Development and Validation of Reagents and Assays for EZH2 Peptide and Nucleosome High-Throughput Screens

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [3H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

[1]  Yong Jiang,et al.  Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27) , 2012, Proceedings of the National Academy of Sciences.

[2]  S. Thrall,et al.  Methyltransferases prefer monomer over core-trimmed nucleosomes as in vitro substrates. , 2011, Analytical biochemistry.

[3]  V. Schramm,et al.  Characterizing DNA methyltransferases with an ultrasensitive luciferase-linked continuous assay. , 2011, Analytical chemistry.

[4]  H. Al‐Hashimi,et al.  Direct Evidence for Methyl Group Coordination by Carbon-Oxygen Hydrogen Bonds in the Lysine Methyltransferase SET7/9* , 2011, The Journal of Biological Chemistry.

[5]  Ryan D. Morin,et al.  Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. , 2011, Blood.

[6]  D. Reinberg,et al.  The Polycomb complex PRC2 and its mark in life , 2011, Nature.

[7]  Chad Quinn,et al.  Assay Development and High-Throughput Screening of Small Molecular c-Abl Kinase Activators , 2011, Journal of biomolecular screening.

[8]  R. Copeland,et al.  Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas , 2010, Proceedings of the National Academy of Sciences.

[9]  E. Hurt,et al.  Clinical significance of Polycomb gene expression in brain tumors , 2010, Molecular Cancer.

[10]  James R. Brown,et al.  Thousands of chemical starting points for antimalarial lead identification , 2010, Nature.

[11]  Anton Simeonov,et al.  A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors. , 2010, Molecular bioSystems.

[12]  Lily Wu,et al.  Comprehensive evaluation of the role of EZH2 in the growth, invasion, and aggression of a panel of prostate cancer cell lines , 2010, The Prostate.

[13]  K. Helin,et al.  Histone methyltransferases in cancer. , 2010, Seminars in cell & developmental biology.

[14]  D. Reinberg,et al.  Chromatin structure and the inheritance of epigenetic information , 2010, Nature Reviews Genetics.

[15]  J. Hevel,et al.  A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates. , 2010, Analytical biochemistry.

[16]  R. Klose,et al.  Histone lysine methylation: an epigenetic modification? , 2010, Epigenomics.

[17]  D. Reinberg,et al.  Role of the polycomb protein EED in the propagation of repressive histone marks , 2009, Nature.

[18]  Emilia Dimitrova,et al.  A Continuous Protein Methyltransferase (G9a) Assay for Enzyme Activity Measurement and Inhibitor Screening , 2009, Journal of biomolecular screening.

[19]  Christodoulos A Floudas,et al.  High Throughput Characterization of Combinatorial Histone Codes* , 2009, Molecular & Cellular Proteomics.

[20]  I. Kariv,et al.  A Simplified Scintillation Proximity Assay for Fatty Acid Synthase Activity: Development and Comparison with Other FAS Activity Assays , 2009, Journal of biomolecular screening.

[21]  A. Shilatifard,et al.  An operational definition of epigenetics. , 2009, Genes & development.

[22]  Gavin Harper,et al.  Process Validation and Screen Reproducibility in High-Throughput Screening , 2009, Journal of biomolecular screening.

[23]  Isabel Coma,et al.  Statistics and decision making in high-throughput screening. , 2009, Methods in molecular biology.

[24]  R. Trievel,et al.  Structural origins for the product specificity of SET domain protein methyltransferases , 2008, Proceedings of the National Academy of Sciences.

[25]  V. Bilim,et al.  Regulation of Pancreatic Tumor Cell Proliferation and Chemoresistance by the Histone Methyltransferase Enhancer of Zeste Homologue 2 , 2008, Clinical Cancer Research.

[26]  J Fraser Glickman,et al.  Scintillation proximity assays in high-throughput screening. , 2008, Assay and drug development technologies.

[27]  Ted M. Lakowski,et al.  A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism* , 2008, Journal of Biological Chemistry.

[28]  D. Reinberg,et al.  Ezh2 Requires PHF1 To Efficiently Catalyze H3 Lysine 27 Trimethylation In Vivo , 2008, Molecular and Cellular Biology.

[29]  R. Trievel,et al.  Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases. , 2007, Biochemistry.

[30]  D. Wasilko,et al.  TIPS: Titerless Infected-Cells Preservation and Scale-Up , 2006 .

[31]  Johan Eide,et al.  Expression of enhancer of zeste homologue 2 is significantly associated with increased tumor cell proliferation and is a marker of aggressive breast cancer. , 2006, Clinical cancer research : an official journal of the American Association for Cancer Research.

[32]  Stephen A Boorjian,et al.  Increased Expression of the Polycomb Group Gene, EZH2, in Transitional Cell Carcinoma of the Bladder , 2005, Clinical Cancer Research.

[33]  Cyrus Martin,et al.  The diverse functions of histone lysine methylation , 2005, Nature Reviews Molecular Cell Biology.

[34]  S. Jacobsen,et al.  Substrate Specificity and Kinetic Mechanism of Mammalian G9a Histone H3 Methyltransferase* , 2004, Journal of Biological Chemistry.

[35]  Paul Tempst,et al.  Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3. , 2004, Molecular cell.

[36]  Debashis Ghosh,et al.  EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Geoff Kelly,et al.  Structure and catalytic mechanism of the human histone methyltransferase SET7/9 , 2003, Nature.

[38]  S. Dhanasekaran,et al.  The polycomb group protein EZH2 is involved in progression of prostate cancer , 2002, Nature.

[39]  G. Schnitzler Isolation of Histones and Nucleosome Cores from Mammalian Cells , 2000, Current protocols in molecular biology.

[40]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.