Target Validation / Biochemical and Cellular Assay Development

TARGET VALIDATION / BIOCHEMICAL AND CELLULAR ASSAY DEVELOPMENT

EVERY STEP OF THE WAY

EVERY STEP OF THE WAY1

David Fischer March 28, 2017

CONTENTS

2

1 Introduction

2 Target validation

3 Biochemical assay development

4 Cellular assay development

Library Design

Analytical &Purification

ProcessChemistryCADD

Synthetic Chemistry

Formulation

Pharmaceutics

Chemo-genomics

Ch

‘END TO END’ INTEGRATED DRUG DISCOVERY

3 EVERY STEP OF THE WAY

B/DMolecular Biology

Cell LineGeneration

FBDDStructuralBiology

CRISPRAdenoviral

Platform Human 1o Cells

Functional Genomics

D/PS/I

Safety Assessment

Safety Pharmacology

Non-GLP/GLP Toxicology

Anatomic &Clinical Pathology

Imaging

Animal ModelDevelopment

Large Animal Efficacy Models

DiscoveryPathology

in vivo Efficacy

In Vivo Validation

PK/PD

Dose to Human Predictions

ADME

Bioanalysis

Targets Clinical Candidate

Pharmacologyin vitro/in vivo

Hit Finding:HTS, HCS

IND EnablingStudies

MedicinalChemistry

BiomarkerDevelopment

Target Discovery

& Validation

DP DP

Discovery Pathway

Chemistry

Biology/ Discovery Technologies

DMPK/Pharmacology/Safety/ In vivo models

DISCOVERY – CENTERS OF EXCELLENCE


CNS

Complex cell biologyIntegrated drug discovery

OncologyMetabolic disease

Inflammation Oncology

Ion channel

ONCOLOGY

CNS PAIN

CARDIOVASCULARMETABOLIC

DISEASE

INFLAMMATIONIMMUNOLOGY

RESPIRATORYDISEASE

RARE AND NEGLECTED

DISEASE

OCULAR DISEASE

BREADTH OF THERAPEUTIC AREA EXPERTISE


Extensive integrated drug discovery expertise across multiple therapeutic areas

ONCOLOGY CNS IMMUNOLOGY CV/ METABOLISM RESPIRATORY

TARGETDISCOVERY AND

VALIDATIONAdenovirus technology ● Human primary cell assays ● High-content platforms ● Mechanism of action studies ● CRISPR gene editing

HIT FINDING Compound screening libraries ● Virtual and Fragment Screening ● Knowledge-based design ● Phenotypic screening

MEDICINAL CHEMISTRY Informatics and molecular modeling ● Chemical synthesis and scale-up ● Analysis and purification

IN VITRO/IN VIVO

PHARMACOLOGY

2D and 3D cultures> 400 PDX modelsSyngeneic models

Humanized modelsXenograft models

NeurologyPsychiatry

Neuropathic painNeuromuscular deficiency

Neurodegenerative disease

PsoriasisT-cell activation

PeritonitisColitis

OsteoarthritisCytokine release

Vaccine assessment

DiabetesDiabetic complications

AtherosclerosisNASH

AsthmaCOPD

Pulmonary inflammationMucocilliary clearance

CoughFibrosis

BIOMARKERDEVELOPMENT Biomarker identification ● Ex vivo development and validation ● Dose-to-man predictions ● Translation into clinic

IND ENABLING STUDIES

In vitro toxicology ● DMPK (non-GLP and GLP) ● Exploratory toxicology ● Genetic toxicology ● Safety pharmacology● Subchronic/chronic toxicology ● Development and reproductive toxicology

COMPELLING SUCCESS RATES INSMALL MOLECULE DISCOVERY

6

74 preclinical candidates to date

1 Nature Drug Discovery, 2010, 9, 203; DDT, 2003, 8(23), 1067; DDT, 2013, 19(3), 3412 There are several candidates whose development status is currently unknown. A number of these may also have achieved clinical PoC or be moving towards that goal

>25% of candidates progressed to clinical PoC or beyond- Better than the industry standard (12-24%)1

- Additional 11 being progressed towards clinical PoC2

- Delivering 5 candidates per year for past 10 years

DISEASE AREANO. OF

CANDIDATES Preclinical Phase I Phase IIa Phase IIb Phase III Registration

Inflammation 13CHEMOKINE, INTEGRIN, GPCR, CYTOKINE, KINASE, ENZYME

Respiratory 26GPCR, PROTEASE, NHR, KINASE

CNS 8GPCR, NHR

Metabolic disease 5ENZYME, KINASE, PROTEASE, NHR

Oncology 16ENZYME, KINASE, PPI, NHR

Anti-bacterial 2UNKNOWN

Anti-viral 1PROTEASE

Cardiovascular 2ION CHANNEL

Secretory diarrhoea 1ION CHANNEL

IN VITRO DISCOVERY PLATFORMS


HIT FINDING

• HTS • Phenotypic screens• Extensive compound

libraries

TARGET DISCOVERY & VALIDATION

• Gene family expertise• Complex biology:

primary/patient-derived cells

MEDICINAL CHEMISTRY

• CADD, Scale-up process

• Crystallography, biophysics

• Pharmaceutics

IN VITRO PHARMACOLOGY

• In vitro safety• ADME/PK

OUR DISCOVERY TEAM


>650 scientists

Strong diverse pharmaceutical

company pedigree

300 patents generated for our partners

Library of peer-reviewed publications

>>1,000 in vivo studies per year

Largest group of certified veterinary pathologists in the

world

Experience guiding drugs into the clinic and onto the market

30 5 27 38

Chemistry ADME in vitro biology in vivo pharmacology

%

‘Melting pot’ of industry expertise with the drive of a professional CRO organization

Target Validation

9EVERY STEP OF THE WAY

TARGET VALIDATION

• The gold standard for a validated drug target is an approved drug with a defined molecular mechanism of action

• Positive Phase 2a data contribute, as does human genetic data (e.g. PCSK9)

• To add confidence to novel drug targets, accumulating pre-clinical evidence is paramount• Interrogate the target with genetic means (RNAi / CRISPR) and tool compounds if available• Show evidence for translational effects in relevant models (primary cells, animal models)• Beware of pitfalls (target engagement / PK)

• Target validation can be done one target at a time, or for multiple targets in parallel

• Target validation can also be performed after the identification of a small molecule through phenotypic drug discovery (target deconvolution)

Build confidence in the target or mechanism of action

EXAMPLE OF LITERATURE VALIDATION


None of the reported small molecule TrkB “agonists” work through TrkB, a monoclonal antibody does work

A reported “pro-drug” is not a pro-drug, but most likely contained a contaminant metabolite

Although in vitro findings were robust, reported in vivo data were most likely off-target. Our findings demonstrate that the physicochemical properties, metabolic and P-glycoprotein substrate liabilities of 4b render it unsuitable as a molecular tool to investigate central Class I HDAC inhibition in vivo in mouse by oral administration

resulting in CRL’s publications with CHDI

CRISPR/CAS9 GENOME EDITING

12 CONFIDENTIAL INFORMATION

Principles

Cas95’3’ Insertion site

gRNA

Double stranded break 5’3’ 5’

3’

5’3’

5’ 5’

5’Indel

Non-homologous end joining (NHEJ)

5’

3’3’

3’3’

5’5’

5’5’

Homology-driven repair (HDR)

3’3’

3’

3’Precise editing

EMT6 BRCA1 KNOCKOUTStrategy using HDR


Nucleofection

• 10ug DNA

• 1x10^6 cells

Brac1 – KO

• conserve Start codon

• remove Exon2

• add stop-tag

• introduce frame shift

• remove splice signal

EMT610 μg DNA

Brightfield 24 hours 48 hours

10x

113 bp deletion (*black)

EMT6 BRCA1 KNOCKOUTResults


Results

• Stop-tag was not inserted in genome (SCR7 not efficient in EMT6 cells)

• Deletion of Exon2, frameshift and deletion of splice signal successful

• Successful BRCA1 Knockout optained

D2

Selection+ SCR7

D0

Seed cells4T1 and

EMT6

D-x

Transform 0.5x10^6 cells with 30 ug DNA

Add SCR7

D3

Nucleofection

StopSelection

G418

Harvest and dilute to

single cell colonies

D4 DX

-dublicatesingle cell colonies

-Lyse

- Start expansion- Lyse lyse

cells

DX

Bank cells

DX

-Isolate DNA- Identify

clones by PCR

-Isolate DNA- Send to

sequencin

Timeline

PCR results

Sequencing results (Homozygous knockout)

SILENCESELECTTM


Target discovery, target validation and MOA

• Knock-down libraries (SilenceSelect®)– small molecule tractable and biologics targets– >22,000 shRNAs– >5,700 genes (drugable genome)– >11,000 transcripts

• Human FL cDNA libraries (FLeXSelect®)– > 2,000 human full-length cDNAs

• Efficient cherry-picking– expanding on demand

• Clear IP position– US pat 6,340,595; 6,413,776; 7,029,848; 7,332,337

• Very efficient transduction and RNAi in many human primary cell types

– no need for selection– multiple fiber types available

Arrayed virus collections

Human drugable genome

Screen

Adenoviruses with cDNA or shRNA

Assay in cellular disease models

Drug targets

Cloning

Production

www.SilenceSelect.com

15

ADENOVIRUSES FOR shRNA DELIVERYAdvantages


Non-integrating (DNA unaffected)

High transduction efficiency• Human primary cells and cell lines• Cells of rodent origin

With two different fiber types transduce ~ 90% of all human cells• Fiber panel expressing green fluorescent protein (GFP) available

Low toxicity seen in different cell types

Replication incompetent thus safe to use

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

shRNA_1 shRNA_2 shRNA_3 shRNA_4 shRNA_5 shRNA_6

mR

NA

kn

ock

-do

wn

gene X

HUMAN PRIMARY FIBROBLASTS

17

Knockdown of mRNA – no selection

Conclusions: • Effective knockdown of most target genes • 96% of the target genes have at least 1 shRNA that inhibits mRNA by >70%• 76% of the shRNA viruses knock down their target by >70%

ADENOVIRAL TRANSDUCTION


Example: Human Primary Cells

Pre-adipocytes Adipocytes Hepatocytes

CASE STUDY TARGET DISCOVERY


Developed an HTS assay in CF patient-derived cells

Screened SilenceSelect library (~5,000 genes)

Validated hits in multiple functional assays

Final target validation in human primary lung epithelial cells from CF patients

Project with the Cystic Fibrosis Foundation

0.0 2.5 5.0 7.5 10.0 12.5 15.0

15

25

405060708090

100110

Hit 8

Empty

Hit 1

10µM Fsk50µM Gst

10µM CFTRinh

10µM amiloride

Time (min)

I sc(µ

A.c

m-2

)

correction of mutant CFTR chloride channel activity

cyto-toxicity counter-assay

efficacy, bioinformatics

expression profiling

354

315

11,334

210

190

139

shRNAs

cell-surface expression

on-target analysis

primary cell cultures in Ussingchambers

19

19 targets validated for drug discovery

19

CASE STUDY TARGET VALIDATION


• GSK identified an adipocyte gene expression profile associated with obesity

• CRL generated a library of 600 shRNAs(100 genes)

• CRL developed five different assays in human primary adipocytes

• CRL screened the library in the five different assays

• GSK published the data in 2013 on a conference poster & CRL Presented at a conference in 2015

Project with GSK

20

USHER III SYNDROMECase study target deconvolution


The Usher syndromes (USHs) are characterized by loss of hearing and vision with varying onset of symptoms depending on the genetic type (I, II or III and subtypes)

Rare disease ~1000 patients in USA

Patients with USHIII experience progressive hearing loss and the onset of retinitis pigmentosa (RP) symptoms usually by the 2nd decade of life

Collaboration between:

TREATMENT HYPOTHESISTowards the identification of a small molecule therapy for Usher III


USHIII caused by single point mutation in Clarin-1 gene: Encodes for Clarin-1 protein ( a four transmembrane protein)

CLRN1N48K mutation leads to loss of glycosylation site

Identify small molecule that inhibits degradation of mutant CLRN1N48K and restores trafficking of mutant CLRN1N48K to the cell surface

ribosome

CLRN1 CLRN1N48K

healthy cell CLRN1N48K CLRN1N48K

+ small molecule

Tian, J Biol Chem 2009

PHENOTYPIC ASSAY DEVELOPMENTHigh Content Assay used


HEK293 Clarin-1 N48K-HA

Treat cells with compound

Fix and stain with DAPI and anti-HA Ab

DAPI-stained nuclei

nuclei

cells

Clarin-1-HA stained cells

HTS SCREEN


Screen ~50,000 compounds

48 compounds selected for secondary screen

Counter screen to eliminate proteosome inhibitors

5001000150020002500300035004000

50 6.4 3.2 0Bortezomib

(nM) BF942 concentration (µM)

50001000015000200002500030000350004000045000

50 6.4 3.2 0

Bortezomib (nM)

BF942 concentration (µM)

Num

ber o

f Cel

lsDe

nsity

/ Ce

ll

NN N

N

Cl

BF942EC50 2.0 µM

STRUCTURE ACTIVITY RELATIONSHIP


NN

R1

NN

ClR3

R2

Cl, Br, F: activeMe, H, OMe, CHO, SMe, CH2Cl: inactive

N

S

F

0.8 µM 1.5 µM 1.2 µM

0.8 µM >15 µM

N

0.8 µM 2.0 µM 2.4 µM

2.0 µM 0.5 µM

NN

N

Cl

NN

N

Cl

Inactive at 25µM

O

N

N

N

O

O NN

0.2 µM 0.63 µM 0.56 µM 0.85 µM

NN

NN

Cl

BF934EC50

0.31 uM

TARGET IDENTIFICATION EXPERIMENTS


Biotin labelled compound prepared

Cell lysate incubated with BF071

Labelled proteins extracted and separated

2 labelled bands identified by MS as HSP60 and HSP90

HSP60

HSP90

Mw(kDa)

250

150

100

75

50

37

25

NN N

N

Cl

NH

O

NHO

SHN

NHO

H

H

BF071EC50

1.6 uM

Alagramam KN et al. Nat Chem Biol. 2016 Jun;12(6):444-51

TARGET IDENTIFICATION EXPERIMENTS


Activity of HSP60 (a) and HSP90 (b) measured in presence of BF844 and related inactive compounds BF066 and BF136

NN

NN

Cl

OH

NN

NN

Cl

NN

NN

Cl

BF844 BF066 BF136

0.36 µM Inactive Inactiveat 26µM at 26µM


A NEW MOUSE MODEL OF USHIII


Transgenic Clrn1N48K/N48K (KI/KI) mice developed expressing wt CLRN1 under control of Atoh1 gene enhancer; this allows normal development of hearing and vision, but is turned off later in life, leaving only N48K to be expressed

Mice show delayed-onset progressive hearing loss compared to Clrn1N48K/N48K (KI/KI) mice

P22 P35 P46 P55 P70


Control KI/KI Tg:KI/KI Control KI/KI Tg:KI/KI Control KI/KI Tg:KI/KI Control KI/KI Tg:KI/KI Control KI/KI Tg:KI/KI

EFFICACY RESULTS


0

10

20

30

40

50

60

70

80

90

100

8 16 32

Med

ian

Thre

shol

d He

arin

g (d

B)

Sound Frequency (kHz)

Median ABR thresholds in BF844 treated versus untreated Tg;KI/KI mice at P55

Control (WT)

Vehicle (Tg;KI/KI)

Regimen I (Tg;KI/KI)

Regimen II (Tg;KI/KI)

NN

NN

Cl

BF844 OH

10,000 foldimprovement


Biochemical assay development

HIT FINDING APPROACHES AT CHARLES RIVER


HTS860,000 compound library

Industry standard automation and informatics

>60 screens since 2014

Fragments2,500 compound library

Fragment to active in silico tools

Orthogonal biophysical platforms

> 20 fragment screens run

PhenotypicHCS platforms

RNA platforms

HT-FACS

Decade of experience of human primary and patient derived cell models

Knowledge-BasedStrong CADD input

Industry standard software and proprietary tools

Significant medicinal chemistry expertise in knowledge-based design and SBDD

Multiple approaches – use the most appropriate (combinations)

ASSAY DEVELOPMENT AND HTS

Highly experienced assay development and HTS teams

Projects supported by cell line generation and protein production

Broad and diverse screening technology base

Comprehensive compound collection ~ 860,000 compounds

Confidence based on experience• > 15 year history of providing HTS services• > 70 HTS completed since 2014

Seamless hit to lead and lead optimization options• 74 development compounds identified • 25% of candidates have achieved clinical PoC


TYPICAL HTS WORKFLOW

33

Potency determination phase against Assay#1 and Assay #2: compounds tested as 10-point curves, n=2

Testing for compounds for purity determination

Assay transfer (or development) and validation for primary HTS assay (Assay#1)

Assay transfer (or development) and validation for counter-screen assay(s) (Assay#2)

AssayI#1 pilot screen:5,000 -10,000 compounds, n=2

Assay#1 primary screen:single concentration, n=1

Go/No-go decision point (Client)

Go/No-go decision point (Client)

Hit Compound Selection (CRL/Client) / Go/No-go decision pointHit Confirmation testing Assay#1 and Assay #2:

single concentration, n=2

Hit Compound Selection (CRL/Client) / Go/No-go decision point

FROM HTS TO CANDIDATE IDENTIFICATION


Primary screening hit confirmation

Potency determination

and LCMS analysis

Assay development /

transferCompound selection, plating, pilot screen Medicinal Chemistry

Computational hit expansion, screening

Full and open data and structure disclosureFlexibility on hit calling criteriaInclusion of interference filters

Frequent hitter analysis

COMPREHENSIVE ASSAY PLATFORM COVERAGE


• Qube, Sophion (x1)

• IonWorks Quattro, MDS (x2)

• IonWorks Baracuda, MDS (x2)

• PatchXpress, MDS (x3)

• Qpatch, Sophion (x2)

• Conventional ephys (x9)

• FLIPRTetra, MDS (x4)

• FDSS6000, Hamamatsu (x1)

• ViewLux, PE (x2)

• Caliper LabChip (x1)

• Envision, PE (x8)

• InCell 2200, GE (x3)

• InCell 6000 confocal (x1)

• Meso Scale Discovery (x3)

• Luminex FlexMAP

• Roche real time Q-PCR (96 & 384)

• Biorad QX 200 digital droplet PCR

• Agilent Tapestation

• Accumen, TTP

• Microbeta Trilux, PE (x4)

• Top Count x2

• HTMS system; ADDA Sciex (x1)

• Biacore T200 (x1) & 4000 (x1)

• LI-COR Odyssey (x2)

• Maxcyte STX transfection platform (x1)

• Labcyte Echo acoustic dispenser (x1)

• FACS (BD FACS Canto) (x1)

• ACEA xCELLigence RTCA Cardio (x1)

• ACEA xCELLigence RTCA CardioECR (x1)

• Axion Maestro Multi-electrode array (MEA) (x1)

• Nanion Technologies CardioExcyte96 (x2)

• Comprehensive range of automatic dispenser tip based and acoustic dispensing systems

• Including LAF housed systems

http://www.google.co.uk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=kKm9FPjY21wPOM&tbnid=y0E1VF8dOOaY7M:&ved=0CAUQjRw&url=http://ttplabtech.com/liquid-handling/mosquito_x1/&ei=JBavUu6BMqar7AbI6YHADw&bvm=bv.57967247,d.ZGU&psig=AFQjCNHk2x2-gKMeYbrMMIxoyRGPJKMURg&ust=1387292508076483

http://www.google.co.uk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=kKm9FPjY21wPOM&tbnid=y0E1VF8dOOaY7M:&ved=0CAUQjRw&url=http://ttplabtech.com/liquid-handling/mosquito_x1/&ei=JBavUu6BMqar7AbI6YHADw&bvm=bv.57967247,d.ZGU&psig=AFQjCNHk2x2-gKMeYbrMMIxoyRGPJKMURg&ust=1387292508076483

HTS SPECIFIC ASSAY CONSIDERATIONSTypical considerations

36

Consideration Common factors

Reagents Availability (batch?)Stability with timeStability of expression (for cellular targets)

Control compounds/conditions Are they available?Are they valid?

Assay robustness Appropriate pharmacologyDMSO toleranceZ’-factor & signal window (under valid control conditions)Signal stability

Assay format 384 or 1536-wellAssay volumeReagent availability and costFalse positive liability

Consideration Common factors

Confidence in hits and hit rate False +ve and -ve ratesAssay noisePredicted activity threshold and hit ratePositional effects on data distributionScreening concentration?

Automation Can the assay be scaled?Liquid handling considerationsLiquid hander QC interval

Data handling Processing volume of dataError trappingPass/fail criteriaReporting

Hit progression Orthogonal assaysSelectivity assays

ASSAY STATISTICS


Z’, kappa statistics

Z-factor Interpretation1.0 Idealbetween 0.5 and 1.0 An excellent assaybetween 0 and 0.5 A marginal assayless than 0 There is too much overlap between the positive and

negative controls for the assay to be useful

When running duplicates:Calculate concordance for hit-calling using kappa statistics

kappa=1: perfect concordance beween duplicateskappa=0: random distributionCriterium: kappa>0.2

HTS 2014-16 SUMMARY

74 HTS campaigns, 39 Clients

14 million compounds screened:

Average number of compounds screened: 180,000 (excluding focused screens)• 384 and 1536-well screening formats

Range of target types:

38

Client Mixed CRL compounds only

16 26 58

0 5 10 15 20 25

AntibacterialEnzyme

EpigeneticGPCR

Ion ChannelPhenotypic

PPIProtein Binding

Transporter

HTS EXPERIENCE (2014-2016)


Target class Biochemical CellularAnti-bacterial 4Enzyme 15 4Epigenetic 4GPCR 4 8Ion Channel 6Kinase 5Other 1Pathway 2Phenotypic 3PPI 11 1Protein binding 1Transporter 4Total in 3 years 42 31

Format # screens# compounds (avg)

# compounds (max)

AlphaScreen 5 255,600 414,000Colorimetric 12 148,192 200,000FLIPR 9 209,200 667,000Fluorescence 2 295,000 500,000FP 3 233,333 300,000FRET 3 223,000 420,000HCS 2 195,000 200,000HT-MS 4 275,625 302,500HTRF 12 175,250 250,000IW Barracuda 1 100,000 100,000Luminescence 6 112,110 200,000Radiometric 11 235,345 800,000Biochemical 42 217,170 800,000Cellular 31 162,400 667,000

Format # screens# compounds (avg)

# compounds (max)

Cellular Assay Development

DISCOVERY ION CHANNELS

41

Electrophysiology: Automated and conventional patch-clamp

Conventional patch-clamp (x9)

QPatch HTX 48 (x2)

PatchXpress (x3)

IonWorks Quattro (x2)Qube with stacker (x1)

IonWorks Barracuda (x2)


UK US UK

UK

US

UK/US Instrument Seal resistance

Recording wells/ cells

(per instrument)

Approx. plates/ repeats per day Wells per week

Manual patch clamp Giga-ohm 1 8 40

PatchXpress Giga-ohm 16 16 1,280

QPatch HTX Giga-ohm 48 16 3,840

IonWorks Quattro Mega-ohm 384 8 15,360

IonWorks Barracuda Mega-ohm 384 8 15,360

Qube (with stacker) Giga-ohm 384 16 30,720

HCS PLATFORM & EXPERIENCE


Routine use of high content read-outs for more than a decade• First generation: in-house built equipment and algorithms, described in Nat Biotechnol. 2002 Nov;20(11):1154-7.• Second generation (2006-2011): GE InCell Analyzer 1000• Third generation (2009-2013: GE InCell Analyzer 2000 and BD Pathway 435• Current generation: InCell 2200 (3x), InCell 6000

Centralized server (36TB) for data storage, four workstations for data analysis

>70 man years HCS expertise

>50 novel HCS assays developed over past 5 years

HIGH CONTENT CAPABILITY


Quantification of events in different cellular populations at the subcellular levelto measure:

Separation of toxicity and on target pharmacology

Nuclear blebbing/ condensation, Micro-nuclei,

mitochondrial function

Differentiation using markers/morphology

Neurite Outgrowth/ retraction

Subcellular biomarker trafficking

Cytosolic to nuclear translocation

Real time events

Calcium signalling in ES cell derived

cardiomyocytes

HUMAN PRIMARY CELL EXPERIENCE


Adipocytes• non-diseased / Type-2 Diabetes

Astrocytes

Basophils• blood-derived

Beta cells (pancreatic islets)

Bronchial epithelial cells• control / COPD / Cystic Fibrosis / IPF

Chondrocytes• non-diseased / RA

Dendritic cells

Endothelial cells

Fibroblasts • synovial / dermal / cardiac / lung • control / COPD / RA / IPF / SSc / HD

Hepatocytes• control / Type-2 Diabetes

Keratinocytes• control / SSc

Macrophages• control / Huntington’s disease

Mast cells

Mesangial cells

Neurons• human stem cell-derived (iPSC/hESC/fetal)• rodent primary neurons

Neutrophils• blood-derived / CD34+-derived

Osteoblasts• human mesenchymal stem cells

Skeletal myoblasts and myotubes• control / muscular dystrophy• human / mouse

CELLULAR ASSAY CASE STUDYApproach for a cytoplasmic-nuclear translocation read-out

• Compound selection (diversity filter)• Phase 1: Assay development by high content imaging• Phase 2: Assay automation

• Phase 3: Pilot screen• Phase 4: HTS

• Phase 5: Confirmation and dose-response curves• Phase 6: Hit expansion

Assay development

Drug Discovery

Screening

Validation

HIGH CONTENT SCREEN CASE STUDYAssay set-up

CRL diverse compound collection

InCell (GE Healthcare)BenchCel®, Bravo™(Agilent )MultiDrop (Thermo Scientific)

D0Seeding Cells

D1Compound addition

DxRead-out: Nuclear translocation

Hit compounds

ASSAY DEVELOPMENT GFP LINE algorithm development for nuclear translocation

Ctrl pos 100 nM pos 5000 nM

• Clear nuclear translocation detected

• red circles: no nuclear translocation• green circles, cells showing translocation

ASSAY DEVELOPMENT ANTIBODY STAININGalgorithm development for nuclear translocation

Ctrl pos 100 nM pos 5000 nM

• Clear nuclear translocation detected

• More background

• Lower throughput

• red circles: no nuclear translocation• green circles, cells showing translocation

ASSAY AUTOMATION - GFP STABLE CELL LINE

Up to 1% DMSO does not affect nuclear count or translocationExcellent assay window with positive control

• intermediate concentration is sufficient for maximal nuclear translocation

Low variation within and between plates

DMSO tolerance and positive control

1.00

%

0.50

%

0.25

%

0.10

%

0.00

%

5µM

2µM

1µM

0.5µ

M

0.1µ

M0

20

40

60

80

100

DMSO Torin1

% N

ucle

ar T

rans

loca

tion

1.00

%

0.50

%

0.25

%

0.10

%

0.00

%

5µM

2µM

1µM

0.5µ

M

0.1µ

M

0

300

600

900

Plate 1

Plate 2

DMSO Torin1N

ucle

ar C

ount

Pos control Pos control

PILOT SCREENFinding optimal library concentration

Source 1 Source 2 Source 3 Source 4

5 µM

10 µM

10 µM

20 µM

SCREEN

Heat map of Normalized Corrected % Nuclear Translocation

Results of a single batch of 47 x 384-well

High % translocation

Low % translocation

No plate positional effect observed

SCREEN Results of a single batch of 47 x 384-well

% nuclear translocation % activity vs controls Robust Z-score (samples)

Nuclear count

DMSO Torin1 Samples0

20

40

60

80

100

Raw

% N

ucle

ar T

rans

loca

tion

DMSO Torin1 Samples0

1000

2000

3000R

awN

ucle

i Cou

nt

DMSO Torin1 Samples-25

0

25

50

75

100

125

Nor

mal

ized

% N

ucle

ar T

rans

loca

tion

DMSO Torin1 Samples

0

20

40

60

Rob

ust

Z-sc

ore

% N

ucle

ar T

rans

loca

tion

Pos Ctrl Pos Ctrl

Pos Ctrl

Pos Ctrl

SCREEN Hit-rate

Batch 1 Batch 2 Batch 3Number of plates 46 46 47

Number of compounds 15853 16035 16474Robust Z’ factor of controls

Average 0.777 0.570 0.829Minimum 0.628 0.249 0.680Maximum 0.896 0.725 0.909

Cutoff ≥2.77Robust Z-score

≥3.84 Robust Z-score

≥2.87 Robust Z-score

Number of hits 159 173 164% Hit rate 1.00% 1.08% 1.00%

% of DMSO as hits 0.95% 0.87% 2.66%% of Pos Ctrl as hits 100% 100% 100%

CONCENTRATION RESPONSE CURVES

DT2000-0102795

log (Conc [M])-9 -7 -5

-20

0

20

40

60

80

100

120

0

100

200

300

400

500

600

700

DT2000-0102795

log (Conc [M])-9 -7 -5

-1

0

1

2

3

4

5

0

100

200

300

400

500

600

700

Ave. EC50 2.923 µM Ave. EC50 3.509 µM

Activity and cytotoxicity at same concentration

% N

ucle

ar T

rans

loca

tion

Ratio

IxA

(Nuc

/Cel

l)

Nuclei Count

Nuclei Count

Two examples

Ave. EC50 2.251 µM Ave. EC50 3.220 µM

DT2009-0225479

log (Conc [M])-9 -7 -5

-20

0

20

40

60

80

100

120

0

100

200

300

400

500

600

700

DT2009-0225479

log (Conc [M])-9 -7 -5

-1

0

1

2

3

4

5

0

100

200

300

400

500

600

700

% N

ucle

ar T

rans

loca

tion

Ratio

IxA

(Nuc

/Cel

l)

Nuclei Count

Nuclei Count

Clear dissociation of activity and cytotoxicity

PRIMARY CELL AND BIOMARKER ASSAYS


• Human Eosinophil Chemotaxis• Confirmation of MOA

• T Cell clone cytokine release• Confirmation of T cell activity• Confirmation of on target activity

• Whole blood eosinophil shape change• Used for routine potency screening • Assessment of plasma protein binding

• Whole blood assay used as clinical biomarker • Transferred to client clinical trials group• Used as efficacy marker and for patient selection • Currently in Phase IIb

0.01 0.1 1 10 100 1000-25

0

25

50

75

100

125

[Compound] (nM)

% In

hibi

tion

T-Cell clone cytokine release

1 10 100 1000-25

0

25

50

75

100

125

[Compound] (nM)

% In

hibi

tion

Whole blood Eosinophil shape change

CONCLUSIONS


• Build confidence in your target / mechanism of action• Decide on best strategy to find novel chemical matter / create an IP position• Make sure you design assays where you understand / capture the pharmacology• Consider selectivity assays and translational / biomarker asssays as early as

possible

Science

Target Validation / Biochemical and Cellular Assay Development