Structure- and Property-Based Optimization of Efficient Pan-Bromodomain and Extra Terminal Inhibitors to Identify Oral and Intravenous Candidate I-BET787.

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.

Identification and Optimization of a Ligand-Efficient Benzoazepinone Bromodomain and Extra Terminal (BET) Family Acetyl-Lysine Mimetic into the Oral Candidate Quality Molecule I-BET432.

The bromodomain and extra terminal (BET) family of proteins are an integral part of human epigenome regulation, the dysregulation of which is implicated in multiple oncology and inflammatory diseases. Disrupting the BET family bromodomain acetyl-lysine (KAc) histone protein-protein interaction with small-molecule KAc mimetics has proven to be a disease-relevant mechanism of action, and multiple molecules are currently undergoing oncology clinical trials. This work describes an efficiency analysis of published GSK pan-BET bromodomain inhibitors, which drove a strategic choice to focus on the identification of a ligand-efficient KAc mimetic with the hypothesis that lipophilic efficiency could be drastically improved during optimization. This focus drove the discovery of the highly ligand-efficient and structurally distinct benzoazepinone KAc mimetic. Following crystallography to identify suitable growth vectors, the benzoazepinone core was optimized through an explore-exploit structure-activity relationship (SAR) approach while carefully monitoring lipophilic efficiency to deliver I-BET432 (41) as an oral candidate quality molecule.

Isoniazid mediates the CYP2B6*6 genotype-dependent interaction between efavirenz and antituberculosis drug therapy through mechanism-based inactivation of CYP2A6.

Efavirenz is commonly used to treat patients coinfected with human immunodeficiency virus and tuberculosis. Previous clinical studies have observed paradoxically elevated efavirenz plasma concentrations in patients with the CYP2B6*6/*6 genotype (but not the CYP2B6*1/*1 genotype) during coadministration with the commonly used four-drug antituberculosis therapy. This study sought to elucidate the mechanism underlying this genotype-dependent drug-drug interaction. In vitro studies were conducted to determine whether one or more of the antituberculosis drugs (rifampin, isoniazid, pyrazinamide, or ethambutol) potently inhibit efavirenz 8-hydroxylation by CYP2B6 or efavirenz 7-hydroxylation by CYP2A6, the main mechanisms of efavirenz clearance. Time- and concentration-dependent kinetics of inhibition by the antituberculosis drugs were determined using genotyped human liver microsomes (HLMs) and recombinant CYP2A6, CYP2B6.1, and CYP2B6.6 enzymes. Although none of the antituberculosis drugs evaluated at up to 10 times clinical plasma concentrations were found to inhibit efavirenz 8-hydroxylation by HLMs, both rifampin (apparent inhibition constant [Ki] = 368 µM) and pyrazinamide (Ki = 637 µM) showed relatively weak inhibition of efavirenz 7-hydroxylation. Importantly, isoniazid demonstrated potent time-dependent inhibition of efavirenz 7-hydroxylation in both HLMs (inhibitor concentration required for half-maximal inactivation [KI] = 30 µM; maximal rate constant of inactivation [kinact] = 0.023 min(-1)) and recombinant CYP2A6 (KI = 15 µM; kinact = 0.024 min(-1)) and also formed a metabolite intermediate complex consistent with mechanism-based inhibition. Selective inhibition of the CYP2B6.6 allozyme could not be demonstrated for any of the antituberculosis drugs using either recombinant enzymes or CYP2B6*6 genotype HLMs. In conclusion, the results of this study identify isoniazid as the most likely perpetrator of this clinically important drug-drug interaction through mechanism-based inactivation of CYP2A6.

Characterization of pristane-induced arthritis, a murine model of chronic disease: response to antirheumatic agents, expression of joint cytokines, and immunopathology.

OBJECTIVE: To characterize chronic murine pristane-induced arthritis (PIA) with regard to the response to antirheumatic agents, expression levels of proinflammatory cytokines, and immunopathologic features. METHODS: Male DBA/1 mice were injected intraperitoneally with pristane oil to induce a chronic polyarthritis, which was monitored by visual scoring. Serum antibody and splenocyte responses to a panel of putative joint-derived autoantigens were measured. Whole paws were evaluated postmortem for changes in the levels of proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 by enzyme-linked immunosorbent assay, and standard histopathology techniques were used to determine joint structural changes. Therapeutic studies were performed for up to 8 weeks of dosing with prednisolone, methotrexate, 3 nonsteroidal antiinflammatory drugs (celecoxib, diclofenac, and indomethacin), a p38 MAPK inhibitor, SB242235, and human soluble TNF receptor (sTNFR; etanercept) and murine sTNFR fusion proteins. RESULTS: Antibody and cellular responses to the putative joint autoantigens revealed a broad extent of autoimmunity in PIA. TNFalpha, IL-1beta, and IL-6 were all persistently up-regulated in PIA joints. Prednisolone, methotrexate, celecoxib, indomethacin, and SB242235 all significantly reduced the arthritis scores. Etanercept was ineffective in reducing the arthritis scores, whereas murine sTNFR produced a significant, but nonsustained, benefit. Only prednisolone significantly reduced the expression of TNFalpha, IL-1beta, and IL-6 in the joints. Prednisolone and methotrexate demonstrated the most effective joint protection. CONCLUSION: We have markedly extended the characterization of PIA as a murine model of chronic inflammatory arthritis by demonstrating cellular and humoral autoantigenicity, elevation of clinically precedented joint cytokines, and variation in the response to several antirheumatic therapies. PIA offers significant potential for the long-term study of immunopathologic mechanisms and novel therapies in rheumatoid arthritis.

Glucuronides of tea catechins: enzymology of biosynthesis and biological activities.

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are major green tea catechins with antioxidant and anticancer activities. In this study, we characterized the glucuronidation of EGCG and EGC in human, mouse, and rat microsomes and by nine different human UGT 1A and 2B isozymes expressed in insect cells. Six EGCG and EGC glucuronides were biosynthesized, and their structures were identified for the first time. (-)-EGCG-4"-O-glucuronide was the major EGCG glucuronide formed in all incubations. The catalytic efficiency (V(max)/K(m)) for (-)-EGCG-4"-O-glucuronide formation followed the order: mouse intestine > mouse liver > human liver > rat liver >> rat small intestine. The UGT-catalyzed glucuronidation of EGC was much lower than that of EGCG. The V(max)/K(m) for (-)-EGC-3'-O-glucuronide followed the following order: mouse liver > human liver > rat liver > rat and mouse small intestine. Human UGT1A1, 1A8, and 1A9 had high activities with EGCG. UGT1A8, an intestine-specific UGT, had the highest V(max)/K(m) for EGCG but low activity with EGC. Mice appeared to be more similar to humans than rats to humans in the glucuronidation of EGCG and EGC. Some of these catechin glucuronides retained the activities of their parent compounds in radical scavenging and in inhibiting the release of arachidonic acid from HT-29 human colon cancer cells. These results provide foundations for understanding the biotransformation and biological activities of tea catechins.