Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor.

The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a K(i) of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anticancer target.

[1]  M. Mann,et al.  Global and site-specific quantitative phosphoproteomics: principles and applications. , 2009, Annual review of pharmacology and toxicology.

[2]  R. Krishna,et al.  Post-translational modification of proteins. , 1993, Advances in enzymology and related areas of molecular biology.

[3]  Ruben Abagyan,et al.  Small molecules block the polymerization of Z alpha1-antitrypsin and increase the clearance of intracellular aggregates. , 2007, Journal of medicinal chemistry.

[4]  J. Workman,et al.  Characterization of the Yeast Trimeric-SAS Acetyltransferase Complex* , 2005, Journal of Biological Chemistry.

[5]  Simon C Watkins,et al.  Current Protocols In Cytometry , 1997 .

[6]  Ruben Abagyan,et al.  Modeling of the aryl hydrocarbon receptor (AhR) ligand binding domain and its utility in virtual ligand screening to predict new AhR ligands. , 2009, Journal of medicinal chemistry.

[7]  R. Kitz,et al.  Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. , 1962, The Journal of biological chemistry.

[8]  Ruben Abagyan,et al.  ICM—A new method for protein modeling and design: Applications to docking and structure prediction from the distorted native conformation , 1994, J. Comput. Chem..

[9]  Tapas K. Kundu,et al.  Small Molecule Modulators of Histone Acetyltransferase p300* , 2003, Journal of Biological Chemistry.

[10]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[11]  Timothy W. Sikorski,et al.  The Human Monocytic Leukemia Zinc Finger Histone Acetyltransferase Domain Contains DNA-binding Activity Implicated in Chromatin Targeting* , 2007, Journal of Biological Chemistry.

[12]  P. Cole,et al.  Transcriptional Coactivator Protein p300 , 2001, The Journal of Biological Chemistry.

[13]  A. Pandey,et al.  Chemical Rescue of a Mutant Enzyme in Living Cells , 2006, Science.

[14]  David A. Band,et al.  Sas4 and Sas5 Are Required for the Histone Acetyltransferase Activity of Sas2 in the SAS Complex* , 2003, The Journal of Biological Chemistry.

[15]  S. Agrawal,et al.  Mechanism of p300 specific histone acetyltransferase inhibition by small molecules. , 2009, Journal of medicinal chemistry.

[16]  R. Goodman,et al.  CBP/p300 in cell growth, transformation, and development. , 2000, Genes & development.

[17]  P. Cole,et al.  Structure and chemistry of the p300/CBP and Rtt109 histone acetyltransferases: implications for histone acetyltransferase evolution and function. , 2008, Current opinion in structural biology.

[18]  E. Seto,et al.  Lysine acetylation: codified crosstalk with other posttranslational modifications. , 2008, Molecular cell.

[19]  S. Agrawal,et al.  Inhibition of Lysine Acetyltransferase KAT3B/p300 Activity by a Naturally Occurring Hydroxynaphthoquinone, Plumbagin* , 2009, The Journal of Biological Chemistry.

[20]  M. J. Barratt,et al.  Phosphoacetylation of histone H3 on c‐fos‐ and c‐jun‐associated nucleosomes upon gene activation , 2000, The EMBO journal.

[21]  M. J. Barratt,et al.  The nucleosomal response associated with immediate‐early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG‐14 kinase , 1999, The EMBO journal.

[22]  Thomas A. Halgren,et al.  Merck molecular force field. IV. conformational energies and geometries for MMFF94 , 1996, J. Comput. Chem..

[23]  R. Roeder,et al.  HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF. , 2000, Molecular cell.

[24]  B. Shoichet,et al.  High-throughput assays for promiscuous inhibitors , 2005, Nature chemical biology.

[25]  J. Yates,et al.  A Fasting Inducible Switch Modulates Gluconeogenesis Via Activator-Coactivator Exchange , 2008, Nature.

[26]  J. Denu,et al.  A continuous, nonradioactive assay for histone acetyltransferases. , 2000, Analytical biochemistry.

[27]  Claudio N. Cavasotto,et al.  Discovery of novel chemotypes to a G-protein-coupled receptor through ligand-steered homology modeling and structure-based virtual screening. , 2008, Journal of medicinal chemistry.

[28]  H. Haisma,et al.  Histone acetyl transferases as emerging drug targets. , 2009, Drug discovery today.

[29]  J. Blanchard,et al.  Bisubstrate inhibition: Theory and application to N-acetyltransferases. , 2006, Biochemistry.

[30]  M. Endo,et al.  Dantrolene analogues revisited: general synthesis and specific functions capable of discriminating two kinds of Ca2+ release from sarcoplasmic reticulum of mouse skeletal muscle. , 2003, Bioorganic & medicinal chemistry.

[31]  J. Licht,et al.  Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein , 2004, Molecular and Cellular Biology.

[32]  Robert A. Copeland,et al.  Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis , 1996 .

[33]  R. Abagyan,et al.  De novo discovery of serotonin N-acetyltransferase inhibitors. , 2007, Journal of medicinal chemistry.

[34]  G. Wilson,et al.  Fluorescent markers for hypoxic cells. A study of novel heterocyclic compounds that undergo bio-reductive binding. , 1991, Biochemical pharmacology.

[35]  L. Mahadevan,et al.  Dynamic histone H3 methylation during gene induction: HYPB/Setd2 mediates all H3K36 trimethylation , 2007, The EMBO journal.

[36]  Ronen Marmorstein,et al.  Structure of the GCN5 histone acetyltransferase bound to a bisubstrate inhibitor , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Wei Gu,et al.  Activation of p53 Sequence-Specific DNA Binding by Acetylation of the p53 C-Terminal Domain , 1997, Cell.

[38]  P. Cole,et al.  p300/CBP-associated Factor Histone Acetyltransferase Processing of a Peptide Substrate , 2000, The Journal of Biological Chemistry.

[39]  E. Olson,et al.  The many roles of histone deacetylases in development and physiology: implications for disease and therapy , 2009, Nature Reviews Genetics.

[40]  Ling Wang,et al.  The structural basis of protein acetylation by the p300/CBP transcriptional coactivator , 2008, Nature.

[41]  R Abagyan,et al.  Flexible protein–ligand docking by global energy optimization in internal coordinates , 1997, Proteins.

[42]  Ronen Marmorstein,et al.  Fungal Rtt109 histone acetyltransferase is an unexpected structural homolog of metazoan p300/CBP , 2008, Nature Structural &Molecular Biology.

[43]  C. Bolm,et al.  Highly modular synthesis of C1-symmetric aminosulfoximines and their use as ligands in copper-catalyzed asymmetric Mukaiyama-aldol reactions. , 2005, Chemistry.

[44]  P. Cole,et al.  Chemical probes for histone-modifying enzymes. , 2008, Nature chemical biology.

[45]  R. Evans,et al.  Nuclear Receptor Coactivator ACTR Is a Novel Histone Acetyltransferase and Forms a Multimeric Activation Complex with P/CAF and CBP/p300 , 1997, Cell.

[46]  Andrew J. Bannister,et al.  The CBP co-activator is a histone acetyltransferase , 1996, Nature.

[47]  B. Howard,et al.  The Transcriptional Coactivators p300 and CBP Are Histone Acetyltransferases , 1996, Cell.

[48]  Ruben Abagyan,et al.  Derivation of sensitive discrimination potential for virtual ligand screening , 1999, RECOMB.

[49]  R. Marmorstein,et al.  Chemistry of acetyl transfer by histone modifying enzymes: structure, mechanism and implications for effector design , 2007, Oncogene.

[50]  C. Allis,et al.  Extraction, purification and analysis of histones , 2007, Nature Protocols.

[51]  R. Roeder,et al.  Regulation of the p300 HAT domain via a novel activation loop , 2004, Nature Structural &Molecular Biology.

[52]  S. M. Ramsh,et al.  Synthesis of 1,4-Dihydropyrano[2,3-c]pyrazole Derivatives , 2005 .

[53]  Yukihiro Itoh,et al.  Isoform-selective histone deacetylase inhibitors. , 2008, Current pharmaceutical design.

[54]  Andrew J. Bannister,et al.  p300 is required for orderly G1/S transition in human cancer cells , 2007, Oncogene.

[55]  Peter J. Alaimo,et al.  Recent advances in chemical approaches to the study of biological systems. , 2001, Annual review of cell and developmental biology.

[56]  Minoru Yoshida,et al.  [Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A]. , 1990, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[57]  N. Desroy,et al.  Discovery of new Gram-negative antivirulence drugs: structure and properties of novel E. coli WaaC inhibitors. , 2008, Bioorganic & medicinal chemistry letters.

[58]  M. Mann,et al.  Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions , 2009, Science.

[59]  Andrew J. Bannister,et al.  Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity , 2005, Molecular Cancer Therapeutics.

[60]  T. Kundu,et al.  Specific inhibition of p300-HAT alters global gene expression and represses HIV replication. , 2007, Chemistry & biology.

[61]  Udaykumar Ranga,et al.  Curcumin, a Novel p300/CREB-binding Protein-specific Inhibitor of Acetyltransferase, Represses the Acetylation of Histone/Nonhistone Proteins and Histone Acetyltransferase-dependent Chromatin Transcription* , 2004, Journal of Biological Chemistry.

[62]  H. Scheraga,et al.  Energy parameters in polypeptides. 10. Improved geometrical parameters and nonbonded interactions for use in the ECEPP/3 algorithm, with application to proline-containing peptides , 1994 .

[63]  P. Cole,et al.  Synthesis and evaluation of a potent and selective cell-permeable p300 histone acetyltransferase inhibitor. , 2005, Journal of the American Chemical Society.

[64]  Y.‐S. Lee,et al.  Peptide Synthesis with Polymer Bound Active Ester. Part 2. Synthesis of Pyrazolone Resin and Its Application in Acylation Reaction , 1992 .