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Alison O'mahony and Dan Treiber Discoverx Corporation, Fremont Novel Assays and Human Model Systems for Epigenetic Drug Discovery Alison O’Mahony and Dan Treiber DiscoveRx Corporation, Fremont, CA 94538-3142 Abstract BioMAP Profiling of Reference BET Family BRD Inhibitors We 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 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 DNA Tagged Bromodomain phenotypic impact of this target class • Highest Pearson correlation (similarity) at this dose is I-BET and I-BET-151 (r = 0.910) Immobilized Ligand At lowest dose tested (370nM), PFI-1 is weakest inhibitor while JQ1 has the strongest effects Test compound • 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 - Test Compound Competition No Competition modulation of matrix-related markers all of which have relevance for oncology + Test Compound BROMOscan and BioMAP Identify and Phenotypically Classify Dual BRD-Kinase Inhibitors 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. Kinase Inhibitor 1 CpdKB HDACi BioMAP Systems Platform BRD BioMAP Reference Predictive JAK Assay Systems Prole Database Informatics Tools Inhibitors Two structurally unrelated compounds (CpdK and CpdKB) that target same p38 MAPK Structure bound to BRD4(1) kinase are linked but only the CpdKB BRD4 K = 12 nM BRD4 K = 150 nM Inhibitors d d (S.Knapp, in collaboration with that has dual activity clusters with Pan- HDACi DiscoverX; manuscript submitted) reference benchmark iBET compounds Human primary cells Biomarker responses to drugs are Specialized informatics tools 72 known kinase inhibitors screened across BROMOscan • CpdKB showed a more complex phenotype across BioMAP com- Disease-models stored in the database are used to predict clinical outcomes 40+ systems >3000 drugs panel – 6% hit rate pared to benchmarks. CpdKB-specific effects include decreased inflammation markers, decreased chemokine/cytokine production • Potent (K = 0.01- 1 uM), diverse Bromodomain profiles d (IL8, MIG, IL-1, IL6 and IL-10) and effects on matrix-related mark- BROMOscan – First In Class Bromodomain Screening Platform • Inhibitors designed to target TKs, STKs and lipid kinases shown to ers including decreased MMPs, tPA and uPA< increased PAI-1 and have bromodomain activity TIMP-1. 34 validated bromodomain assays III • Pairwise correlation analysis of the BioMAP profiles showed that • Over 50% coverage distinct targets BioMAP Systems while benchmark inhibitors are confined to specific clusters, the BRWD3(2) PHIP(2) BRD2(1) II CpdKB compound clustered with both their BET benchmarks and • 7/8 families represented BRD3(1) The dual kinase/BRD inhibitor, CpdKB, was tested at mul- their respective selective kinase inhibitor. CREBBP WDR9(2) BRD4(1) EP300 tiple doses across BioMAP and compared with benchmark BRD2(2) • Putative therapeutic targets (BET, ATAD2, TRIM24) BAZ1B bromodomain (BET) and Kinase (JAK and PLK) inhibitors. ATAD2B BRD8(1) BRDT(1) BRD8(2) BRD3(2) ATAD2A • CpdKB could be differentiated at the lowest dose tested based on • All BET family domains plus 24 non-BET assays BRD4(2) IV BRD9 BAZ1A BRDT(2) PCAF activities that correlate with bromodomain activity versus the kinase BRD7 Bromodomain Targets BRPF1 GCN5L2 I only inhibitor, CpdK. ATAD2A BRD4(1) BRPF3 SMARCA2 BRPF3 FALZ BRD1 CECR2 PB1(2) ATA2B BRD4(2) CECR2 TAF1(2) PB1(3) BAZ2A PB1(1) PB1(4) HDACi Signatures – Vorinostat, Entinostat and Panobinostat BAZ2A BRD4(1,2) CREBBP TAF1L(2) BAZ2B PB1(6) VIII BAZ2B BRD4(full length, short-iso) EP300 TRIM24(Bromo.) PB1(5) TRIM66 SMARCA4 MLL ASH1L BRD1 BDR7 FALZ TRIM24(PHD, Bromo.) TRIM24 TRIM28 BRWD3(1) SMARCA2 ZMYND11 PHIP(1) TRIM33 BRD2(1) BRD9 GCN5L2 TRIM33(PHD, Bromo.) TAF1L(2) WDR9(1) V SP110 TAF1(2) BRD2(2) BRDT(1) PBRM1(2) WDR9(2) SP100 PRKCBP1 TAF1(1) BRD3(1) BRDT(2) PBRM1(5) SP140 LOC93349 TAF1L(1) BRD3(2) BRPF1 PCAF VII VI Agreement Between BROMOscan and SGC Tm Shift Data 11 Bromosporine-induced Tm shift measurements 10 • Validated fluorescence-based thermal shift assay developed at 9 the SGC 8 7 • Magnitude of bromodomain Tm shift in the presence of inhibitor 6 predicts affinity 5 • Data collected by S. Knapp and S. Muller-Knapp, SGC, 4 personal communication 3 2 BROMOscan and Tm Shift data in outstanding agreement While Vorinostat and Entinostat have similar profiles (370 nM dose), Panobinostat is most similar at ~25X lower dose (13.7 nM) 1 Shift (100 uM Bromosporine) m • May relate to pan-HDAC selectivity versus the more selective effects of Vorinostat (HDAC-3) and Entinostat (HDAC-1, -3) T • Striking agreement between two very different formats 4 10 40 100 400 1000 4000 10000 40000 • Cross-validates the formats Common activities that could indicate an HDAC-3 selective signature include decreased TNFα (LPS) and sIL-10 (Mphg), Bromosporine K (nM) d increased E-sel, IL-8 (SAg), increased PAI-1, tPA (BE) and TF (CASMC) and TIMP-1 • Further quantifies Bromosporine binding promiscuity • Such activities could guide compound selection using above sentinels for screening Outstanding agreement between BROMOscan and Tm Shift data • Cross-validates different assay formats • Further quantifies Bromosporine binding promiscuity BET Family of BRD Family II (BET) Validation: Kd Data • HDAC Inhibitors cluster independent of Bromodomains • Selective HDACi. including Vorinostat and Entinostat, cluster at Pear- BROMOscan Published ITC* HDAC Inhibitors Inhibitor Bromodomain Kd (nM) Kd (nM) son ≥ 0.8 indicating similar BioMAP profiles at these doses BRD2(1) BRD2(2) BRD3(1) BRD3(2) BRD2(1) 79 61 • Profiles for Panobinostat, the pan-HDACi, cluster only across its own BRD2(2) 23 dose range I-BET BRD3(1) 34 51 (active enantiomer) BRD3(2) 27 • HDACi in BioMAP Database include clinical and tool compounds BRD4(1) 59 55 including structural analogs BRD4(2) 11 BRDT(1) 160 nd Pan-HDAC BRDT(2) 45 nd Inhibitor BRD2(1) 9700 nd BRD2(2) 1500 I-BET BRD3(1) 2600 nd Assay Signal (inactive enantiomer) BRD3(2) 1300 BRD4(1) 5000 Interaction not detected Summary & Conclusions BRD4(2) 840 BRDT(1) 15000 nd Several challenges need to be addressed to enable epigenetic drug discovery: [I-BET], nM BRDT(2) 4200 nd *Nature (2010) 468: 1119. • Epigenetic targets are structurally complex with limited options for high-throughput screening BROMOscan data consistent with published ITC data • Toxicity and serious adverse effects have led to low uptake of epigenetic therapies • Potency, rank order and lack of potent activity for inactive I-BET enantiomer Rapid screening tools such as BROMOscan as well as the identification of signatures in BioMAP that are predictive for Accurate BROMOscan data collected for multiple inhibitors efficacy and safety will significantly support compound discovery, lead optimization and pre-clinical development in this • JQ1, I-BET, PFI-1 & other known inhibitors (not shown) emerging therapeutic space. © 2013 DiscoveRx Corporation. All Rights Reserved. 091013 DiscoveRx Corporation 42501 Albrae Street, Fremont, CA 94538 United States tel | 510.979.1415 (Fremont, CA) tel | 800.644.5687 (San Diego, CA) e | [email protected] Europe tel | +44.121.260.6142 e | [email protected] www.discoverx.com.
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