Fig. 5.

Compound 4j is a potent and selective ABHD3 inhibitor. a, b ABPP gels of HEK 293T cells overexpressing human ABHD3 treated with compound 4j (a) in vitro (30 min, 37 °C) or (b) in situ (2 h, 37 °C). c IC₅₀ curves for the aforementioned in vitro and in situ 4j treatments. The data represent average values ± S.E.M. values for three independent experiments per group. d Quantitative ABPP-SILAC analysis of SW620 cells treated in situ with DMSO (heavy-isotopically labeled cells) or 4j (0.5 or 10 µM, 2 h; light-isotopically labeled cells). After in situ treatments, heavy and light cells were lysed, treated with the broad-spectrum, serine hydrolase-directed activity-based probe FP-biotin (6 µM, 1 h, RT), combined and processed for quantitative MS-based analysis, as described^[41]. ABHD3 shows complete (95+%) inhibition at both concentrations and 4j appears to have no major off-targets. The data represent average values ± S.E.M. values for quantified peptides per protein derived from two independent experiments