Stephen V. Frye, Ph.D. Jian Jin, Ph.D. Center for Integrative Chemical Biology & Drug Discovery

1. Carolina Innovations SeminarCenter for Integrative Chemical Biology and Drug DiscoverySeptember 2nd, 2010 Stephen V. Frye, Ph.D. Jian Jin, Ph.D. Center for Integrative Chemical Biology & Drug Discovery University of North Carolina Chapel Hill http://www.pharmacy.unc.edu/labs/center-for-integrative-chemical-biology-and-drug-discovery

2. Genetic Medicine Building

3. Drug Discovery –vs- Development Drug Discovery: Target identification, Hit to Lead Chemistry, Target Validation, Candidate Selection Hypothesis generation, revision & exploration at the chemistry/biology interface chemistry biology >10 years $1 billion Drug Development: Scale-up, Formulation, Toxicology, Phase I-III Clinical trials Regulated process to establish efficacy & safety of a potential new medicine

4. Why Academic Drug Discovery? Biotech no more successful per $ spent than majors less able to tackle ‘big problems’ predictive toxicology refinement of HTS – compounds and technology Academic Drug Discovery a growing enterprise but… fit with academic mission? skill sets missing resources limited where to focus? CICBDD July 2009 • Global • Many diseases are not adequately addressed • Understanding of both molecular targets and disease pathways is accelerating • Pharmaceutical Drug Discovery • Industry’s reputation and sustainability in question • Trend to rely on ‘external’ discovery • Organizational challenges as big as the scientific ones

5. Drug Discovery – what can be done better in academia? Current industrial paradigm for drug discovery = A linear process with little patience for deviation, “FAIL FAST” mentality Target selection – the disease connection hypothesis Efficacy in treating disease in Humans When the target’s role in human disease is unprecedented, this rarely works (<5% success rate) CICBDD July 2009

6. Drug Discovery – what we are trying to do better at UNC. UNC can excel at small molecule driven elucidation of biology relevant to Drug Discovery biology Target selection – the disease connection hypothesis Small molecule tool to enhance target understanding & validation Efficacy in treating disease in Humans chemistry RESEARCH: hypothesis generation, revision & exploration at the chemistry/biology interface

7. Center for Integrative Chemical Biology and Drug Discovery Vision: The Center will be an innovative and sustainable scientific force in the creation of new small molecule therapeutics to improve public health, to transform the drug discovery paradigm and to place UNC at the forefront of translational medicine.

8. CENTER Capabilities Medicinal Chemistry (Jian Jin & Xiaodong Wang) 8internal chemists can support 3-5 projects 4 external chemistry FTEs to support projects and tool compound synthesis Computational Chemistry (Dmitri Kireev) design of focused screening sets, virtual screening structure-based design IT infrastructure • Assay Development (Bill Janzen) • 6 scientists • protein expression & purification • cell-based or in vitro assays • multiple platforms & readouts possible • Compound Screening • screens up to 100K compounds possible • Three HTS run since 4Q09 • Small tube store installed and now loaded

9. CENTER Mission Center Initiated Projects in Chemical Biology “prospective science” Target Proposals from UNC Faculty “responsive collaborations” Core Staff + joint faculty small molecule ‘probes’ target validation drug leads

10. Envisioned process at UNC for responsive projects 1 year, 50% success 1 year, 80% 2-3 yrs, 50% Target I.D. & Validation Investigator driven target proposals with validation data from HT-genomics, HT – siRNA, In vivo models Hit I.D. Assay develop- ment MTS, focused sets, knowledge-based design, HTS Lead I.D. SAR dev., compd optimiz., Cellular - in vivo efficacy, initial dmpk, selectivity profiling, target validation Candidate I.D. lead opt., In vivo efficacy & safety, 7 day tox., scale-up, dmpk in 3 species, target validation External Resources? CICBDD RESOURCES

11. Portfolio Model Number of items Assay Develop ment Chemistry(H2L) Preproject 10 projects 5 projects 15-20 projects Work in progress Effort Project Progress

12. Portfolio Goals

13. How do faculty currently establish a collaboration with the CICBDD? • Drop by & chat • Reviewed by Center Faculty • General Criteria: • Disease relevance of target/pathway • Potential scientific impact of small molecule tools • Tractability of assay development and ligand discovery • Portfolio balance and fit with center expertise • Funding status & prospects

14. Mer Kinase Background • Mer kinase – a member of the Tyro3/Axl/Mer RTK family • Expressed in monocytes, functions to clear apoptotic material • Never expressed in normal T or B lymphocytes • Capable of sending anti-apoptotic signals • Mer kinase expressed in most T and B cell ALL lines • Mer expression in childhood leukemias • Mer mRNA expressed in 30-40% T cell ALL (ClinCanc Res 2006 12:2662) • New data :Mer protein expressed in 41% B ALL (16 of 16 E2A-PBX1 ALLs) • Mer protein expressed 54% T cell ALLs and 68% pediatric AML • Mer protein expressed in 50% of recurrent/induction failures • Protein structure with small molecule bound published by the Structural Genomics Consortium • Huang, X., et al., Structural insights into the inhibited states of the Mer receptor tyrosine kinase. J StructBiol, 2009. 165(2): p. 88-96.

15. Inhibition of Mer Expression Alters Chemosensitivity and In Vivo Outcome In vivo leukemia model: injection of 5x105 697 cells in Nod/SCID mice. Enhanced survival with Mer shRNA knockdown. 697 B cell (E2A-PBX) chemosensitivity altered by Mer knockdown Target validation with shRNA,Linger et al., Blood, 2009 114:2678

16. Mer Kinase Status at UNC Goal: Chemosensitization of ALL patients i.v. initial formulation, oral secondary Project has been underway for 1.5 years – now funded by the NCI (1st year = $1.614M) Structure-based hit generation has yielded one lead series: low nanomolar Ki’s, robust structure-activity relationships Promising initial dmpk (UNC569, mouse, 4.4h t1/2, 56% F) Broad kinase profiling underway Cellular assays being optimized – compounds appear to have <1 mM IC50’s Compounds suitable for in vivo testing are in hand Additional hit generation is ongoing via focused screening and further structure-based design Initial crystals of the Mer kinase domain have been obtained – optimizing conditions to develop a system for routine co-crystal structures Confidential UNC CBC Plasma concentration-time data of UNC 569A(mouse, dose:3mg/kg).

17. CENTER Mission Center Initiated Projects in Chemical Biology “prospective science” Target Proposals from UNC Faculty “responsive collaborations” Core Staff + joint faculty small molecule ‘probes’ target validation drug leads

18. Chemical Biology of Chromatin Regulation PKMT Royal PKDM Frye, S. V.; Heightman, T.; Jin, J. Targeting Methyl Lysine. Annu. Rep. Med. Chem. 2010, in press, DOI: 10.1016/S0065-7743(10)45020-4. HAT Bromo HDAC GOAL = Chemical Probes

19. Why chemical probes? Weiss, W.A., S.S. Taylor, and K.M. Shokat, Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat ChemBiol, 2007. 3(12): p. 739-44. • Temporal resolution • rapid exposure and elimination of effects are possible • Mechanistic flexibility • can potentially target separate functions of a protein, as opposed to ablating them all • Ease of delivery • freely cell permeable, potential for oral activity • Applicability to drug discovery • transition from target validation to therapeutic intervention is more direct

20. What is a quality chemical probe? • Molecular Profiling: Sufficient in vitro potency and selectivity data to confidently associate its in vitro profile to its cellular or in vivo profile. • Mechanism of Action: Activity in a cell-based or cell-free assay influences a physiologic function of the target in a dose-dependent manner. • Identity of the Active Species: Has sufficient chemical and physical property data to permit interpretations of results to be attributed to its intact structure or a well characterized derivative. • Proven Utility as a Probe: Cellular activity data available to confidently address at least one hypothesis about the role of the molecular target in a cell’s response to its environment. • Availability: Is readily available to the academic community with no restrictions on use. • Frye, S. V. The art of the chemical probe. Nat ChemBiol2010, 6, 159-161.

21. PKMTs as a Target Class H3K4 H3K27 H3K9 H3K9 H3K9 H3K9 H3K9 H3K9 • > 50 human PKMTs and > 10 PRMTs identified since the first PKMT discovered in 2000 • Growing evidence suggests PKMTs involved in various human diseases including cancer • Lack of chemical probes: only 2 selective PKMT inhibitors reported (BIX-01294 and chaetocin) H3K4 H3K36 H3K36 H3K4 H4K20H3K9 H4K20 H3K4 H3K27 H3K4 H3K4 H3K4 H3K79 H3K4 H3K4 H3K4 H3K36 H4K20 H4R3 H3R8 H3K4 H4R3 H4K20 H4R3 H4K20 H3K36 H3R2 H3R17 H3R26 H4R3 H3K4 H3K9 Wu et al, PLOS One, 2010, 5, e8570 Copeland et al, Nat. Rev. Drug Discovery, 2009, 8, 724 H3K4

22. 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)

23. Structure-based Design of UNC0224 Array-based Optimization* UNC0123 G9a (ThioGlo): IC50 = 330 nM G9a (AlphaScreen): IC50 = 230 nM Reduced MW while maintaining potency BIX01294 G9a (ThioGlo): IC50 = 180 nM G9a (AlphaScreen): IC50 = 250 nM Kubicek, et al. 2007, Mol Cell, 473 UNC0224 Synthesized in 10 steps G9a (ThioGlo): IC50 = 43 nM G9a (AlphaScreen): IC50 = 57 nM G9a (ITC): KD = 23 nM GLP-BIX01294 complex Adopted from Chang, et al. 2009, Nat. Stru. Mol. Bio., (16), 316 Liu et al, J. Med. Chem.2009, 52, 7950

24. 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

25. Most Potent G9a Inhibitors to Date • Key SAR findings • Longer side chain (up to 5 atoms) or bigger N-capping group increases potency • Basicity of the nitrogen necessary for high potency • Although UNC0224 and UNC0321 are more potent than BIX01294 in biochemical assays, they are less potent in the cell-based assay Liu et al, J. Med. Chem.2010, 53, 5844

26. 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 properties Feng Liu & Xin Chen

27. UNC0638 More Potent Than BIX01294 UNC0638 at 250 & 500 nM reduced cellular H3K9Me2 levels close to, but not equivalent to G9a/GLP knockdown Dalia Barsyte (SGC), unpublished results

28. Quantitative MS Analysis of Effects of UNC0638 on Histone PTMs • UNC0638 ↓H3K9me2, –H3K9me3, ↓H3K9me1, ↑H3K9un & ↑H3K14ac • Similar to G9a and GLP Knockdown Ben Garcia (Princeton) unpublished results

29. UNC0638 Less Toxic Than BIX01294 UNC0638 BIX01294 H3K9m2 MTT 110 H3K9m2 100 MTT 90 80 70 60 % response 50 40 30 20 10 0 10 0 10 1 10 2 10 3 10 4 10 5 nM Poor separation of functional and toxic effects Good separation of functional and toxic effects Dalia Barsyte (SGC), unpublished results

30. UNC0638 Molecular Profile • Ki = 3 nM and IC50 < 15 nM vs G9a • IC50 = 19 nM vs GLP • Competitive with peptide substrate and non-competitive with cofactor SAM • Co-crystal structure of G9a-UNC0638-SAH confirms MOA • Rapid and on and off rate – reversible inhibitor • At least 100-fold selective over a broad range of epigenetic targets including PKMTs, PRMTs, PKDMs, HATs, and DNMTs, and a wide range of non-epigenetic targets including GPCRs, ion channels, transporters, & kinases

31. UNC - SGC Probe Consortium Released UNC0638 as a Chemical Probe on June 1st http://www.thesgc.org/chemical_probes/UNC0638/#overview • Data released prior to publication. Living document that is updated as new data is generated

32. Collaborators Who Are Using UNC0638 • Mark Minden (OCI) • Test on EVI-1 expressing • leukemia cells • Jayne Danska(Sick Kids, Toronto) • Test on large panel of ALL cells Jay Bradner (Dana Farber/Broad Inst) Test on MLL dependent leukemic cells Effects on epigenetic markers • Rob Bristow (OCI) • Test on prostate cancer • cell lines Stuart Schreiber (Broad) Profile against >1000 genetically defined cancer cell lines Alex Tarakhovsky (Rockefeller) IFN production after viral infection Viral resistance Changes of epigenetic markers James Ellis (Sick Kids, Toronto) iPS cell studies David Margolis (UNC) Effects on HIV-1 latency Angelique Whitehurst (UNC) Synergy with chemo therapies Rod Bremner (TWRI) Assess effect on IFN expression Michael Taylor (Sick Kids Hosp) Test on DAOY cell line (medulloblastoma) Martin Hirst (UBC) ChIP-seq Jing Huang (NCI, NIH) Bill Janzen (UNC) p53 methylation Apoptosis of MCF7 cells

33. Summary • Discovered potent, selective, and cellularly active G9a/GLP chemical probes via structure-based inhibitor design in combination with SAR exploration • UNC0638, which has significantly improved tox/func ratio, available to research community without restrictions on use for investigating the biology of G9a/GLP and their role in chromatin remodeling in health and disease.

34. Acknowledgements UNC CICBDD Feng Liu Xin Chen Julianne Yost Tim Wigle Sam Pattenden Dmitri Kireev Jacqueline Norris Cathy Simpson Martin Herold Emily Hull-Ryde Bill Janzen Stephen Frye Bryan Roth Lab (UNC) Tom Mangano Jon Evans Cheryl Arrowsmith Lab (SGC) Dalia Barsyte Peter Brown Masoud Vedadi Matthieu Schapira AbdellahAllali-Hassani Gregory Wasney Aiping Dong Aled Edwards Anton Simeonov Lab (NCGC) Amy Quinn Ajit Jadhav Ben Garcia Lab (Princeton) Peter DiMaggio Funding: NIH RC1GM090732, UNC Carolina Partnership