Characterization of Cytosolic Glutathione S-Transferases Involved in the Metabolism of the Aromatase Inhibitor, Exemestane

Abstract

Exemestane (EXE) is a hormonal therapy used to treat estrogen receptor–positive breast cancer by inhibiting the final step of estrogen biosynthesis catalyzed by the enzyme aromatase. Cysteine conjugates of EXE and its active metabolite 17β-dihydro-EXE (DHE) are the major metabolites found in both the urine and plasma of patients taking EXE. The initial step in cysteine conjugate formation is glutathione conjugation catalyzed by the glutathione S -transferase (GST) family of enzymes. The goal of the present study was to identify cytosolic hepatic GSTs active in the GST-mediated metabolism of EXE and 17β-DHE. Twelve recombinant cytosolic hepatic GSTs were screened for their activity against EXE and 17β-DHE, and glutathionylated EXE and 17β-DHE conjugates were detected by ultra-performance liquid chromatography tandem mass spectrometry. GST α (GSTA) isoform 1, GST μ (GSTM) isoform 3 and isoform 1 were active against EXE, whereas only GSTA1 exhibited activity against 17β-DHE. GSTM1 exhibited the highest affinity against EXE with a Michaelis-Menten constant (K M ) value that was 3.8- and 7.1-fold lower than that observed for GSTA1 and GSTM3, respectively. Of the three GSTs, GSTM3 exhibited the highest intrinsic clearance against EXE (intrinsic clearance = 0.14 nl·min −1 ·mg −1 ). The K M values observed for human liver cytosol against EXE (46 μM) and 17β-DHE (77 μM) were similar to those observed for recombinant GSTA1 (53 and 30 μM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 composed 4.3% and 0.57%, respectively, of total protein in human liver cytosol; GSTM3 was not detected. These data suggest that GSTA1 is the major hepatic cytosolic enzyme involved in the clearance of EXE and its major active metabolite, 17β-DHE. SIGNIFICANCE STATEMENT Most previous studies related to the metabolism of the aromatase inhibitor exemestane (EXE) have focused mainly on phase I metabolic pathways and the glucuronidation phase II metabolic pathway. However, recent studies have indicated that glutathionylation is the major metabolic pathway for EXE. The present study is the first to characterize hepatic glutathione S -transferase (GST) activity against EXE and 17β-dihydro-EXE and to identify GST α 1 and GST μ 1 as the major cytosolic GSTs involved in the hepatic metabolism of EXE.

Influence of glutathione-S-transferase polymorphisms on the metabolism of the potent aromatase inhibitor, exemestane

“…The rate of GS conjugate formation with EXE ranged from 0.16 to 1.44 nmol•min − 1 •mg cytosolic protein − 1 and with DHE from 0.062 to 0.23 nmol•min − 1 •mg cytosolic protein − 1 . Since GSTA1 was the only enzyme previously shown to exhibit DHE-GS formation activity (23), only EXE-GS rates of formation were analyzed against the GSTM1 null genotype. After stratifying by GSTM1 genotype, no signi cant differences in EXE-GS formation were observed in the same human liver specimens (Fig.…”

“…Pre-incubation at 37°C for 3 min was followed by the addition of 5 mM GSH to initiate the reaction (1 h, 37°C). Reactions were quenched with a mixture of ice-cold acetonitrile (25 µL) and deuterium labeled S-(androsta-1,4-diene-3,17-dion-6α-ylmethyl)-L-glutathione (D 3 -EXE-GS ) and S-(androsta-1,4-diene-17β-ol-3-on-6α-ylmethyl)-L-gutathione (D 3 -DHE-GS) internal standards (23).…”

“…GSH conjugate metabolites of EXE and DHE were quanti ed against standard curves with known concentrations of EXE-GS or DHE-GS. Chemical synthesis of the EXE-GS and 17β-DHE-GS standards were previously described (23). Rates of EXE-GS and 17β-DHE-GS conjugation were calculated in nmol•min -1 •mg cytosolic protein -1 .…”

“…A validated method for monitoring EXE-GS and 17β-DHE-GS conjugate formation was used to quantify GSH conjugates (23). Brie y, GSH conjugates were detected using a LC-MS system (Waters Acquity UHPLC / XEVO G2-S QToF) with analyte separation achieved using a UHPLC BEH C18 column (2.1 x 100 mm) with a ow rate of 0.4 mL/min, a column temperature of 35°C, and a sample temperature of 8°C.…”

“…17β-DHE is further metabolized by phase II glucuronidation via UDP-glucuronosyltransferase (UGT) 2B17 (21,22). In addition, both EXE and 17β-DHE can be glutathionylated by glutathione-S-transferase (GST) enzymes, primarily GSTs A1 and M1 (23), with the resulting glutathione (GSH) conjugates of EXE and 17β-DHE further metabolized to cysteine conjugates by γ-glutamyl transferase (GGT) and dipeptidases (24)(25)(26). In post-menopausal patients with ER + breast cancer, cysteine conjugates of EXE and 17β-DHE comprise 77% and 35% of the total urinary and plasma EXE metabolites, respectively, indicating that glutathionylation is a major metabolic pathway for EXE (26).…”