Place your BETs: the therapeutic potential of bromodomains

Review

Place your BETs: the therapeutic potential of bromodomains R.K. Prinjha, J. Witherington and K. Lee Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline PLC, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK

Therapeutic targeting of the processes that regulate histone modification is a growing area of scientific exploration. Although most interest has concentrated on the various families of enzymes that contribute to these processes, this review focuses on emerging data demonstrating the chemical tractability and therapeutic potential of a hitherto underexplored family of proteins, namely the bromodomain (BRD) family of reader proteins. These proteins perform a crucial role in translating histone modifications with powerful transcriptional consequences. We review current knowledge of the biology of this emergent target class and highlight recent breakthroughs that now make the BRD family of reader proteins attractive for drug discovery. BRDs and epigenetics Epigenetics is a term used to describe heritable gene regulation or transcriptional silencing across generations of cells and even organisms [1]. This process is mediated through dynamic and reversible changes in chromatin accessibility and post-translational modifications of histone tails [2–4]. To date, several physiological processes have been proposed to contribute to epigenetic regulation, including DNA methylation, non-coding RNA-mediated scaffolding and complex formation, and histone modification [2,5]. The term ‘histone modification’ relates to the post-translational covalent modification of histone proteins that markedly influences the ability of associated DNA to be transcribed. This is a rapidly expanding area of research and a wide range of modifications have been described, together with a large repertoire of enzymes and associated proteins that serve to direct the placement and removal of these modifications or so-called marks. These observations have led to the concept of the ‘histone code’ and the idea of writers, erasers and readers (Box 1) [6]. The BRD-containing family of proteins (Figure 1) is an important class of histone modification reader proteins that recognize acetylated lysine residues within histone proteins. BRDs were first described in 1992 as domains of approximately 110 amino acids that are conserved in several genes from humans, Drosophila and yeast, each with crucial roles in transcriptional regulation [7,8]. BRDs mediate this control by recognizing acetylation marks on histones and functioning as a scaffold for the assembly of macromolecular complexes that alter chromatin accessibility to transcription Corresponding author: Lee, K. ([email protected]).

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factors and also allowing the recruitment or activation of the RNA polymerases. The total number of unique individual human BRDs is now 57, and these have recently been clustered into nine subgroups based on sequence identity [9]. Although BRD-containing proteins were initially considered to be undruggable, in the current review we will describe the recent developments that have shown these proteins not only to be good targets for drug development but also to possess an unpredicted and selective effect on gene expression. We will outline the emerging biological roles of many of these proteins to highlight their potential in a range of disease settings. An ability to selectively modulate gene expression with small molecule inhibitors has been a clinical aspiration that now appears feasible for the first time with these BRD inhibitors. Discovery of BRD inhibitors Dhalluin and colleagues solved the first three-dimensional structure of a BRD using NMR to resolve the structure of the transcriptional co-activator p300/CBP associated factor (PCAF) [10]. BRDs share a conserved fold comprising a left-handed bundle of four amphipathic helices (aZ, aA, aB and aC) linked by two diverse loop regions that contribute to ligand specificity (ZA and BC loops). At one end, the N and C termini come together, emphasizing the modular architecture of this domain and underscoring the idea that the BRD could act as a functional unit for protein interactions. At the opposite end, the ZA loop packs against the BC loop, forming a hydrophobic pocket that recognizes the acetylated ligand (Figure 2). Employing chemical shift mapping using titration data and NMR structural analysis of the BRD in complex with acetyl-histamine, Dhalluin et al. demonstrated that the methyl and methylene groups of the ligand made significant contacts with the side chains of Val 752, Ala 757, Tyr 760, Tyr 802, Asn 803 and Tyr 809, which are highly conserved residues across the BRD family. This was subsequently confirmed with a crystal structure of the GCN5 H4KAc16 complex [11]. In addition to binding to the conserved hydrophobic and aromatic residues, the acetylated lysine residue formed a hydrogen bond with a highly conserved asparagine. Based on these findings, Zhou conducted an NMR-based chemical screening campaign to identify small molecules that bound to the BRD of PCAF [12]. The acetyl-lysine (AcK) binding pocket is hydrophobic in almost all BRDs. However, the electrostatics at the opening of the AcK binding pocket display significant variations, so the emphasis was on identifying compounds that selectively

0165-6147/$ – see front matter ß 2012 Published by Elsevier Ltd. doi:10.1016/j.tips.2011.12.002 Trends in Pharmacological Sciences, March 2012, Vol. 33, No. 3

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Box 1. The histone code For more than 40 years, it has been known that DNA associates with various histone proteins to form so-called nucleosomes that themselves then bundle to form chromatin fibres which facilitate the organization and packing of the DNA in the nucleus. Histones are globular proteins with a flexible N terminus (often referred to as the histone tail) that protrudes from the nucleosome and is subject to a wide range of potential covalent modifications that are believed to have an important influence on chromatin structure and the accessibility of the proximal DNA to transcription (Figure I). From these observations arose the notion that these covalent marks act to create a code laid down and removed by specific families of enzymes [6,79]. The code is read by specific families of proteins such as bromodomains, which can then act to form assembly points on which other proteins such as the pTefb complex associate to direct gene transcription [80]. More recently, the observation that the code is also context dependent, with the generation of one specific histone mark potentially influencing the placement of others, has led to the concept that the process should be considered a language rather than a code [81].

HISTONES

Writing

Erasing

Reading

Acetylases

Deacetylases

Bromodomain

Methylases

Demethylases

Chromodomain

Kinases

Phosphatases

PHD finger WD40 repeat

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Figure I. DNA associates with histone proteins to form nucleosomes. The Nterminal regions of the histone associated proteins project from the nucleosome and are subject to the activity of the various groups of proteins indicated (categorized by function as writing, erasing and reading).

bound in and around the AcK binding pocket, because it was postulated this may lead to selectivity. This approach identified compounds with affinities comparable to that of the Tat-AcK50 peptide (IC50 5 mM). The lead compound did not bind the BRDs of CREB binding protein (CBP) or transcriptional intermediary factor 1 beta (TIF1b), indicating for the first time that specificity within the BRD family is achievable. Zhou and colleagues went on to identify a series of molecules that blocked the K382-acetylated p53 association with CBP utilizing a structure-based NMR spectroscopy screen [13]. Given the structural differences at the AcK binding pocket and the fact that most known drug molecules contain one aromatic ring, the Zhou research team constructed a knowledge-based library of approximately 200 compounds from a collection of

approximately 14,000 small molecules. One criterion for inclusion was that compounds required the presence of one aromatic ring connected to a -NHCOCH3 group either directly or via a two-three carbon chain to act as an acetyl lysine mimetic or biostere. This combination was postulated to mimic the binding of the acetylated lysine residue into the BRD pocket, and it led to the identification of four structurally distinct clusters of low molecular weight compounds. In a subsequent larger study, Zhou and colleagues conducted a 3000 compound NMR diversity screen for CBP [14]. This led, after optimization, to MS120 (Kd 19 mM), which could inhibit doxorubicin-induced apoptosis in cardiomyocytes. The molecular basis for recognition was highlighted to be the phenoxy group forming a hydrogen bond to the conserved Asn1168 of CBP [14]. Although the Zhou research team demonstrated the key principle that small-molecule inhibitors of BRDs could be identified, these efforts led only to the identification of compounds with micromolar affinities, which have limited utility in biological investigation. Recently, two independent groups reported the first selective nanomolar inhibitors for the tandem BRD-containing family of transcriptional regulators known as the BET proteins (Brd2, Brd3, Brd4 and BrdT), suggesting for the first time that BRDs may be appropriate for small-molecule drug discovery [15,16]. The GlaxoSmithKline (GSK) group employed a phenotypic assay and subsequent chemoproteomics, siRNA, biophysical assays and X-ray crystallography to elucidate the BET proteins as the molecular target of novel apolipoprotein A-1 (ApoA1) upregulators [15,17]. The compounds bind to the AcK recognition pocket and directly antagonize the interaction between the BRDs and acetylated histone peptide (Figure 3) [15]. The crystal structures offer insights into how selectivity for the BET family is achieved and the basis of their mimicry of the native AcK peptide. The 1,2,4-Me triazolyl ring mimics both the hydrophobic and the hydrogen bonding characteristics of the acetylated peptide (Figure 3). No interaction was observed between the inhibitor and two other BRDs, CBP and ATAD2, which the authors hypothesized was due to the ligand making distinct contacts outside the AcK cavity. By contrast, differences between the BET domains are relatively minor and are tolerated by the inhibitor compounds. BD1 of Brd2, 3 and 4 have an isoleucine at the position analogous to residue 162 in Brd2. This ‘gatekeeper’ residue, which varies in size across the BRD family, controls access to a lipophilic region comprising a tryptophan-proline-phenylalanine sequence (known as the WPF shelf) which is present in a number of BRDs. The importance of this shelf was also highlighted in a recent publication that disclosed the X-ray structures of the BD1 and BD2 BRDs from BrdT in complex with histone peptides from H4 and H3, respectively [18]. A key finding was the cooperative binding mode of a diacetylated histone H4 tail to the single BRD of BrdT-BD1 in which the AcK pocket binds the first H4AcK mark at position 5. The second AcK at position 8 lies across the WPF shelf and leads to increased binding affinity. Using an anti-inflammatory phenotypic profiling assay, researchers from Mitsubishi Pharmaceuticals [19] discovered that thienodiazepines could also bind to BET proteins. This finding was exploited by the SGC research group and collaborators, who modified the generic 147

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BRPF1 BRD1 BPRF3 BRD9 BRD7 KIAA1240 ATAD2

CREBBP EP300

BRD2_1 BRD3_1 BRD3_2 BRD4_1

BRDT_1 BRDT_2 BAZ1A

BRD2_2

BRD4_2

BRD8

WDR9_2 PHIP_2 BRWD3_2

TAF1_1

BAZ1B

PRKCBP1

TAF1L_1 TAF1_2 CECR2 TAF1L_2 FALZ GCN5L2 PCAF

BAZ2B BAZ2A

TRIM33

ZMYND11

T

TIF1 TRIM66 Y SP140

T

MLL TRIM28

Y

SP110

Y

SP100 LOC93349

WDR9_1 BRWD3_1

ASH1L

T

PHIP_1 T PB1_1 PB1_3 PB1_5 PB1_2 PB1_4

Y

SMARCA4 SMARCA2

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Figure 1. The bromodomain family of histone modification reader proteins. Phylogenic tree showing the structural homology between known bromodomain-containing proteins. Those marked with a green circle indicate where the structure of the bromodomain has been resolved and those marked with a red circle indicate that the acetyllysine recognition pocket is structurally atypical.

structure disclosed in the Mitsubishi patents to identify (+)JQ-1 [16]. Similar to the interactions observed with the GSK inhibitor, the triazole ring of JQ-1 formed a hydrogen bond with the evolutionarily conserved asparagine, and the selectivity profile suggested similar minimal interactions outside the BET family [16]. The compounds reported independently by the GSK and SGC groups represent novel chemical templates that have been added to recently (Figure 4) [20,21]. All are distinct from the previously reported simple acetyl-containing templates, with a clear mode of action and demonstrated ability to produce inhibitors with high (nM) affinity, specificity, cell permeability and in vivo efficacy (see below). Role of BRD-containing proteins in disease and their emerging therapeutic potential As outlined above, the remarkable conservation of the BRD module from yeast to human underlines the importance of acetyl-lysine recognition in fundamental biological processes [7]. Until recently, the crucial embryonic developmental role of many of these proteins in gametogenesis, cell specification or differentiation have, unsurprisingly, resulted in embryonic lethality that has precluded workers from gaining an understanding of their role in post-natal biology and disease pathogenesis. The discovery of selective small-molecule inhibitors of the BET family BRDs described above has for the first time alerted us to the 148

immense potential of this family and caused us to reevaluate the rationale for these proteins as potential therapeutic targets. We have looked at a range of data sources including expression patterns, phenotypes following gene knockout or knockdown, genetic associations with disease and, where available, effects of small-molecule modulators. A full representation of all the supporting data for 42 proteins in all disease areas is clearly beyond the scope of this review but selected examples are given that best illustrate the remarkable therapeutic potential role for this novel class of previously intractable protein targets. BRD roles in inflammation Early evidence for the role of BET proteins in lymphocytes came from studies of transgenic mice overexpressing Brd2. These mice developed splenic follicular B cell lymphomas, which had a transcriptional profile overlapping that seen in human-derived samples and a propensity for transplantable leukemia [22]. These observations suggested that the BET proteins had a critical role in the specification, expansion and maintenance of lymphocyte lineages known to mediate many autoimmune diseases. Insights into the role of genes in disease can often be gained from human genetic studies; however, the location of the Brd2 gene within the MHC complex has hampered genetic analysis of its role in immune mediated diseases. Nevertheless, variations in the frequency of

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ZA BC

A

B Z C

N-Terminus

C-Terminus TRENDS in Pharmacological Sciences

Figure 2. Three-dimensional structure of BRD-2(N) as determined by X-ray crystallography.

single nucleotide polymorphisms (SNPs) mapping to the Brd2 gene in rheumatoid arthritis (RA) patients were first observed in high resolution mapping studies by Chissoe and colleagues at GSK [23] and were subsequently independently confirmed in a multivariate study of the interaction of smoking, genotype and citrullinated peptide levels in RA cohorts [24]. These results suggest that variations in Brd2 function or expression may contribute in part to the development of RA disease. At the biochemical and molecular level, Huang and colleagues illustrated the role of Brd4 in the regulation of nuclear factor kappa B (NFkB) dependent genes in response to endotoxin-mediated activation of the innate immune system [25]. Furthermore, Medzhitov and colleagues were able to identify a role for Brd4 in the regulation of inducible gene expression in macrophage primary response genes. They found that it acted by affecting an acetylation and pTEFb dependent switch from basal transcription of immature unspliced transcripts to high levels of active mature mRNA [26]. These studies were extended by Nicodeme et al. [15]. In mouse bone marrow-derived macrophages, they demonstrated that a subset of lipopolysaccharide (LPS)-induced genes was differentially inhibited by the active inhibitor at both the mRNA and protein levels. Using chromatin immunoprecipitation (ChIP), this transcriptional inhibition was correlated with the prevention of signal-induced Brd-2, -3 and -4 protein recruitment to affected gene promoters. Using ChIP-Seq to further investigate the mechanisms by which this transcriptional selectivity was achieved, the authors observed that the small-molecule inhibitor IBET selectively attenuated the induction of secondary response genes characterized by low CpG content, low basal H3 and H4 acetylation levels, low K3K4me3 methylation and low RNA polymerase II occupancy [15,26,27]. This work suggested that the BET proteins are involved in the recognition of gene promoters containing a combination of post-translational histone marks characteristic of poised but inactive secondary response genes [26,28].

Gatekeeper residue

I-BET WPF shelf

ZA channel

N N Acetyl-lysine pocket

N

N

MeO

O

N H “N” side

Cl “C” side TRENDS in Pharmacological Sciences

Figure 3. Structure of I-BET (orange) bound to the acetyl-binding pocket of BRD4-BD1 overlaid with acetylated histone H4 peptide (H4ac, green).

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WPF shelf

W81 N O

P82

N NH

O

N140 F83

N

O N

M105

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Figure 4. Structure of I-BET151 (magenta) bound to the acetyl-lysine recognition pocket of BRD4-BD1 (cyan) overlaid with the H3K14 acetyl-peptide (green).

The physiological consequences of I-BET were tested in mice treated with lethal doses of LPS in a model of endotoxic shock routinely used to study aspects of sepsis in humans [29]. In these studies, both prophylactic and, more remarkably, therapeutic dosing of I-BET was able to suppress serum cytokine expression and survival (Figure 5). Indeed, increased survival was also observed in models of heat-killed Salmonella and caecal ligation and puncture models of bacterial sepsis [15], suggesting that I-BET affected some fundamental regulatory systems, underlining the enormous anti-inflammatory therapeutic potential of this class of inhibitor. Emerging data are now also starting to link other nonBET family BRD-containing proteins with immune-mediated diseases. Rare variants in the BRD-containing promyelocytic leukaemia (PML) nuclear body-associated speckled protein SP110 provide the first intriguing insight into its role in immune disease [30]. Studies of Lebanese migrants now resident in Sydney, Australia have identified a cohort of individuals displaying high penetrance of a

LPS 100

Key:

Survival (%)

80

Control I-BET only

60

I-BET (preventative) 40

I-BET (therapeutic)

20

0 0

25

50

75

100

Time (h) TRENDS in Pharmacological Sciences

Figure 5. GSK I-BET prophylactic and therapeutic treatment protects mice from lethal doses of LPS-induced endotoxic shock. Reproduced with permission from [15].

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hepatic vascular occlusion, fibrosis and immunodeficiency syndrome (hVODI) [31]. These individuals are identified either through a family history of vascular occlusive disease (VOD) or through childhood presentation with severe combined T and B cell immunodeficiency. They display susceptibility to a range of pathogens including Pneumocystis jerovici and opportunistic fungal (but surprisingly not mycobacterial) infections. The characteristics of the immunodeficiency caused by the autosomal missense mutations in exon 2 or 5 causing loss of protein expression are particularly immunologically interesting because the total number of circulating lymphocytes appears wholly unaffected, as is their response to activation by PMA/ ionomycin. By contrast, they show a dramatic loss of memory T and B cells and concomitant severe hypogammaglobulinaemia [30]. The related protein SP140, which shares domain structure and significant sequence homology with SP110, was identified as a PML nuclear body component that was highly induced by interferon (IFN) gamma and other inflammatory stimuli in lymphocytes [32]. Differential binding of SP140 to the HIV-1 viral infectivity factor (Vif) in infection-permissive (but not in non-permissive) cultured cells further suggests a role in host defence [33]. It has also been shown to be a relatively common auto-antigen in the autoimmune disease primary biliary cirrhosis (PBC) [34]. More recently, a well-powered meta-analysis of SNP variation in Crohn’s disease identified the SP140 locus as a novel disease susceptibility gene [35]. Although the functional significance of the variation remains unclear, it is further suggestive of a role of this BRD-containing protein in the function of the immune system. SMARCA4 (BRG) is another BRD-containing protein and is a crucial component of the BAF complex that has been linked to several immune functions (comprehensively reviewed in [36]. It was found, through yeast two-hybrid (Y2H) screening, to bind to STAT2 [37] and regulate the basal and induced expression of IFN-regulated genes, and is postulated to be responsible for perhaps 90% of the

Review ‘interferon signature’ that characterizes diseases such as systemic lupus erythematosus (SLE) [38]. Its activity has been linked to T cell differentiation [39] and more recently in the selective development of Treg cells in conditionally Brg1 gene-ablated mice [40,41]. BRD roles in oncology The fundamental role for BET family proteins in cell division has been highlighted by many observations [9]. Work from several laboratories has elucidated the role of BET proteins in chromatin binding and macromolecular complex formation and function, pointing to a central role in oncogenesis. This was definitively demonstrated by the finding that transgenic overexpression of Brd2 in lymphocytes produced B cell lymphomas that were transcriptionally identical to activated B cell lymphoma isolates from patients [22,42,43]. The expression of Brd4 was found to be deregulated in breast cancer biopsies, and the Brd4 transcriptional signature was found to discriminate rate of disease progression [44]. The functional effects of Brd4 on metastasis and stem cell transformation have been recently determined to reside in the C terminus prolinerich domain of the protein [45]. Brd4 (and more rarely Brd3) gene translocation with the otherwise testis- and nuclear-restricted protein NUT has been shown to result in a rare but rapidly fatal condition termed NUT midline carcinoma [46]. In cell lines carrying the translocation, administration of siRNA to the Brd4 protein or treatment with JQ1 is sufficient to reduce proliferation and induce differentiation of the cells into keratinocytes. Consistent with these in vitro observations, JQ1 was able to significantly inhibit tumour growth in mice in two xenograft models of NUT midline carcinoma [16]. Recently, I-BET has been used in an elegant chemoproteomics strategy to map the proteins that interact with BET proteins. This comprehensive BET interactome complements recent studies looking at the Brd4 C terminus [47], but additionally has provided a rationale for the potential of these BET inhibitors in many oncological states including lymphomas, multiple myeloma SET domain (MMSET) myelomas and mixed-lineage (MLL) leukaemias. These predictions were confirmed with a novel inhibitor I-BET151 in two independent mouse xenograft models of AML leukaemia [20]. Consistent with these findings, an independent small hairpin RNA (shRNA) screen identified Brd4 as a key leukaemia target [48]. Furthermore, testing of I-BET and JQ-1 in myeloma cells was found to regulate myc expression and inhibit disease progression in mouse xenograft models [49]. Although space restrictions prevent a full listing of all BRD-containing proteins that have some biological association with cancer, we have selected a small number with compelling links. In addition to the study mentioned above linking to Crohn’s disease [35], a separate genetic study has also identified SNPs near the SP140 gene that were associated with increased incidence of B-cell chronic lymphocytic leukaemia (CLL) (odds ratio 1.41). Although the available sample numbers were small, a subsequent encouraging analysis of SP140 levels in circulating lymphocytes appeared to show a correlation of disease-associated genotypes and mRNA expression [50].

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The ATPase AAA domain containing protein ATAD2 (ANCCA) has been widely studied, and has been shown to be regulated by the pRb-E2F and MLL pathways and be responsible for coupling with myc in the regulation of proliferation and senescence associated target genes [51] including B-Myb and Ezh2 [52]. The BRD-mediated recognition of acetylated histone H3K14 was found to be necessary for the anchoring and function of the E2F-MLL complex in transcriptional coactivation and cell proliferation [53]. ATAD2 overexpression was found in breast cancer [52] and lung and testis cancer biopsies, among others [54], with a strong negative correlation with patient survival and prognosis. The large mixed lineage leukaemia (MLL) gene is characterized by the presence of PHD, BRD and histone lysine methyltransferase (KMT-SET) domains, and is often involved in pathogenic chromosomal translocations with a large number of other genes [55]. These translocations are found to cause human acute leukaemias that can be classified as myeloid (AML), lymphoblastic (ALL) or mixed lineage (MLL) in nature. These MLL translocations have been described most commonly in infant leukaemias (70%), but have also been found in adult (10%) and increasingly in topoisomerase inhibitor chemotherapy treatment-induced disease (tAML) [55]. As mentioned above, these conditions are appearing to be extremely sensitive to BET BRD inhibition [20]. Bromodomain roles in infectious disease In addition to the BRD-containing proteins involved in modulating responses to viral or bacterial infection discussed above, a growing body of literature highlights the role of BET family proteins in the life cycle of infectious agents including HIV, herpes and papilloma viruses [56– 58]. These diverse biological effects extend through the activity of assembling transcriptional complex components such as pTEFb [59–61], interacting directly with viral proteins such as papilloma E2 [62] or LANA [63] and mediating chromosomal anchoring to ensure infection of dividing daughter cells [64]. Other emerging areas of therapeutic interest The role of the BET family proteins (particularly Brd2) in metabolic disease has been well reviewed recently [42,65,66] and I-BET molecules have been shown to upregulate ApoAI [17], a protein known to be involved in antiinflammatory and metabolic processes. The recent demonstration of Brd3 binding to the acetylated GATA1 transcription factor significantly extends the function of these proteins beyond histone binding alone [67,68]. The demonstration in these studies that Brd3 has a critical role in haematopoietic erythroid gene regulation was examined using GSK I-BET819X and suggests a potential role in the treatment of associated diseases [17]. Defects in epigenetic processes in mental retardation disease [69] and other CNS diseases has been reviewed recently [70]. Brd2 haplo-insufficiency has been linked to neuronal deficits and epilepsy [71]. Genetic studies have linked SNPs in the single BRD-containing gene Brd1 (BRPF1; BRL) with both schizophrenia and bipolar disorder in Europe [72,73] but not in Japan [74]. Human 151

Review genetics has also linked SNPs in SMARCA2 (BRM1; SWI2) to schizophrenia [75–77]. CBP, which has long been linked with the processes of learning and memory, has been found to be mutated in individuals with Rubinstein– Taybi syndrome [78], a genetic disease that involves broad thumbs and toes, short stature, distinctive facial features and varying degrees of intellectual disability. The CBP BRD inhibitor ischemin was found to prevent doxorubicin-mediated DNA damage-induced apoptosis in cultured rat cardiomyocytes [14]. This activity was postulated to highlight the potential for p53-CBP inhibitors to provide therapeutic benefit in ischemia-reperfusion injury situations, including myocardial infarction. Concluding remarks It is clear from the above that the BRD family of histone reader proteins plays a diverse range of roles that are crucial for normal physiological function. Interference with these processes during development is often lethal, perhaps underlying their fundamental importance and thereby hindering a full evaluation of their biological significance until now. It had previously appeared that this BRD family of proteins was intractable to small-molecule drug discovery due to the seemingly complex nature of the protein–protein interactions known to take place at the chromatin level. However, through the pioneering work of Zhou and colleagues, together with the rapid exploitation of serendipitous findings by several groups, we are now in an exciting position where small-molecule inhibitors can be developed to fully dissect the biological and medical importance of this emerging class of protein. Acknowledgements We would like to thank our colleagues at the Structural Genomics Consortium and at GSK for their critical review and comments.

Conflict of interest R.K.P., J.W. and K.L. are employees of GlaxoSmithKline.

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