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Pjb Probes 2009

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Pjb Probes 2009

  1. 1. Discovery of Epigenetic Chemical Probes Peter J. Brown Project Manager, Epigenetics Nov 19 th 2009 SGC Oxford SGC Toronto SGC Stockholm
  2. 2. Outline <ul><li>What is a chemical probe? </li></ul><ul><li>What are the goals of the Epigenetics Project? </li></ul><ul><li>How are we going to find chemical probes? </li></ul><ul><li>What is the progress to date? </li></ul>
  3. 3. What is a chemical probe? <ul><li>Chemical probes are potent, selective ligands which are used to validate a target using in vivo or cell-based experiments. </li></ul><ul><li>Probes do not have to contain novel chemical templates or meet rigorous criteria for clinical use in humans. </li></ul><ul><li>However, probes need to be sufficiently bioavailable and/or cell penetrable for experiments to be meaningful. </li></ul>
  4. 4. Why do we need chemical probes? <ul><li>Number of pioneer drugs (Priority Reviewed NCEs) has not increased from 1993-2008 </li></ul><ul><li>Investment in pharmaceutical R&D has risen dramatically over this period </li></ul><ul><li>>90% failure rate in clinical trials for pioneer drugs due to lack of efficacy—EXPENSIVE! </li></ul><ul><li>Need better data with which to make target-selection decisions </li></ul>Public Data from Center of Drug Evaluation and Research: www.fda.gov/cder/ 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 13 12 10 18 9 16 19 9 7 7 9 17 13 6 7 7 0 20 40 60 80 100 120 Yearly FDA Approvals New Drug Approvals New Chemical Entities Priority Reviewed NCEs
  5. 5. Open Access Chemical Probes <ul><li>Why do we need more? </li></ul><ul><ul><li>Low productivity of drug discovery </li></ul></ul><ul><ul><li>High failure rate for pioneer targets </li></ul></ul><ul><li>What did we learn from other protein families? </li></ul><ul><ul><li>Quality chemical probes = quality target validation </li></ul></ul><ul><ul><li>Protein family approach is efficient and effective </li></ul></ul><ul><li>What are our plans? </li></ul><ul><ul><li>Open access probes = freely available </li></ul></ul>
  6. 6. Histone DNA Lysine Modification Write Read Erase Acetyl (Ac) HAT Bromo HDAC Methyl (Me n ) HMT Royal KDM Chemical Biology of Epigenetics
  7. 7. Structural Genomics Consortium Epigenetics Project <ul><li>Global SGC Goal—to deliver 37 chemical probes in 4 years and place these in public domain for use in target validation experiments. </li></ul><ul><ul><li>Fully characterize probes in terms of </li></ul></ul><ul><ul><ul><li>Potency (IC 50 <100 nM) </li></ul></ul></ul><ul><ul><ul><li>selectivity across protein family (30-fold inter-branch selectivity) </li></ul></ul></ul><ul><ul><ul><li>activity in secondary and/or cell-based assays (<1  M) </li></ul></ul></ul><ul><ul><ul><li>determine structure of ligand-protein complex </li></ul></ul></ul><ul><li>Toronto group primarily focused on HMTs and HATs with some effort in the Royal family (12 probes). </li></ul><ul><li>Oxford group primarily focused on Demethylases and Bromo domains (25 probes). </li></ul>
  8. 8. Epigenetics Partners (Toronto) <ul><li>Pharma partners </li></ul><ul><ul><li>GlaxoSmithKline (GSK) </li></ul></ul><ul><ul><ul><li>Medicinal chemistry </li></ul></ul></ul><ul><ul><ul><li>Encoded Library Technology (ELT) Screening </li></ul></ul></ul><ul><li>Academic/Government </li></ul><ul><ul><li>University of North Carolina (UNC) </li></ul></ul><ul><ul><ul><li>Screening and medicinal chemistry </li></ul></ul></ul><ul><ul><li>NIH Chemical Genomics Center (NCGC) </li></ul></ul><ul><ul><ul><li>High throughput screening and chemistry </li></ul></ul></ul><ul><ul><li>Ontario Institute for Cancer Research (OICR) </li></ul></ul><ul><ul><ul><li>Medicinal chemistry </li></ul></ul></ul>
  9. 9. Discovery Strategies <ul><li>High Throughput Screening </li></ul><ul><ul><li>NCGC (440K), UNC (110K), OICR (150K). Little overlap (670K unique compounds) </li></ul></ul><ul><li>Fragment-based Design </li></ul><ul><ul><li>Screening of low MW fragments at high concentration. X-ray follow-up. </li></ul></ul><ul><li>Structure-based Design </li></ul><ul><ul><li>Low/medium throughput screening of designed compounds or small arrays </li></ul></ul><ul><li>Virtual Screening </li></ul><ul><ul><li>Assessment of 6M diverse compounds using GLIDE software suite. </li></ul></ul><ul><ul><li>Additional virtual arrays built around HTS hits . </li></ul></ul>
  10. 10. HMT Assays in production Struc solved by SGC Struc solved by others Assay in Production BIX-01294
  11. 11. HAT Assays in production Struc solved by SGC Struc solved by others Assay in Production
  12. 12. Assessing HMT Activity Evys Collazo & Raymond C. Trievel et al. (2005) Analytical Biochemistry 342: 86–92
  13. 13. Progress on EHMT2 (G9a) 2007 : first publication: discovered by HTS – mild cell activity at 4 µM ( Mol Cell . 2007;25(3):473-81) 2008 : available from Sigma 2009 : co-crystal structure with GLP ( Nat Struct Mol Biol . 2009 ), using SGC clone and protocol. <ul><li>G9a catalyses the methylation of Lys 9 of Histone 3 (H3K9) </li></ul><ul><li>Assays use shortened peptide H3 1-20 or H3 1-25 </li></ul>BIX-01294
  14. 14. Design of UNC0224 as a Potent G9a Inhibitor BIX-01294 G9a (Thioglo): IC 50 = 0.11  M GLP (Thioglo): IC 50 = 0.062  M Feng Liu & Xin Chen, CICBDD Abdellah Allali-Hassani, SGC UNC0123 G9a (Thioglo): IC 50 = 0.33  M Reduced MW and lipophilicity while maintaining potency Array-based optimization GLP-BIX-01294 complex Adapted from Chang, et al. 2009, Nat. Stru. Mol. Bio., (16), 316 UNC0224 G9a (Thioglo): IC 50 = 0.015  M GLP: IC 50 = 0.020  M, inactive vs SETD7 & SETD8 Selective over a panel of 30 non-Epi targets (except hitting M 2 at 82% inh at 1  M)
  15. 15. UNC0224 is a Potent G9a inhibitor BIX-01294 IC50 = 106 nM UNC0224 IC50 = 15 nM
  16. 16. Binding Studies ITC was performed in duplicate with average K d values of 130 ± 18 and 23 ± 8 nM for BIX-01294 and UNC0224 respectively. BIX-01294 UNC0224 0.0 0.5 1.0 1.5 -14 -12 -10 -8 -6 -4 -2 0 2 -0.8 -0.6 -0.4 -0.2 0.0 -10 0 10 20 30 40 50 60 70 80 Time (min) µcal/sec Molar Ratio kcal/mole of injectant 0.0 0.5 1.0 1.5 -14 -12 -10 -8 -6 -4 -2 0 2 -0.8 -0.6 -0.4 -0.2 0.0 -10 0 10 20 30 40 50 60 70 80 Time (min) µcal/sec Molar Ratio kcal/mole of injectant 0.0 0.5 1.0 1.5 -8 -6 -4 -2 0 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 -10 0 10 20 30 40 50 60 70 80 Time (min) µcal/sec Molar Ratio kcal/mole of injectant 0.0 0.5 1.0 1.5 -8 -6 -4 -2 0 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 -10 0 10 20 30 40 50 60 70 80 Time (min) µcal/sec Molar Ratio kcal/mole of injectant
  17. 17. Co-crystallization of G9a and UNC0224 Crystals diffracted to better than 1.7Å Deposited in PDB (3K5K)
  18. 18. BIX-01294 co-crystallized with GLP [3fpd] UNC0224 co-crystallized with G9a [3k5k] H3K9me2 co-crystallized with GLP [2rfi]
  19. 19. What are Bromodomains? <ul><li>Small domain (~110 residues) that selectively binds to acetylated lysine residues </li></ul><ul><li>Bundle of four  -helices, Z, A, B and C plus two loops forming a pocket with a conserved Asn residue </li></ul><ul><li>A recognition domain forming part of numerous chromatin modifying proteins, including histone acetyltransferases (eg CREB & PCAF) and transcriptional coactivators/repressors </li></ul><ul><li>55 unique bromodomains identified to date </li></ul>
  20. 20. Bromodomain Goals <ul><li>At least 1 probe from each major subfamily </li></ul><ul><li><100 nM Kd by ITC or displacement assay </li></ul><ul><li>>30-fold selectivity vs representative proteins from other subfamilies (highlighted in red) </li></ul><ul><li>Demonstration of interaction with target protein in cells at <1uM </li></ul>
  21. 21. Tm shift data for Bromodomains <ul><li>Rapid, low protein consumption </li></ul><ul><li>Partially validated by comparison with ITC </li></ul><ul><li>Screened ~10K compounds: fragments, VLS, pharmacophores </li></ul><ul><li>Hits for >10 bromodomains </li></ul>Thermal stability (DSF) Bromo Subfamily 1 2 3 5 6 7 8 9 13 14 Bromodomain SMARCA2 LOC93349 BAZ2B ATAD2 BRD9 BRPF1 CREBBP BRD2 BRD4 CECR2 PCAF FALZ Most potent hit  Tm / ˚C 5.6 12.9 3.0 3.8 5.6 12.1 6.7 4.8 8.4 3.6  5.3 3.9
  22. 22. Temperature A Potent BRD4 Ligand  Tm=7˚C <ul><li>Identified through focused screening in Tm shift assay </li></ul><ul><li>High potency revealed by ITC </li></ul>K D 16.20 ± 0.4 nM
  23. 23. Selectivity for the BET subfamily <ul><li>BDGBJ000086 shows affinity only for closely related bromodomains in the BET subfamily, and shows no interaction with bromodomains across the remaining subfamilies </li></ul>
  24. 24. BDGBJ000086 selectively displaces H4KAc 4 from BRD2 <ul><li>BDGBJ000086 shows good affinity and selectivity – progressed into exploratory cellular assays </li></ul>Compound Bromodomain Tm shift / °C Kd (ITC) / nM Alphascreen IC 50 / nM BDGBJ000086 BRD2_1 4.2 70 <41 CREBBP 0.8 16200 7500 BAZ2B 0 nd 202000
  25. 25. Summary <ul><li>Outlined strategy for the discovery of Epigenetic Chemical Probes. </li></ul><ul><li>Shown how collaborators are key components in the project. </li></ul><ul><li>Significant progress made in assay development. </li></ul><ul><li>Exciting progress towards probes for G9a (EHMT2) and BRD BET subfamily. </li></ul>
  26. 26. Acknowledgements <ul><li>Toronto </li></ul><ul><li>Cheryl Arrowsmith </li></ul><ul><li>Masoud Vedadi </li></ul><ul><li>Matthieu Schapira </li></ul><ul><li>Jinrong Min </li></ul>Anton Simeonov Oxford Chas Bountra Tom Heightman Stefan Knapp Brian Marsden Stephen Frye Bill Janzen Jian Jin Tim Willson Ryan Trump

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