drugs could be the result of the induction of apoptosis or cell cycle arrest or their combination. Complexes 1, 2 and 3 induce arrest in the G2/M phase on PC-3 cells.

220

Conclusions:

Our results showed that 1-substituted- Ru-benzimidazole derivatives to induce toxicity in human prostate cancer cell lines, and to suggest that their mechanisms of action appears to involved apoptosis. Keywords: DAPI staining; Ruthenium; p-cymene; Cell cycle arrest

Figure 1. Cell cycle arrest properties of compounds on PC-3 human prostate cancer cells. Cells were treated with IC50 values of compound 1, for 24 h, 48h and 72 h. DMSO treated cells were used as vehicle control. Data are representative of the mean ± SEM of three separate experiments. (**p < 0.0001 vs control) References: 1. Xiang P, Zhou T, Wang L, Sun CY, Hu J, Zhao YL, Yang L. Novel Benzothiazole, Benzimidazole and

Benzoxazole Derivatives as Potential Antitumor Agents: Synthesis and Preliminary in Vitro Biological Evaluation. (2012) Molecules; 17:873-883.

2. Wan D, Tang B, Wang YJ, Guo BH, Yin H, Yi QY, Liu YJ. Synthesis and anticancer properties of ruthenium (II) complexes as potent apoptosis inducers through mitochondrial disruption. (2017) Eur J Med Chem.; 139: 180-190.

3. Akhtar MJ, Khan AA, Ali Z, Dewangan RP, Rafi M, Hassan MQ, Akhtar MS, Siddiqui AA, Partap S, Pasha S, Yar MS. Synthesis of stable benzimidazole derivatives bearing pyrazole as anticancer and EGFR receptor inhibitors. (2018) Bioorg Chem.; 78:158-169.

4. Ali M, Ali S, Khan M, Rashid U, Ahmad M, Khan A, Al-Harrasi A, Ullah F, Latif A. Synthesis, biological activities, and molecular docking studies of 2-mercaptobenzimidazole based derivatives.(2018) Bioorg Chem.; 80:472-479.

5. Ajani OO, Tolu-Bolaji OO, Olorunshola SJ, Zhao Y, Aderohunmu DV. Structure-based design of functionalized 2-substituted and 1,2-disubstituted benzimidazole derivatives and their in vitro antibacterial efficacy. (2017) J Adv Res.; 8(6):703-712.

6. Özdemir İ, Şahin N, Çetinkaya B. Transfer hydrogenation of ketones catalyzed by 1-alkylbenzimidazole ruthenium (II) complexes. (2007) (Monatshefte für Chemie-Chemical Monthly, 138(3): 205-209.

7. Cummings BS, Schnellmann RG. Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. (2002) J Pharmacol Exp Ther.; 302:8-17.

8. Cummings BS, Lasker JL, Lash LH. Expression of glutathione-dependent enzymes and cytochrome P450s in freshly isolated and primary cultures of proximal tubular cells from human kidney. (2000) J Pharmacol Exp Ther.; 293: 677–685.

221

P-80

Formulation and Evaluation of Gemcitabine Loaded Polymeric Nanoparticles as a Radiolabeled Drug Delivery System

Serap Teksöza, Çiğdem İçhedefa, İbrahim Sarıkavaka, Selin Yılmaza, Yasemin Parlakb,

Elvan Sayıt Bilginb aEge University, Institute of Nuclear Sciences, Department of Nuclear Applications,

Bornova-İzmir bCelal Bayar University, Faculty of Medicine, Department of Nuclear Medicine, Manisa

E-mail: steksoz@hotmail.com

Background:

The main objective of this study is to design multi-functional drug carrier system as a prototype product. For this purpose, Gemcitabine that used for multiple kinds of tumor therapy, and technetium(I)-tricarbonyl core [99mTc(CO)3]+, was incorporated to the PLGA-PEG-COOH (polylactic acid-polyethylene glycol-carboxyl) polymeric structure. It is thought that cytotoxic side effects of drug in non-target tissues may be reduced and in specific tissues both imaging and therapy can be performed. A nucleoside analog gemcitabine is a chemotherapeutic agent which has increasing the use in pancreatic, bladder, ovarian and breast cancers. Nowadays, polymeric structures are preferred drug delivery systems. Technetium-99m has got ideal half-life and gamma energy that is why it is widely used in nuclear medicine as diagnostic purposes. Methods: First of all, PLGA-PEG-COOH co-polymer was synthesized using esterification reaction and the product was characterized by Fourier transform infrared (FTIR) spectroscopy and Nuclear Magnetic Resonance (1H-NMR). Gemcitabine loaded PLGA-PEG nanoparticles were prepared by double solvent diffusion method. The particle size, zeta potential and the nanoparticle morphology was characterized with Dynamic Light Scattering (DLS), Zeta Potential Analysis and Scanning Electron Microscopy (SEM) imaging, respectively. And then, radiolabelling efficiency of gemcitabine loaded radiolabeled PLGA-PEG nanoparticles (99mTc(CO)3-PLGA-PEG-Gem) were observed by Thin Layer Radiochromatography(TLRC) method. Results:

The synthesis of PLGA-PEG copolymer was performed successfully according to the 1H-NMR and FTIR results. The particles size of the Gemcitabine loaded nanoparticles were found as 100-250 nm. The radiolabeling of drug loaded nanoparticles was efficiently carried out. Conclusions: Systems formed by drug, polymer and radionuclide combination offer the most promising opportunities in the diagnosis and treatment of many diseases. Thus, it will bring the connection of organometallic and coordination chemistry with medical chemistry and biochemistry into the forefront. Keywords: Gemcitabine, drug delivery system, radiolabeling

222

Acknowledgement: This study was carried out by funding support of Celal Bayar University Scientific Research project numbered 2017-001. References:

1. Alberto R, Schibli R, Egli A, Schubiger AP. A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules: Synthesis of [99mTc(OH2)3(CO)3]+ from [99mTcO4]- in Aqueous Solution and Its Reaction with a Bifunctional Ligand. (1998) Journal of the American Chemical Society; 120(31): 7987-7988.

2. Betancourt T, Byrne JD, Sunaryo N, Crowder SW, Kadapakkam M, Patel S, Casciato S, Peppas LB. PEGylation strategies for active targeting of PLA/PLGA nanoparticle. (2008) Journal of Biomedical Materials Research Part A; 263-276.

3. Caruthers, SD, Samuel AW, Gregory ML. Nanotechnological applications in medicine. (2007) Current Opinion in Biotechnology; 18:26–30.

4. Hong H, Zhang Y, Sun J, Cai W. Molecular imaging and therapy of cancer with radiolabeled nanoparticles. (2009) Nano Today; 4:399-413.

223

P-81

Investigation of Tramadol Hydrogel with ultrasound on Rats

Sitem Merve Şahina , Gülengül Dumanb , Alper Yamanc, Veysel Yaman Akgünd, Ece Gençe aProf.Dr. Ece Genç-1; bDr. Gülengül Duman-2;c Dr. Alper Yaman-3

E-mail: mervebiyomedikal@gmail.com Background: Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injection. However, the stratum corneum acts as a barrier that limits the penetration of substances through the skin. Tramadol hydrogel is potential candidates as analgesic drug delivery vesicles using ultrasonic stimulation as a release trigger. It is a novel approach that increases of skin permeability of Tramadol and also enables to enhance the analgesia. In this study, an ultrasonic transducer with using power supply was used to enhance releasing of Tramadol. Tramadol dosage for rats was calculated to weights of rats. ‘Hot plate’ sensitivity test was used to determine the analgesic effect of Tramadol on rats. At this study, twentyone Spraque Dawley rats were used to determine the analgesia effect in four groups. Those are Placebo, Gel alone, Tramadol gel, Tramadol gel with ultrasonic applications. Forthy KiloHertz ultrasonic transducer was used as a trigger effect. The hot plate sensitivity test was used for the analgesia effect. The ‘Hot plate’ sensitivity test was applied to rats. The time was recorded the rat’s licking their paws to determine the analgesia effect of the different rat groups. According to results, thus far have reinforced that Tramadol hydrogel with using ultrasonic stimulation as a release trigger was dramatically increased of skin permeability of Tramadol and also enables to enhance the analgesia effect (Figure 1). Methods:

Ultrasound was applied at frequencies of 1 or 3 MHz, and intensities of up to 2 W/cm2 using an ultrasound generator. 21 Spraque Dawley rats were used to determine the analgesia effect in four groups. Those are Placebo, Gel alone, Tramadol gel, Tramadol gel with ultrasonic applications. The paws of the rats were recorded by hot plate sensitivity test for 10 minutes, 20 minutes, 30 minutes, 40 minutes and 60 minutes without any application in the placebo group. In the sonication application, 40 KiloHertz ultrasonic transducer was used as a trigger effect for Tramadol. The hot plate sensitivity test was used for the research of analgesia effect. The ‘Hot plate’ sensitivity test was applied to rats. The time was recorded the rat’s licking their paws to determine the analgesia effect of the different rat groups. Results:

Continuous application of ultrasound for total time of 60 minutes at 40 kiloHertz enhanced the transdermal transport of Tramadol permeability. We deduced cavitation involves the generation and oscillation of gaseous bubbles in a medium and may be induced by the exposure to ultrasound. Cavitation occurs due to the nucleation of small gaseous cavities during the negative pressure cycles of ultrasound, followed by the growth of these bubbles throughout subsequent pressure cycles.17 Ultrasound waves helped longitudinal pressure wave inducing sinusoidal pressure variations in the skin. It based on the discussions presented

224

in the previous sections, it appears that ultrasound exposure in the therapeutic range causes cavitation in the keratinocytes of the SC. Oscillations of the ultrasound- induced cavitation bubbles near the keratinocyte-lipid bilayer interfaces may, in turn, cause oscillations in the lipid bilayers, hence causing structural disorder of the SC lipids. Shock waves generated by the collapse of cavitation bubbles at the interfaces may also promote the structure-disordering effect. Because the diffusion of permeants through a disordered bilayer phase can be considerably faster than that through a normal bilayer, transdermal transport in the presence of ultrasound is expected to be higher than passive transport. Conclusions:

Various phenomena, including cavitation, thermal effects, and me- chanical effects, have been analyzed as possible mechanisms of sonophoresis. Cavitation-induced lipid bilayer disordering was thought to be the most important cause for ultrasonic enhancement of transdermal transport. Hot plate sensitivity test provided to increase sonophoretic transdermal transport enhancement. Keywords: Ultrasonic stimulation, Controlled drug delivery, Tramadol, Transdermal, ‘Hot plate’sensitivity test

Figure 1: Caption for Figure 1. ( Control; GT: Tramadol Gel; GTS: Tramadol Gel +Sonication application (10 minute) References:

1. W. G Pitt, G. A. Husseini, B. J. Staples,(2004) Expert Opinion Drug Delivery ;1(1):37-56. 2. B. W. Barry,(2001), European Journal of Pharmaceutical Sciences ; 14(2):101-114.

0

5

10

15

20

25

30

35

40

10 20 30 40 60

Hot p

late

late

ncy

(sec

onds

)

Time (min)

Control GT GTS

225

P-82

Understanding the Role of Phosphatidylinositol-3,5-Biphosphate

in Cell Division

Şeyma Nur Erkan, Ayşe Koca Çaydaşı Department of Molecular Biology and Genetics, Koç University, Istanbul, TURKEY 34450

E-mail: serkan18@ku.edu.tr

Background: Mitosis is the cell division that produces two daughter cells with same genetic material as the parental cell. Among stages of mitosis we are specifically interested in understanding events that occur while finishing mitosis. These events are best understood in the unicellular model eukaryote Saccharomyces cerevisiae, the budding yeast. A signaling pathway called the mitotic exit network (MEN) is essential for mitotic exit of budding yeast cells. Yet, it remains elusive how yeast cells coordinate mitotic exit with chromosome segregation. Our preliminary data identifies phosphotidylinositol (3,5) biphosphate (PtdIns (3,5) P2) as a potential mitotic exit regulator in yeast. Among other phosphotidylinositol derivatives, PtdIns (3,5) P2 has relatively low abundance in the cell. Formation of PtdIns (3,5) P2 is regulated by Vac7, Vac14, Fab1 and Fig4 proteins. Lack of PtdIns (3,5) P2 is associated with diseases such as Amyothropic Lateral Sclerosis and Charcot-Marie-Tooth disease. However, molecular functions of PtdIns (3,5) P2 are not well established. Here, we aim to reveal the molecular role of PtdIns (3,5) P2 in mitotic cell division. Elucidating this function of PtdIns (3,5) P2 may lead a model for designing drugs against those neurological disorders in the future (1). Methods: To understand the possible function of PtdIns (3,5) P2; Vac7, Vac14 and Fab1 are deleted from S. cerevisiae genome. Using these knock out strains, we performed genetic and biochemical analysis that will help understand how those proteins effect mitotic exit and the cell cycle. In addition, to elucidate the cell cycle phase at which PtdIns (3,5) P2 affects mitotic exit, aforementioned knockout yeast strains are analyzed via fluorescence microscopy. All in all; a combination of genetic, biochemical and microscopy techniques are used to address these points. Results: Our genetic data show that deletion of Vac7 and Vac14 are synthetic lethal in strains with mitotic exit defects. Conclusions: Outcome of this study will reveal the mechanism by which PtdIns (3,5) P2 controls mitosis. Our studies state that PtdIns (3,5) P2 contributes MEN pathway for exit from mitosis so far. Understanding this novel function of PtdIns (3,5) P2 might bring possible solutions for specific neurological diseases. Keywords: Mitosis, Phosphotdylinositol (3,5) biphosphate, mitotic exit, cell cycle, Saccharomyces cerevisiae

References: (1) Sullivan, Matt, and David O. Morgan. “Finishing Mitosis, One Step at a Time.” Nature Reviews

Molecular Cell Biology, vol. 8, no. 11, 2007, pp. 894–903., doi:10.1038/nrm2276.

226

P-83

Identification of Small Organic Molecules as Potent Catalyst

Sezen Alsancaka, Yeşim Çamlısoya, Nihan Çelebi-Ölçüma

aDepartment of Chemical Engineering, Yeditepe University, İstanbul,Turkey

E-mail: sezen.alsancak@yeditepe.edu.tr

Background: Quantum mechanical calculations have successfully uncovered numerous organocatalytic reaction mechanisms and explained the observed reaction outcomes; yet, the use of computations for the design of new organocatalysts is still limited to a small number of examples. Not surprisingly, most computational predictive organocatalyst design strategies are challenged by synthetic feasibility, and suffer from being non-practical. Proposed new computational approach1 that combines the quantitative power of quantum mechanical calculations with drug design tools provides an opportunity to uncover practically catalytic activities of commercially-available chiral organic compounds with no previously determined catalytic activity. Methods: The catalytic active site models were generated using quantum mechanical calculations. The functional groups of cinchona alkaloid based organocatalyst which can catalyze the target reaction determined as pharmacophore groups. Catalytic group maps were generated using arrangement of these pharmacophore groups in the active site models. The resulting catalytic group maps were screened against database for commercially available molecule. The catalytic activity and selectivity of determined lead molecule were demonstrated using quantum mechanical calculations. Results: This work will describe how the quantitative power of quantum mechanical calculations is coupled with drug design tools to shed light on catalytic activities of readily available small organic molecules for reactions of oxindoles with nitrosobenzene to address the challenge of catalysis of one of the two competing pathways in a pharmaceutically important target reaction (Figure 1). Conclusions: The readily available small organic molecules was identified as potent catalyst for reactions of oxindoles with nitrosobenzene. Keywords: oxindoles, nitrosobenzene, quantum mechanic, pharmacophore search

Figure 1: Hydroxyamination and Aminoxylation Reactions of 2-oxindoles References: Tübitak 1001, “Determination of Active Organocatalysts Using Computational Methods”, 114Z791.

NPG

O

R

PhNO

Cinchona Alkaloids NPG

O

RNOH

Ph

NPG

O

ROHNPh

+

hydroxyamination aminoxylation

227

P-84

An Investigation on Aggregation Status of Macrogol-Bound Zinc Phthalocyanine in Different Solvents

Songül Şahin, Erbil Ağar Ondokuz Mayıs University, Department of Chemistry, Samsun, Turkey

songul.sahin@omu.edu.tr, erbagar@omu.edu.tr

Background: Phthalocyanines(Pcs) are macrocyclics bearing four isoindoline groups. Thanks to their constitutional electronic delocalization, they have found application area in different fields[1]. Using as photosensitizers in photodynamic theraphy is one of the fields [2]. Photodynamic therapy(PDT) is a treatment method that includes administration of a photosensitive drug which is names as photosensitizer, and later on light irradiation at the appropriate wavelength. In the presence of tissue oxygen, cytotoxic free radicals that are produced at the target tissue causes cell death[3]. One phthalocyanine(Pc) (known as "photosens" or "aluminium phthalocyanine") has been approved for clinical application, two phthalocyanine (photocyanine, zinc Pc and Pc 4, silicon Pc) are still under clinical trials for different cancer treatments[4]. Due to the phthalocyanines’ planarity, a high self-aggregation in polar environments is observed. Aggregation is imbrigation state of molecules in solution and at that state physical and chemical properties of molecules vary[5]. Methods: In this study, macragol-containing new zinc phthalocyanine compound was synthesized and investigated for its aggregation behaviour. The syntheses were done three steps. All compounds were characterized by FT-IR, 1H-NMR, 13C-NMR, elemental analysis, UV-Vis, TGA. Aggregation behaviours were determined by UV-vis spectroscopy in different solvents.

Step 1

Step 2

Step 3

228

Figure 1: UV-vis spectrum of A13 ZnPc in different solvents at 5x10-5 M concentration.

Results: There is no ZnPc aggregation in chloroform, dimethylsulfoxide, dimethylformamide. There is H-type aggregation formation in methanol (MeOH), ethanol (EtOH) and J-type aggregation formation in water (H2O). Chloroform (CHCl3), N,N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO) suitable for A13 ZnPc’ photophysical and photochemical studies . Conclusions: A13 zinc phthalocyanine protects its monomeric state (non-aggragated) in chloroform, N,N-dimethylformamide and dimethylsulfoxide solutions. But, the compound shows polymerization tendency in polar solvents like methanol, ethanol, water. It have ultimate polimerization(J aggregation) degree in water. For this reason, to using of chloroform, N,N-dimethylformamide or dimethylsulfoxide solutions are more suitable than other solvents in photodynamic therapy activity studies. Keywords: Phthalocyanine, Macrogol , Aggregation, Water Solubility, UV-vis spectra. This study is supported by Ondokuz Mayıs University PYO. FEN.1904.16.012 numbered BAP Project References: 1. de la Torre, G., M. Nicolau, and T. Torres, Chapter 1 - Phthalocyanines: Synthesis, Supramolecular

Organization, and Physical Properties A2 - Nalwa, Hari Singh, in Supramolecular Photosensitive and Electroactive Materials. 2001, Academic Press: San Diego. p. 1-111.

2. Dougherty, T.J., et al., Photodynamic therapy. Journal of the National Cancer Institute, 1998. 90(12): p. 889-905.

3. Saini, R., et al., Prospects in the Application of Photodynamic Therapy in Oral Cancer and Premalignant Lesions. Cancers, 2016. 8(9): p. 83.

4. Li, X., et al., Phthalocyanines as medicinal photosensitizers: Developments in the last five years. Coordination Chemistry Reviews, 2017.

5. Snow, A.W., 109 - Phthalocyanine Aggregation, in The Porphyrin Handbook, K.M. Kadish, K.M. Smith, and R. Guilard, Editors. 2003, Academic Press: Amsterdam. p. 129-176.

229

P-85

Targeting the Interactions Between Immune Checkpoint Receptor TIGIT and Its Ligand CD112

Tiyodora Akbulut, Cansu Uygun and Ozlem Ulucan

Istanbul Bilgi University tiyodora.akbulut@bilgiedu.net, cansu.uygun@bilgiedu.net

Background:

Immune checkpoints are immune system regulators that maintain immune homeostasis and prevent autoimmunity. Cancer cells often manipulate immune checkpoint mechanisms to escape anti-tumor immune response by overexpressing the immune checkpoint ligands. Binding of those ligands to their receptors on T cells will cause constitutive activation of immune checkpoints which will result in T cell exhaustion. For this reason, the interactions between the immune checkpoint receptors and their ligands caught attention and were proven to be effective targets in treating cancer [1,2]. Methods:

In this project we employed computational methods (see Figure 1) to target the interaction between negative immune check point receptors TIGIT and its ligand CD112 (Nectin 2) by designing small molecules that bind effectively and specifically to the ligand CD112. The main reason for choosing these systems is that it is frequently used by cancer cells to suppress the immune response, and that crystal structure of the complex is known. Results:

The residues Thr55, Gln56, Tyr113, Pro114 and Asp115 on TIGIT at the TIGIT:CD112 interface were found to be important in binding. Triple combinations of these important residues were used to construct several pharmacophores which used later to screen the ZINC database (3). More than 2000 small molecules were retrieved from the ZINC database using the pharmacophores. These molecules were docked to the CD112 using Autodock 4.2 program [4,5]. Analysis of the docking results revealed several micromolar binders. Conclusions: Using computational methods we have discovered several drug candidates that bind to CD112 at micromolar level. However, further computational and experimental investigations are required for validation. Keywords: Immune checkpoints, TIGIT, CD112, small molecules, drug design

230

P-86

BAG-1 mediated HSF1 phosphorylation regulates gene expression of heat shock proteins in breast cancer cells

Tugba Kızılboga Akguna, Nisan Denizce Cana, Ezgi Basturka, Baran Dingiloglua, Evren Onay

Ucarb, Gizem Dinler Doganaya

a Istanbul Technical University, Molecular Biology and Genetics Department, Istanbul, Turkey b Istanbul University, Molecular Biology and Genetics Department, Beyazıt, Istanbul, Turkey

E-mail: tugbakizilboga@hotmail.com

Background:

BAG-1 is an adaptor protein expressed as four different isoforms that modulates cell survival and apoptosis pathways. By knowing the details of interaction networks of BAG-1 and other chaperone proteins, it is possible to understand how protein homeostasis is regulated in cancer. Our study was designed to determine the effects of BAG-1 overexpression on HSF-1 and HSPs in HER-2 positive (BT-474) and HER-2 negative (MCF-7) cell lines. Previous studies have shown that BAG-1 overexpression changes the pathway in order to induce the cell survival. In this context, it was investigated how BAG-1 and HSP (Hsp90, Hsp70 and Hsp27) interactions lead to HER-2 dependent survival in breast cancer cells.

Methods:

In this study, the interactions between HER-2 (Epidermal Growth Factor Receptor) and HSF-1 (Heat shock factor-1) will be first evaluated and then checked if they play a role in selection between cell survival and apoptosis mechanisms. In this purpose, the presence of BAG-1 isoforms will be shown in BAG-1 upregulated and downregulated BT-474 and MCF-7 breast cancer cells by using immunoblotting. Afterwards, the prospective changes in expression levels of Hsp90, Hsp-70 and Hsp27 proteins caused by differential BAG-1 expression will be analyzed. For the purpose of showing any potential effect on HSF-1, some proteins on this pathway will be checked in BAG-1 upregulated and downregulated breast cancer cells. BAG-1 and Hsp70 (also Hsp27 and HSF-1) interactions were demonstrated by immunoprecipitation. At the same time, the cellular localizations of BAG-1 related proteins were detected by immuno-staining. In order to demonstrate the different interactions of each BAG-1 isoform, protein purification was carried out by TAP tag purification. Purified proteins from the BAG-1L, BAG-1M and BAG-1S isoforms were analyzed by LC-MS/MS (Waters Synapt G2-Si HDMS). The obtained findings were evaluated in PRISM protein-protein interaction program and interaction surfaces were tried to be determined.

Results:

As a result of our study, it was observed that cell survival was increased in breast cancer due to BAG-1 expression. Especially when the HER-2 expression is examined, it has been shown that the expression of BAG-1 directly or indirectly increases the expression of heat shock

231

proteins by affecting the HSF-1 transcription factor on the downstream pathway, thereby protecting the cell against stress. We provide evidence that overexpression of BAG-1 and HER2 in breast cancer leads to concomitant constitutive activation of the HSF1-HSP axis, with subsequent stabilization of numerous tumor-promoting HSP clients such as BAG-1, AKT, mTOR and HSF-1 itself, thereby causing a robust acceleration in tumor growth. We show that BAG-1 siRNA inhibiting HER2 leads to inhibition of phosphoactivated Ser326 HSF1, and subsequently blocks the activity of the Hsp27 chaperone machinery.

Conclusions:

The identification of the interaction surfaces of the BAG-1 isoforms and HSP (Hsp90, Hsp70 and Hsp27) will provide significant contributions to the literature to understand the role of protein folding, cell survival, cell stress and drug resistance mechanisms. By using advanced techniques (HDX-MS method) of previously undefined surfaces, it will be possible to develop new target molecules and explain the structural mechanisms.

Keywords: Bag-1, Hsp70, HER-2, chaperones, breast cancer This work is supported by TUBITAK 115Z169 project.

Figure 1: BAG-1 mediated cell survival with HSPs.

References: 1. Alberti, S., J. Höhfeld, et al. (2002). “Ubiquitylation of BAG-1 Suggests a Novel Regulatory

Mechanism during the Sorting of Chaperone Substrates to the Proteasome.” The Journal of Biological Chemistry 277(48): 45920- 45927.

2. Meng, L., Hunt, C., Yaglom, J. A., Gabai, V. L., & Sherman, M. Y. (2011). Heat shock protein Hsp72 plays an essential role in Her2-induced mammary tumorigenesis. Oncogene, 30(25), 2836-2845.

232

P-87

Investigation of Potential Usage of Some Natural Products

on Various Enzyme Targets

Turgut Mesut Yılmaza, İsmail Hakkı Akgüna aEge University, Faculty of Engineering, Bioengineering Department, Bornova, İzmir, TURKEY

E-mail: turgutmesutyilmaz@gmail.com, ismail.hakki.akgun@ege.edu.tr Background: Natural products have been used for many years in medicine and alternative medicine sectors. Today, drug discovery studies related to natural products are still going on. In order to evaluate natural products as active substances, synthesis, purification and analysis processes are carried out by conventional methods (1). This process takes a long time and requires high cost. With molecular simulation methods, natural products can interact with many enzymes or receptor structures at the same time. At this rate, it is possible to obtain approximate results with faster and lower cost (2). Methods: Natural products screened in this work obtained from AfroDb Natural Products (AfroNp) (3) and Indofine Natural Products (IndoNp) databases indexed in ZINC15 database (4). Duplicate molecules in the databases were filtered and only one of the copies of them was used. Then, oral bioavailability and drug-like filters were applied on these molecules respectively. All of this filter were applied using Mona software (5). Crystal structures of targets obtained from Protein Data Bank (PDB). Ligand preparations were performed by using Marvin Standardizer software (6). Molecular docking and local docking experiments were performed using Autodock Vina (7). Results: Totally 993 structures were obtained from AfroNp and IndoNp databases. This number was slimmed down to 517 after application of filters (Table 1). We have examined the RCSB database whether the molecules in the filtered natural product library were indexed. Our examination showed that 7 of the molecules in the IndoNP library were totally indexed with 24 target and 9 of the molecules in AfroNp database were indexed with 78 targets. Randomly selected ten targets were investigated. However, we have only reported results of two of them in here. 2ZJW is the PDB code of the crystal structure of ellagic acid complex with human CK2 and 4YUA is the crystal structure of glycogen phosphorylase with again ellagic acid. Our docking simulation results indicated that some of the molecules in the database can bind these targets stronger than ellagic acid (Figure 1). Results were summarized in Table 2. Conclusions: Our results show that some of the natural products which are not screened against some targets show lower binding affinities with docking targets. This lower binding energy reveals to the potential of natural products. Along with the studies carried out, it is anticipated that natural resources will be evaluated as active substance in the following periods and it will give a light to the studies to be carried out. Keywords: Natural products, docking, virtual screening

233

References: 1. Harvey, A. L., Natural products in drug discovery. (2008). Drug Disc. Today, 13(19-20), 894-901. 2. Reddy, A. S., Pati, S. P., Kumar, P. P., Pradeep, H. N., & Sastry, G. N., Virtual screening in drug discovery-a

computational perspective. (2007), Curr Protein Pept Sci , 8(4), 329-351. 3. Ntie-Kang, F., Zofou, D., Babiaka, S. B., Meudom, R., Scharfe, M., Lifongo, L. L., ... & Efange, S. M. (2013).

AfroDb: a select highly potent and diverse natural product library from African medicinal plants. PLoS One, 8(10), e78085.

4. Sterling, T., & Irwin, J. J., ZINC 15–ligand discovery for everyone. (2015) J. Chem. Inf. Model., 55(11), 2324-2337.

5. Hilbig, M., & Rarey, M. MONA 2: a light cheminformatics platform for interactive compound library processing. (2015). J. Chem. Inf. Model., 55(10), 2071-2078.

6. Standardizer was used for structure canonicalization and transformation, JChem 15.11.9, 201 5, ChemAxon (http://www.chemaxon.com).

7. Trott, O., & Olson, A. J., AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. (2010), J. Chem. Inf. Model., 31(2), 455-461.

Table 1. Numbers of the molecules in after application of filters.

Library Name Zinc15 Mona -Duplication Mona –

Oral-Bioavailability Mona –

Drug-Like AfroNP 880 832 731 482 IndoNp 113 97 72 35 TOTAL 993 929 803 5170

Target: 2ZJW Binding Energy: -11.6

kcal/mol Target: 4YUA Binding Energy: -11.0

kcal/mol Figure 1. Docking poses of molecules with lowest binding energies

Table 2. Summary of the docking simulation results of obtained against human CK2 and glycogen phosphorylase targets

2ZJW 4YUA The simulated local docking binding energy of the starting ligand (kcal/mol) -8,5 -8,2 The simulated binding energy of the starting ligand (kcal/mol) -9,2 -9,4 The simulated lowest binding energy (kcal / mol) -11,6 -11,0 The simulated highest binding energy (kcal / mol) -5,9 -5,2 The number and percentage of molecules with higher local docking binding energies than the starting material

278 %54

271 %52

The number and percentage of molecules with higher binding energies than the starting material

154 %30

60 %12

234

P-88

Identification and targeting of the molecular determinants of breast cancer lung metastasis in triple-negative breast cancer

Ünal Metin Tokata, Pelin Gülizar Ersana, Erol Eyüpoğlu, Umar Razab, Denis Thieffryc, Daniel Gautheretd, Özgür Şahina,e,f

a Bilkent University, Department of Molecular Biology and Genetics, 06800, Ankara, Turkey; b National University of Medical Sciences (NUMS), Department of Multidisciplinary Studies, Abid Majeed Road,

Rawalpindi, Pakistan; cIBENS – UMR ENS – CNRS 8197 – INSERM 1024 Ecole Normale Supérieure, Département de Biologie, 46 rue d'Ulm, 75230 Paris Cedex 05, France; dUniversité Paris-Sud, Institute

for Integrative Biology of the Cell, CNRS, CEA, Bâtiment 400 91405 Orsay Cedex, France, e Bilkent University, UNAM – National Nanotechnology Research Center and Institute of Materials Science and

Nanotechnology, 06800, Ankara, Turkey E-mail: umtokat@gmail.com

Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women worldwide. Triple-negative breast cancer (TNBC) is the most aggressive subtype accounting for nearly 15% of all breast cancers and mostly affects young women. Metastasis is a complex, multistep process responsible for approximately 90% of cancer-associated deaths. Similarly, metastasis of TNBC to the lungs is the number one cause of death among TNBC patients. Still, even the latest therapeutic interventions against metastasis remain insufficient at its best to cure patients. Therefore, it is of vital importance to find and target new molecular mediators of lung metastasis to reduce mortality rates among TNBC patients. In line with this, we are aiming to (i) identify new genes contributing to the lung metastasis of triple negative breast cancer, (ii) elucidate the molecular mechanisms and/or cellular processes through which the identified gene function, and identify novel compounds targeting this alteration, and (iii) investigate the clinical relevance of the identified gene in “patient datasets”. Methods: To identify molecular mediators of lung metastasis, we developed Human-in-Mouse (HiM) and Mouse-in-Mouse (MiM) primary and experimental metastatic tumor models of TNBC. We orthotopically or via tail vein injected MDA-MB-231-Luc2-GFP cells to nude mice or 4T1-Luc cells to Balb/c mice in HiM and MiM models, respectively. We delineated global transcriptome profile of both models by RNA sequencing and identified a candidate gene by combining RNA-seq with GEO dataset analyses. We performed pathway and gene set enrichment analyses to pinpoint key cellular processes and molecular mechanisms involved in lung metastasis. We also utilized matching primary tumors and spontaneous lung metastatic nodules from MMTV-PyMT transgenic mice to examine if the alterations are also observed (1) in spontaneous metastasis and (2) in a model which reflects pathological progression of metastasis. We used RNAi and overexpression vector of the candidate gene, and we will perform extravasation and co-culture experiments to evaluate the effect of “loss of function (LOF)” and “gain of function (GOF)”. We are also planning to do lead compound

235

screening on tumor cells to discover novel and highly selective inhibitors against our candidate working in a clinically relevant concentration. Lastly, we checked the clinical relevance of the candidate gene through Kaplan-Meier survival analysis in multiple patient datasets. Results: We first showed by qRT-PCR and Western blot that our candidate gene is upregulated in lung metastasis in both HiM and MiM models as well as in transgenic mouse model. Second, GSEA analyses revealed that “high candidate gene”-enriched processes include several well-characterized “metastasis-associated” processes such as extracellular matrix organization, angiogenesis, EMT, hypoxia and integrin signaling. Last, high candidate gene expression in chemotherapy-treated basal breast cancer patients predicts worse DMFS. Conclusions: We found a promising candidate to overcome lung metastasis in TNBC through our in vivo and in silico analyses. We will complement these approaches by in vitro and more elegant in vivo models, and lead compound screening to test prognostic potential of the candidate gene and offer a better inhibitor against the candidate gene, respectively. Keywords: triple negative breast cancer, metastasis, mouse model, drug screen, DMFS

236

P-89

Synthesis of Murryaquinone-A Derivatives and Investigation of Their Anticancer Activity

Volkan Akyıldızc, Elçin Çağatayd, Hülya Ayar - Kayalıa,b, Yavuz Ergünc

aIzmir Biomedicine and Genome Center (IBG), 35340 İzmir, TURKEY bDepartment of Chemistry, Division of Biochemistry, Faculty of Science, Dokuz Eylul University, 35160

İzmir, TURKEY cDepartment of Chemistry, Division of Organic chemistry, Faculty of Science, Dokuz Eylul University,

35160 İzmir, TURKEY dIzmir International Biomedicine and Genome Institute (IBG), 35340 İzmir, TURKEY

E-mail: hulya.kayali@ibg.edu.tr

Background: In recent years colon cancer incidence is raising alarmingly. Every year, 1.36 million new patients are diagnosed with colon cancer and this type of cancer corresponds to 40% of the cases of cancer diagnosed annually with over half a million deaths (Banerjee et al. 2017). Development of various therapeutic agents has a great importance for fighting against cancer. Many quinone derivates obtained by natural or synthetic way have anti-tumor and cytotoxic activity on various cancer cell lines (Itoigawa et al. 2000). Therefore, new derivatives of the carbazolequinone alkaloid Murrayaquinone-A have synthesized to study cytotoxic effect on the healthy, primer and metastatic colonal cancer cell line. Methods: With this context we have designed the synthesis of seven Murrayaquinone molecules and named these molecules as “5a, 5b, 5c, 5d, 5e, 5g, 5h” (Scheme 1). We observed the effects of Murrayaquinone molecules on the healthy colonal cell line CCD-18Co and also primer colonal cancer cell line Caco-2 and metastatic colonal cancer cell line DLD-1. By in vitro experiments, we have focused on ability of Murrayaquinone molecules to inhibit colon cancer cell proliferation by the binding to albumin to transport, importing nucleus, and then binding to DNA and inducing apoptosis as well as arresting cell cycle. In addition, we assessed the levels of superoxide, peroxide and reactive oxygen species level (ROS) produced by murrayaquinone molecules in the cells.

Scheme 1: The Synthesis of Murrayaquinone-A Derivatives Reagent and Conditions: i) Ethanol, N2, reflux, 6h.; ii) periodic acid, methanol-water(1:1), N2, rt, stirred, 24h; iii) DDQ, dioxane (%90), reflux, 24h (for 5c 4 days).

237

Results: MTT analysis demonstrate that four type of newly synthesized seven Murrayaquinone molecules have a cytotoxic effects in a low concentration especially on metastatic colonal cancer cell line when compared commonly used cancer drug. Our results showed that ROS levels in cancer cell lines treated with DMSO and cisplatin were much higher than those of Murrayaquinone molecules. Also, the highest ROS level was obtained in the metastatic cell line DLD-1. When the superoxide and hydrogen peroxide levels produced in the cells are examined, it is observed that the level of superoxide is decreased and the level of hydrogen peroxide is increased. This can be explained by the induction of the superoxide anion by the superoxide dismutase enzyme. According to histogram of cell cycle, treatment with newly synthesized Murrayaquinone molecules arrested the cell cycles at different phases. It was observed that Caco-2 and DLD-1 cells arrested cell division in S phase while CCD-18Co cell arrested at G2/M phase. DNA binding results showed that intrinsic binding constant (Kb) values of four Murrayaquinone molecules were correlated with each other and they showed high binding affinity. As a concequences of their binding to DNA, Murrayaquinone molecules caused apoptosis in low level. The results of BSA binding study also showed that the molecules may be transported by BSA. Conclusions: In summary, the cytotoxic effects of newly synthesized Murrayaquinone molecules have been studied and some significant data were obtained. These findings suggest the potential use of newly synthesized Murrayaquinone molecules for colon cancer. Keywords: Murrayaquinone-A, colon cancer, ROS, apoptosis. References: 1. Banerjee A, Pathak S, Devi S.V, Vasan D, Murugesan R, Verma R.S. Strategies for targeted drug delivery in treatment of

colon cancer: current trends and future perspectives. (2017) Drug Discov Today.; 22(8):1224-1232. 2. Itoigawa, M., Kashiwada, Y., Ito, C., Furukawa, H., Tachibana, Y., Bastow, K.F., Lee, K.-H. 2000. “Antitumor Agents. 203. Carbazole Alkaloid

Murrayaquinone A and Related Synthetic Carbazolequinones as Cytotoxic Agents”, Journal of Natural Products, 63(7), 893-897.

238

P-90

Efficiency of Computationally Selected Cinchona-Derived Organocatalysts in

the Reaction of Oxindole with Nitrosobenzene

Yeşim Çamlısoya, Sezen Alsancaka, Semin Funda Oğuza, Nihan Çelebi-Ölçüma A Department of Chemical Engineering, Yeditepe University, İstanbul, Turkey

E-mail: yesim.camlisoy@yeditepe.edu.tr The common use of small chiral organic molecules as organocatalysts in organic synthesis and the special interest of pharmaceutical industry in organocatalytic methods clearly demonstrate the impact of the prediction of active organocatalysts using computational methods in science and industry. Additionally the determination of the mode of action of cinchona alkaloids and the development of new organocatalysts for the asymmetric synthesis of the hydroxyamination and aminoxylation products1,2, which are privileged motifs owing to their biological activities, in the reaction of oxindole with nitrosobenzene will constitute an important step towards the catalytic steering of product distribution. In this work3, a cinchona derived organocatalyst, selected based on the results of computations, was synthesized and the efficiency in the presence of the catalyst was determined by NMR and HPLC analyses.

Keywords: oxindoles, nitrosobenzene, organocatalyst, cinchona alkaloids

References:

1. Hibino S., Choshi T. Simple Indole Alkaloids and Those with a Nonrearranged Monoterpenoid Unit. (2001) Nat Prod Rep; 18: 66-87. 2. Peddibhotla S. 3-Substituted-3-hydroxy-2-oxindole, an Emerging New Scaffold for Drug Discovery with Potential Anti-Cancer and other Biological Activities. (2009) Curr Bioact Compd; 5:20-38. 3. Tübitak 1001, “Determination of Active Organocatalysts Using Computational Methods”, 114Z791

239

P-91

Scorpion venom components trigger apoptosis of colorectal tumor cells

through DNA signaling damage

Louisa Béchohra, Fatima Laraba-Djebari, Djelila Hammoudi-Triki USTHB, University of Science and Technology Houari Boumediene, Laboratory of Cellular and

Molecular Biology, Bab Ezzouar 16111, Algeria Email: hammoudid@gmail.com, Louizabechohra@hotmail.fr

Background: Scorpion venom was a rich source of bioactive peptides and toxins which exhibit an antitumor effect in vitro and in vivo. Their targets regulate cancer cell behaviors such as proliferation and migration through ion functions. The aim of the present study was to investigate the propensity of F3 fraction isolated from Androctonus australis hector venom to induce antitumor and apoptosis on colorectal tumor cell line HCT116. Methods: The effect of F3 fraction on HCT116 cytotoxicity and cell proliferation were analyzed by MTT and clonogenic survival assays. Apoptosis were evaluated by Hoechst 33258 staining. DNA damage was estimated by comet assay and western blot analysis along with assessment of ROS (reactive oxygen species) generation by fluorescent dye (DCFH-DA). Results: The obtained results showed that F3 fraction induced a concentration-dependent cytotoxic effect on colon tumor cells which was in accordance with significatif decrease in colony surviving fraction. In addition, fraction components mediated apoptosis in colon tumor cells through the enhancement of apoptotic cells that exhibited condensed bright chromatin and increased percent of DNA fragmentation. These effects were associated with a DNA damage effect particularly double strands break due to phosphorylation of histone γH2AX. Moreover, enhancement in intracellular ROS level was detected and can be a key early signal to F3 fraction-induced cytotoxicity and genotoxicity on HCT116 cell line. Furthermore, the expression of molecular target of scorpion toxins in colorectal membrane tumor cells can be also incriminated in this antitumor activity. Conclusion: Hence, these properties make F3 fraction a promising candidate for development of new antitumor peptides. Keywords: Scorpion venom, F3 fraction, DNA damage, Apoptosis, HCT116 cells.

240

P-92

Efficient Synthesis of Benzimidazole-Cyclohexanone Derivatives

M. Fodili1, N. Belkheiri1, M. Amari1,2, P. Hoffmann3

1Laboratoire de Chimie Organique et des Substances Naturelles, Université de Djelfa, Algérie 2 Faculté de Chimie – USTHB – BP32, El-Alia, 16111 Bab Ezzouar, Alger, Algerie 3Laboratoire de

Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, UMR 5068, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, Cedex 4, France

E-mail: fodilimokhtar@hotmail.com The benzimidazole and benzimidazolothione rings are present in some clinically used drugs, such as proton pump inhibitors, the antiviral enviroxime and theantihistaminic astemizole, but it may also display anti-inflammatory, anti-diabetic, antioxidant.1,2 This work reports the synthesis and characterization of new benzimidazole-cyclohexanone derivatives 3a-d, 4a-d

and 5a-d under different reaction conditions. The intermediates and final compounds were purified and their chemical structures were elucidated using 1H-NMR, 13C-NMR and mass spectral data.3 Keywords: Benzimidazole, Cyclohexanone, NMR, Reaction intermediates References: 1. Yadav G, Ganguly, S,.(2015) Eur. J. Med. Chem. ; 97 : 419-443. 2. Bouaziz O, Amari M, Bachar R, Khier N, Fodili M, Almeida Paz F A,Talhi O, Silva A M S,.(2015) Tetrahedron Letters ; 56: 1020-1024 3. Belkheiri N, Fodili M, Amari M, Hoffmann P,.(2018) J. Fundam. Appl. Sci.; 10(2): 213- 221. O NH NH2 R2 R1 2a-d DMSO, Cat. NEt3, rt, 48h CS2 DMF-DMA CH2Cl2, NEt3, reflux, 10-15h triphosgene, CH2Cl2, NEt3, 0°C, 3h O 3a-d N N R2 R1 O 4a-d N NH R2 R1 O O 5a-d N NH R2 R1 S Scheme 1. Synthesis of benzimidazoles 3a-d, benzimidazolones 4a-d and benzimidazolothiones 5a-d.

241

P-93 Entrapment efficiency, in vitro release testing and release kinetics of chitosan

nanoparticles for prolonged antihistaminic treatment

Gülsel Yurtdaş Kırımlıoğlua, Ahmet Alper Öztürk a a Department of Pharmaceutical Technology, Faculty of Pharmacy,

Anadolu University, Eskişehir, Turkey

E-mail: gyurtdas@anadolu.edu.tr Background: Levocetirizine dihydrochloride (LCD) is an orally active, third generation non-sedative antihistaminic agent widely used in the treatment of chronic idiopathic urticaria, seasonal allergic rhinitis and hay fever [1]. LCD is rapidly absorbed after oral administration, with the peak plasma concentration attained in 0.9-1h [2]. The fast disintegration behavior of conventional LCD formulations may result in initial burst-released kinetics, permitting the drug to be absorbed very quickly and resulting in high serum concentrations. Under these conditions, local irritation to gastrointestinal mucous membranes and other side-effects are inevitable [1,3]. Therefore, it is desirable to design and develop a rational delivery system for LCD. Methods: In this study, LCD was successfully incorporated into the chitosan nanoparticles (F-1 and F-2 contains 0.05 g and 0.1g LCD, respectively) by spray drying method aiming oral application. In order to determine the amount of LCD in drug loaded nanoparticles, drug entrapment efficiency (EE) study was performed by validated HPLC method. Release characteristics of the nanoparticles were evaluated with dialysis membrane method at 37°C ±1°C in freshly prepared simulated intestinal fluid (SIF, pH 6.8) during 72 hours. Release kinetics were investigated using DDSolver software in order to evaluate the mechanism of drug release from nanoparticles. Results: LCD was successfully loaded to the polymeric matrices by spray drying method. In this study, EE % was determined to be 71.37 ± 1.32 and 67.328 ± 2.09 (EE % ± SE) for F-1 and F-2, respectively. LCD release from nanoparticles was much slower than its pure form showing time dependent release manner in release media. Also there was no significant difference between F-1 and F-2 formulations as stated by the release profiles (p> 0.05). Moreover, drug release was extended more than 2-fold with nanoparticles which enhances the possible use of them for better treatment. In vitro release profiles indicated prolonged release of LCD from nanoparticles which followed Korsmeyer-Peppas kinetic model. Conclusions: In vitro studies showed that cationic chitosan nanoparticles containing LCD are effective carrier candidates for reducing local irritation to GI membranes and other side-effects and optimizing plasma drug concentration that can improve bioavailability and protection the sensitive drug against light, thus also improving the stability. Keywords: Levocetirizine dihydrochloride, chitosan, polymeric nanoparticle, in vitro release, release kinetic

242

References:

1. Goindi S, Kumar G, Kaur A. Novel flexible vesicles based topical formulation of levocetirizine: in vivo evaluation using oxazolone-induced atopic dermatitis in murine model. (2014) J Liposome Res.; 24(3):249-257.

2. Takahashi H, Ishida-Yamamato A, Iizuka H. Effects of bepotastine, cetirizine, fexofenadine, and olopatadine on histamine-induced wheal-and flare-response, sedation, and psychomotor performance. (2004) Clin Exp Dermatol.; 29:526-532.

3. Li F, Ji R, Chen X, You B, Pan Y, Su J. Cetrizine dihydrochloride loaded microparticles design using ionotropic cross-linked chitosan nanoparticles by spray-drying method. (2010) Arch Pharm Res; 33(12):1967-1973.

243

P-94

THERMODYNAMIC FIRST LAW ANALYSIS OF PROTEINS

Mert GÜR1, ‡, Mert GÖLCÜK2,*, Sema Zeynep YILMAZ2,*, Elhan TAKA2,*

1Department of Mechanical Engineering, Faculty of Mechanical Engineering, Istanbul Technical University (ITU), Istanbul, Turkey

2Department of Molecular Biology-Genetics & Biotechnology, Istanbul Technical University, Istanbul, Turkey

*All of these authors contributed equally ‡Correspondence: Mert Gür E-mail: gurme@itu.edu.tr

Background: Proteins are nature’s biomolecular machines. Proteins such as transporters, pumps, and motors, have complex function/operating machinery/mechanisms which are comparable to the macro-leveled machines that we encounter in our daily life. These proteins, as it is for their macro scaled counterparts, convert various forms of energy into work and operate on mechanic/thermodynamic cycles. Yet, a thermodynamic first law analysis of proteins is still missing in the literature. In this study, the first law analysis of a protein was performed for the first time in literature (to the best of our knowledge). A set of membrane proteins, including the dopamine transporter, glycine transporter I and II, glutamate transporter, sodium-potassium pump and Ca2+ ATPases, were selected to perform the first law analysis and introduce the concept. Methods: In this study, protein is selected as the thermodynamic system of interest. Conservation of energy principle was applied to the thermodynamic/mechanic cycle of each protein. Thermodynamic first law efficiencies per functional cycle were evaluated; showing how much of the energy input per cycle was converted into useful work. Results: Close to basal ion concentrations, the lowest observed first law efficiency among all investigated membrane proteins was 50%. Moreover, our analysis suggest that membrane proteins can operate on a range of efficiency values based on the extracellular and intracellular ion concentrations. Conclusions: Compared to the efficiency of the macro-leveled machines we encounter in our daily lives; membrane proteins are able to operate with very high efficiencies. Keywords: Thermodynamics, First Law Efficiency, Proteins, First Law Analysis, Biomolecular Machines

244

P-95 Pharmacophore-Based Identification of Potent and Safe Ubiquitin Specific Protease 7 (USP7) Inhibitors Duaa Kanana,b, Tarek Kanana,b, Serdar Durdagib,c,* aSchool of Medicine, Bahcesehir University, Istanbul, Turkey; bComputational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey; cNeuroscience Program, Institute of Health Sciences, Bahcesehir University, Istanbul, Turkey

Email: duaa.kanan@stu.bahcesehir.edu.tr Background: The deubiquitinases or deubiquitinating enzymes, responsible for the reversibility of the process of ubiquitination, have an essential role in coordinating numerous cellular processes including DNA repair, cell-cycle control, epigenetics, tumorigenesis and immunity. USP7 (herpesvirus-associated ubiquitin-specific protease [HAUSP]) is considered a highly promising well-validated target for various malignancies including multiple myeloma, neuroblastoma and ovarian cancer. USP7 is critical in regulating the tumor suppressor p53 along with numerous epigenetic modifiers and transcription factors such as FOXP3. Previous studies showed that USP7 inhibitors led to increased levels of p53 and anti-proliferative effects in hematological and solid tumor cell lines. The aim of this study is to identify potent and safe Ubiquitin Specific Protease 7 (USP7) inhibitors as potential cancer drugs via an integrated computational approach combining pharmacophore screening, QSAR models, MD simulations and MM/GBSA free energy analysis. Methods: In this study, the crystal structure of USP7 was comprehensively explored using a combination of fragment-based and receptor-ligand complex e-pharmacophores as well as structure-based pharmacophore modelling. We used three fragment libraries as well as 46 known USP7 inhibitors to develop these pharmacophores. The fragment libraries were docked via a virtual screening protocol using Glide- HTVS, -SP and -XP, and the top sequential 20% fragments were used to develop the pharmacophore models. We then screened the SPECS library (~220,000 drug-like molecules), and docked the top ligands by Glide-SP. Ligands that demonstrate effective binding, structural stability and low predicted toxicities will be further examined via MD simulations. Results: Seven, six and five featured e-pharmacophores were generated using both the vector and projected points methodologies. In total, 24 e-pharmacophore models were generated of which 9 were explored further. 3 structure-based pharmacophores were selected based on the QSAR statistical analysis. Ligands that will be further analyzed from the pharmacophore screening will be selected statistically based on their docking and fitness scores. Conclusion: The identification of potent and safe ubiquitin specific protease 7 small molecules serves an important contribution to the field of cancer drug design and development, enabling further research in this promising area.

245

Keywords: Ubiquitin Specific Protease 7; deubiquitinating enzymes; deubiquitinases; proteasome; HAUSP; USP7 inhibitors; p53; Mdm2; anti-cancer target; pharmacophores; drug design References:

1. Gopinath, P.; Ohayon, S.; Nawatha, M.; Brik, A. Chemical and semisynthetic approaches to study and target deubiquitinases. Chemical Society Reviews 2016, 45, 4171-4198.

2. Lamberto, I.; Liu, X.; Seo, H. S.; Schauer, N. J.; Iacob, R. E.; Hu, W.; Das, D.; Mikhailova, T.; Weisberg, E. L.; Engen, J. R.; Anderson, K. C.; Chauhan, D.; Dhe-Paganon, S.; Buhrlage, S. J.

3. Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7. Cell Chem Biol 2017, 24, 1490-1500.e1411.

4. Ndubaku, C.; Tsui, V. Inhibiting the Deubiquitinating Enzymes (DUBs). Journal of Medicinal Chemistry 2015, 58, 1581-1595.

5. Nicholson, B.; Marblestone, J. G.; Butt, T. R.; Mattern, M. R. Deubiquitinating enzymes as novel anticancer targets. Future oncology (London, England) 2007, 3, 191-199.

246

P-96 Solid state characterization and cytotoxicity potential of montelukast sodium loaded polymeric nanoparticles Gülsel Yurtdaş Kırımlıoğlua, Şennur Görgülüb a Department of Pharmaceutical Technology, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey

bPlant, Drug and Scientific Researches Center, Anadolu University (AUBİBAM), Eskişehir, Turkey

E-mail: gyurtdas@anadolu.edu.tr Background: Montelukast sodium (MS) is a leukotriene receptor antagonist is used for the maintenance treatment of asthma, chronic asthma attacks and to relieve symptoms of seasonal allergies [1]. The purpose of this study was to formulate and characterize nanoparticles in terms of solid state and evaluate cytotoxicity potential of nanoparticles by MTT. Methods: MS loaded Kolidon® SR nanoparticles were prepared by spray-drying method. FTIR and NMR studies were carried out for the solid state characterization of nanoparticles. For nanoparticle formulations to be acceptable for the human use low toxicity profile is a prerequisite. Hence to confirm the safety, the developed nanoparticles were tested by MTT assay considering both dose and time dependency on human normal lung fibroblast cells (CCD-19Lu) which was obtained from ATCC. Results: According to FTIR spectra, no chemical interaction between polymer and MS could be found due to the absence of new peaks in FTIR spectra of nanoparticles. It was observed that the distinctive MS peaks were disappeared and MS crystallinity was decreased for all nanoparticle formulations. 1H-NMR analysis was applied effectively to characterize the form of MS within the polymeric matrix, molecular mobility and molecular interactions between drug and polymer. Some of distinctive peaks of MS were found also in spectra of nanoparticles indicating successful incorporation of drug into the nanoparticles. These results confirmed that MS molecularly dispersed and encapsulated with in the polymeric matrix. The three day results has been shown that MS caused a decrease in the mitochondrial activity by means of MTT assay applied to CCD-19Lu cells related to the increase of the concentration. The cell viability of CCD-19Lu cells was significantly decreased 2 fold at the end of the 72 h incubation period. When the IC50 and cell viability values of pure MS was compared to nanoparticles prepared, it seems that incorporation of MS into polymeric nanoparticles has enhanced cell viability nearly 4 fold that makes the nanoparticles promising carrier systems for MS. Also, the reduced toxicity of MS in the form of formulations can also be attributed to its association with nanoparticles. Conclusions: As indicated with this study, FTIR studies showed no drug-polymer interaction while NMR studies analyses ascertained the presence of the active agent within the amorphous polymeric structure. Cytotoxic evaluation revealed that the safety and biocompatibility of the particles relatively high cell viability data. Hence, it could be concluded that MS loaded KSR nanoparticles seem to be a promising delivery system for the oral application for the sustained delivery of the active agent to target cells, tissues and organs. Keywords: Montelukast sodium, cytotoxicity, nanoparticle, spray drying, solid state characterization References 1. Raghavendra, NG, Suryakar VB. Design and development of mucoadhesive drug delivery system of montelukast sodium. (2010) IJRAP.; 2(2):491-497.