Archived Content
Targeting Histone Methyltransferases and Demethylases
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WEDNESDAY, OCTOBER 3
8:00 am Interactive Breakfast Breakout Discussion Groups
Cell-Based Methylation Assays for HMT Drug Discovery
Moderator: Konrad T. Howitz, Ph.D., Director, Epigenetics, Reaction Biology Corporation
• Current HTS-suitable cell assays measure methylation state, not HMT activity per se—Good enough??
• Will cell-based histone methylation assays be useful for sites targeted by multiple HMTs, e.g. H3K4?
• Do assays relying on incorporation of modified histones (e.g. H3-GFP) risk artifacts by perturbing chromatin structure?
Challenges Associated with Developing HMTi’s as Cancer Therapeutics
Moderator: Roy Pollock, Ph.D., Director, Biological Sciences, Epizyme
• How do we identify patients in which deregulated HMT activity is a driver of tumorigenesis?
• What is the state of the field with respect to predictive and pharmacodynamic biomarkers?
• What are clear-cut and feasible opportunities?
Tools and Approaches for the Identification of Methyltransferase Substrates
Moderator: Zhaohui Sunny Zhou, Ph.D., Faculty Fellow, Barnett Institute of Chemical and Biological Analysis; Associate Professor, Department of Chemistry and Chemical Biology, Northeastern University
• What are the current limitations of our knowledge of enzyme substrates?
• What advances have we achieved regarding chemical probes for labeling?
• What approaches exist for proteomic analysis and activity-based profiling?
• How do we assess off-targets and downstream events of inhibitions?
Issues Related to Developing Biochemical Methyltransferases Assays for Modulators of Protein and Nucleic Acids Methyltransferases
Moderator: Said A. Goueli, Ph.D., Research Fellow, Cell Signaling Group, Research and Development, Promega Corporation; Clinical Professor, University of Wisconsin School of Medicine
• What is the permissible rate of false hits?
• Does the detection system (fluorescence, luminescence) matter with the choice of an assay?
• Will the principle of the assay such as antibody based assays vs. enzyme based assays influence the decision of assay choice? How much reliability can be placed on the selective anti mono, di, and trimethylated antibodies?
• How important is the cost of the assay and does this affect the choice of what assay to be used?
• What are the preferred screening formats for modulators of methyltransferases and what is the maximum number of steps that can be tolerated in a screening project?
• Is the choice of a broad range of substrates desirable or even absolutely required when a decision of which assay platform will be used for screening?
Homogeneous (AlphaScreen®) and ELISA-Type Assays for Histone Demethylases: Development and Applications for Inhibitor Screening
Moderator: Pavel N. Shashkin, Ph.D., Group Leader, Assay Group, BPS Bioscience, Inc.
• What tools or advancements in the field would help demethylase research move forward?
• Does choice of demethylase substrate affect inhibition parameters?
• What are the limitations of homogeneous assays in inhibitor screens?
• What bottlenecks are being encountered in identifying and qualifying new demethylase inhibitors?
• How do storage conditions affect demethylase activity?
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Understanding Histone Methyltransferases and Demethylases for Targeted Therapy
9:05 Chairperson’s Remarks
Roy Pollock, Ph.D., Director, Biological Sciences, Epizyme
9:10 FEATURED PRESENTATION
Quantitative Proteomics for Examining Histone Modifications
 Benjamin A. Garcia, Ph.D., Presidential Associate Professor, Department of Biochemistry and Biophysics; Director, Quantitative Proteomics, Epigenetics Program, University of Pennsylvania Perelman School of Medicine
Robust high-throughput methods for quantitative characterization or even qualitative identification of combinatorial Histone Codes by any standard biological, immunological or physical technique do not readily exist. We plan to specifically address this deficiency by developing novel mass spectrometry based proteomic methods and accompanying bioinformatics to quantitatively characterize molecular level descriptions of combinatorial Histone Codes, and apply these methods to study how these dynamic Histone Codes influence gene expression under different biological conditions. Here we present initial proteomics data that describes: (i) high-throughput comparison of histone modifications from multiple cellular states (ii) developing mass spectrometry methods for quantitative tracking of combinatorial Histone Codes (iii) monitoring in vivo Histone Code dynamics, and (iv) investigating the role of Histone Code interpreting proteins in recognizing distinct Histone Codes. Ultimately, we will work towards the goal of taking any defined part of the genome and accurately quantifying the Histone Codes, detecting all the non-histone proteins that reside on these distinct pieces of chromatin, and then mapping this proteomic data back to specific genomic locations, therefore taking a proteomic snapshot of what that chromosome landscape looks like during any nuclear event.
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9:40 Biochemical Considerations in HMT Drug Discovery
Ryan Kruger, Ph.D., Manager, Cancer Epigenetics Discovery Performance Unit, Oncology R&D, GlaxoSmithKline Pharmaceuticals
Drug discovery in the field of epigenetics poses unique challenges. The enzymes are large multi-domain proteins that have both epigenetic reader and writer functionalities. Many of these proteins work in the context of large multicomponent complexes where other members of the complex affect enzyme activity. Often times the most physiologically relevant substrate is poorly understood or is completely unknown. This presentation will cover biochemical considerations to overcome these challenges.
10:10 Coffee Break in the Exhibit Hall with Poster Viewing
10:55 Profile Substrates and Inhibitors of Protein Methyltransferases
Minkui Luo, Ph.D., Assistant Member & Professor, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center
Epigenetic regulations are involved in establishing cell-lineage diversity and the errors in these processes have been linked to many diseases including cancer. Among the key biochemical modifications in epigenetics is protein methylation, a process orchestrated by over 60 human protein methyltransferases (PMTs). Profiling targets and inhibitors of the PMTs is pivotal toward elucidating their roles in normal physiology and disease states. Unfortunately, conventional approaches are inefficient in these aspects. To address this challenge, our laboratory has developed a novel technology, which we termed as Bioorthogonal Profiling of Protein Methylation (BPPM). Here we engineered the SAM-binding pockets of designated PMTs to accommodate bulky SAM analogues. The engineered PMTs then tag distinct chemical moieties to the substrates of the designated PMTs, in conjunction with click chemical ligation, for target identification. To develop PMT inhibitors aiming at both potency and specificity, we combined rational design and high throughput screening approaches. Our transition-state analogue inhibitors show high potency against multiple PMTs. The biological evaluations of these inhibitors as chemical probes or anti-cancer reagents are ongoing.
11:25 Targeting the JARID1 Demethylases in Cancer
Qin Yan, Ph.D., Assistant Professor, Pathology, Yale University School of Medicine
My laboratory focuses on the roles and regulatory mechanisms of the JARID1/KDM5 HDMs. Since JARID1A/B demethylases are highly expressed in various cancer types and play critical roles in drug resistance, they are novel targets for cancer treatment. We showed recently with genetically engineered mouse cancer models that JARID1A loss inhibits endocrine tumor formation. We will discuss the mechanisms by which JARID1 proteins promote tumorigenesis and our effort to identify their small molecule inhibitors.
11:55 The Antagonistic Dance between Demethylation and Heterochromatin Sheds Light on Development and Disease
Johnathan R. Whetstine, Ph.D., Assistant Professor, Medicine, Harvard Medical School; Massachusetts General Hospital Cancer Center
Our studies have uncovered important roles for the KDM4 histone demethylase family in modulating chromatin structure, and in turn, cell cycle progression. KDM4A impacts S phase and DNA replication timing by displacing heterochromatin. Two major questions emerge from these molecular insights: Is this antagonism confined to replication? What role does this antagonism play in development and human disease? We have begun uncovering answers to these tquestions and will discuss our most recent findings.
12:25 Design and Enantioselective Synthesis of Metabolically Stable SAM/SAH MimeticsDmitry Genis, Ph.D., CEO, ASINEXSAH is a natural inhibitor of SAM which is a cofactor of DNA and protein methyl transferases but it is susceptible to fast metabolism and non-selective. We will present the design and synthesis of a diverse library of metabolically stable analogues of SAM/SAH which are a useful tool for affecting SAM-dependent processes and have strong potential as epigenetic drugs. Regio-Stereo-and Enantioselective synthesis of C-cyclic and N-cyclic analogs of SAM/SAH is challenging but leads to a broad and innovative set of diverse SAM/SAH mimetics with high IP value.
12:55 Luncheon Workshop (Sponsorship Opportunity Available) or Lunch on Your Own
Evaluating Efficacy and Toxicity of Selective Inhibitors
1:55 Chairperson’s Remarks
Ryan Kruger, Ph.D., Manager, Cancer Epigenetics Discovery Performance Unit, Oncology R&D, GlaxoSmithKline Pharmaceuticals
2:00 FEATURED PRESENTATION
Discovery of Chemical Probes for Histone Methyltransferases
 Jian Jin, Ph.D., Associate Professor and Director, Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill
Histone methyltransferases have received great attention because of the essential function of histone methylation in many biological processes. In this talk, the discovery of substrate-competitive inhibitors of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets, robust on-target activities in cells, and low cell toxicity will be described. In addition, progress toward discovering allosteric inhibitors of PRMT3 and non-nucleoside-based cofactor-competitive inhibitors of HMTs will also be presented.
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2:30 A Novel, Selective EZH2 Inhibitor Exhibits Anti-Tumor Activity in Lymphoma with Activating Mutations of EZH2
Michael McCabe, Ph.D., Investigator, Biology, Cancer Epigenetics Discovery Performance Unit, Oncology R&D, GlaxoSmithKline Pharmaceuticals
The EZH2 histone methyltransferase is recurrently mutated at a limited number of residues in diffuse large B-cell lymphomas resulting in altered substrate preferences and increased tri-methylation of histone H3 lysine 27. We have identified a highly-potent, selective, reversible, SAM-competitive EZH2 inhibitor that targets both WT and mutant forms of EZH2. Biochemical and cellular activities of this inhibitor will be discussed in the context of B-cell lymphomas.
3:00 Refreshment Break in the Exhibit Hall with Poster Viewing
3:40 Targeting Histone Methyltransferases in Cancer Therapy
Roy Pollock, Ph.D., Director, Biological Sciences, Epizyme
Histone methyltransferases are a promising new class of therapeutic targets for oncology indications where specific genetic alterations affecting HMT activity drive tumorigenesis. Epizyme has synthesized potent and selective small molecule inhibitors of several HMTs, including DOT1L, as a step towards developing targeted therapeutics for cancers bearing defined genetic lesions. The properties of such inhibitors, including their ability to selectively kill tumor cells bearing specific genetic alterations in cell culture and animal models, will be discussed.
4:10 Targeting Histone Lysine Methylation
Patrick Trojer, Ph.D., Director, Biology, Constellation Pharmaceuticals
The presence of a recurrent mutation of a single tyrosine residue in the EZH2 catalytic domain in diffuse large B-cell lymphoma and follicular lymphoma suggests that these cancers might be dependent on the altered EZH2 molecular function. We have developed potent, selective and reversible small molecule inhibitors and explored potential phenotypic and molecular effects of EZH2 inhibitors across a panel of DLBCL cells as well as on DLBCL xenograft models.
4:40 Structural Basis of Substrate Methylation and Inhibition of SMYD2
Andrew Ferguson, Ph.D., Principal Scientist, DECS Structural Chemistry, AstraZeneca Pharmaceuticals
The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target. Here we report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. The crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505 are also presented. These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class.
5:10 Close of Conference
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