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.
Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for approximately 40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.
Fatty acid amide hydrolase (FAAH), an intracellular serine hydrolase enzyme, participates in the deactivation of fatty acid ethanolamides such as the endogenous cannabinoid anandamide, the intestinal satiety factor oleoylethanolamide, and the peripheral analgesic and anti-inflammatory factor palmitoylethanolamide. In the present study, we report on the design, synthesis, and structure-activity relationships (SAR) of a novel class of potent, selective, and systemically active inhibitors of FAAH activity, which we have recently shown to exert potent anxiolytic-like effects in rats. These compounds are characterized by a carbamic template substituted with alkyl or aryl groups at their O- and N-termini. Most compounds inhibit FAAH, but not several other serine hydrolases, with potencies that depend on the size and shape of the substituents. Initial SAR investigations suggested that the requirements for optimal potency are a lipophilic N-alkyl substituent (such as n-butyl or cyclohexyl) and a bent O-aryl substituent. Furthermore, the carbamic group is essential for activity. A 3D-QSAR analysis on the alkylcarbamic acid aryl esters showed that the size and shape of the O-aryl moiety are correlated with FAAH inhibitory potency. A CoMSIA model was constructed, indicating that whereas the steric occupation of an area corresponding to the meta position of an O-phenyl ring improves potency, a region of low steric tolerance on the enzyme active site exists corresponding to the para position of the same ring. The bent shape of the O-aryl moieties that best fit the enzyme surface closely resembles the folded conformations observed in the complexes of unsaturated fatty acids with different proteins. URB524 (N-cyclohexylcarbamic acid biphenyl-3-yl ester, 9g) is the most potent compound of the series (IC(50) = 63 nM) and was therefore selected for further optimization.
