Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9 þ acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers. Cancer Res; 75(23); 5106-19. Ó2015 AACR.

Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials.CBP/p300 | bromodomain | epigenetic inhibitors | Th17 | ankylosing spondylitis

Bromodomains, protein modules that recognize and bind to acetylated lysine, are emerging as important components of cellular machinery. These acetyl-lysine (KAc) "reader" domains are part of the write-read-erase concept that has been linked with the transfer of epigenetic information. By reading KAc marks on histones, bromodomains mediate protein-protein interactions between a diverse array of partners. There has been intense activity in developing potent and selective small molecule probes that disrupt the interaction between a given bromodomain and KAc. Rapid success has been achieved with the BET family of bromodomains, and a number of potent and selective probes have been reported. These compounds have enabled linking of the BET bromodomains with diseases, including cancer and inflammation, suggesting that bromodomains are druggable targets. Herein, we review the biology of the bromodomains and discuss the SAR for the existing small molecule probes. The biology that has been enabled by these compounds is summarized.

The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation–π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells.

We describe potent and selective inhibitors of the BRD7 and BRD9 bromodomains intended for use as chemical probes to elucidate the biological roles of BRD7 and BRD9 in cells.

Simple 1-substituted 5-and 6-isoxazolyl-benzimidazoles have been shown to be potent inhibitors of the BET bromodomains with selectivity over the related bromodomain of CBP. The reported inhibitors were prepared from simple starting materials in two steps followed by separation of the regioisomers or regioselectively in three steps.Bromodomains are discrete protein domains that selectively recognize acetyl lysine in proteins. 1 There are 61 bromodomains in proteins that have a variety of functions including histone acetyl transferases such as CBP (cyclic AMP response element-binding protein, binding protein), methyl transferases, transcriptional regulators such as BRD4 (bromodomain-containing protein 4) and chromatin remodelling complexes. 2 The BET family of bromodomain containing proteins is comprised of BRDT, BRD2, BRD3 and BRD4 each of which has two bromodomains that bind to acetylated histone tails. 3 Recently BET inhibitors have been shown to have potential for use in inflammatory disease, atherosclerosis, NUT midline carcinoma, acute leukaemia and lymphoma. [4][5][6][7][8][9] Triazoloazepines such as (+)-JQ1 1, iBET762 (structure not shown) and isoxazoles such as compound 2 have been identified as potent BET inhibitors (Fig. 1). 4,9,10 Since then, a number of other templates incorporating the privileged isoxazole moiety such as in compounds 3 and 4 have been identified by researchers in the EpiNova group at GlaxoSmithKline. 6,11,12 As most known BET inhibitors are complex stereogenic molecules it would be advantageous to find simple, rapidly accessible inhibitors that would be selective † This article is part of a MedChemComm 'New Talents' issue highlighting the work of outstanding rising scientists in medicinal chemistry research. It was thought that fusing a 5-membered ring to the 4-aryl-3,5-dimethylisoxazole moiety of compound 2 (ref. 10) would give access to previously unexploited substitution patterns in known isoxazole-containing bromodomain inhibitors. Simple 5,6-bicyclic bromides 5a-c were transformed into isoxazoles by either direct arylation of 3,5-dimethylisoxazole or Suzuki reaction of the isoxazolylboronic acid to give compounds 6-8 (Scheme 1). 10,13 When tested in an AlphaScreen® assay using isolated bromodomains, compounds 6 and 7 were modest inhibitors of the first bromodomain of BRD4 (BRD4(1)) with no affinity for the CBP bromodomain whereas compound 8 had comparable affinity for both bromodomains (Table 1). 14 The indanone 6 presented an attractive intermediate for further derivitization (Scheme 2). Reduction to the racemic indanol followed by alkylation with benzyl bromide or 2-bromomethyl quinolone gave compounds 9 and 10. The amines 10-14 were prepared by S N 1 alkylation of the indanol with 3-bromo-n-propanol followed by bromide substitution. The basic centres of varying pK a in compounds 10-14 were designed with the potential to interact with an acidic residue on the edge of the BRD4(1) binding pocket, D145.Addition of an O-benzyl group in compound 9 did not increase the affi...

Significant progress has been made in discovering inhibitors and chemical probes of bromodomains, epigenetic readers of lysine acetylation.