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.

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.chemical biology ͉ crystallography ͉ human S tructural, functional, and chemical genomics (proteomics) are disciplines that aim to determine the biochemical, cellular, and physiological functions of proteins on a genome scale. Many of the central, important experimental approaches that are involved, such as protein-based screens for small-molecule inhibitors, depend on the availability of purified and active proteins. To meet this demand, many large projects are devoted to developing methods to generate large numbers of purified proteins. However, the task is proving challenging: on average, for proteins from prokaryotes, only 50-70% of soluble proteins and 30% of membrane proteins can be readily expressed in recombinant form, and only 30-50% of these expressed proteins can be purified to homogeneity (1, 2). The success rates for human proteins are predicted to be significantly lower.To improve the general rates of protein purification, efforts have focused largely on alterations of the recombinant host, the expression conditions, changes of the construct encoding the protein, and the purification conditions. It is also known that the expression and purification of a protein can be improved significantly by the addition of a specific ligand, which serves to stabilize the protein, thereby reducing its propensity to unfold, aggregate, or succumb to proteolysis. This parameter has not been studied systematically, although in individual cases the addition of a specific ligand has had dramatic effects. For example, the recombinant expression of the guinea pig and human forms of the enzyme 11␤-hydroxysteroid dehydrogenase-1 in bacteria was increased dramatically by the addition of an inhibitor of the enzyme to the growing cells (3) Wu, K. L. Kav...

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multi-target profile has, however, necessitated the application of combination therapies, which can pose significant clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as novel targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase/bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, clinical PLK1 and JAK2/FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a novel strategy for rational single agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase/bromodomain inhibitors.