ARTICLE doi:10.1038/mature0950 Selective inhibition of BET bromodomains aagisppkopouuna PicaudYao Shen'Wlm B.m edoroy Elizabeth M.Morse? Christopher A.French,Olaf Wiest Andrew L KungStefan knapp&JamesE.Bradner modules have not yet b ule (JQ that bind by co-crystal main and extra-tern al(BET）family nd provide a versatile chemical Gene regulation is fund. rned by eversible functionin ption elongation facto OP-TEF ble o erpre ting the post-an 学 d ha ffector mo ess th 04 as an in- ing protei ns are of subst the -called N NMC).Functio BRD4-NUT onc in maint ining the char cteristic prolifera d b and prompts terminal squam 1t with peptidic subst nty and Th eotentbrofamily amino-terminal b ergent carboxy-terminal recruitment for the tran tion of d h for targeting ped g the MGI transiti n-tro 44 Bir s02115.U5ARTICLE doi:10.1038/nature09504 Selective inhibition of BET bromodomains Panagis Filippakopoulos1 *, Jun Qi2 *, Sarah Picaud1 *, Yao Shen3 , William B. Smith2 , Oleg Fedorov1 , Elizabeth M. Morse2 , Tracey Keates1 , Tyler T. Hickman4 , Ildiko Felletar1 , Martin Philpott1 , Shonagh Munro5 , Michael R. McKeown2,6, Yuchuan Wang7 , Amanda L. Christie8 , Nathan West2 , Michael J. Cameron4 , Brian Schwartz4 , Tom D. Heightman1 , Nicholas La Thangue5 , Christopher A. French4 , Olaf Wiest3 , Andrew L. Kung8,9, Stefan Knapp1,5 & James E. Bradner2,6 Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic ‘writers’ and ‘erasers’. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein–protein interactions of epigenetic ‘readers’, and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family. Gene regulation is fundamentally governed by reversible, non-covalent assembly of macromolecules1 . Signal transduction to RNA polymerase requires higher-ordered protein complexes, spatially regulated by assembly factors capable of interpreting the post-translational modifica￾tion states of chromatin2 . Readers of epigenetic marks are structurally diverse proteins each possessing one or more evolutionarily conserved effector modules, which recognize covalent modifications of histone proteins or DNA. The e-N-acetylation of lysine residues (Kac) on histone tails is associated with an open chromatin architecture and transcriptional activation3 . Context-specific molecular recognition of acetyl-lysine is principally mediated by bromodomains. Bromodomain-containing proteins are of substantial biological interest, as components of transcription factor complexes and deter￾minants of epigenetic memory4 . There are 41 diverse human proteins containing a total of 57 bromodomains. Despite large sequence var￾iations, all bromodomain modules share a conserved fold comprising a left-handed bundle of four a helices (aZ, aA, aB, aC), linked by diverse loop regions (ZA and BC loops) that contribute to substrate specificity. Co-crystal structures with peptidic substrates showed that the acetyl-lysine is recognized by a central hydrophobic cavity and is anchored by a hydrogen bond with an asparagine residue present in most bromodomains5 . The BET family (BRD2, BRD3, BRD4 and BRDT) shares a common domain architecture featuring two amino-terminal bromodomains that exhibit high levels of sequence conservation, and a more divergent carboxy-terminal recruitment domain (Supplementary Fig. 1)6 . Recent research has established a compelling rationale for targeting BRD4 in cancer. BRD4 remains bound to transcriptional start sites of genes expressed during the M/G1 transition, influencing mitotic pro￾gression4 . BRD4 is also a critical mediator of transcriptional elongation, functioning to recruit the positive transcription elongation factor complex (P-TEFb)7,8. Cyclin-dependent kinase-9, a core component of P-TEFb9–11, is a validated target in chronic lymphocytic leukaemia12, and has recently been linked to c-Myc-dependent transcription13. Thus, BRD4 recruits P-TEFb to mitotic chromosomes resulting in increased expression of growth-promoting genes14. Importantly, BRD4 has recently been identified as a component of a recurrent t(15;19) chromosomal translocation in an aggressive form of human squamous carcinoma15,16. Such translocations express the tandem N-terminal bromodomains of BRD4 as an in-frame chimaera with the NUT (nuclear protein in testis) protein, genetically defining the so-called NUT midline carcinoma (NMC). Functional studies in patient-derived NMC cell lines have validated the essential role of the BRD4–NUT oncoprotein in maintaining the characteristic prolifera￾tion advantage and differentiation block of this uniformly fatal malig￾nancy17. Notably, RNA silencing of BRD4–NUT arrests proliferation and prompts terminal squamous differentiation. These observations underscore the broad utility and immediate therapeutic potential of a direct-acting inhibitor of human bromodomain proteins. A selective and potent inhibitor of BET family bromodomains A major collaborativefocus of our research groups concerns the develop￾ment of chemical probes18,19 and the optimization of therapeutic leads for the translation of small-molecule modulators of epigenetic targets as cancer therapeutics. Motivated by the above rationale, we have developed biochemical platforms for the identification of new inhibitors of bromodomain isoforms using high-throughput screening, as well as the annotation of putative ligands emerging from collaborative and published research. In the course of these studies, we learned of an *These authors contributed equally to this work. 1 Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK. 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA. 3 Walther Cancer Research Center and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA. 4 Department of Pathology, Brigham & Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, Massachusetts 02115, USA. 5 Department of Clinical Pharmacology, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK. 6 Department of Medicine, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, USA. 7 Department of Imaging, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA. 8 Lurie Family Imaging Center, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA. 9 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children’s Hospital, Boston, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA. 23/30 DECEMBER 2010 | VOL 468 | NATURE | 1067 ©2010 Macmillan Publishers Limited. 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