AbstractWe have developed a comprehensive suite of in vitro biochemical, cellular assays and human model systems to support compound screening and

development by evaluating target-specific physio-chemical binding properties and compound effects on complex biological signaling networks.

In this study, we determine inhibitor potency and selectivity using BROMOscanSM, the industry’s largest panel of bromodomain targets and evaluate

the phenotypic impact of these inhibitors on human primary cell-based BioMAP® Systems. We profiled a number of benchmark inhibitors that

target kinases, BET family bromodomain reader proteins and Histone deacetylases (HDACs) to generate binding profiles and phenotypic signatures

for each target class when compared to profiles for over 3000 clinical, failed pharmaceutical or tool compounds in the BioMAP database.

Interestingly, a number of reported kinase inhibitors were shown to bind to bromodomains with high potency (Kd = 0.01 – 1 uM) and also demon-

strated more complex phenotypic signatures when compared to selective benchmark inhibitors, consistent with their dual kinase/bromodomain

activities. These data highlight the potential for unforeseen, high affinity synergistic inhibition of these important epigenetic regulators in addition to

their original target kinase. DiscoveRx assays can provide a comprehensive evaluation of epigenetic inhibitors with respect to target potency,

selectivity and impact on signaling mechanisms and resultant phenotypes in human cells. Taken together, these findings can be used to guide

compound prioritization, indication selection and highlight potential safety issues to thereby improve the probability of clinical success.

BROMOscan Core Technology Platform

Competition No Competition

+ Test Compound

- Test Compound

DNA Tagged Bromodomain

Immobilized Ligand

Test compound

BROMOscan provides a direct measure of the amount of bromodomain bound to an immobilized ligand in the presence or absence of test com-pound using an ultrasensitive quantitative PCR (qPCR) readout.

BioMAP Systems Platform

BioMAPAssay Systems

ReferencePro�le Database

Predictive Informatics Tools

Human primary cells Disease-models40+ systems

Biomarker responses to drugs are stored in the database>3000 drugs

Specialized informatics tools are used to predict clinical outcomes

BROMOscan – First In Class Bromodomain Screening Platform

34 validated bromodomain assays • Over 50% coverage distinct targets

• 7/8 families represented

• Putative therapeutic targets (BET, ATAD2, TRIM24)

• All BET family domains plus 24 non-BET assays

Bromodomain TargetsATAD2A BRD4(1) BRPF3 SMARCA2

ATA2B BRD4(2) CECR2 TAF1(2)

BAZ2A BRD4(1,2) CREBBP TAF1L(2)

BAZ2B BRD4(full length, short-iso) EP300 TRIM24(Bromo.)

BRD1 BDR7 FALZ TRIM24(PHD, Bromo.)

BRD2(1) BRD9 GCN5L2 TRIM33(PHD, Bromo.)

BRD2(2) BRDT(1) PBRM1(2) WDR9(2)

BRD3(1) BRDT(2) PBRM1(5)

BRD3(2) BRPF1 PCAF

I

II

III

IV

V

VI VII

VIII

BRWD3(1)PHIP(1)ZMYND11

MLLASH1L

PB1(2)PB1(3)

PB1(1) PB1(4)

PB1(6)

PB1(5)

SMARCA2

TAF1L(2)

TAF1(2)PRKCBP1

TAF1L(1)TAF1(1)

TRIM28

SMARCA4

WDR9(1)

BAZ2A

BAZ2B

TRIM66

TRIM24

TRIM33

SP110

SP100

LOC93349SP140

BRD8(2)

BRD7

BRD1BRPF3

BRPF1

BRD9

BRD8(1)BAZ1B

ATAD2B

EP300CREBBP

ATAD2A

WDR9(2)

BAZ1A

PHIP(2)

BRWD3(2)

BRDT(2)

BRDT(1)

CECR2

BRD4(2)

GCN5L2FALZ

BRD3(2)

BRD2(2)

PCAF

BRD2(1)BRD3(1)

BRD4(1)

Agreement Between BROMOscan and SGC Tm Shift Data

Bromosporine Kd (nM)

Tm S

hift

(100

uM

Bro

mo

spo

rine

)

11

10

9

8

7

6

5

4

3

2

1

4 10 40 100 400 1000 4000 10000 40000

Bromosporine-induced Tm shift measurements

• Validated fluorescence-based thermal shift assay developed at the SGC

• Magnitude of bromodomain Tm shift in the presence of inhibitor predicts affinity

• Data collected by S. Knapp and S. Muller-Knapp, SGC, personal communication

BROMOscan and Tm Shift data in outstanding agreement

• Striking agreement between two very different formats

• Cross-validates the formats

• Further quantifies Bromosporine binding promiscuity

Outstanding agreement between BROMOscan and Tm Shift data

• Cross-validates different assay formats

• Further quantifies Bromosporine binding promiscuity

Family II (BET) Validation: Kd Data

Ass

ay S

igna

l

[I-BET], nM

BRD2(1) BRD2(2) BRD3(1) BRD3(2)Inhibitor Bromodomain

BROMOscanKd (nM)

Published ITC*Kd (nM)

I-BET(active enantiomer)

BRD2(1) 79 61

BRD2(2) 23

BRD3(1) 34 51

BRD3(2) 27

BRD4(1) 59 55

BRD4(2) 11

I-BET(inactive enantiomer)

BRDT(1) 160 nd

BRDT(2) 45 nd

BRD2(1) 9700 nd

BRD2(2) 1500

BRD3(1) 2600 nd

BRD3(2) 1300

BRD4(1) 5000 Interaction not detected

BRD4(2) 840

BRDT(1) 15000 nd

BRDT(2) 4200 nd

*Nature (2010) 468: 1119.

BROMOscan data consistent with published ITC data• Potency, rank order and lack of potent activity for inactive I-BET enantiomer

Accurate BROMOscan data collected for multiple inhibitors• JQ1, I-BET, PFI-1 & other known inhibitors (not shown)

Alison O’Mahony and Dan TreiberDiscoveRx Corporation, Fremont, CA 94538-3142

DiscoveRx Corporation 42501 Albrae Street, Fremont, CA 94538 United States tel | 510.979.1415 (Fremont, CA) tel | 800.644.5687 (San Diego, CA) e | info@discoverx.com Europe tel | +44.121.260.6142 e | euroinfo@discoverx.com www.discoverx.com

© 2013 DiscoveRx Corporation. All Rights Reserved. 091013

BioMAP Profiling of Reference BET Family BRD Inhibitors

Anti-proliferative effects Cytotoxcity (SRB ≤ -0.3Log ≈ >50% reduction in total cell protein)

BET inhibitors exhibit very similar BioMAP profiles over a broad dose range indicating signature activities that classify phenotypic impact of this target class• Highest Pearson correlation (similarity) at this dose is I-BET and I-BET-151 (r = 0.910)

At lowest dose tested (370nM), PFI-1 is weakest inhibitor while JQ1 has the strongest effects • JQ1 is overtly cytotoxic in B cells (BT), PFI-1 has similar profile at 1.1 uM dose (~3X less potent)

• I-BET, JQ1 and I-BET-151, but not PFI-1, are broadly anti-proliferative at this dose

BioMAP profiling reveals a broad range of activities including broad anti-proliferative effects, anti-inflammatory effects and modulation of matrix-related markers all of which have relevance for oncology

BROMOscan and BioMAP Identify and Phenotypically Classify Dual BRD-Kinase Inhibitors

Kinase Inhibitor 1 CpdKB

BRD4 Kd = 12 nM BRD4 Kd = 150 nM

Two structurally unrelated compounds (CpdK and CpdKB) that target same kinase are linked but only the CpdKB that has dual activity clusters with reference benchmark iBET compounds

Structure bound to BRD4(1)(S.Knapp, in collaboration with DiscoverX; manuscript submitted)

BRD

HDACi

JAKInhibitors

Pan- HDACi

p38 MAPK Inhibitors

72 known kinase inhibitors screened across BROMOscan panel – 6% hit rate• Potent (Kd = 0.01- 1 uM), diverse Bromodomain profiles

• Inhibitors designed to target TKs, STKs and lipid kinases shown to have bromodomain activity

BioMAP Systems

The dual kinase/BRD inhibitor, CpdKB, was tested at mul-tiple doses across BioMAP and compared with benchmark bromodomain (BET) and Kinase (JAK and PLK) inhibitors.• CpdKB could be differentiated at the lowest dose tested based on

activities that correlate with bromodomain activity versus the kinase only inhibitor, CpdK.

• CpdKB showed a more complex phenotype across BioMAP com-pared to benchmarks. CpdKB-specific effects include decreased inflammation markers, decreased chemokine/cytokine production (IL8, MIG, IL-1, IL6 and IL-10) and effects on matrix-related mark-ers including decreased MMPs, tPA and uPA< increased PAI-1 and TIMP-1.

• Pairwise correlation analysis of the BioMAP profiles showed that while benchmark inhibitors are confined to specific clusters, the CpdKB compound clustered with both their BET benchmarks and their respective selective kinase inhibitor.

HDACi Signatures – Vorinostat, Entinostat and Panobinostat

While Vorinostat and Entinostat have similar profiles (370 nM dose), Panobinostat is most similar at ~25X lower dose (13.7 nM) • May relate to pan-HDAC selectivity versus the more selective effects of Vorinostat (HDAC-3) and Entinostat (HDAC-1, -3)

Common activities that could indicate an HDAC-3 selective signature include decreased TNFα (LPS) and sIL-10 (Mphg), increased E-sel, IL-8 (SAg), increased PAI-1, tPA (BE) and TF (CASMC) and TIMP-1• Such activities could guide compound selection using above sentinels for screening

BET Family of BRD

HDAC Inhibitors

Pan-HDAC Inhibitor

• HDAC Inhibitors cluster independent of Bromodomains

• Selective HDACi. including Vorinostat and Entinostat, cluster at Pear-son ≥ 0.8 indicating similar BioMAP profiles at these doses

• Profiles for Panobinostat, the pan-HDACi, cluster only across its own dose range

• HDACi in BioMAP Database include clinical and tool compounds including structural analogs

Summary & Conclusions

Several challenges need to be addressed to enable epigenetic drug discovery:

• Epigenetic targets are structurally complex with limited options for high-throughput screening

• Toxicity and serious adverse effects have led to low uptake of epigenetic therapies

Rapid screening tools such as BROMOscan as well as the identification of signatures in BioMAP that are predictive for efficacy and safety will significantly support compound discovery, lead optimization and pre-clinical development in this emerging therapeutic space.

Novel Assays and Human Model Systems for Epigenetic Drug Discovery