Presented By : HARDIK PARIKH Department of Medicinal Chemistry

Presented By:HARDIK PARIKH

Department of Medicinal ChemistryInstitute for Structural Biology and Drug Discovery

Virginia Commonwealth Universityemail: parikhhi@mymail.vcu.edu

CDC25 PHOSPHATASE:A POTENTIAL TARGET FOR NOVEL

ANTICANCER AGENTS

30/10/2009

For more presentations and information visit http://www.pharmaxchange.info

Outline

Timeline of Cancer

Cell Cycle Regulation of Cdc25 Phosphatases

Structure of Cdc25 Phosphatases

Catalytic Mechanism of Cdc25 Phosphatases

Small Molecule Inhibitors of Cdc25 Phosphatases

Future Prospects

What is Cancer?

According to NCI, “Cancer is a term used for diseases in which abnormal

cells divide without control and are able to invade other tissues.”

NCI Website - http://www.cancer.gov/cancertopics/what-is-cancer 3

Timeline of Cancer

3000BC: Earliest observations of cancerBone remains of mummies have

revealed growths suggestive of the bone cancer.The Edwin Smith Papyrus, oldest descriptions of cancer known, described 8 cases of tumors.

Origin of word CancerCredited to Greek physician Hippocrates

(460-370 BC). He used the terms ‘carcinos’ and ‘carcinoma’.

ACS Website- http://www.cancer.org/docroot/CRI/content/CRI_2_6x_the_history_of_cancer_72.aspImages adapted from – http://www.cancerquest.org (accessed 10/22/09)

1914: Mutation theory of cancerTheodor Boveri proposed the Somatic

Mutation Theory of Cancer. He believed that cancer was caused by abnormal chromosomes.

Timeline of Cancer

1761: Giovanni Morgagni of Padua was the first to perform autopsies to relate the patient's illness to the pathologic findings after death.

1890 : First Cancer TreatmentWilliam Halsted, the first professor of surgery at John Hopkins, Harvard, and Yale, performed the first radical mastectomy.

Timeline of Cancer

2003: Human Genome ProjectIdentified ~25,000 genes in human DNA.

1940s: Era of Cancer Chemotherapy Goodman and Gilman suggested that

nitrogen mustards could be used to treat lymphoma.

2006: First cancer vaccineFDA approved Gardasil, a vaccine that

protects against HPV – Human papillomavirus, major cause for cervical cancer.

1971: War on Cancer declared by President NixonThe National Cancer Act was signed into law; additional

$100 million funds released to find a cure for cancer.

WHO website - http://www.who.int/mediacentre/factsheets/fs297/en/index.html (accessed 10/22/09).Jemal, A. et al. CA Cancer J Clin. 2009, 59, 225-249. 7

Current Scenario Cancer – the second leading cause of deaths worldwide.

WHO has estimated 12 million deaths due to cancer worldwide in 2030.

According to American Cancer Society, About 1.5 million new cancer cases and more than

500,000 deaths are expected in USA alone in 2009.

Half of all men and one-third of all women in the United States will develop cancer during their lifetimes.

Cancer is the reason of 1 out of every 4 deaths in USA.

CANCER

Targeting Cancer

Normal Cell Cycle

All cancers share a common feature – rapid and uncontrolled cell proliferation.

Cancerous Cell Cycle

CANCER

Misregulation/

Over activationCdc25Phosphatase Activates

CycCdk

Cell Cycle Regulator

Regulat

activit

Cell Division Cycle 25 (Cdc25) Phosphatase

Control the progression of cell cycle through activating Cyclin-dependent Kinase(Cdk) – Cyclin complexes

In the event of DNA damage – Key targets of the checkpoint machinery that ensures genetic stability

They are Dual Specificity Phosphatases (DSP), a subfamily of Protein Tyrosine Phosphatases (PTPs).

9Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.

Cdc25 Isoforms In mammalian cells,three Isoforms have been identified :

Cdc25A, Cdc25B, Cdc25C

10Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.

Cell Cycle Regulation byCdc25 Phosphatases

Cell cycle progression requires activation of the cyclin-dependent kinases(Cdk).

Activation of the Cdk/cyclin complex

Myt1/Wee1

(Inactive)

12Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

CycCdk

Cyclin-Dependent Kinase

Cyclin

CAK Cdk Activating Kinase

CycCdk

CAKPhosphorylation

(Active)

CycCdk

T161Dephosphorylation

• Cdc25B activates Cdk1-CyclinB at the centrosomeduring the G2/Mtransition.

• Cdc25C activates The Cdk1-CyclinB complex in the nucleus at the onset of mitosis.

Cdc25B

Cdc25C

Cdc25A

• Cdc25A mainly controls the G1/S Transitions via the dephosphorylation

and activation of the Cdk2/CyclinE and

Cdk2/CyclinAcomplexes.

Regulation of Cell Cycle Transition

Different isoforms activate different complexes -

CycBCdk1

The Checkpoint Response

Checkpoint Kinase 1

Checkpoint Kinase 2

Mitogen-activated Protein Kinase Activated Protein Kinase 2 /MAPKAP Kinase2

Cdc25 Phosphatases

14Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

DNA Damage

Degradation via proteosome

The Checkpoint Response DNA Damage DNA Damage DNA Damage

Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

Degradation via proteosome

Cytoplasmic Sequestration

Cdc25B

Cdc25C

Cdc25A

CycBCdk1

The Checkpoint Response

Cell cycle arrest -

Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

Cdc25 overexpression causes Tumors

Over-activation of Cdk-cyclin complexes – pushes cell cycle in untimely manner.

Cdc25A overexpression accelerates entry

into S-phase

Cdc25B over-expression rapidly pushes the S or G2phase cells into mitosiseven with incompletely replicated DNA.

Cdc25A

CycBCdk1

Cdc25B

Cdc25C

Cdc25 overexpression: A recurring theme in Cancer

Structure ofCdc25 Phosphatases

Structure

C-terminal regionCatalytic Domain

N-terminal regionRegulatory Domain

N-terminal regions are highly divergent

Contains sites for phosphorylationubiquitinationwhich regulate phosphataseactivity.

Contains signals to control the intracellular localization

C-terminal regions are highly homologous

(~60% pairwise identity over ~200 amino acids)

Contains the Catalytic Site

The HCX5R motifHis – Cys – XXXXX – Arg

conserved within the PTP family

20Rudolph, J. Biochemistry, 2007, 46, 3595-3604.

Structure

Cdc25A(PDB ID: 1c25)

Cdc25B(PDB ID: 1qb0)

Crystal Structure of Catalytic Domain of Cdc25B

Side-view Top-view(PDB ID : 1qb0)

Red – Active siteloop (HCX5R)Blue - Sulfate

Top-view

Red – Active siteloop (HCX5R)Blue - Sulfate

Crystal structure of the catalytic domain of Cdc25B was solved by X-ray Crystallography at 1.9Å resolution.

The active site loop contains the signature HCX5R sequence.

Crystal Structure of Catalytic Domain of Cdc25BFor more presentations and information visit http://www.pharmaxchange.info

HCX5R motif Histidine 472

H Cysteine 473 C Glutamic acid 474 Phenylalanine 475 Serine 476 X Serine 477 Glutamic acid 478 Arginine 479 R

Backbone amides of five X resides along with arginine form multiple H-bonds with the bound sulfate. 24Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.

HCX5R motif Histidine 472

H Cysteine 473 C Glutamic acid 474 Phenylalanine 475 Serine 476 X Serine 477 Glutamic acid 478 Arginine 479 R

The thiolate anion of cysteine lies directly below the bound sulfate.

Active site The active site pocket is

small and extremely shallow.

Gets filled up completely by the phosphoryl group of the substrate alone.

Allows access to both pThr and pTyr containing substrates, in accord with its dual-specificity nature.

A large cavity adjacent to the catalytic pocket was identified

Called “swimming-pool” for the abundance of well ordered water molecules

Active site

Yellow – Active site cysteineRed – Water molecules

SwimmingPool

27Rudolph, J. Mol Pharmacol. 2004, 66, 780-782.

Cys473

Phe475

Arg544

H2N NH

Tyr428

Phe543

Thr547 CH3

Trp550

Arg482

Swimming poolCatalytic pocket

Arg479

28Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.

Catalytic Mechanism ofCdc25 Phosphatases

Catalytic Mechanism

Reaction mechanism for PTPs -

OSubstrate

Substrate

HO Substrate

30Chen, W. et al. Biochemistry, 2000, 39, 10781-10789.

Catalytic Mechanism

Identity of catalytic acid –

No sequence conservation with other PTPs

Asp383 of Cdc25A was implicated as catalytic acid on the basis of reduction of activity of D383N mutant.

Glu474 of Cdc25B (corresponding to Glu431 in Cdc25A), the first of the five X residues, could serve the role of the catalytic acid.

Glu478 of Cdc25B (corresponding to Glu435 in Cdc25A), the last of the five X residues, is a more likely candidate for the catalytic acid.

31Chen, W. et al. Biochemistry, 2000, 39, 10781-10789.

Catalytic Mechanism

Enzyme uses a monoprotonated substrate

The protein might use as its substrate a monoprotonated phosphate in contrast to the typical bisanionic phosphate, because of higher intrinsic reactivity.

HOSubstrateP

OSubstrate

32Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.

HOSubstrate

Catalytic Mechanism

Enzyme uses a monoprotonated substrate

Substrate

HO Substrate

33Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.

Small MoleculeInhibitors of

Cdc25 Phosphatases

Translation

Post – Translation

Protein-Protein Interaction

Subcellular Localization

Degradation

Transcription

Potential Druggable Targets for Cdc25

Enzyme Activity

35Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

Inhibitors of Cdc25

Natural products

Lipophilic acids

Quinones

Electrophiles

Sulfonylated aminothiazoles

Phosphate mimics

Inhibitors of Cdc25

Natural products

Lipophilic acids

Quinones

Electrophiles

Phosphate mimics

Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

Inhibitors of Cdc25OH

Natural products

Lipophilic acids

Quinones

Electrophiles

Phosphate mimics

Natural products

Lipophilic acids

Quinones

Electrophiles

Phosphate mimics

Inhibitors of Cdc25

Natural products

Lipophilic acids

Quinoids

Electrophiles

Phosphate mimics

Inhibitors of Cdc25

Natural products

Lipophilic acids

Quinoids

Electrophiles

Phosphate mimics

Inhibitors of Cdc25

Natural products

Lipophilic acids

Quinoids

Electrophiles

Phosphate mimics

Inhibitors of Cdc25

Natural products

Lipophilic acids

Quinones as Inhibitors of Cdc25B

Electrophiles

Phosphate mimics

Electrophilic properties of quinones suggest two possbile

interactions with enzyme : a sulfhydryl arylation of cysteine an ether linkage of serine

Can also oxidize the catalytic thiolate group of Cys473

37Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.

Naphthoquinones

Quinolinediones

Indolyldihydroxy-quinone

RX = S,O

Benzothiazole/Benzoxazole –

diones

Naphthoquinones

IC50 = 3.8 μM*

* in-vitro IC50 values

NSC672121O

IC50 = 0.125 μM*

NSC95397

Covalently inhibits enzyme by arylating the catalytic

cysteine

Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.

Naphthoquinones

IC50 = 3.8 μM*

IC50 = 0.125 μM*

IC50 = 4.13 μM*

IC50 = 1.75 μM*

Naphthoquinones

IC50 = 12.9 μM*

IC50 = 10.3 μM*

IC50 = 4.1 μM*

IC50 = 1.8 μM*

* Growth inhibitory IC50 values for MCF7 human breast cancer cell lines

41Peyregne, V. P. et al. Mol. Cacncer Ther., 2005, 4, 595-602.

Naphthoquinones

Hydrogen bonding between the enolic anion and the hydroxy group

NaphthoquinonesBinding Mode

Ligand Ntot foccΔGbind

(kcal/mol)GOLD score

NSC 128981 11 11 -7.89 52.28

Result of 50 independent Autodock and GOLD docking runs –

NSC 128981IC50 = 0.62 μM

43Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.

Quinolinediones

IC50 = 0.21 μM

NSC 663284

* in-vitro IC50 value

Inhibits enzyme in both reversible and irreversible manner.

Chlorine moiety is not required

Quinolinediones

IC50 = 0.21 μM

Decreased activity when - substituted with different groups shifted to 6-position (IC50 = 20μM)

2-morpholin-4-ylethylamino moiety increases activity

Quinolinediones

IC50 = 0.21 μM

2-morpholin-4-ylethylamino moiety increases activity

Small groups are tolerated

Quinolinediones

R = 2-Me : IC50 = 4.6 μM

R = 4-Me : IC50 = 4.6 μM

R = 2-CN : IC50 = 3.7 μM

Aza analogues are less active

IC50 = 0.21 μM

QuinolinedionesBinding Mode

NSC 663284 10 26 -8.12 42.97

NSC 663284IC50 = 0.21 μM

Two modes were observed – Autodock placed the

quinolinequinone ring into the “swimming pool” cavity

GOLD placed quinolinequinone ring into the catalytic site

QuinolinedionesBinding Mode

Autodock GOLD

Indolyldihydroxyquinones

Mode of action different from other quinones – Reversible and non-covalent inhibitors.

Two electron donating hydroxy groups and elctron donating indole substituent, making them much less likely to accept nucleophiles.

48Sohn, J. et al. J. Med. Chem., 2003, 46, 2580-2588.

Methyl group is tolerated

Substituents of size greater than propyl increase

potency

Halides and benzyloxy increase potency

Methyl is deleteriousIC50 = 18 μM

Substitution reduced activity

Binding Mode

Compound 1 11 11 -7.89 52.28

Compound 1IC50 = 1 μM

Two modes were observed – Autodock placed the quinone ring

into the “swimming pool” cavity

GOLD placed quinone ring into the catalytic site

Binding Mode

Autodock GOLD

Benzothiazole- and Benzoxazole- diones

RX = S,O

R = EtR = Ph

IC50 = 0.15 to 0.44 μM

NR = EtR = Ph

IC50 = 0.25 μM

Irreversible inhibition

Acceptor H-bond group

Cys473

Phe475

Arg544

H2N NH

Tyr428

Phe543

Thr547 CH3

Trp550

Arg482

Arg479

Pharmacophoric Model for Cdc25B Reversible Inhibition

Arg544

H2N NH

Arg482

Pharmacophoric Model

Group B : Core structure, mostly quinone

Tyr428

Phe543

Thr547 CH3

Trp550

Group A : A bulky aromatic system 53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.

Cys473

Phe475A B C

Arg479

Group C : An aromatic ring or acceptor H-bond group 53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.

Cys473

Phe475

Arg544

H2N NH

Tyr428

Phe543

Thr547 CH3

Trp550

Arg482

Arg479

Linker : An alkylic chain of 3-4 units 53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.

Future Prospects

Lack of any apparent substrate recognition site in the catalytic loop.

The C473S mutant binds tightly to Cdk2-pTpY – CycA.

Three hotspot residues located >20Å from the active site, mediate protein substrate recognition.

55Sohn, J. et al. PNAS, 2004, 101, 16437-16441.

Future Prospects

Substrate Recognition Site -

R488L and Y497A mutants reduced the kcat/Km for Cdk2-pTpY – CycA, while retaining the activity towards the small-molecule substrates.

R492L mutation showed similar results.

Arg 488

Arg 492

Tyr 497

56Sohn, J. et al. PNAS, 2004, 101, 16437-16441.Rudolph, J. Biochemistry, 2007, 46, 3595-3604.

Future Prospects

Substrate Recognition Site -

Future Prospects

Substrate Recognition Site –

Docking model of Cdc25B with its protein substrate Cdk2-pTpY–CycA showed the three hotspot residues – Arg488, Arg492 and Tyr497 interacting with the two aspartate residues of Cdk2.

Cdc25B: magentaCdk2-pYpY–CycA : blue

Rudolph, J. Biochemistry, 2007, 46, 3595-3604.

Future Prospects

A potential binding pocket

Binding of suitable ligandscould engage the substrates involved in substrate recognitionand interfere in enzyme/substrateassociation.

Arg 488

Arg 492

Tyr 497

Rudolph, J. Biochemistry, 2007, 46, 3595-3604.

Future Prospects

Peptide Derived Inhibitors

Inhibitors designed based on sequence homology with the protein substrate.

Active site peptide ligand

Future Prospects

Is activating Cdc25 Phosphatase a feasible approach?

Moderate increase in levels of Cdc25B have shown to

significantly increase the sensitivity of tumor cells to doxorubicin or ionizing radiations.

Idea would be to radiosensitize or chemosensitize cancer cells and push them to commit suicide.

High risk factor to patient.

Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.

CONCLUSIONS

Cdc25 Phosphatases represent a good target for developing novel anticancer drugs.

Scope for developing novel strategies to target them.

Crystal structures of Cdc25A and Cdc25B provide a rational basis for the design of potent and selective inhibitors.

Further improvement of these inhibitory compounds is likely to lead to their introduction in human clinical trials.

ACKNOWLEDGEMENT

Dr. Glen Kellogg

Kellogg’s Molecular Modeling & Drug Design Group

Department of Medicinal Chemistry

Presented By : HARDIK PARIKH Department of Medicinal Chemistry

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