Role of glutathione‐S‐transferases in the metabolism of the anti‐cancer agent and aromatase inhibitor, exemestane

Breast cancer is the most commonly diagnosed cancer in women, with approximately 1 in 8 women in the U.S. developing breast cancer over their lifetime. The majority of breast cancers diagnosed each year are estrogen receptor positive (ER+). Drugs targeting the ER pathway are a major treatment modality for ER+ patients and are additionally prescribed for the prevention of breast cancer. Exemestane (EXE) is an inhibitor which blocks the enzyme aromatase in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventative agent demonstrated a reduction in breast cancer incidence by more than 65%. However, response and toxicity varied widely among individuals, which may be attributed to genetic polymorphisms in enzymes that metabolize EXE. Data from our lab demonstrate that a cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) make up 77% of the total metabolite in urine. Cysteine conjugates are formed from metabolism of glutathione (GSH) conjugates in a reaction catalyzed by the glutathione‐S‐transferases (GST) family of the enzymes. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE and to determine kinetic parameters for the identified enzymes. In a screening of the Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. EXE‐GSH conjugation activity in human liver samples was screened by incubating GSH with EXE or 17β‐DHE (0.125–1.0 mM) in cytosol (HLC) or microsomes (HLM) prepared from human livers. GSH conjugates were formed for both cellular fractions, indicating that both cytosolic and microsomal GST enzymes are active against EXE and 17β‐DHE. GSH conjugates were detected by UPLC‐MS and quantified using synthesized EXE‐GS and 17β‐DHE‐GS standards. The purity and identity of both standards and internal standards were confirmed using NMR and UPLC‐MS and found to be > 95% pure. Individual recombinant human GSTs were then screened to determine which enzyme is primarily responsible for the metabolism of EXE. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene. Results from the individual EXE‐GSH conjugation assays indicate the cytosolic enzymes GSTA1, GSTM3 and GSTP1 are active against EXE and 17β‐DHE; studies of microsomal GSTs are on‐going. These data suggest that several GSTs, both cytosolic and microsomal, may be important in the phase II conjugation of EXE and its major active metabolite, 17β‐DHE. Support or Funding Information This study is supported by National Institutes of Health (NIH) (Grant R01‐ES025460 and R01‐CA164366) and the Health Sciences and Services Authority of Spokane This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .