Potent and Selective Inhibitors of Glutathione S-Transferase Omega 1 That Impair Cancer Drug Resistance

Abstract

Glutathione S-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. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in the detoxification of chemo-therapeutic agents

Efficacy and Pharmacology of the NLRP3 Inflammasome Inhibitor CP-456,773 (CRID3) in Murine Models of Dermal and Pulmonary Inflammation

“…We tested KT53, a potent and selective inhibitor of GST Omega 1-1, in our NLRP3 inflammasomedependent IL-1b release assay (25). KT53 weakly inhibited LPS plus nigericin-induced IL-1b release from PBMCs with an IC 50 of 3505 nM, but KT53 was approximately equipotent for inhibiting LPS-induced IL-6 with an IC 50 of 4181 nM (data not shown).…”

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

“…Using the HPLC-based procedure, we studied the effect of specific GSTO1 inhibitors, namely KT53 [11] and CMFDA [12], as well as the non-specific GSTO1 inhibitor zinc [13] on 4-NPG reduction by rat liver cytosol. Importantly, each of these inhibitors inactivates GSTO1 by reacting with the active site cysteine.…”

“…Indeed, CMFDA is deacetylated by esterases and then conjugated to GSH through its electrophilic chloromethyl group by GST to form methylfluorescein-SG [20]. KT53 contains an electrophilic a-chloroacetamide group [11], rendering it a potential GST substrate. Thus, rapid inactivation of CMFDA and KT53 by conjugation with GSH in the cytosol could explain why GSTO1 preserves its activity to reduce 4-NPG when this substrate is supplied after pre-incubation of the cytosol with these electrophilic GSTO1 inhibitors in the presence of GSH.…”

“…Therefore, we set out to develop a high-performance liquid chromatography (HPLC)-based procedure, anticipating that it will be sensitive enough to quantify the conversion of 4-NPG to 4-NAP in the presence of GSH as a measure of GSTO1 activity. In this endeavor, our main objectives were (i) to identify and quantitate the product(s) of 4-NPG reduction; (ii) to determine the conditions under which product formation, in the presence of GSH, 2-ME, or the disulfide reducing agent tris(2-carboxyethyl)phosphine (TCEP), is linear with respect to time and cytosolic protein concentration; and (iii) to ascertain the inhibitory effect on product formation of chemicals that inactivate GSTO1, such as the specific GSTO1 inhibitor KT53 [11] and 5-chloromethylfluorescein diacetate (CMFDA) [12], as well as zinc ion [13]. Development of the spectrophotometric GSTO1 assay into a more sensitive HPLC-based procedure is reported below, whereas studies on the involvement of GSTO1 in reduction of dimethylarsinic acid are intended to be communicated separately.…”

Exploring Metabolic Pathways and Regulation through Functional Chemoproteomic and Metabolomic Platforms

“…These include selective inhibitors of anti-cancer targets protein methyl esterase 1 (PME1), glutathione transferase-omega (GSTO), and RBBP9, as well as an inhibitor for the anti-inflammatory target protein arginine deaminase 4 (Bachovchin et al, 2009, 2011; Knuckley et al, 2010; Tsuboi et al, 2011). …”