Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes

Abstract

High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein.

Figure 1

Schematic representation of the FluoPol-ABPP platform. An enzyme is dispensed into a 384-well plate, and a different test compound is added to each well. Shown are representative wells where the test compound is an inhibitor of the enzyme (a) or is inactive (b). A fluorescent ABPP probe is then dispensed to all wells, and the plate incubated for a fixed time interval. The reaction of the probe with uninhibited enzyme (b), but not inhibited enzyme (a), will greatly increase the apparent mass of the probe, resulting in the maintenance of a strong FluoPol signal.

Figure 2

Optimization and validation of the FluoPol-ABPP platform for RBBP9. (a) RBBP9 (2 μM) and FP-rhodamine (75 nM) generated a strong, time-dependent increase in FluoPol signal. No labeling was observed in the absence of enzyme or with the catalytically-dead S75A mutant RBBP9. The indicated 45-minute time point (Z' = 0.71) prior to reaction completion was selected for HTS. Error bars represent s.d.. (b) Under these conditions, FP-biotin (30 min pre-incubation) inhibited FP-rhodamine labeling of RBBP9 with an IC₅₀ value of 6.3 μM as determined by gel-based competitive ABPP. Left panel, fluorescent gel is shown in gray scale. Right panel, error bars represent s.e.m. (c) FP-biotin (5 μM) gave an ∼50% reduction in the RBBP9-generated FluoPol signal. Error bars represent s.d.

Figure 3

Identification of RBBP9 primary hits. (a) A screen of 18,974 compounds identified 35 hits that reduced the FluoPol signal of FP-rhodamine labeling of RBBP9 by > 50% relative to control reactions run in the absence of added compound. (b) Structures of representative primary hits. (c) Inhibition of FP-rhodamine labeling of RBBP9 by representative compound hits (20 μM, 30 min preincubation) as determined by gel-based competitive ABPP (upper panel). Compound 4 covalently dimerized RBBP9 as determined by protein staining (Coomaissee blue) (lower panel, asterisk). (d and e) Structures of emetine analogues (d) and IC₅₀ curves for RBBP9 (e) as determined by gel-based competitive ABPP. Asterisk in d notes compound insolubility at high concentrations (> 300 μM) precluded accurate IC₅₀ determination. Error bars in e represent s.e.m.

Figure 4

Competitive ABPP in proteomes identifies emetine (1) as a selective inhibitor of RBBP9. (a) Evaluation of representative hits (20 μM, 30 min preincubation) by competitive ABPP in the mouse brain membrane proteome (1 mg/mL proteome; 1 μM FP-rhodamine, 10 min, room temperature). Recombinant human RBBP9 (400 nM) was doped into this proteome for comparison. (b) Competitive ABPP of representative hits in RBBP9-transfected COS-7 cytosolic proteomes (1 mg/mL protein). RBBP9-transfected cells expressed high levels of active RBBP9 compared to mock-transfected cells, as judged by ABPP (Supplementary Fig. 3). (c) Primary hits can be segregated into four general categories based on performance in competitive proteomic ABPP assays. (d) Concentration-dependent effects of emetine (1) on FP-rhodamine labeling of mouse brain serine hydrolases. Note that emetine selectively inhibits RBBP9 labeling at concentrations up to 1 mM.

Figure 5

Mechanistic characterization of RBBP9 inhibitors. (a) RBBP9 (2 μM) was incubated (30 min) with DMSO, emetine (500 μM), or compound 2 (50 μM). Each reaction was then split into two fractions - one fraction was reacted directly with FP-rhodamine (left panels), and the other fraction was passaged over a Sephadex G-25M column and then reacted with FP-rhodamine (right panels) to assess the reversibility of inhibition. (b and c) IC₅₀curves for compound 2 (b) and emetine (c) with human RBBP9 (wt), the C163R mutant of human RBBP9, and mouse RBBP9 as determined by gel-based competitive ABPP. Error bars represent s.e.m.

Figure 6

FluoPol-ABPP platform identifies a selective inhibitor of GSTO1. (a) GSTO1 (1 μM) and SE-rhodamine (75 nM) generated a strong, time-dependent increase in FluoPol signal. No labeling was observed in the absence of enzyme, with a catalytically-dead C32A mutant GSTO1 (1 μM), or in the presence of glutathione (1 mM), which has been shown to react with C32. The indicated 90-minute time point (Z' = 0.67) prior to reaction completion was selected for HTS. Error bars represent s.d. (b) Structures of representative primary hits and IC₅₀ values for inhibition of GSTO1 as determined by competitive gel-based ABPP (400 nM GSTO1, 1 μM SE-rhodamine, 20 min, room temperature). (c) Assessing the reversibility of GSTO1 inhibition. GSTO1 (2 μM) was incubated with DMSO or the indicated compound for 30 min 6 (280 μM), 7 (28 μM), 8 (28 μM) or 9 (280 μM). Each reaction was then split into two fractions - one fraction was reacted directly with SE-rhodamine (left panels), and the other fraction was passaged over a Sephadex G-25M column and then reacted with SE-rhodamine (right panels). (d and e) Evaluation of compounds at 20 μM (d) or 1 μM (e) by competitive ABPP in the soluble proteome of MDA-MB-231 breast cancer cells (2 mg/mL protein, 5 μM FP-rhodamine, 1 hr, room temperature). Lower panels in d and e represent reduced-intensity images of the 30 kDa region of the upper panels where endogenous GSTO1 migrates.