Mechanistic studies of inactivation of glutathione S-transferase Pi isozyme by a haloenol lactone derivative.

Cancer chemotherapy often fails due to acquired drug resistance. One of the most critical biochemical changes observed in drug-resistant tumor cells is over-expression of glutathione S-transferase Pi isozyme (GSTP1). Glutathione S-transferase inhibitors have been used as potentiating agents of chemotherapeutic drugs. Earlier we reported haloenol lactone 1 as a site-directed GSTP1 inactivator. We proposed that enzymatic hydrolysis of the haloenol lactone may be the initial step of GSTP1 chemical modification, resulting in the inactivation of the enzyme. Enzyme inactivation is initiated through addition of Cys-47 to the lactone ring, which is opened in the process to form an alpha-bromoketone adduct. The acidity of Cys-47 confers good leaving group properties, and rapid hydrolysis occurs to generate an alpha-bromoketoacid intermediate. The reaction may proceed via alkylation of the transient thioester to form a six-membered ring episulfonium ion intermediate which would be yet more reactive toward hydrolysis, with either process leading to the observed mass increase of 230 Da. To probe the importance of the bromine of the lactone in GST inactivation, we designed and synthesized compound 2. Unlike lactone 1, lactone 2 did not show time-dependent inhibitory effect on GSTP1. Incubation of compounds 1 and 2 with excess of N-acetyl cysteine produced the corresponding di-N-acetyl cysteine conjugate and mono-N-acetyl cysteine conjugate, respectively. To probe the role of Cys-47 in the inactivation of GSTP1 by compound 1, we prepared mutant C47A GSTP1. The mutant GSTP1 still showed good activity toward CDNB, but it lost susceptibility to the inactivation by compound 1. In addition, LC-MS/MS technique allowed us to identify the modified Cys-47 after the enzyme was exposed to compound 1.