Epigenetic proteins are intently pursued targets in ligand discovery. To date, 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) which binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity toward a subset of human bromodomains is explained by co-crystal structures with 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 anti-proliferative 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.

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch-NRF2 interaction. X-ray crystallographic screening identified three distinct "hot-spots" for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1-NRF2 interaction.

Not “from above”, but “from the side”: Configuration‐retaining β‐glycosidases protonate their substrate either anti or syn to the endocyclic C1−O bond as the first step in the enzymic cleavage of the glycosidic bond (see schematic drawing). Insights into the mechanism of action of glycosidases have been gained by a combination of the synthesis of inhibitors, the study of the kinetics of their inhibition, and the analysis of the crystal structures of glycosidases and glycosidase–ligand complexes.

The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation.

BackgroundSmall molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. Nε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors.Principal FindingsHigh-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay.ConclusionsThese studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb, and has been proposed to release Pol II from promoter-proximal pausing. We show that BRD4 occupied widespread genomic regions in mouse cells, and directly stimulated elongation of both protein-coding transcripts and non-coding enhancer RNAs (eRNAs), dependent on the function of bromodomains. BRD4 interacted physically with elongating Pol II complexes, and assisted Pol II progression through hyper-acetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonized BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by assisting transcript elongation both at enhancers and on gene bodies.

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.

Selective degradation of proteins by proteolysis targeting chimeras (PROTACs) offers a promising potential alternative to protein inhibition for therapeutic intervention. Current PROTAC molecules incorporate a ligand for the target protein, a linker, and an E3 ubiquitin ligase recruiting group, which bring together target protein and ubiquitinating machinery. Such hetero-bifunctional molecules require significant linker optimization and possess high molecular weight, which can limit cellular permeation, solubility, and other drug-like properties. We show here that the hetero-bifunctional molecule can be formed intracellularly by bio-orthogonal click combination of two smaller precursors. We designed a tetrazine tagged thalidomide derivative which reacts rapidly with a trans-cyclo-octene tagged ligand of the target protein in cells to form a cereblon E3 ligase recruiting PROTAC molecule. The in-cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully used to degrade two key oncology targets, BRD4 and ERK1/2. ERK1/2 degradation was achieved using a CLIPTAC based on a covalent inhibitor. We expect this approach to be readily extendable to other inhibitor-protein systems because the tagged E3 ligase recruiter is capable of undergoing the click reaction with a suitably tagged ligand of any protein of interest to elicit its degradation.