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Layout of the presentation

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✓ Basic principles of cell-based assaysSources of cellsQualtitative and (semi)quantitative studiesImportant considerations in quantitative assaysCell-based assays in drug discovery & medicinal chemistry

✓ ApplicationsDrug discovery & medicinal chemistryMaterial scienceNatural products

✓ Challenges and basic references in cell culture and assays

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Cell and biobanks

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❖ Mammalian cells and microorganismsBanco de Células do Rio de Janeiro (BCRJ)Coleção Brasileira de Microrganismos de Ambiente e Indústria (CBMAI)American Type Cell Culture (ATCC)Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbHPublic Health England ... and many others...

❖ A reliable source of cells is essential to the development of a study

❖ Missidentification of cell linesShort tandem repeat (STR) for cell authentication

❖ Contamination with other cells

Mycoplasma is one of the most important problems

Methods: Biouminescence Polymerase chain reaction (PCR)Fluorescence Scanning Electron Mycroscopy (SEM)

Invivogen data http://bcrj.org.br/

Qualitative, (semi)quantitative studies

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✓ Check the blank

✓ Choose your controls with care

✓ Check the error of the experiments

✓ Analyse the cell response variations , samples, etc.

Reproducibility

✓ Design the layout of the experiment

Qualitative: yes/no answer using a cutoffSemi-quantitative: range of response (weak, medium, strong)Quantitative: potency

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Qualitative, (semi)quantitative studies

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Qualitative results

? !

Jones, G. J. Comput. Aided Mol. Des. 2015, 29, 1.

Effectiveconcentration

May be used forAgonist or antagonist

Inhibitoryconcentration

Cytotoxicconcentration

GrowthInhibition

Cytostaticactivity

Quantification: Concentration-response curve

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GI50 IC50 EC50 CC50

GI90 IC90 EC90 CC90

IC50 = 0.711 µM 0.713 µM 0.715 µM

SE = 0.0975 0.143 0.138R2 = 0.995 0.987 0.988S = 3.31 3.22 2.82

LogIC50 -6.15HillSlope -0.704IC50 7.11x10-7

Span 104

95% Confidence IntervalsBottom -7.40 to 3.76Top 97.7 to 106LogIC50 -6.27 to -6.03HillSlope -0.843 to -0.564IC50 5.34x10-7 to 9.45x10-7

Span 95.5 to 112Goodness of FitDegrees of Freedom 14R square 0.995Absolute Sum of Squares 154Sy.x 3.31Number of pointsAnalyzed 18

Std. ErrorBottom 2.60Top 1.90LogIC50 0.0577HillSlope 0.0650Span 3.74

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Quantitative study

Importance of the Hill slope

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✓ Formula: Y= Bottom +(Top -Bottom)(1+10((LogIC50-X).HillSlope))

Hill = -1

Hill = -10

log(inhibitor) vs. responseVariable slope (four parameters) Ambiguous

Best-fit values

Bottom 2.33

Top 99.5

LogIC50 ~ -6.99

HillSlope ~ -7.46

IC50 ~ 1,01x10-7

Span 97.2

?

Caldwell, G.W.; et al. Curr. Top. Med. Chem. 2012, 12, 1282.

Quantitative study

Potency versus efficacy

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❖ Potency: amount of a compound that leads to a defined effect.

❖ Efficacy: maximum effect that a compound produces regardlessof the dose.

https://step1.medbullets.com/pharmacology/107007/efficacy-vs-potency

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Factors that influence the assays

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❖ Many problems by bias the assay, like problem with the cell passage,compound degradation or precipitation, plates with problems, amongothers.

❖ For colorimetric assays: Compound can absorb in the same wavelenght.

❖ For fluorescence assays:❖ Compound can supress fluorescence (quencher)❖ Compound can absorb and emmit in the same conditions❖ Cells have green fluorescence background

Always check your system

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Cell lines have a specific size and shape

Round or odd-shape cellsVacuolationGranulationSize and shape of the nucleusetc.

Temporal progression of VERO cellvacuolation (phase-contrast lightmicroscopy; magnification, ×200).

Control cells(negative control)

Concanamycin(Positive control)

1 h incubation

V. cholerae hemolysin1-2 h incubation

Concanamycin +V. cholerae hemolysin

2 h incubation

V. cholerae hemolysinlonger incubation

Figueroa-Arredondo, P.; et al.Infect Immun. 2001, 69, 1613.

c-Myc-knockdown cells (HeLa-630)Confocal microscopy

Cui, F.; et al. Oncol. Lett. 2017, 14, 2878.

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Always check your system

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Negative control Estradiol

Ethanol/water (80:20) Ethanol/water (95:5)

Granulation of breast cancercells (MCF-7) is observed for

Morus alba extracts high ethanol proportion

Manuscript in preparation

Compound precipitation(green and cells (blue)

[cpd] = 50.0 µM

[cpd] = 6.25 µM

Drug discovery process

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Cell-based assays are essential to link the biochemical to in vivo assays via in vitro studies

DrugLigandsBiological

targetPrototype

compound

Disovery and

development

Identification

and

optimization

Clinical

trialsApproval

Clinical trials:

phase IVPhases

I, II e III

0 5 15

Estimated time (years)

Previously to

drug discovery

Cell-based assays

Leitão, A.; Montanari, M. L. C. ; Montanari, C. A. Desenvolvimento de fármacos. In: Montanari, C. A. (Org.) QUÍMICA MEDICINAL: Métodos e fundamentos em planejamento de fármacos. 1ed. São Paulo: EDUSP, 2011, v. , p. 94-127.

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Medicinal Chemistry & cell-based assays

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Known target and ligand Known target only

SBDD(VS) and LBDD(VS)SBDD

de novo

Known ligand only Ligand and target unknown

LBDDLigand diversity (chemical library)

Use compounds with known MoA

LBDD: ligand-based drug discovery VS: virtual screeningSBDD: structure-based drug discovery MoA: mechanism of actionHTS: High-throughput assay HCS: high-content assays

Cell-based assaysCompound(s) bioactive profiling

based on reference ligand

Cell-based assaysPhenotypic linked to mechanism

Compound profiling based onreference ligand

Cell-based assaysCompound(s) bioactive profiling

HTS and HCS

Cell-based assaysMechanism-based assay

Importance of the cell-based assays

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Applications vary from early drug discovery to development

➢ Phenotypic assays – mechanism-based or not➢ Pharmacodynamics

➢ Mechanism of action

➢ Pharmacokinetics & toxic (ADME-Tox) studies➢ Permeability➢ Metabolism➢ Toxicity

➢ Applications under development: 3D technologies andmicrofluidics to replace animal models

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Context

✓ mTOR: master regulator in physiopathotological processesHosking, R. Cell 2012, 149, 957

✓ Roles in nutrient regulation, life span, cancer growth, etc.Maiese, K.; Molecules to Medicine with mTOR. Academic Press, 2016.

✓ Rapamycin and rapalogs: allosteric inhibitors

Waldner, M.; et al. Br. J. Clin. Pharmacol. 2016, 82, 1158

✓ In this work: competitive inhibitors (ATP binding site)

✓ Like other kinase inhibitors: polypharmacological response

(PI3K and mTOR)Smith, M.C.; et al. Mol Cancer Ther. 2016, 15, 2344

Benjamin, D.; et al.

Nat. Rev. Drug Discov. 2011, 10, 868

ATP

HEAT HEAT FAT FRB Kinase FATC

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Cheminformatics Approach

✓ Database: mTOR and PI3K compounds

✓ Filter analysis with 2D parameters

✓ ROCS model

✓ Docking: (a) PI3K and (b) mTOR

Anchoring hydrogen bonds

eMolecules database(5.9 M)

Filter (756 056)

ROCS(2 996)

HB analysis & Clustering(32)

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Chemical set – main scaffolds

Cytotoxicity study

Cell-based screening

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P C -3

Neq

0437

Neq

0438

Neq

0439

Neq

0440

Neq

0441

Neq

0442

Neq

0443

Neq

0444

Neq

0445

Neq

0446

Neq

0449

Neq

0450

Neq

0451

Neq

0453

Neq

0459

Neq

0460

Neq

0461

Neq

0469

Neq

0470

Neq

0474

Neq

0475

Neq

0476

Neq

0481

Neq

0482

Neq

0490

Neq

0495

Neq

0497

Neq

0498

Neq

0503

Neq

0505

Neq

0506

Rap

am

ycin

Bic

alu

tam

ide

MD

V3100

YM

155

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

1 1 0

1 2 0

1 3 0

C o m p o u n d s [1 0 0 M ]

% C

ell

via

bil

ity

PC3: metastatic prostate cancer cell

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Cell-based screening

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D U 1 4 5

Neq

0437

Neq

0438

Neq

0439

Neq

0440

Neq

0441

Neq

0442

Neq

0443

Neq

0444

Neq

0445

Neq

0446

Neq

0449

Neq

0450

Neq

0451

Neq

0453

Neq

0459

Neq

0460

Neq

0461

Neq

0469

Neq

0470

Neq

0474

Neq

0475

Neq

0476

Neq

0481

Neq

0482

Neq

0490

Neq

0495

Neq

0497

Neq

0498

Neq

0503

Neq

0505

Neq

0506

Rap

am

ycin

Bic

alu

tam

ide

MD

V3100

YM

155

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

1 1 0

1 2 0

1 3 0

C o m p o u n d s [1 0 0 M ]

% C

ell

via

bil

ity

DU145: metastatic prostate cancer cell

Cell-based screening

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B a lb -c 3 T 3 c lo n e A 3 1

Neq

0437

Neq

0438

Neq

0439

Neq

0440

Neq

0441

Neq

0442

Neq

0443

Neq

0444

Neq

0445

Neq

0446

Neq

0449

Neq

0450

Neq

0451

Neq

0453

Neq

0459

Neq

0460

Neq

0461

Neq

0469

Neq

0470

Neq

0474

Neq

0475

Neq

0476

Neq

0481

Neq

0482

Neq

0490

Neq

0495

Neq

0497

Neq

0498

Neq

0503

Neq

0505

Neq

0506

Rap

am

ycin

Bic

alu

tam

ide

MD

V3100

YM

155

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

1 1 0

1 2 0

1 3 0

C o m p o u n d s [1 0 0 M ]

% C

ell

via

bil

ity

Balb-C 3T3 clone A31: mouse fibroblast cell (non-tumorogenic)

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Concentration-response data

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Compound PC-3

EC50 (µM)

Balb/C 3T3

%viability (100 µM)

SI

Neq0438 75.0 (±4.3) 68.1(±8.89) >1.33

Neq0440 60.7(±0.95) 95.4(±9.19) >1.65

Rapamycin 43.1(±0.92) 58.3(±7.00) >2.32

SI = selectivity index

Neq0438 Neq0440

Pathway analysis

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PI3K-AKT-mTOR pathwayPC-3 cells

Rapamycin

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Structural features

25Takeuchi, C.S.; et al. J. Med. Chem. 2013, 56, 2218 Grasso, C.S.; et al. Nat. Med. 2015, 21, 555

Neq0438

XL388mTOR

BuparlisibPI3K, mTOR

logKw

E64 E64d Neq0438 Odanacatib-0.77 0.57 1.6 2.0

Samelyn Martinsunpublished result

Fang, Y.; Eglen, R. M. SLAS Discov. 2017, 22, 456.

The most common methods to produce spheroids

Ultra-lowattachment well

Hanging dropplate

Suspension ina bioreactor

Pilar ormagnetic levitation

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Cytostatic activity

3D (spheroid) cultures for liver (HepG2) andprostate (DU145) cancer cells

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Cytostatic activity

2D and 3D (spheroid) cultures for liver (HepG2) andprostate (DU145) cancer cells

Manuscript in preparation

HepG2 (2D) Resazurin standardization

HepG2 (3D)

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Context

✓ Morus alba extracts is commonly used for menopause

(etnopharmacological use)

✓ Challenge: Lack of standardization of extract is a limitation

and challenge

✓ In this work: study the standardized extracts for the

estrogenic activity

✓ Approach: standardize the extract according to the

estrogenicity using cells (bioguided optimization)

✓ Collaboration USP/UFSCar: Dra. Patrícia Bergo, Prof. Dr.

Moacir Forim

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Hormone replacement therapybased on phytotherapeutic compounds

Poluzzi, E.; et al. F. Curr. Med. Chem. 2014, 21, 417.Menezes, I. R. A. ; Leitão, A. ; Montanari, C. A. . Steroids 2006, 71, 417.

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31Turbolysis (A) and ethanol contant (B) are the most important variables.

Analysis of the yield of the extraction

• main

▪ interactions

Chemometric analysis of the extracts using variables

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Effect of the ethanol &screening of the estrogenic activity

Agonist activity Cytotoxic activity

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Estrogen stimulation for low extracts

Days Days

Days Days

E2

Neg. control

Ethanol content (% v/v)

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Potency of the best extracts

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Analysis of the extracts

RRE = relative response for estrogenicity

No correlation was observed between

Yield of the extract

RR

E (%

)

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Cytotoxicity for extracts with high ethanol content

RRE = relative response for estrogenicity

75% (v/v) ethanol

95% (v/v) ethanol

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Analysis of the extracts for the estrogenic activity

RRE = relative response for estrogenicity

Use of non-estrogenic cell line: MDA-MB 231

Ethanol content (% v/v)

RR

E (%

)

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Standardization of the extract

➢ Quantitative analysis of the following parameters:

• Seazon;

• Sun exposure;

• Time of the day to collect the material;

• Stage of the plant development;

• Raining and humidity;

• Distribution of markers of the plant leaves at different hights

➢ According to ANVISA, it is allowed a variation of theconcentration up to 15% when the active marker is used and20% when it is the analytical marker.

Manuscript in preparation

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Context

✓ Propyl and butylparabens are widely used as food preservatives

✓ These are known estrogenic compounds and could lead to cancer

✓ These chemicals contaminate the environment (water)

✓ Exposition can be harmful to humans

Action

✓ Degradation of parabens by photolysis reaction

✓ Analysis of the bioactivity of the degradation product

PrP BuP

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Kinetics of propylparaben alone and in a mixture

Effect of the pH for propylparaben

Degradation studies

As expected, propyl and butylparabens had almost the same results.

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PrP BuP PrP +BuP NC PC

MCF-7 breast cancer cells are estrogen-dependent

Growth promoted by the activation of estrogenic compounds

Estrogenic profile of the material:

t0 = 50 mM; t1/2 = 20 mM; t1 < 1 mM

Phenotypic cell-based studies linked to mechanism

Degradation of thematerial leads to inactive

compounds for MCF-7

Samples were notcytotoxic for mouse fibroblast cell line

Balb/C3T3 clone A31

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Adhesion of fibroblast cells on a biomaterial

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24 h incubation

Control Chitosan Chitosan +1% laponite

Chitosan +2.5% laponite Chitosan +5% laponite Chitosan +10% laponite

➢ Novel chitosan-modified biomaterial was prepared by Dr. Virginia/Prof. Carla➢ Assays were performed to detect the biocompatibility and adhesion

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Cell-based systems for permeability are commonly used for pharmacokinetisassays

Brief overview of a permeability study

Press, B.; Di Grandi, D.Curr. Drug Metabol. 2008, 9, 893.

Challenges

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❑ Use of primary cellsProblem: heterogeneity

❑ Lack of reproducibility❑ Check your controls!

❑ Importance of good laboratory practices

❑ Live cell imaging

❑ Organ-on-chipSpontaneous contractions in mouse embryonic stem cell–derived cardiomyocytes, transduced with CellLight® Actin-GFP.

Live-cell fluorescence imaging (red and green channels) capturing mitotic division in HeLa cells. Cells were transduced with CellLight® Histone 2B-GFP and Mitochondria RFP.

Live cell imagingOrgans-to-human-on-chip

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Basic references

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Books

Scientific society & MeetingSociedade Brasileira de Biologia Celular (SBBC)Congress of SBBC

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