Determining the identity of prodrug activating enzyme(s) is key to understanding the mechanistic basis for enhanced cellular delivery, biodistribution, and prodrug stability. In addition, understanding species-specific prodrug sensitivities is critical for evaluating pre-clinical animal models and drug-drug interactions. Competitive Activity Based Protein Profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class within native proteomes, and has proven to be a powerful approach for activity-guided enzyme annotations. Here we describe a modified ABPP approach using direct substrate competition to determine prodrug binding enzymes of the serine hydrolase (SH) class. The ABPP approach was validated by confirming and validating that CES1 is an oseltamivir binding enzyme in intestinal cell homogenates by gel-based fluorophosphonate (FP) competition. Activation was then confirmed with recombinant hCES1. The competitive binding between oseltamivir and the FP ABPP probe was kinetically analyzed on-gel. Addition of WWL50, a mechanism-based specific carbamate inhibitor of CES1, blocked oseltamivir hydrolysis, and demonstrated exceptional selectivity across >50 active human serine hydrolases by SILAC-ABPP utilizing mass spectrometry. A second reported CES1 inhibitor, WWL79, was shown to inhibit the mouse but not human CES1. Further, complete inhibition of the hydrolysis of several additional ethyl ester prodrugs by WWL50 indicates human CES1 as their dominant activating enzyme in Caco-2 and Hep G2. Overall, we have presented a substrate-competitive activity-based protein profiling (scABPP) approach to broadly survey potential prodrug hydrolyzing enzymes, and determined a very specific hCES1 inhibitor (WWL50). The scABPP approach for surveying the SH class of hydrolase enzymes appears to be a promising methodology for new ester prodrug design and preclinical evaluation.
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