Histone–lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone–bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC50 values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.

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.

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 non-BET 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.

The bromodomain protein module, which binds to acetylated lysine, is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodomain and extra terminal domain) bromodomain family with good ligand efficiency. X-ray crystal structures of the most potent compounds reveal key interactions required for high affinity at BRD4(1). Cellular studies demonstrate that the phenol and acetate derivatives of the lead compounds showed strong antiproliferative effects on MV4;11 acute myeloid leukemia cells, as shown for other BET bromodomain inhibitors and genetic BRD4 knockdown, whereas the reported compounds showed no general cytotoxicity in other cancer cell lines tested.

The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology.Abbreviations ABP, activity-based probe; ABPP, activity-based protein profiling; AOMK, acyloxymethyl ketone; Dha, dehydroalanine; DUB, deubiquitinase; HECT, homologous to the E6-AP carboxyl terminus; MS, mass spectrometry; NEDD8, neural precursor cell expressed, developmentally down-regulated 8; OTU, ovarian tumor domain protease; RBR, RING-between-RING; RING, really interesting new

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

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...

Formaldehyde is universally employed to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, and avoiding high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5–2.4 fold using a catalyst under optimized conditions, and by 7–25 fold compared to a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.