Discovery and Characterization of Small Molecule Inhibitors of the BET Family Bromodomains

Chun-wa Chung*†, Hervé Coste§, Julia H. White†, Olivier Mirguet§, Jonathan Wilde†, Romain L. Gosmini§, Chris Delves†, Sylvie M. Magny§, Robert Woodward†, Stephen A. Hughes†, Eric V. Boursier§, Helen Flynn†, Anne M. Bouillot§, Paul Bamborough†, Jean-Marie G. Brusq§, Françoise J. Gellibert§, Emma J. Jones†, Alizon M. Riou§, Paul Homes†, Sandrine L. Martin§, Iain J. Uings‡, Jérôme Toum§, Catherine A. Clément§, Anne-Bénédicte Boullay§, Rachel L. Grimley†, Florence M. Blandel§, Rab K. Prinjha‡, Kevin Lee‡, Jorge Kirilovsky§, and Edwige Nicodeme§

^† Molecular Discovery Research, GlaxoSmithKline R&D, Stevenage SG5 4HT, U.K.

^‡ Epinova, Discovery Performance Unit, GlaxoSmithKline R&D, Stevenage SG5 4HT, U.K.

^§ Lipid Metabolism Discovery Performance Unit, GlaxoSmithKline R&D, Les Ulis, France

J. Med. Chem., 2011, 54 (11), pp 3827–3838

DOI: 10.1021/jm200108t

Publication Date (Web): May 13, 2011

*Phone: +44 1438 763342. Fax: +44 1438 763352. E-mail: Chun-Wa.H.Chung@gsk.com.

Cite this:J. Med. Chem. 54, 11, 3827-3838

Dedication

This paper is dedicated to the memory of Dr. François Hyafil, who died on 26 May 2006.

Abstract

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein–protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.

Additional figures, experimental data, material and methods. Chemical synthesis and characterization of all intermediate compounds to 1, 2, 3: biological reagents and plasmids details; luciferase reporter assay methods; chemoproteomic and siRNA: chemoproteomic and siRNA results, example gels from chemoproteomics experiments, table of proteins identified by MS on analysis of gels, chemoproteomic material and methods, siRNA material and methods; biophysical characterization: constructs and production of single and tandem bromodomain proteins, example ITC data, KD determination of FRET H4 peptide for the tandem BET proteins, thermal shift and ITC selectivity data, matrix of sequence identify between BET bromodomains and CREBBP, ATAD2, biacore methods and analysis of cmpd1 binding to Brd-BD1/2; fluorescence anisotropy assay: correlation of FA BET binding activity with Apo-A1 upregulation, FA material and methods; X-ray crystallography: X-ray refinement statistics, Omit maps of the ligands, crystallization and crystallography material and methods, electrostatic surface of Brd2. This material is available free of charge via the Internet at http://pubs.acs.org.

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Discovery and Characterization of Small Molecule Inhibitors of the BET Family Bromodomains

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