A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity

Abstract

The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely even in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided dual potency/selectivity structure-activity relationships from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 (DPP4) inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling, and suggest such profiling can be incorporated into the earliest stages of drug discovery.

DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis

“…2). To assess the selectivity of Val-boroPro more broadly across the serine hydrolase family, we profiled Val-boroPro across ~100 serine hydrolase enzymes using EnPlex 15 and by competitive activity-based protein profiling in THP-1 and RAW 264.7 lysates (Fig. 2a, Supplementary Fig.…”

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling

“…Many groups have found that the protein targets of reactive small molecules can differ substantially when identified in situ (Bachovchin et al, 2014; Martinez Molina et al, 2013; Speers et al, 2003). Accordingly, we treated A549 cells with thioester 1 , isolated proteomes, and analyzed protein labeling via SDS-PAGE (Figure 2).…”

LACTB is a tumour suppressor that modulates lipid metabolism and cell state

“…LACTB is inhibited by Z-AAD-CMK 27 , a known granzyme-B inhibitor, which led us to predict that it might cleave peptide bonds adjacent to aspartic-acid residues. Indeed, LACTB efficiently cleaved the fluorogenic peptide Ac-YVAD-AMC, but not the A-AMC or VP-AMC peptides, indicating that LACTB possesses proteolytic activity and can cleave peptide bonds adjacent to aspartic acid residues (Fig.…”