Part 1 of Ohio State's Drug Development Bootcamp

1. TARGET VALIDATION /
BIOCHEMICAL AND CELLULAR
ASSAY DEVELOPMENT
EVERY STEP OF THE WAY
EVERY STEP OF THE WAY1
David Fischer March 28, 2017

2. CONTENTS
2
1 Introduction
2 Target validation
3 Biochemical assay development
4 Cellular assay development

3. Library
Design
Analytical &
Purification
Process
ChemistryCADD
Synthetic
Chemistry
Formulation
Pharmaceutics
Chemo-
genomics
Ch
‘END TO END’ INTEGRATED DRUG DISCOVERY
3 EVERY STEP OF THE WAY
B/D
Molecular
Biology
Cell Line
Generation
FBDDStructural
Biology
CRISPR
Adenoviral
Platform Human 1o Cells
Functional
Genomics
D/P
S/I
Safety
Assessment
Safety
Pharmacology
Non-GLP/GLP
Toxicology
Anatomic &
Clinical Pathology
Imaging
Animal Model
Development
Large Animal
Efficacy Models
Discovery
Pathology
in vivo
Efficacy
In Vivo
Validation
PK/PD
Dose to Human
Predictions
ADME
Bioanalysis
Targets
Clinical
Candidate
Pharmacology
in vitro/in vivo
Hit Finding:
HTS, HCS
IND Enabling
Studies
Medicinal
Chemistry
Biomarker
Development
Target
Discovery
& Validation
DP DP
Discovery Pathway
Chemistry
Biology/ Discovery Technologies
DMPK/Pharmacology/Safety/
In vivo models

4. DISCOVERY – CENTERS OF EXCELLENCE
4 EVERY STEP OF THE WAY
CNS
Complex cell biology
Integrated drug discovery
Oncology
Metabolic disease
Inflammation Oncology
Ion channel
ONCOLOGY
CNS PAIN
CARDIOVASCULAR
METABOLIC
DISEASE
INFLAMMATION
IMMUNOLOGY
RESPIRATORY
DISEASE
RARE AND
NEGLECTED
DISEASE
OCULAR
DISEASE

5. BREADTH OF THERAPEUTIC AREA EXPERTISE
5 EVERY STEP OF THE WAY
Extensive integrated drug discovery expertise across multiple therapeutic areas
ONCOLOGY CNS IMMUNOLOGY CV/ METABOLISM RESPIRATORY
TARGET
DISCOVERY AND
VALIDATION
Adenovirus 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 models
Syngeneic models
Humanized models
Xenograft models
Neurology
Psychiatry
Neuropathic pain
Neuromuscular deficiency
Neurodegenerative
disease
Psoriasis
T-cell activation
Peritonitis
Colitis
Osteoarthritis
Cytokine release
Vaccine assessment
Diabetes
Diabetic complications
Atherosclerosis
NASH
Asthma
COPD
Pulmonary inflammation
Mucocilliary clearance
Cough
Fibrosis
BIOMARKER
DEVELOPMENT
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

6. COMPELLING SUCCESS RATES IN
SMALL MOLECULE DISCOVERY
6
74 preclinical candidates to date
1 Nature Drug Discovery, 2010, 9, 203; DDT, 2003, 8(23), 1067; DDT, 2013, 19(3), 341
2 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 AREA
NO. OF
CANDIDATES Preclinical Phase I Phase IIa Phase IIb Phase III Registration
Inflammation 13
CHEMOKINE, INTEGRIN, GPCR, CYTOKINE, KINASE, ENZYME
Respiratory 26
GPCR, PROTEASE, NHR, KINASE
CNS 8
GPCR, NHR
Metabolic disease 5
ENZYME, KINASE, PROTEASE, NHR
Oncology 16
ENZYME, KINASE, PPI, NHR
Anti-bacterial 2
UNKNOWN
Anti-viral 1
PROTEASE
Cardiovascular 2
ION CHANNEL
Secretory diarrhoea 1
ION CHANNEL

7. IN VITRO DISCOVERY PLATFORMS
7 EVERY STEP OF THE WAY
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

8. OUR DISCOVERY TEAM
8 EVERY STEP OF THE WAY
>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

9. Target Validation
9EVERY STEP OF THE WAY

10. 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

11. EXAMPLE OF LITERATURE VALIDATION
11 EVERY STEP OF THE WAY
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

12. CRISPR/CAS9 GENOME EDITING
12 CONFIDENTIAL INFORMATION
Principles
Cas9
5’
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

13. EMT6 BRCA1 KNOCKOUT
Strategy using HDR
13 EVERY STEP OF THE WAY
Nucleofection
• 10ug DNA
• 1x10^6 cells
Brac1 – KO
• conserve Start codon
• remove Exon2
• add stop-tag
• introduce frame shift
• remove splice signal
EMT6
10 μg DNA
Brightfield 24 hours 48 hours
10x
113 bp deletion (*black)

14. EMT6 BRCA1 KNOCKOUT
Results
14 EVERY STEP OF THE WAY
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 cells
4T1 and
EMT6
D-x
Transform
0.5x10^6 cells
with 30 ug DNA
Add SCR7
D3
Nucleofection
Stop
Selection
G418
Harvest and
dilute to
single cell
colonies
D4 DX
-dublicate
single 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)

15. SILENCESELECTTM
EVERY STEP OF THE WAY
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

16. ADENOVIRUSES FOR shRNA DELIVERY
Advantages
16 EVERY STEP OF THE WAY
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
mRNAknock-down
gene X

17. 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%

18. ADENOVIRAL TRANSDUCTION
18 EVERY STEP OF THE WAY
Example: Human Primary Cells
Pre-adipocytes Adipocytes Hepatocytes

19. CASE STUDY TARGET DISCOVERY
EVERY STEP OF THE WAY
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
40
50
60
70
80
90
100
110
Hit 8
Empty
Hit 1
10µM Fsk
50µM Gst
10µM CFTRinh
10µM amiloride
Time (min)
Isc(µA.cm-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 Ussing
chambers
19
19 targets validated for drug discovery
19

20. CASE STUDY TARGET VALIDATION
EVERY STEP OF THE WAY
• 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

21. USHER III SYNDROME
Case study target deconvolution
21 EVERY STEP OF THE WAY
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:

22. TREATMENT HYPOTHESIS
Towards the identification of a small molecule therapy for Usher III
22 EVERY STEP OF THE WAY
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

23. PHENOTYPIC ASSAY DEVELOPMENT
High Content Assay used
23 EVERY STEP OF THE WAY
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

24. HTS SCREEN
24 EVERY STEP OF THE WAY
Screen ~50,000 compounds
48 compounds selected for secondary screen
Counter screen to eliminate proteosome inhibitors
500
1000
1500
2000
2500
3000
3500
4000
50 6.4 3.2 0
Bortezomib
(nM)
BF942 concentration (µM)
5000
10000
15000
20000
25000
30000
35000
40000
45000
50 6.4 3.2 0
Bortezomib
(nM)
BF942 concentration (µM)
NumberofCellsDensity/Cell
N
N N
N
Cl
BF942
EC50 2.0 µM

25. STRUCTURE ACTIVITY RELATIONSHIP
25 EVERY STEP OF THE WAY
N
N
R1
N
N
Cl
R3
R2
Cl, Br, F: active
Me, 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
N
N
N
Cl
N
N
N
Cl
Inactive at 25µM
O
N
N
N
O
O N
N
0.2 µM 0.63 µM 0.56 µM 0.85 µM
N
N
N
N
Cl
BF934
EC50 0.31 uM

26. TARGET IDENTIFICATION EXPERIMENTS
26 EVERY STEP OF THE WAY
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
N
N N
N
Cl
NH
O
NH
O
S
HN
N
HO
H
H
BF071
EC50 1.6 uM
Alagramam KN et al. Nat Chem Biol. 2016 Jun;12(6):444-51

27. TARGET IDENTIFICATION EXPERIMENTS
27 EVERY STEP OF THE WAY
Activity of HSP60 (a) and HSP90 (b) measured in
presence of BF844 and related inactive
compounds BF066 and BF136
N
N
N
N
Cl
OH
N
N
N
N
Cl
N
N
N
N
Cl
BF844 BF066 BF136
0.36 µM Inactive Inactive
at 26µM at 26µM
Alagramam KN et al. Nat Chem Biol. 2016 Jun;12(6):444-51

28. A NEW MOUSE MODEL OF USHIII
28 EVERY STEP OF THE WAY
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
Alagramam KN et al. Nat Chem Biol. 2016 Jun;12(6):444-51
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

29. EFFICACY RESULTS
29 EVERY STEP OF THE WAY
0
10
20
30
40
50
60
70
80
90
100
8 16 32
MedianThresholdHearing(dB)
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)
N
N
N
N
Cl
BF844
OH
10,000 fold
improvement
Alagramam KN et al. Nat Chem Biol. 2016 Jun;12(6):444-51

30. Biochemical assay
development

31. HIT FINDING APPROACHES AT CHARLES RIVER
31 EVERY STEP OF THE WAY
HTS
860,000 compound
library
Industry standard
automation and
informatics
>60 screens since 2014
Fragments
2,500 compound
library
Fragment to active in
silico tools
Orthogonal biophysical
platforms
> 20 fragment screens
run
Phenotypic
HCS platforms
RNA platforms
HT-FACS
Decade of experience of
human primary and
patient derived cell
models
Knowledge-Based
Strong 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)

32. 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
32 EVERY STEP OF THE WAY

33. 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 point
Hit Confirmation testing Assay#1 and Assay #2:
single concentration, n=2
Hit Compound Selection (CRL/Client) / Go/No-go decision point

34. FROM HTS TO CANDIDATE IDENTIFICATION
34 EVERY STEP OF THE WAY
Primary screening
hit confirmation
Potency
determination
and LCMS analysis
Assay
development /
transfer
Compound selection,
plating, pilot screen Medicinal Chemistry
Computational hit
expansion,
screening
Full and open data and structure disclosure
Flexibility on hit calling criteria
Inclusion of interference filters
Frequent hitter analysis

35. COMPREHENSIVE ASSAY PLATFORM COVERAGE
35 EVERY STEP OF THE WAY
• 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

36. HTS SPECIFIC ASSAY CONSIDERATIONS
Typical considerations
36
Consideration Common factors
Reagents Availability (batch?)
Stability with time
Stability of expression (for cellular
targets)
Control compounds/conditions Are they available?
Are they valid?
Assay robustness Appropriate pharmacology
DMSO tolerance
Z’-factor & signal window (under valid
control conditions)
Signal stability
Assay format 384 or 1536-well
Assay volume
Reagent availability and cost
False positive liability
Consideration Common factors
Confidence in hits and hit rate False +ve and -ve rates
Assay noise
Predicted activity threshold and hit rate
Positional effects on data distribution
Screening concentration?
Automation Can the assay be scaled?
Liquid handling considerations
Liquid hander QC interval
Data handling Processing volume of data
Error trapping
Pass/fail criteria
Reporting
Hit progression Orthogonal assays
Selectivity assays

37. ASSAY STATISTICS
37 EVERY STEP OF THE WAY
Z’, kappa statistics
Z-factor Interpretation
1.0 Ideal
between 0.5 and 1.0 An excellent assay
between 0 and 0.5 A marginal assay
less 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
duplicates
kappa=0: random distribution
Criterium: kappa>0.2

38. 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
Antibacterial
Enzyme
Epigenetic
GPCR
Ion Channel
Phenotypic
PPI
Protein Binding
Transporter

39. HTS EXPERIENCE (2014-2016)
39 EVERY STEP OF THE WAY
Target class Biochemical Cellular
Anti-bacterial 4
Enzyme 15 4
Epigenetic 4
GPCR 4 8
Ion Channel 6
Kinase 5
Other 1
Pathway 2
Phenotypic 3
PPI 11 1
Protein binding 1
Transporter 4
Total in 3 years 42 31
Format # screens
# compounds
(avg)
# compounds
(max)
AlphaScreen 5 255,600 414,000
Colorimetric 12 148,192 200,000
FLIPR 9 209,200 667,000
Fluorescence 2 295,000 500,000
FP 3 233,333 300,000
FRET 3 223,000 420,000
HCS 2 195,000 200,000
HT-MS 4 275,625 302,500
HTRF 12 175,250 250,000
IW Barracuda 1 100,000 100,000
Luminescence 6 112,110 200,000
Radiometric 11 235,345 800,000
Biochemical 42 217,170 800,000
Cellular 31 162,400 667,000
Format # screens
# compounds
(avg)
# compounds
(max)

40. Cellular Assay
Development

41. 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)
EVERY STEP OF THE WAY
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

42. HCS PLATFORM & EXPERIENCE
42 EVERY STEP OF THE WAY
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

43. HIGH CONTENT CAPABILITY
43 EVERY STEP OF THE WAY
Quantification of events in different cellular populations at the subcellular level
to 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

44. HUMAN PRIMARY CELL EXPERIENCE
44 EVERY STEP OF THE WAY
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

45. CELLULAR ASSAY CASE STUDY
Approach 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

46. HIGH CONTENT SCREEN CASE STUDY
Assay set-up
CRL diverse compound collection
InCell (GE Healthcare)BenchCel®, Bravo™(Agilent )MultiDrop (Thermo Scientific)
D0
Seeding Cells
D1
Compound addition
Dx
Read-out: Nuclear translocation
Hit
compounds

47. 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

48. ASSAY DEVELOPMENT ANTIBODY STAINING
algorithm 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

49. ASSAY AUTOMATION - GFP STABLE CELL LINE
Up to 1% DMSO does not affect nuclear count or translocation
Excellent 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
0.5µM
0.1µM0
20
40
60
80
100
DMSO Torin1
%NuclearTranslocation
1.00%
0.50%
0.25%
0.10%
0.00%
5µM
2µM
1µM
0.5µM
0.1µM
0
300
600
900
Plate 1
Plate 2
DMSO Torin1NuclearCount
Pos control Pos control

50. PILOT SCREEN
Finding optimal library concentration
Source 1 Source 2 Source 3 Source 4
5 µM
10 µM
10 µM
20 µM

51. 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

52. SCREEN
Results of a single batch of 47 x 384-well
% nuclear translocation % activity vs controls Robust Z-score (samples)
Nuclear count
DMSO Torin1 Samples
0
20
40
60
80
100
Raw
%NuclearTranslocation
DMSO Torin1 Samples
0
1000
2000
3000Raw
NucleiCount
DMSO Torin1 Samples
-25
0
25
50
75
100
125
Normalized
%NuclearTranslocation
DMSO Torin1 Samples
0
20
40
60
RobustZ-score
%NuclearTranslocation
Pos Ctrl Pos Ctrl
Pos Ctrl
Pos Ctrl

53. SCREEN
Hit-rate
Batch 1 Batch 2 Batch 3
Number of plates 46 46 47
Number of compounds 15853 16035 16474
Robust Z’
factor of
controls
Average 0.777 0.570 0.829
Minimum 0.628 0.249 0.680
Maximum 0.896 0.725 0.909
Cutoff
≥2.77
Robust 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%

54. 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
%NuclearTranslocation
RatioIxA(Nuc/Cell)
NucleiCount
NucleiCount
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
%NuclearTranslocation
RatioIxA(Nuc/Cell)
NucleiCount
NucleiCount
Clear dissociation of activity
and cytotoxicity

55. PRIMARY CELL AND BIOMARKER ASSAYS
55 EVERY STEP OF THE WAY
• 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)
%Inhibition
T-Cell clone cytokine release
1 10 100 1000
-25
0
25
50
75
100
125
[Compound] (nM)
%Inhibition
Whole blood Eosinophil shape change

56. CONCLUSIONS
56 EVERY STEP OF THE WAY
• 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