Optimization and Characterization of an Inhibitor for Glutathione S-Tranferase Omega 1 (GSTO1)

Glutathione transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Using activity-based proteomic methods, glutathione S-tranferase omega (GSTO1) has been shown to be overexpressed in human cancer cell lines that show enhanced aggressiveness. Other studies have implicated GSTO1 in chemotherapeutic resistance. Cancer remains one of the most life-threatening diseases for which effective treatments and cures are lacking. Recently, targeted therapeutics (i.e., selective inhibitors that block individual enzymes) have shown great promise for the treatment of cancer. Inhibiting GSTO1 may thus offer a new therapeutic strategy for cancer. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective GSTO1 inhibitor probe, ML175, by high-throughput screening using fluorescence polarization-activity-based protein profiling (FluoPol-ABPP), and several rounds of gel-based competitive activity-based protein profiling (ABPP) secondary assays employing SAR analysis of selected compounds. ML175, a hindered alpha-chloroacetamide, was identified as a highly potent and selective inhibitor of the target enzyme GSTO1. ML175 has an IC50 of 28 nM, greater than 350-fold selectivity against potential anti-targets as assessed by competitive ABPP profiling, and has been shown to be active in situ, completely inhibiting GSTO1 at 250 nM compound concentration. As determined from LC-MS/MS analysis, ML175 is an activity-based inhibitor; it covalently labels the active site cysteine nucleophile of GSTO1. ML175 has favorable stability, solubility, and cytotoxicity profile, and improves on the current state of the art for GSTO1 inhibitors. Taken together, our findings suggest that it is very possible to develop potent and selective probes based on tempered electrophilic scaffolds, and that ML175 will be a highly successful probe for biochemical investigation of GSTO1.