Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

摘要: Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific sensitivities necessary evaluating preclinical animal models potential drug-drug interactions. Despite significant adoption methodologies enhanced pharmacokinetics, functional annotation activating enzymes laborious often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase family broadly reactive with reporter-linked fluorophosphonates, which have shown provide mechanism-based covalent labeling strategy state cellular amidases, esterases, thioesterases. Here we describe modified ABPP using direct substrate competition identify ethyl ester prodrug, influenza neuraminidase inhibitor oseltamivir. Substrate-competitive analysis identified carboxylesterase 1 (CES1) as oseltamivir-activating intestinal cell homogenates. Saturating concentrations oseltamivir lead four-fold reduction observed rate constant CES1 inactivation by fluorophosphonates. WWL50, reported carbamate mouse CES1, blocked hydrolysis activity human homogenates, confirming primary liver lines. related WWL79 inhibited but not providing series probes analyzing mechanisms different models. Overall, present substrate-competitive activity-based surveying candidate hydrolyzing outline kinetic parameters discovery, design, development prodrugs.