Abstract 760: Sensitizing tumors to chemotherapy by inhibition of gamma-glutamyl transpeptidase

Abstract

Abstract Tumors that express the cell surface enzyme gamma-glutamyl transpeptidase (GGT) have increased resistance to chemotherapy and radiation. GGT cleaves the gamma-glutamyl bond of extracellular glutathione, initiating the cleavage of glutathione into glutamate, cysteine and glycine. Glutathione can not be taken up intact by tumor cells. Cleavage of extracellular glutathione increases the supply of cysteine to the cell, which is essential to increasing intracellular glutathione levels. Glutathione detoxifies chemotherapy drugs, and elevated levels of intracellular glutathione can block apoptosis. We are developing inhibitors of GGT that can be used to sensitize tumors to therapy. The standard assay for GGT activity uses p-nitroanilide derivatives as substrates, millimolar concentrations of dipeptides as acceptors and is buffered at pH 8.6. Under these assay conditions several groups of investigators have shown that GGT cleaves the gamma-glutamyl bond of the substrate and transfers the gamma-glutamyl group to an acceptor by a Ping-Pong mechanism. However, the standard assay for GGT activity does not measure the reaction catalyzed by GGT in vivo. We have assayed GGT activity under physiologic conditions. We have found that some compounds that inhibit GGT activity in the standard reaction do not inhibit under physiologic conditions. Our data reveal that, at physiologic pH and osmolarity with reduced concentrations of acceptor, the enzyme does cleave the gamma-glutamyl bond by a Ping-Pong mechanism, but hydrolysis of the intermediate of the gamma-glutamyl enzyme intermediate is significant, and competition between water and the peptide acceptor for the intermediate makes it appear Sequential. Several compounds that inhibit GGT in the standard assay actually accelerate activity as the concentration of the acceptor is reduced to physiologic levels. To identify inhibitors that are optimized for clinical use, we have developed an assay that evaluates GGT cleavage of glutathione, the physiologic substrate, at physiologic pH and osmolarity. We are using our previously reported inhibitor OU749 and other lead compounds to further characterize the physiologic GGT reaction and to identify essential substructural components for inhibition of GGT activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 760.