ABSTRACT
Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 µM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.
Subject(s)
Nuclear Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Transcription Factors/antagonists & inhibitors , Benzodiazepinones/chemistry , Benzodiazepinones/pharmacology , Cell Cycle Proteins , Crystallography, X-Ray , HeLa Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/chemistry , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Mitogen-Activated Protein Kinase 7/antagonists & inhibitors , Mitogen-Activated Protein Kinase 7/chemistry , Mitogen-Activated Protein Kinase 7/metabolism , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Polypharmacology , Structure-Activity Relationship , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state.
