Small Molecule Inhibitors of BromodomainâAcetyl ... - ACS Publications

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Small Molecule Inhibitors of Bromodomain−Acetyl-lysine Interactions Michael Brand,† Angelina M. Measures,† Brian G. Wilson,† Wilian A. Cortopassi,†,‡ Rikki Alexander,§ Matthias Höss,§ David S. Hewings,† Timothy P. C. Rooney,† Robert S. Paton,†,‡ and Stuart J. Conway*,† †

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K. Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, U.K. § UCB, 208 Bath Road, Slough, Berkshire SL1 3WE, U.K. ‡

ABSTRACT: Bromodomains are protein modules that bind to acetylated lysine residues. Their interaction with histone proteins suggests that they function as “readers” of histone lysine acetylation, a component of the proposed “histone code”. Bromodomain-containing proteins are often found as components of larger protein complexes with roles in fundamental cellular process including transcription. The publication of two potent ligands for the BET bromodomains in 2010 demonstrated that small molecules can inhibit the bromodomain−acetyl-lysine protein−protein interaction. These molecules display strong phenotypic effects in a number of cell lines and affect a range of cancers in vivo. This work stimulated intense interest in developing further ligands for the BET bromodomains and the design of ligands for nonBET bromodomains. Here we review the recent progress in the field with particular attention paid to ligand design, the assays employed in early ligand discovery, and the use of computational approaches to inform ligand design.

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Lysine acetylation is now recognized as a protein PTM that is involved in a multitude of fundamental cellular functions and might be viewed as analogous to protein phosphorylation in terms of its prevalence and importance.9,10 This view is supported by the emergence of the “histone code” hypothesis, which proposes that PTMs, including lysine acetylation, on histones form a combinatorial code that affects gene transcription. This hypothesis suggests that a given pattern of marks on histone tails gives rise to specific downstream functions, presenting new targets for therapeutic intervention.11,12 This idea has resulted in the terms “writers” and “erasers” being coined for enzymes that add or remove a PTM, respectively. Similarly, protein modules, including bromodomains, which bind PTMs, are termed “readers” of the histone code.13 There are 61 bromodomains that have been identified in 46 different proteins in the human genome.14 Recent work, described below, has revealed that, despite mediating PPIs,15 potent and drug-like small molecules that inhibit the KAc− bromodomain interaction can be developed. These small molecules have been instrumental in elucidating the biology of several families of bromodomain-containing proteins (BCPs). This work has resulted in five bromodomain ligands in clinical

ysine acetylation was identified as a protein post-translational modification (PTM) by Allfey et al., in 1964, who observed both acetylation and methylation of histones and suggested a role for these modifications in the regulation of RNA synthesis.1 Subsequently, the same laboratory showed that acetylation was occurring on the ε-nitrogen of lysine.2 This PTM is, however, not limited to histones, with one study identifying over 3600 sites of acetylation on 1750 proteins derived from epithelial (A549) and lymphoid (Jurkat) human cell lines.3 The fundamental importance of acetyl-lysine residues (KAc) and dynamic nature of this PTM was further underlined by the identification of enzymes that alter the lysine acetylation state, in the form of GCN5 as the first lysine/histone acetyltransferase (KAT/HAT)4 and HDAC1 the first histone deactylase.5 The effect of lysine acetylation in histones was first recognized as causing neutralization of the positive charge on lysine, resulting in a weaker association with negatively charged DNA and a more relaxed chromatin structure (euchromatin) that promotes transcriptional activation. However, the identification of a protein module, the bromodomain, which binds to KAc,6 suggested a second role for these residues, in which they mediate protein−protein interactions (PPIs) involving histones through KAc−bromodomain binding.7 It has subsequently been shown that this mechanism enables the assembly of protein complexes that affect transcription. Non-histone proteins also bind to bromodomains; for example, p53 is acetylated on lysine 382 and subsequently binds to cAMP response element binding protein (CREB) binding protein (CREBBP) via its bromodomain.8 © 2014 American Chemical Society

Special Issue: Post-Translational Modifications Received: December 5, 2014 Accepted: December 30, 2014 Published: December 30, 2014 22

DOI: 10.1021/cb500996u ACS Chem. Biol. 2015, 10, 22−39

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Figure 1. (A) Histone tails are subjected to multiple PTMs, including lysine acetylation. Lysine acetylation state is “read” by bromodomains, protein modules that invariably exist as part of a more complex protein architecture. (B) The X-ray crystal structure of the BCP BRD4(1) bromodomain in complex with the histone H4-mimicking peptide H41−12KAc5KAc8 (carbon = purple, PDB code 3UVW). The KAc residue binds in a well-defined pocket and, in BRD4(1), forms interactions with N140 and a second interaction with Y97, via a structured water molecule. The peptide binds across the WPF shelf, which is a hydrophobic region adjacent to the KAc-binding pocket. The ZA channel is typically occupied by two structural water molecules, at least in the BET bromodomains and CREBBP. ET, extra terminal domain; CTM, C-terminal motif.

between the two bromodomains found in each BCP. BRD4 and BRDT have a central role in the regulation of transcriptional elongation, which is mediated by their interaction with the positive transcription elongation factor B (P-TEFb), ultimately resulting in activation of RNA polymerase II.20,21 While this association indicates that BRD4 has a general role in transcription, inhibition of BRD4 bromodomain function affects only a small subset of, usually lineage-specific, genes. Consequently, BRD4 has been associated with a wide range of diseases and conditions.13 The biology of BRD2 and BRD3 is less well understood but is seemingly not redundant with respect to that of BRD4.

trials for a range of indications. Here we describe the state of the art in the development of bromodomain ligands and the application of these molecules to understand the biology of BCPs.

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STRUCTURE OF BROMODOMAINS Bromodomains are compact protein modules comprising approximately 110 amino acids that are found as part of larger protein architectures (Figure 1A). Despite mediating PPIs, bromodomains contain a defined and largely hydrophobic pocket that binds to acetylated lysine residues on histones and also many other proteins (Figure 1B). The carbonyl oxygen atom of KAc binds to a highly conserved asparagine residue at the top of helix B [N140 in BRD4(1)] and forms a water-mediated hydrogen bond with a conserved tyrosine residue [Y97 in BRD4(1)]. An interesting characteristic of those bromodomains that have been investigated structurally is the four water molecules that form the base of the KAc-binding pocket. In BRD4, the histone peptide binds in a hydrophobic groove on the surface of the bromodomain. Part of this groove is flanked by the residues W81, P82, and F83 and is consequently termed the WPF shelf (Figure 1B). The ZA channel is a region of the bromodomain that is framed by the ZA loop (Figure 1B). In the bromodomain and extra terminal domain (BET) family of BCPs and the CREBBP bromodomain, this channel contains two structural water molecules. However, the size, nature, and water content of this channel vary significantly between bromodomains.

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DISCOVERY AND DEVELOPMENT OF BET BROMODOMAIN LIGANDS Despite pioneering work by the Zhou group on the development of ligands for the PCAF22 and CREBBP23 bromodomains, it was the 2010 publication of two diazepine-based potent pan-BET selective ligands, (+)-JQ1 (3)24 and I-BET762 (1)25 that ignited significant interest in the bromodomains as therapeutic targets. It is notable that the diazepine scaffold has a long history as the basis of drug molecules and is viewed as a privileged structure that is particularly effective at engaging with proteins.26,27 It is perhaps significant that the lead molecules for both ligands were discovered using a phenotypic screening approach and that their cellular target was initially unknown. Diazepine-Based BET Bromodomain Ligands. I-BET762 (1). The discovery of I-BET762 (1, Figure 2) was based on a phenotypic screening approach to discover small molecule upregulators of apolipoprotein A1 (ApoA1), which is involved in protection from atherosclerosis progression and with antiinflammatory effects. When these studies commenced, no molecular mechanism to achieve this up-regulation was known. A luciferase reporter-based screen was used to identify smallmolecule enhancers of ApoA1 expression. Subsequent compound optimization led to the development of I-BET762 (1), with an EC50 value of 700 nM in the reporter gene assay. The benzodiazepine core was essential for activity, as was the aryl

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BROMODOMAIN AND EXTRA TERMINAL DOMAIN (BET) BROMODOMAIN-CONTAINING PROTEINS The BET family of BCPs, which comprises BRD2−4 and the testes specific BRDT, has been the focus of most work in the area of bromodomain ligand development.13,16−19 The BET BCPs are all tandem bromodomain-containing proteins with the first and second bromodomains located next to each other toward the N-terminal of the proteins. There is greater similarity between the first or second bromodomains of the BET family than 23


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Figure 2. Structures of key BET bromodomain inhibitors (1−18) and their IC50 or KD values with the method used to obtain these values. The KAcmimicking component of the molecule, as determined by X-ray crystallography, is shown in red. * indicates that BRD4(1) incorporates a L94A mutation.

group extending from the 6-position.28 The C(4) stereochemistry of the molecule had a significant influence on its potency, with only the (S)-enantiomer showing activity, suggesting a defined protein target for the molecule. A chemoproteomic approach was used to identify the targets for the compound, which was found to be the bromodomains of BRD2, BRD3, and BRD4. This proposed interaction was confirmed by in vitro isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) experiments. I-BET762 (1) bound to the tandem bromodomains of BRD2−4 [KD(BRD4) = 55 nM by ITC] and displaced a tetra-acetylated histone H4 peptide [IC50(BRD4) = 36 nM by fluorescence resonance energy transfer (FRET)] while showing no affinity for bromodomains outside the BET family.25,29 Mitsubishi Compounds and (+)-JQ1 (3). The patent literature on compounds with the general core structure exhibited by (+)-JQ1 (3, Figure 2) can be traced back to at least 1996, when a patent30 from Yoshitomi Pharma (later Welfide and then Mitsubishi Pharma) details thienotriazolodiazepine-based compounds for use in the treatment of autoimmune diseases including ulcerative colitis and Crohn’s disease.

A patent published by the same company in 2009 identified the BET bromodomains as the targets for these compounds, leading to suppression of cellular proliferation in tumor models.31 The triazolobenzodiazepine-based BET ligand, (+)-JQ1 (3), was developed by Filippakopoulos et al. based on compounds disclosed in the Mitsubishi patents.24,31,32 Optimization of the benzodiazepine core led to the highly potent compound (+)-JQ1, which was selective for the BET BCPs over other families of BCPs and displayed an in vitro IC50 of 77 nM (AlphaScreen). Other Diazepine-Based BET Bromodomain Ligands. OTX015 (5, Figure 2) is a thienotriazolodiazepine-based BET bromodomain inhibitor developed by OncoEthix. This compound is very similar to (+)-JQ1, but has the tert-butyl ester replaced by a (4-hydroxy)phenyl-substituted carboxamide.33 Researchers at Bayer have recently sought to optimize the pharmacokinetic properties and solubility of I-BET762 by replacing the ethylamide group. Analysis of a number of substituents in this position revealed a compound (8) with a 2methyl-1,3,4-oxadiazole replacement. This compound has an IC50 value of 20 nM against BRD4(1) by AlphaScreen.34 MS417 24


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mediated hydrogen bond with the carbonyl oxygen atom of N140 (Figure 4). Compound 6 was shown to be a potent BET bromodomain probe in cellular assays, and pharmacokinetic profiling in rat and dog demonstrated suitable characteristics for further in vivo experiments. Sharp et al. appended a range of KAc-mimicking head groups to the cyclopentylphenylsulfonamide scaffold to compare their ability to bind to BRD2−4, using an AlphaScreen assay.46 The previously identified 3,5-dimethylisoxazole and the structurally related pyrazole showed the greatest affinity for the BET bromodomains. The microsomal stability of the compounds was investigated with the 3,5-dimethylisoxazole-based compound showing rapid metabolism. However, the 3,5-dimethylisoxazolebased I-BET151 showed a much longer half-life in the same assay, indicating the rapid metabolism is not an inherent property of the 3,5-dimethylisoxazole motif. RVX-208 (7). Like I-BET762 and I-BET151, RXV-208 (7) was originally identified as an upregulator of ApoA1 and progressed to Phase II clinical trials; subsequently two studies showed that it binds to the BET bromodomains.47,48 This compound is currently undergoing clinical development as a treatment for atherosclerosis.49,50 Unlike previously described BET inhibitors, RVX-208 (7) shows modest selectivity for the second over the first bromodomain of the BETs. For example, ITC experiments carried out by Picaud et al. gave a KD of 140 nM for BRD4(2), but 1.1 μM for BRD4(1).47 It is currently unclear whether this selectivity significantly affects the physiological action of the compound. PFI-1 (14). The dihydroquinazolinone fragment was independently identified as a ligand efficient KAc mimic by a number of groups.37,51,52 Fish et al.53 employed this fragment to develop the potent and selective BET-bromodomain probe, PFI-1 (14). Structure-based fragment assessment and optimization techniques were used to develop a potent (BRD4(1) IC50 = 220 nM; AlphaScreen) and ligand efficient compound with good selectivity over a broad panel of phylogenetically diverse bromodomains determined by differential scanning fluorimetry (DSF) analysis. Furthermore, 14 showed antiproliferative effects on several leukemia cell lines in clonogenic growth assays. Studies in rats showed promising pharmacokinetic results despite modest oral bioavailability. Data reported in a separate publication showed that incubation of sensitive cell lines with PFI-1 resulted in cell-cycle arrest and induction of apoptosis.54 Down-regulation of the well-known oncology target Aurora B kinase in vitro was also achieved with this compound, potentially providing a valuable alternative strategy to inhibit this important cancer target. Thiazol-2-one-Based Bromodomain Ligands. Zhao et al. at the State Key Laboratory of Drug Research in the Chinese Academy of Sciences used a fragment-based approach to identify novel chemotypes that form the basis of BET bromodomain inhibitors.55 Forty-one putative BRD4-binding fragments were identified using docking studies, and these hits were progressed to crystallographic studies. Structure-based optimization of a 4phenyl-thiazol-2-one-based hit identified a number of compounds with submicromolar IC50 values. Compound 15 had an IC50 value of 230 nM in an FA assay for the displacement of fluorescein-tagged (+)-JQ1 from BRD4(1). The selectivity of this compound over other bromodomains was not discussed. Activity in cellular assays, including growth inhibition and MYC mRNA downregulation in HT-29 colon cancer cells, showed little correlation with inhibitory activity in the FA assay, perhaps as a result of limited membrane permeability. The most potent of

(4, Figure 2) is another diazepine-based BET bromodomain ligand, which was developed by the laboratory of Ming−Ming Zhou. This compound contains a methyl ester in place of the tertbutyl ester of (+)-JQ1 (3) and binds to BRD4(1) and BRD4(2) with similar affinities to (+)-JQ1.35 An interesting extension to the existing diazepine-based BET bromodomain ligands, disclosed by Coferon, attempts to capitalize on the tandem nature of the bromodomains in the BET BCPs.36 Two I-BET762 derivatives were synthesized, which contain complementary diol and boronic acid moieties that link covalently once both ligands are bound to one of the tandem bromodomains. This linkage effectively forms a divalent ligand in situ and results in increased affinity for the target bromodomains on the BET proteins. The drug would be administered in the form of covalently linked dimers designed for optimal binding to the target protein. The drug dissociates to monomers under physiological conditions, undergoing reassembly upon target engagement. 3,5-Dimethylisoxazole-Based Bromodomain Ligands. The 3,5-dimethylisoxazole moiety has been independently discovered by a number of groups as an excellent KAc mimic. Ligands based on this KAc mimic have been developed for both the BET37−43 and CREBBP37,44,45 bromodomains, with evidence emerging to suggest this group will also be an effective KAc mimic in other phylogenetically diverse bromodomains. Using a fragment-based approach, Hewings et al. identified 3,5-dimethylisoxazole-based compounds as ligands for the BET BCPs with low micromolar affinity.37 These compounds were of low molecular weight and had good ligand efficiency and hence were suitable for further development to give more potent ligands. By optimizing the interaction of these compounds with the ZA channel and the WPF shelf of the BET bromodomains, a more potent set of ligands was developed (9−10, Figure 2) that caused reduced viability of lung adenocarcinoma cell lines.42 Scientists at GSK independently developed the 3,5-dimethylisoxazole motif as a KAc mimic. The 3,5-dimethylisoxazolecontaining BET-bromodomain inhibitor, I-BET151 (11), was identified by phenotypic screening and developed in parallel to IBET762 (1).38 This highly potent compound [BRD4 KD = 100 nM by SPR, and KD = 36 nM by fluorescence anisotropy (FA)] selectively binds to bromodomains of the BET subfamily and has improved pharmacokinetic properties compared to triazolobenzodiazepine scaffolds.40 Researchers at GSK also took a structure-based design approach to developing 3,5-dimethylisoxazole-based BET bromodomain inhibitors. Starting from 4-phenyl-3,5-dimethylisoxazole, it was shown that a sulfonamide-linked lipophilic ring was effective at occupying the WPF shelf, improving bromodomain affinity. The addition of a cyclopentyl group gave a compound (12) with reasonable affinity for the BET bromodomains.39 Researchers at Constellation Pharmaceuticals reported an isoxazole-based BET bromodomain ligand that was developed using hits from a fragment screen.43 This screen identified a number of compounds with potencies in the micromolar range including the 5-amino-3-methyl-4-phenylisoxazole fragment. Analysis of cocrystal structures of this fragment with BRD4(1) ultimately led to the development of the carboxamide (6), which has an IC50 value of 26 nM against BRD4(1), similar to the values displayed by (+)-JQ1 (3) and I-BET151 (11) in the same assay. In compound 6, the isoxazole resides in the KAc-binding pocket and acts as a KAc mimic, and the 4-chlorophenyl group binds to the WPF shelf. The carboxamide NH2 group forms a water25


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Figure 3. Dual kinase−bromodomain inhibitors. The structures of reported dual kinase−bromodomain inhibitors (19−23) and their IC50 or KD values with the method used to obtain these values. The KAc-mimicking component of the molecule, as determined by X-ray crystallography, is shown in red. Note that 19 shows different binding modes when crystallized with BRD4(1) and BRDT, perhaps reflecting its low affinity for these bromodomains. The classic hinge-binding motif interacts with N140 in BRD4(1) (PDB ID: 4O70) whereas the pyridine N-oxide interacts with N109 in BRDT (PDB ID: 4KCX).

recently published by Wyce et al.61 and Gosmini et al.62 IBET726 binds to the tandem bromodomains of BRD4 with a KD of 4 nM by ITC (IC50 value of 22 nM in a TR-FRET assay). It shows excellent selectivity against the closely related CREBBP bromodomain with a KD value of 6.3 μM (ITC) and no significant activity against a panel of other bromodomains. IBET726 has been extensively profiled in cells, and shows potent antiproliferative activity in many, but not all, solid tumor cell lines tested, with IC50 values in a 6-day growth inhibition assay of less than 50 nM in some neuroblastoma cell lines. Dual Kinase−Bromodomain Inhibitors. A number of kinase inhibitors have been identified as binding to the KAcbinding pocket of some bromodomains, often with affinities that have implications for their in vivo activity. Martin et al. showed that Dinaciclib (19, Figure 3), a cyclin-dependent kinase (CDK) inhibitor currently in Phase III trials for chronic lymphocytic leukemia, was observed crystallographically to occupy the KAc binding pocket of BRDT(1).63 However, analysis in the BROMOscan assay revealed only weak binding, with a KD value of 37 μM. LY294002 (20), a phosphoinositide 3-kinase (PI3K) inhibitor and widely used chemical probe, also interacts with BET bromodomains. Titrating LY294002 to compete bromodomains away from an immobilized LY294002 derivative gave an apparent KD value of 2 μM for BRD4. LY294002 also showed cellular effects consistent with BET bromodomain inhibition, including suppression of cytokine release after LPS stimulation.64 Neither study investigated whether these compounds could inhibit the reported kinase activity of BRD4.65 Interestingly, BRD4 was identified as a cellular target of LY294002 in 2007 by a similar chemoproteomic approach using an immobilized inhibitor.66 Furthermore, a study in 2005 showed that LY294002 inhibited NF-κB-dependent transcription and nitric oxide production, while another PI3K inhibitor, wortmannin, did not.67 It seems likely that these effects result from BRD4 inhibition.68,69 Ciceri et al. screened 628 kinase inhibitors against BRD4(1) (AlphaScreen) to identify kinase inhibitors that displayed potent

the compounds in the cellular assay was only moderately active [GI50 of 37 μM in HT-29 proliferation assay, cf. 2.3 μM for (+)-JQ1].55 Diazobenzene-Based Bromodomain Ligands. The laboratory of Zhou and co-workers identified a diazobenzene-based compound 27, ischemin, as a ligand for the CREBBP bromodomain (Figure 5).56 The same group has more recently employed a related scaffold to develop potent BET bromodomain inhibitors.57 MS436 (17) inhibited the binding of fluorescein-tagged MS417 (4) to BRD4(1) in an FA assay with a Ki value of 65% compared to placebo. This finding is important as the majority of diabetes mellitus patients die from cardiovascular diseases. Diabetes mellitus patients given RVX-208 tended to have lower blood glucose compared to those patients given a placebo. In diabetes mellitus patients who had low levels of high-density lipoprotein, the blood glucose was significantly lower following treatment with RVX-208 compared to placebo. It was noted that the time required for RVX-208 to reduce blood glucose was at least 12 weeks following initiation of treatment. Phase 1 clinical trials for the effect of OTX015 (5) in acute leukemia and other hematological malignancies (ClinicalTrials.gov identifier: NCT01713582) and selected solid tumors (ClinicalTrials.gov identifier: NCT02259114) are currently recruiting. Trials are also scheduled to assess the effect of OTX015 in patients with recurrent glioblastoma multiform (ClinicalTrials.gov identifier: NCT02296476) and the effect of OTX015, in combination with azacitidine, in patients with acute myeloid leukemia (ClinicalTrials.gov identifier: NCT02303782).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.B. thanks the Berrow Foundation for studentship support. A.M. thanks Pfizer Neusentis and the EPSRC for studentship support. B.W. thanks UCB and the BBSRC for studentship support. D.S.H. thanks Cancer Research U.K. for studentship support. W.A.C. is supported by a Science Without Borders (CAPES) scholarship. S.J.C. thanks St. Hugh’s College, Oxford, for research support, and the BBSRC, the EPSRC, GSK, Pfizer Neusentis, and UCB for supporting research in the area of bromodomain ligands.

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ABBREVIATIONS ApoA1, apolipoprotein A1; BCP, bromodomain-containing protein; BET, bromodomain and extra terminal domain; CDK, cyclin-dependent kinase; CREB, cAMP response element binding protein (CREB) binding protein (CREBBP); DFT, density functional theory; DHQ, 3,4-dihydro-3- methyl-2(1H)quinazolinone; DSF, differential scanning fluorimetry; ELISA, enzyme-linked immunosorbent assay; FA, fluorescence aniso34


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ACS Chemical Biology tropy; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; HDAC, histone deactylase; ITC, isothermal titration calorimetry; KAc, acetyllysine residues; KAT/HAT, lysine/histone acetyltransferase; MD, molecular dynamics; MM, molecular mechanics; NMC, NUT midline carcinoma; PI3K, phosphoinositide 3-kinase; PPI, protein−protein interaction; PTEFB, positive transcription elongation factor B; PTM, protein post-translational modification; QM, quantum mechanical; SPR, surface plasmon resonance

coactivator CBP in p53 transcriptional activation. Mol. Cell 13, 251− 263. (9) Kouzarides, T. (2000) Acetylation: a regulatory modification to rival phosphorylation ? EMBO J. 19, 1176−1179. (10) Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E., and Mann, M. (2014) The growing landscape of lysine acetylation links metabolism and cell signalling. Nat. Rev. Mol. Cell Biol. 15, 536−550. (11) Strahl, B. D., and Allis, C. D. (2000) The language of covalent histone modifications. Nature 403, 41−45. (12) Gardner, K. E., Allis, C. D., and Strahl, B. D. (2011) Operating on chromatin, a colorful language where context matters. J. Mol. Biol. 409, 36−46. (13) Filippakopoulos, P., and Knapp, S. (2014) Targeting bromodomains: epigenetic readers of lysine acetylation. Nat. Rev. Drug Discovery 13, 337−356. (14) Filippakopoulos, P., Picaud, S., Mangos, M., Keates, T., Lambert, J.-P., Barsyte-Lovejoy, D., Felletar, I., Volkmer, R., Müller, S., Pawson, T., Gingras, A.-C., Arrowsmith, C. H., and Knapp, S. (2012) Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149, 214−231. (15) Wilson, A. J. (2009) Inhibition of protein-protein interactions using designed molecules. Chem. Soc. Rev. 38, 3289−3300. (16) Hewings, D. S., Rooney, T. P. C., Jennings, L. E., Hay, D., Schofield, C. J., Brennan, P. E., Knapp, S., and Conway, S. J. (2012) Progress in the development and application of small molecule inhibitors of bromodomain-acetyl-lysine interactions. J. Med. Chem. 55, 9393−9413. (17) Jennings, L. E., Measures, A. R., Wilson, B. G., and Conway, S. J. (2014) Phenotypic screening and fragment-based approaches to the discovery of small-molecule bromodomain ligands. Future Med. Chem. 6, 179−204. (18) Garnier, J.-M., Sharp, P. P., and Burns, C. J. (2014) BET bromodomain inhibitors: a patent review. Expert Opin. Ther. Pat. 24, 185−199. (19) Gallenkamp, D., Gelato, K. A., Haendler, B., and Weinmann, H. (2014) Bromodomains and their pharmacological inhibitors. ChemMedChem 9, 438−464. (20) Jang, M. K., Mochizuki, K., Zhou, M., Jeong, H.-S., Brady, J. N., and Ozato, K. (2005) The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase IIdependent transcription. Mol. Cell 19, 523−534. (21) Gaucher, J., Boussouar, F., Montellier, E., Curtet, S., Buchou, T., Bertrand, S., Hery, P., Jounier, S., Depaux, A., Vitte, A.-L., Guardiola, P., Pernet, K., Debernardi, A., Lopez, F., Holota, H., Imbert, J., Wolgemuth, D. J., Gér ard, M., Rousseaux, S., and Khochbin, S. (2012) Bromodomain-dependent stage-specific male genome programming by Brdt. EMBO J. 31, 3809−3820. (22) Mujtaba, S., He, Y., Zeng, L., Farooq, A., Carlson, J. E., Ott, M., Verdin, E., and Zhou, M.-M. (2002) Structural basis of lysine-acetylated HIV-1 Tat recognition by PCAF bromodomain. Mol. Cell 9, 575−586. (23) Pan, C., Mezei, M., Mujtaba, S., Muller, M., Zeng, L., Li, J., Wang, Z., and Zhou, M.-M. (2007) Structure-guided optimization of small molecules inhibiting human immunodeficiency virus 1 Tat association with the human coactivator p300/CREB binding protein-associated factor. J. Med. Chem. 50, 2285−2288. (24) Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro, S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J., Schwartz, B., Heightman, T. D., La Thangue, N., French, C. a, Wiest, O., Kung, A. L., Knapp, S., and Bradner, J. E. (2010) Selective inhibition of BET bromodomains. Nature 468, 1067− 1073. (25) Nicodeme, E., Jeffrey, K. L., Schaefer, U., Beinke, S., Dewell, S., Chung, C.-W., Chandwani, R., Marazzi, I., Wilson, P., Coste, H., White, J., Kirilovsky, J., Rice, C. M., Lora, J. M., Prinjha, R. K., Lee, K., and Tarakhovsky, A. (2010) Suppression of inflammation by a synthetic histone mimic. Nature 468, 1119−1123.

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KEYWORDS Acetyl-lysine: Post-translational acetylation of lysine results in acetyl-lysine, which has no charge and hence a weaker association with negatively charged DNA. Acetyl-lysine residues also bind to bromodomains BET: The bromodomain and extra terminal domain family of bromodomain-containing proteins, comprising BRD2−4 and BRDT BRD4: Bromodomain-containing protein 4 Bromodomain: A protein module that mediates protein− protein interactions by binding to an acetyl-lysine residue in the partner protein. Bromodomains are viewed as epigenetic reader domains Chromatin: In eukaryotic cells DNA is packaged around histone proteins to form a nucleosome particle. Many nucleosome particles form a macromolecular complex that is known as chromatin and in turn forms chromosomes Epigenetic: An epigenetic trait is generally recognized as a stably heritable phenotype resulting in changes in a chromosome without alterations in the DNA sequence Probe: Typically a small molecule that has well-characterized biological activity and has been designed to interact with one target or a small number of targets. Such molecules can be used in vitro or in vivo and are powerful tools for understanding the role of the target(s) in a more intact biological setting Reader: A protein domain that binds to a defined posttranslational modification on a partner protein and hence mediates a protein−protein interaction

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