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 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 structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 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 that such profiling can be incorporated into the earliest stages of drug discovery.

Figure 1

The EnPlex platform. (a) Schematic representation. Purified enzymes are coupled to Luminex microspheres, with a different bead color for each enzyme. Multiplexed bead complexes are incubated with a compound before being treated with a biotinylated activity-based probe and a streptavidin R-phycoerythrin conjugate (SAPE). The mixtures are scanned on a Luminex flow cytometer, where one laser detects the bead color (enzyme identity) and a second laser detects the R-phycoerythrin signal (enzyme activity). (b) A strong Luminex signal was observed with wild-type (WT) bead-coupled PME1 at low enzyme concentrations. No signals were observed with the catalytically dead S156A mutant PME1. Error bars represent the s.d of three independent experiments. The enzyme concentration was calculated assuming 100% of the protein was coupled to the beads. (c) IC₅₀ curve for the ABL127-PME1 interaction determined by EnPlex. Note that the exact enzyme concentration is not important for EnPlex, as both PME1 concentrations gave identical IC₅₀ values. Data are means ± s.e.m of three independent experiments. (d) Luminex signals for WT and inactive (SA) LYPLA1, LYPLA2, and RBBP9, and for WT lysozyme (1 nM of each enzyme was used). Error bars represent the s.d of three independent experiments.

Figure 2

Large-scale profiling of serine hydrolase-inhibitor interactions. (a) Complete dose-response profiles of sitagliptin, clasto-lactacystin β-lactone, and bromoenol lactone, which represent varying degrees of selectivity. Compounds were assayed at nine doses (from 5 nM to 33 μM) in duplicate or triplicate. The percent inhibition at each concentration relative to DMSO controls is shown. (b) Two-dimensional hierarchical cluster analysis of IC₅₀ values obtained by EnPlex. Values are listed in Supplementary Data Set 1.

Figure 3

Comparative selectivity profiling of telaprevir and boceprevir. (a) Complete EnPlex profiles. The blue arrows indicate the enzymes inhibited by telaprevir. (b) Structures of telaprevir, boceprevir, and PRCP inhibitor “compound 8o”. Similar portions of these compounds are colored red. (c) Gel-based competitive ABPP analysis with the FP-biotin probe against the chymotrypsin-like elastases. Full gel images are shown in Supplementary Figure 11. IC₅₀ values determined from three independent experiments are shown. (d) PRCP IC₅₀ curves obtained by EnPlex and by a fluorometric substrate assay. Data are means ± s.e.m of three independent experiments.

Figure 4

JZL195 inhibits AOAH deacylation of LPS in vitro and in vivo. (a) Structure of JZL195. (b) Confirmation of the interaction between JZL195 and AOAH by gel-based competitive ABPP. Full gel images are shown in Supplementary Figure 11. (c) IC₅₀ curves for the JZL195-AOAH interaction as determined by EnPlex, gel-based ABPP, and in a radioactivity-based LPS deacylation assay. Data are means ± s.e.m of three independent experiments. (d) Mice treated with vehicle or JZL195 (20 mg kg⁻¹, i.p., 6 h) were sacrificed and AOAH activity was measured in the indicated tissue lysates using a LPS deacylation assay. ***p < 0.001 for vehicle versus JZL195 groups. Data are presented as mean values ± s.e.m.; n = 4–6/group.

Figure 5

EnPlex analysis of a boronic acid and nitrile compound library. (a) Primary screening of all compounds (numbers correspond to Supplementary Table 6) against all enzymes at three concentrations. DPP4, RBBP9, and APEH are colored as indicated, and all other enzymes are colored gray. (b) Hit compounds for DPP4, APEH, and RBBP9 are displayed according to potency (x-axis) and selectivity (y-axis). “# Targets” refers to the number of enzymes inhibited >50% at the indicated concentration. Complete EnPlex profiles for (c) the DPP4 inhibitor ARI-2408 (from 1.7 nM to 33 μM) and (d) the APEH inhibitor 226 (from 5 nM to 33 μM) are shown. (e) EnPlex profiles for the enzymes inhibited by the most potent RBBP9 inhibitors. These inhibitors have similar potencies against RBBP9, but widely varying selectivities.