Global SGC Goal—to deliver 37 chemical probes in 4 years and place these in public domain for use in target validation experiments.
Fully characterize probes in terms of
Potency (IC 50 <100 nM)
selectivity across protein family (30-fold inter-branch selectivity)
activity in secondary and/or cell-based assays (<1 M)
determine structure of ligand-protein complex
Toronto group primarily focused on HMTs and HATs with some effort in the Royal family (12 probes).
Oxford group primarily focused on Demethylases and Bromo domains (25 probes).
Medicinal chemistry and Encoded Library Technology (ELT).
Medicinal Chemistry and high throughput screening.
Medicinal Chemistry and cell-based assay development.
Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina (UNC)
Screening and medicinal chemistry
NIH Chemical Genomics Center (NCGC)
High throughput screening and chemistry
Ontario Institute for Cancer Research (OICR)
High Throughput Screening
Little overlap (670K unique compounds)
Multiple screening formats available
NCGC 440K UNC 110K OICR 150K
Of all the target classes tested using this approach, the highest success rate is for enzyme inhibitors.
Screening of 2040 low MW fragments at high concentration.
84% of collection MW between 150 and 250.
Screen at 300 M in activity assay.
Low/medium throughput screening of designed compounds or small arrays
Assessment of 6M diverse compounds using MolSoft software suite.
Additional virtual arrays built around HTS hits .
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
HAT Assays in production Struc solved by SGC Struc solved by others Assay in Production B1 B2 B3 B4 B5 B6
Assessing Activity for HMTs Evys Collazo & Raymond C. Trievel et al. (2005) Analytical Biochemistry 342: 86–92
Protein Lysine Methyltransferase G9a
First identified as a H3K9 MT in 2002 (Tachibana et al, Genes Dev. 2002 , 16, 1779) . Shares 80% sequence identity with GLP (AKA EHMT1) (Chang et al, Nat. Struct. Mol. Biol. 2009, 16, 31). G9a and GLP work cooperatively via forming a heterodimer.
Overexpressed in cancer and knockdown inhibits cancer cell growth (McGarvey et al, Cancer Res. 2006, 66, 3541) (Kondo et al, PLoS ONE 2008, 3, e2037)
Also dimethylates K373 of p53, which results in the inactivation of p53 (Huang et al, J. Biol. Chem. 2010 , 285, 9636)
Plays an important role in the development of cocaine addition (Maze et al, Science 2010, 327, 213) and mental retardation (Schaefer et al, Neuron 2009, 64, 678), and in maintenance of HIV-1 latency (Imai et al, J. Biol. Chem. 2010 , 285, 16538)
BIX01294, a G9a inhibitor, efficacious as a replacement for c-Myc and Sox2 for reprogramming of mouse fetal neural precursor cells into iPS cells (Shi et al, Cell Stem Cell 2008, 3, 568)
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
First Co-crystal Structure of G9a + small molecule: G9a-UNC0224 complex PDB code: 3K5K
7-Dimethylaminopropoxy side chain binds in the lysine binding channel, validating the binding hypothesis, but does not fully fill available space
Liu et al, J. Med. Chem. 2009 , 52, 7950
Assay Measures Cellular Levels of H3K9me2 Dalia Barsyte (SGC), unpublished results
In Cell Western assay in MDA-MB-231 cells
Treated with inhibitors for 48 h
Cellular H3K9me2 levels measured by immunostaining using anti-H3K9me2 & IR800
Simultaneously assess cell viability via DNA staining using DRAQ5
Although UNC0224 is more potent than BIX01294 in biochemical assays, it is less potent in the cell-based assay
Compounds Designed to Improve Cellular Potency
Exploit newly identified SAR to increase lipophilicity, thus cell membrane permeability while maintaining high potency
Prepared > 50 combination compounds. Aiming to achieve balanced in vitro potency & physical chemical propertie s
Feng Liu & Xin Chen
UNC0638 More Potent Than BIX01294 Dalia Barsyte (SGC), unpublished results UNC0638 at 250 & 500 nM reduced cellular H3K9Me2 levels close to G9a/GLP knockdown
Quantitative MS Analysis of Effects of UNC0638 on Histone PTMs Ben Garcia (Princeton) unpublished results
Outlined strategy for the discovery of Epigenetic Chemical Probes.
Shown how collaborators are key components in the project.
Significant progress made in identifying multiple chemical starting points for optimization
Two probes declared for G9a/GLP and BRD BET subfamily.
Oxford Chas Bountra Tom Heightman Stefan Knapp Brian Marsden F UNDING P ARTNERS Canadian Institutes for Health Research, Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Knut and Alice Wallenberg Foundation, Merck & Co., Inc., Novartis Research Foundation, Ontario Innovation Trust, Ontario Ministry for Research and Innovation, Swedish Agency for Innovation Systems, Swedish Foundation for Strategic Research, and Wellcome Trust. Stephen Frye Bill Janzen Jian Jin Bryan Roth Tim Willson Ryan Trump Anton Simeonov James Bradner Jun Qi Ben Garcia