Integrated Solution for Epigenetic Drug Discovery and Development

Elizabeth Quinn, PhDDirector, LeadHunter Discovery Services

equinn@discoverx.com12th September 2013

DiscoveRx  Platform Technologies 

Product & Service Solution Portfolio

LeadHunter Services

Quantitative Binding

Enzyme Complementation

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Assay Development

Kits and Cell Lines

Primary Human Cell Profiling

>80% Coverage of Druggable Targets + Primary Cell Phenotypic Assays 

HDAC/HMT

Dep

th of C

overage

100%

0%284

394 21 23

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GPCRs Kinases NHRs Pathways Bromodomain

1,118 assays covering 751 druggable targets• All major drug target classes covered• Multiple assays for single target

Primary Cell Profiling 

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Challenges: • Limited HTP compound screening options• Cellular toxicity and adverse effects have resulted in low uptake of epigenetic therapies in the clinic

Opportunities: • Proven therapeutic potential for epigenetic targets 

• Innovative approaches combined with “lessons‐learned” can help guide therapeutic development in the epigenetic space

Targeting Epigenetics For Therapeutic DevelopmentHDAC, Demethylases and Bromodomain Target Classes

Integrated Strategy for Therapeutic Development

CellularActivity

Primary Cell Model Systems

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“…knowing a target is not the same as knowing what the target does, let alone knowing the effects of a chemical inhibitor in diverse disease settings.”   Nature Biotech 22, 1253‐1259 (2004).

HTSSAR

Integrated Strategy for Therapeutic DevelopmentKey Questions

• Hit identification• Is my compound potent and selective?

• What is the phenotypic impact of my compound in the context of  disease?

• What efficacy and safety biomarkers are detected?

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• Does my compound bind the target in a cellular environment?

Relevant assays + optimized tool compounds  Increased probability of success

CellularActivity

Primary Cell Model Systems

HTSSAR

Integrated Strategy for Therapeutic DevelopmentTarget Biochemistry: SAR

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Quantitative Binding

• Hit identification• Is my compound potent and selective?

CellularActivity

Primary Cell Model Systems

HTSSAR

Epigenetic Proteins

Modification Write Erase Read

Acetyl HAT HDAC Bromo

Methyl HMT HDM Chromo, PHD, Tudor, MBT

Enzyme Enzyme Binding8

KINOMEscan® Technology ‐ The Solution

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KINOMEscanapplied to BRDs

World’s Largest Kinase Panel• Cover >80% human kinome• Now 456 assays – lipid, TK, mutants• Enables new drug discovery paradigms

Our Goal for Bromodomains• Leverage our expertise • Build a first in class panel of bromodomain binding assays

BROMOscan Technology: How it Works

Measure amount of bromodomain bound to immobilized ligand in the presence and absence of test compound

Competition No Competition

‐ Test Compound + Test Compound

Ultrasensitive qPCR readout (6‐log range)

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Bromodomain Assay Validation

• Small Molecules‐ Measure Kds consistent with known ITC values (when available)

‐ Inhibitors: • JQ1 (active enantiomer)• I‐BET (active and inactive enantiomers)• Other known BRD inhibitors

• Acetylated Histone Tail Peptide Ligands‐ Measure Kds consistent with published values (when available)

‐ Demonstrate selectivity for acetylated over non‐acetylated peptides

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Bromodomain Menu

Bromodomain Targets

ATAD2A BRD3(1,2) BRDT(2) PBRM1(5)ATAD2B BRD4(1) BRDT(1,2) PCAFBAZ2A BRD4(2) BRPF1 SMARCA2BAZ2B BRD4(1,2) BRPF3 TAF1(2)

BRD1BRD4(full‐length, short‐iso) CECR2 TAF1L(2)

BRD2(1) BRD7 CREBBP TRIM24(Bromo.)BRD2(2) BRD8(1) EP300 TRIM24(PHD,Bromo.)BRD2(1,2) BRD8(2) FALZ TRIM33(PHD,Bromo.)BRD3(1) BRD9 GCN5L2 WDR9(2)BRD3(2) BRDT(1) PBRM1(2)

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Roman numerals indicate SGC‐defined families

Human Bromodomain Dendrogram

•39 bromodomain assays •Almost 60% coverage of the entire family •Multiple putative therapeutic targets: BET, ATAD2, CECR2, and TRIM24(PHD,Bromo.)

Current BROMOscan assays

New assays available October 2013

*     Tandem BRD2, BRD3, BRDT and full‐length, short‐iso BRD4 assay also available.

Bromosporine Kd (nM)

T mSh

ift (1

00 u

M B

rom

ospo

rine)

Comparison of BROMOscan and SGC Tm Shift Data for the Promiscuous Inhibitor Bromosporine

• 24/25 BROMOscan assays analyzed• Each square represents a unique bromodomain • Outstanding agreement between BROMOscanand Tm Shift data− Cross‐validates the formats− Further quantifies Bromosporine promiscuity

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Striking agreement between two very different assay readouts

Global Validation of BROMOscan Panel

SGC Tm shift data provided by S. Knapp, PhD in collaboration with DiscoverX

BET Family Assay Validation  JQ1 Kds Consistent with Reported Values

Data Comparison• Similar Kd values measured with BROMOscan & ITC

‐ Domain affinity rank order identical• Optimized conditions ensure true thermodynamic Kd  

measurements *Nature (2010) 408: 1067

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BET Family ValidationI‐BET

Nature (2010) 468: 1119.

BROMOscan data consistent with published ITC data• Potency• Rank order• Lack of potent activity for inactive I‐BET enantiomer

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Bromodomain Inhibitor Profiles

I‐BET Bromosporine

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BET‐Selective Promiscuous

PFI‐1

Semi‐BET‐Selective

72 known kinase inhbitors tested (6% hit rate)• Potent (Kd = 0.01‐ 1 uM) & diverse BRD profiles• Inhibitors that target TKs, STKs and lipid kinases have BRD activity

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Kinase & Bromodomain Polypharmacology

BROMOscan Services Portfolio

Service Number of Assays Description

24Set of 24 human bromodomainsvalidated for single point testing

1-24 Personalized or custom panels

1-34 Quantitative binding constants

34 Kd profile of entire BROMOscanmenu

1-24Compound library screening against a single target or against the full panelLIBRARY

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14 additional assays available in October 2013

BROMOscan ‐ Drive Your Epigenetic Programs Forward

Your Collection of  Drug Candidates

Phenotypic Screens

Chemoproteomics

Biophysical assays

Crystallography

Single Target HTSFollow upKd Determinations

Selectivity Profiling

Jumpstart Your Program – Only 2 Weeks to Results!19

BROMOscan Summary

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• BROMOscan is a robust and highly validated panel of 39 bromodomain assays

• Quantitative binding information• Rigorously validated, state‐of‐the‐art reference inhibitors• Accurate, precise & reproducible

• BROMOscan can be used to rapidly generate inhibitor profiles across the entire bromodomain family 

• Identify lead compounds • Optimize potency & selectivity

• BROMOscan can be used as a discovery tool to screen existing chemical assets 

• Gain starting points for new programs• Uncover off‐target liabilities

Integrated Strategy for Therapeutic DevelopmentCellular Assays

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Quantitative Binding

• Is my compound potent and selective?

• Does my compound bind the target in a cellular environment?

Enzyme Complementation

CellularActivity

Primary Cell Model Systems

HTSSAR

• Bromodomains are not enzymatic • Cell‐based assays are typically difficult and 

low‐throughput• FRAP assays commonplace• Limited quantitation, impractical for dose 

response analysis

Dey et al. MBC 2009

Is my compound cell‐permeable? Does it interact specifically with the target? How does the biochemical potency relate to 

the cellular potency?

Challenges of Targeting BromodomainsUsing Cell‐Based Assays

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InCELL Hunter™ – Ligand Induced Stabilization

‐ In vitro compound binding enhances protein stability 

Protein PurificationStructural Biology Thermal Shift Assays

TargetConcept:

Altered Half‐lifeMaintain Steady State

+ compound

‐ Can the same principles be applied to cell‐based assays?  

‐ compound

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InCELL Hunter: How It Works

Cell‐based Bromodomain Assays  Measure compound permeability Binding to intracellular protein Determine cellular potency

• Compound activity in a native cellular environment

• Simple chemiluminescent protocol

• Bridges gap between biochemical and cellular phenotypic assays

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Ideal for studying bromodomain protein:protein interactions

Compound Binding to BRD4 in Live CellsJQ1 and IBET

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JQ1IBET

JQ1 > IBET

CMPD Lit* InCELL

JQ1 120 230

C 100n

M

1000

nM

Western Blot Confirmation

Concentration of JQ1

*Filippakopoulos, et al. Nature 468: 1067‐1073, 2010.

Ligand [M]

RLU

-12 -10 -8 -6 -40

100000

200000

300000

400000

Target Specificity and Selectivity

• Differentiate target‐selective stereoisomers and determine target specificity

• Multiple target classes available, ideal for counter‐screening

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Enantiomer Testing Counter Screening

BRD4A

Ligand [M]

RLU

-12 -10 -8 -6 -40

100000

200000

300000

400000

JQ-1 (+)

JQ-1 (-)

BRD4(1)

BRD4A

Ligand [M]

RLU

-12 -10 -8 -6 -40

100000

200000

300000 IBET (+)

IBET (-)

BRD4(1)

G9A

UNC0638 [M]

RLU

10- 1 1 10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 40

500000

1000000

1.5100 6

UNC0638JQ1(+)JQ(-)

G9A

Expansion to Tandem Domains

Domain Compound Kd (nM)BRD3(1) I‐BET 34BRD3(1) JQ1 25BRD3(1) PFI‐1 87

BRD3(2) I‐BET 27BRD3(2) JQ1 26BRD3(2) PFI‐1 72

Organization of Tandem Bromodomains

Anna C. Belkina & Gerald V. DenisNature Reviews Cancer 12, 465-477 (July 2012)

BROMOscan

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BRD3 Tandem

[M]

RLU

10- 1 1 10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 40

200000

400000

600000

800000(+)JQ-1I-BETPFI-1

BRD9 Assay Development

Kd (nM)E12348 40JQ1 >100000

Wild‐Type Sequence Binding Deficient Mutant

Point mutant studies confirm the specificity of the compound binding reaction

RLU

BottomTopLogEC50HillSlopeEC50

E12348 188537600771-5.7981.1291.594e-006

JQ1 165184191889-7.35714.874.392e-008

Compound [M]10-10 10-9 10-8 10-7 10-6 10-5 10-40

100000

200000

300000

400000

500000E12348JQ1(+)

Best-fit values

TopLogEC50HillSlopeEC50

E12348

156000220747-5.2503.1625.624e-006

JQ1(+)

173137209674-5.9342.4511.165e-006

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TAF1L(2) Assay Development

Kd (nM)E12348 15BDOIA383 14,000

Wild‐Type Sequence Binding Deficient Mutant

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RLU

BottomTopLogEC50HillSlopeEC50

E12348 [M]163561744973-5.6521.2552.231e-006

PF1 BD01A383156116200123-5.46918.853.399e-006

Mutant Studies in progress

InCELL Hunter ‐ Summary

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• InCELL Hunter is novel, ligand‐induced stabilization approach to cellular assay development

• Proven EFC technology• Robust & quantitative

• InCELL Hunter assays can be used to guide selection of lead compounds based on:

• Cell permeability• Intracellular protein binding• Cellular potency

• InCELL Hunter technology can be applied to druggable target classes including:

• Bromodomains• Methyltransferases• Kinases

Integrated Strategy for Therapeutic DevelopmentPrimary Human Cell Systems

Quantitative Binding

• Is my compound potent and selective?

• Does my compound bind the target in a cellular environment?

Enzyme Complementation

CellularActivity

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Primary Human Cell Systems

• What is the phenotypic impact of my compound in the context of  disease?

• What efficacy and safety biomarkers are detected?

Primary Cell Model Systems

HTSSAR

BioMAP® Technology PlatformOverview

BioMAPAssay Systems

ReferenceProfile Database

Predictive Informatics Tools

Human primary cells Disease‐models30+ systems

Biomarker responses to drugs are stored in the database

>3000 drugs

Specialized informatics tools are used to predict clinical outcomes

Human Biology Integrated into a Robust, Scalable Platform

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Each drug induces a signature profile

3C

SAg

BE3C

HDF3CGF

Fibroblasts (HDF)

PBMC

HT-29 Cell Line (Colon adenocarcinoma)

TCR stimulation

Tissue Primary Cell System BioMAP Profile

BioMAP® Systems Model Complex BiologyPrimary Human Cell Systems

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BioMAP Technology PlatformReference Database

BioMAP database contains profiles of >3000 agents• Drugs – Clinical stage, approved, and failed

• Experimental Chemicals ‐ Research tool compounds, environmental chemicals

• Biologics ‐ Cytokines, factors, peptides, antibodies, soluble receptors

Compound TargetI‐BET BRDJQ1 BRD

I‐BET‐151 BRDPFI‐1 BRD

Panobinostat HDACVorinostat, HDACEntinostat HDACScriptaid, HDACTrichostatin HDAC

M344 HDACUNC0624 G9a/GLP methyltransferaseGSK‐J4 Lysine Demethylase (KDM)GSK2801 BAZ2B/A BRDSGC‐CBP30 CREBBP (CBP) and EP300 

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BET Inhibitors Have Unique BioMAP Signature Common Activities of I‐BET Inhibitors at Lowest Active Dose

• BET inhibitors have highly similar BioMAP profiles (r ≥ 0.91)• Overlay signature shows broad anti‐proliferative effects on EC, T cells, CASMC 

and Fibs indicating impact on vascular, stromal and immune biology• Additional anti‐inflammatory and matrix modulating activities support potential 

for development in oncology• May also have anti‐fibrotic potential but likely not suitable for autoimmune 

diseases

TF, HLA-DR, Proliferation

VCAM VCAM IL-8, Proliferation

sIgG, IL-6 uPAR, uPA VCAM, MIG, Proliferation

EGFR, PAI-1, MMP-1, TIMP-1, Proliferation

PAI-1, SRB MMP-1, TIMP-1 VCAM, sIL-10

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Clinical HDACi Class Signature in BioMAPVorinostat, Entinostat and Panobinostat

• HDACi signature in BioMAP at lower more clinically relevant doses reveals activities related to efficacy and safety including• Strong immune suppressive effects in SAg and BT with anti‐proliferation activity 

detected on HDF• Decrease in TNFα (LPS) shows strong anti‐inflammatory effects at these low 

doses plus matrix modulation effects also indicate Potential for hematological oncology application

• Safety : Increased SAA and TF detected at higher doses  CV risk

SAA

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Similarity Clustering Confirms Target PhenotypeCompounds Compared To Database Profiles for Signature Match 

p38MAPK

BET

JAK

HDAC

• Statistical measures of profile similarity are used to cluster compounds with shared phenotypic impact irrespective of their molecular target(s). • Dot color and size depicts

compound name and dose respectively.

• Compound profiles for compounds that are statistically significantly similar (Pearson 0.9) are represented by dots connected by a line to form a cluster over multiple doses.

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BioMAP Summary of Epigenetic Inhibitors 

• BioMAP profiles can be used to classify compounds based on phenotypic impact on primary human cell systems• Determine target or mechanism related BioMAP signatures

• BioMAP activities can be used to predict efficacy to guide indication selection and identify potential safety and efficacy related biomarkers • Dose response effects can also guide therapeutic window for on‐

versus off‐target effects

• BioMAP can detect activities that are associated with adverse effects or safety concerns early in development program

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DiscoveRx Epigenetics Solution Portfolio

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• Is my compound potent and selective?

• What is the phenotypic impact of my compound in the context of  disease?

• What efficacy and safety biomarkers are detected?

• Does my compound bind the target in a cellular environment?

CellularActivity

Primary Cell Model Systems

HTSSAR

InCELL Hunter Product Menu

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Available Now Coming SoonBRD2(1) BRD9BRD3(1) TAF1L(2)BRD3(1,2) TAF1(2)BRD4(1)BRD2(1)BRDT(1)BRDT(1,2)

Assay Development Services

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Options A B  C

Timeline 8‐10 weeks 10‐14 weeks 18‐24 weeks

Deliverable Expression validated cell pool

Functionally validated cell pool

Functionally validated clonal cell line

What You Get• Select target• Select cell background• EFC expression validation

• All Option A deliverablesPlus

• Functional validation and optimization of compound/ target interaction

• Option A and B deliverablesPlus

• Isolation of single cell clone

• Clonal stability testing

25 Bromodomain pools now offered through CAD Services

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Complete Bromodomain Menu

• 34 BROMOscan targets‐ 24 MAX panel‐10 Kd mode‐Tandems‐Other isoforms

• 6 ICH clones• 25 ICH pools

Thank You 

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