As regulators of transcription, epigenetic proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, “reader” proteins become drivers of disease. In the case of bromodomains, which recognize N-ε-acetylated lysine, selective inhibition of individual bromodomain-and-extra-terminal (BET)-family bromodomains has proven challenging. We describe the >55-fold N-terminal-BET bromodomain selectivity of 1,4,5-trisubstitutedimidazole dual kinase−bromodomain inhibitors. Selectivity for the BRD4 N-terminal bromodomain (BRD4(1)) over its second bromodomain (BRD4(2)) arises from the displacement of ordered waters and the conformational flexibility of lysine-141 in BRD4(1). Cellular efficacy was demonstrated via reduction of c-Myc expression, inhibition of NF-κB signaling, and suppression of IL-8 production through potential synergistic inhibition of BRD4(1) and p38α. These dual inhibitors provide a new scaffold for domain-selective inhibition of BRD4, the aberrant function of which plays a key role in cancer and inflammatory signaling.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300LSECΔ/Δ mice compared to p300fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

In memory of FranÅoisD iederich who inspired ag eneration of scientists for tackling problems in molecular recognition Abstract: Bromodomain and extra-terminal (BET) family proteins,BRD2-4 and T, are important drug targets;however, the biological functions of each bromodomain remain illdefined. Chemical probes that selectively inhibit asingle BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here,w e develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays,w es how that our lead compound is 9-33 fold selective for BRD4 D1 over the other BET bromodomains.X -rayc rystallography supports ar ole for the selectivity based on reorganization of an onconserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers,B RD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation mayshed light on differential BET bromodomain functions.

Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. RNA-seq and histone modification ChIP-seq of human liver explants show upregulation of multiple CXCL chemokines in AH. Liver sinusoidal endothelial cells (LSEC) are identified as an important source of CXCL expression in human liver, regulated by TNFα/NF-κB signaling. A super-enhancer is identified for multiple CXCL genes by multiple approaches. dCas9-KRAB-mediated epigenome editing or pharmacologic inhibition of Bromodomain and Extraterminal (BET) proteins, transcriptional regulators vital to super-enhancer function, decreases chemokine expression in vitro and decreases neutrophil infiltration in murine models of AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment.

The Bromodomain and Extra Terminal (BET) family of proteins recognize post-translational N-ε-acetylated lysine modifications, regulating transcription as "reader" proteins. Bromodomain inhibitors are interesting targets for the development of potential cancer, inflammation, and heart disease treatments. Several dual kinase-bromodomain inhibitors have been identified by screening kinase inhibitor libraries against BET proteins. Although potentially useful from a polypharmacology standpoint, multitarget binding complicates deciphering molecular mechanisms. This report describes a systematic approach to mitigating kinase activity in a dual kinase-bromodomain inhibitor based on a 1,2,3-triazole-pyrimidine core. By modifying the triazole substituent and altering the pyrimidine core, this structure−activity relationship study enhanced BET activity while reducing the p38α kinase activity >90,000-fold. A BRD4-D1 cocrystal structure indicates that the 1,2,3triazole is acting as a N-ε-acetylated lysine mimic. A BRD4 sensitive cell line, MM.1S, was used to demonstrate activity in cells, which is further supported by reduced c-Myc expression.