The document outlines the goals and progress of the Epigenetics Project run by the Structural Genomics Consortium to discover chemical probes that target epigenetic enzymes. The project aims to deliver 37 probes within 4 years that are potent, selective, and validated for use in target validation experiments. Significant progress includes probes discovered for histone methyltransferases (HMTs) and bromodomains through various screening and structure-based design approaches.

1. Discovery of Epigenetic Chemical Probes Peter J. Brown Project Manager, Epigenetics Structural Genomics Consortium Sept 29 th 2010 SGC Oxford SGC Toronto SGC Stockholm

4. Histone DNA Lysine Modification Write Read Erase Acetyl (Ac) HAT Bromo HDAC Methyl (Me n ) HMT Royal KDM Chemical Biology of Epigenetics

10. HMT Assays in production Struc solved by SGC Struc solved by others Assay in Production Assay in Development BIX-01294 B2 B3 B4 B5 B6 B7 B1

11. HAT Assays in production Struc solved by SGC Struc solved by others Assay in Production B1 B2 B3 B4 B5 B6

12. Assessing Activity for HMTs Evys Collazo & Raymond C. Trievel et al. (2005) Analytical Biochemistry 342: 86–92

14. Structure-based Design of UNC0224 BIX01294 G9a (ThioGlo): IC 50 = 180 nM G9a (AlphaScreen): IC 50 = 250 nM Kubicek, et al. 2007, Mol Cell, 473 G9a (ThioGlo): IC 50 = 43 nM G9a (AlphaScreen): IC 50 = 57 nM G9a (ITC): K D = 23 nM UNC0123 G9a (ThioGlo): IC 50 = 330 nM G9a (AlphaScreen): IC 50 = 230 nM Reduced MW while maintaining potency Array-based Optimization* UNC0224 GLP-BIX01294 complex Adopted from Chang, et al. 2009, Nat. Stru. Mol. Bio., (16), 316 Liu et al, J. Med. Chem. 2009 , 52, 7950 Synthesized in 10 steps

18. UNC0638 More Potent Than BIX01294 Dalia Barsyte (SGC), unpublished results UNC0638 at 250 & 500 nM reduced cellular H3K9Me2 levels close to G9a/GLP knockdown

20. UNC0638 Less Toxic Than BIX01294 Dalia Barsyte (SGC), unpublished results In Vitro G9a IC 50 (nM) H3K9me2 48h IC 50 (nM) Cell Tox 48h EC 50 (nM) Tox/Func Ratio BIX-01294 133 ± 15 500 ± 43 2805 5.6 UNC0638 <15 81 ± 9 11190 138 Poor separation of functional and toxic effects BIX01294 10 0 10 1 10 2 10 3 10 4 10 5 0 10 20 30 40 50 60 70 80 90 100 110 H3K9m2 MTT nM % response Good separation of functional and toxic effects UNC0638 H3K9m2 MTT

21. Effects of UNC0638 in Other Cell Lines Dalia Barsyte (SGC), unpublished results Cell Lines IC 50 (nM) Toxic/Func Ratio H3K9me2 MTT Breast carcinoma MDA231 81 11,000 138 MCF7 70 7,600 109 Prostate carcinoma PC3 59 14,000 233 22RV1 48 4,500 95 Colon carcinoma HCT116 wt 210 11,000 55 HCT116 p53-/- 240 11,000 47 Human fibroblast IMR90 120 2,300 19

22. UNC0638 Mechanism of Action Tim Wigle, unpublished results UNC0638 is competitive with peptide, not SAM

30. Interaction Confirmed by ITC

31. Selectivity for the BET subfamily Thermal Shift Data

33. Cell-based Assay James Bradner NUT-BRD4 GFP NUT-GFP

34. BETsOFF inhibits Proliferation James Bradner

35. BETsOFF inhibits Tumour Growth James Bradner Day 1 Day 5 Day 11 Day 18