Louis S. Chupak

Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry

The present study describes a novel methodology for the detection of reactive compounds using in vitro peptide-trapping and liquid chromatography–high-resolution accurate mass spectrometry (LC-HRMS). Compounds that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be associated with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addition, reactive compounds identified in biological screening can be associated with data that can be misinterpreted if the reactive nature of the compound is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compounds suspected to be chemically reactive. The assay consists of in vitro incubations of test compounds (under conditions of physiological pH) with synthetically prepared peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixtures were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS analysis is a useful approach for screening of intrinsically reactive compounds identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochemical activity assays.

Investigation of the mode of binding of a novel series of N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamides to the hepatitis C virus polymerase.

Structure based rationales for the activities of potent N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamide inhibitors of the hepatitis C viral polymerase are described herein. These compounds bind to the hepatitis C virus non-structural protein 5B (NS5B), and co-crystal structures of select examples from this series with NS5B are reported. Comparison of co-crystal structures of a potent analog with both NS5B genotype 1a and genotype 1b provides a possible explanation for the genotype-selectivity observed with this compound class and suggests opportunities for the further optimization of the series.

Structure activity relationship studies on chemically non-reactive glycine sulfonamide inhibitors of diacylglycerol lipase

N-Benzylic-substituted glycine sulfonamides that reversibly inhibit diacylglycerol (DAG) lipases are reported. Detailed herein are the structure activity relationships, profiling characteristics and physico-chemical properties for the first reported series of DAG lipase (DAGL) inhibitors that function without covalent attachment to the enzyme. Highly potent examples are presented that represent valuable tool compounds for studying DAGL inhibition and constitute important leads for future medicinal chemistry efforts.

SAR studies on a series of N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamides: potent inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus.

Described herein is the initial optimization of (+/-) N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamide (1), a hit discovered in a high throughput screen run against the NS5B polymerase enzyme of the hepatitis C virus. This effort resulted in the identification of (S)-N-sec-butyl-6-((R)-3-(4-(trifluoromethoxy)benzylcarbamoyl)-4-(4-(trifluoromethoxy)phenylsulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (2), that displayed potent replicon activities against HCV genotypes 1b and 1a (EC(50) 1b/1a=7/89 nM).