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Unbiased Ligand Discovery for Histone Deacetylases Using Small Molecule Microarrays

Unbiased Ligand Discovery for Histone Deacetylases Using Small Molecule Microarrays Nicole M. Martinez, Summer Research, Chemical Biology Program. HDACs Participate in Regulation of Gene Expression. Histone Deacetylases HDACs Acetylation regulates gene expression

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Unbiased Ligand Discovery for Histone Deacetylases Using Small Molecule Microarrays

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  1. Unbiased Ligand Discovery for Histone Deacetylases Using Small Molecule Microarrays Nicole M. Martinez, Summer Research, Chemical Biology Program

  2. HDACs Participate in Regulation of Gene Expression • Histone Deacetylases HDACs • Acetylation regulates gene expression • HAT transcription activation complex • HDAC transcription repression complex de Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van Kuilenburg, A. B. Biochem. J. 2003, 370, 737-749.

  3. HDACs Impact Cancer Biology • gene expression • oncoprotein stability • cell migration • protein catabolism • cell cycle control http://targethdac.com

  4. HDACs and Cellular Functions Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer. 2006, 6, 38-51.

  5. HDAC Inhibitors (HDACi) • Anti-cancer agents • Few HDACi are isoform selective • Allosteric inhibitors are highly desirable • Tools for assigning function to HDACs

  6. Existing HDACi Synthetics Natural Products

  7. Small-Molecule Microarrays (SMMs) print stocks onto functionalized slides DMSO stock solutions A647 α-GST GST HDAC protein-small molecule interaction on a microarray fluorescent features reveal putative binding interactions

  8. Biophysical Assays *10,800 compounds per library DIV: Diversity Oriented Synthesis Small Molecules library NPC: Natural Products and commercials (Including FDA approved drugs) library

  9. SMM Unbiased Approach Approach: • Small Molecule Microarrays to determine unbiased protein-ligand interactions Benefit: • High-throughput • Quick and cheap • Small amount of protein analyte

  10. From Raw Data to Hits f = microarray featuremedian pixel intensity b = local backgroundmedian pixel intensity xcpd = f - b Z* 1.25 3.75 6.25 Overlay of GAL File cpd- µmock Z* = ( ) 0.96 cpd mock 1+ http://chembank.broad.harvard.edu

  11. Representative Data: HDAC6 ZScoreA DIV library ZScoreB ZScoreC CompositeZ Rounded

  12. Structures of Common HDAC Hits

  13. Common HDAC Biased Hits

  14. Structures of HDAC6 Specific Hits HDAC biased plates 2131-2134

  15. What Happens Next? • Screen with small molecule microarrays against the“Forma Collection” in order to find allosteric binders • Surface plasmon resonance (SPR) to evaluate binding kinetics • in vitro activity assays • Phenotypic assays

  16. ChemBank: A Tool for the Community DSA-ChemBank:796,063 curated compounds, 1,963 assays, 149 projects, 16,942,065 well measurements. ChemBank:528,062 curated compounds, 529 assays, 45 projects, 5,764,724 well measurements 43,651 users at 8,309 organizations in 154 countries http://chembank.broad.harvard.edu

  17. Mentors Angela Koehler, Ph.D. Jason Fuller Collaborators James Bradner, M.D. Stephen Haggarty, Ph.D. Katie Doud Summer Research Program in Genomics Shawna Young (Program Coordinator) Bruce Birren, Ph.D. Acknowledgments

  18. HDAC Cross Comparison HDAC biased plates 2131-2134

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