Xu_2015_Mol.Pharm_12_3399

Reference

Title : Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes - Xu_2015_Mol.Pharm_12_3399
Author(s) : Xu H , Majmudar JD , Davda D , Ghanakota P , Kim KH , Carlson HA , Showalter HD , Martin BR , Amidon GL
Ref : Mol Pharm , 12 :3399 , 2015
Abstract :

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

PubMedSearch : Xu_2015_Mol.Pharm_12_3399
PubMedID: 26262434
Gene_locus related to this paper: human-CES1

Related information

Inhibitor WWL50
Substrate WWL50    Oseltamivir
Gene_locus WWL50    Oseltamivir    human-CES1

Citations formats

Xu H, Majmudar JD, Davda D, Ghanakota P, Kim KH, Carlson HA, Showalter HD, Martin BR, Amidon GL (2015)
Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes
Mol Pharm 12 :3399

Xu H, Majmudar JD, Davda D, Ghanakota P, Kim KH, Carlson HA, Showalter HD, Martin BR, Amidon GL (2015)
Mol Pharm 12 :3399