Cytosolic glutathione‐S‐transferase A1 (GSTA1) plays a primary role in the metabolic clearance of the aromatase inhibitor, Exemestane

Abstract

Exemestane (EXE) is used to treat estrogen receptor positive (ER+) breast cancer in postmenopausal women by inhibiting the enzyme aromatase in the final step of estrogen biosynthesis. Variability exists in both overall patient response and adverse events, which may be attributed to inter‐individual variation in EXE metabolism. EXE is metabolized primarily to the active metabolite, 17β‐dihydro‐EXE (17β‐DHE), and preliminary studies have demonstrated that cysteine conjugates of EXE and 17β‐DHE comprise 77% of total EXE metabolites in the urine of patients. Cysteine conjugation is a multi‐step process, the first of which is glutathione (GSH) conjugation, catalyzed by the glutathione‐S‐transferase (GST) family of the enzymes. The goal of the present study was to identify the hepatic GSTs active in the metabolism of EXE and 17β‐DHE. In a screening of the Human Protein Atlas, 12 cytosolic GSTs were found to be hepatically expressed and were initially screened by incubating 0.5 μg of pure recombinant GST enzyme with EXE or 17β‐DHE (125 μM) and reduced glutathione (GSH). The resulting GS‐conjugates of EXE and 17β‐DHE were detected and quantified using UPLC‐MS/MS. Results from the conjugation assays indicated that the cytosolic enzyme GSTA1 is highly active against both EXE and 17β‐DHE, while GSTM3 and GSTM1 are only moderately active against EXE. No other recombinant cytosolic GST enzymes tested (GSTA2, GSTA4, GSTK1, GSTM2, GSTM4, GSTO1, GSTP1, GSTZ1 and GSTT1) exhibited activity against either EXE or 17β‐DHE. KM values for human liver cytosol (HLC) and purified recombinant GSTA1 were similar for both EXE (45 μM vs. 77 μM, respectively) and 17β‐DHE (54 μM and 30 μM, respectively) as substrate, suggesting that GSTA1 may be the main driver for EXE and 17β‐DHE glutathione conjugation in human liver cytosol. Using EXE as a substrate, Vmax value for GSTA1 was >7‐fold higher than that observed for GSTM3 (9.8 nmol/min/mg vs. 1.3 nmol/min/mg, respectively). The EXE intrinsic clearance (ClINT) for GSTA1 (130 nl/min/mg) was >10‐fold higher than that observed for GSTM3 (11 nl/min/mg). These data suggest that GSTA1 may be an important component in the metabolic pathway contributing to the elimination of EXE in breast cancer patients.Support or Funding InformationThis work is supported by grants from NIH, National Cancer Institute (grant R01‐CA164366; to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)