Bifunctional alkylating agents (e.g., melphalan, cyclophosphamide, chlorambucil) and platinum antitumor compounds (e.g. cisplatin, carboplatin) are used to treat a wide range of human malignancies. The antitumor activity of such compounds is due to their ability to crosslink chromosomal DNA of rapidly dividing cells thereby preventing tumor cell proliferation and inducing tumor cell death. The therapeutic efficacy of such agents is believed to be reduced by their reaction with glutathione (g-glutamylcysteinylglycine, GSH) a peptide present in all cells including tumor cells. Studies published over the past several years show that the intrinsic or acquired resistance of many tumors to bifunctional alkylating agents or platinum antitumor compounds is associated with elevated levels of GSH in the tumor. To reverse such resistance, tumor-bearing animals have been administered buthionine sulfoximine (BSO), and inhibitor of gamma-glutamylcysteine synthetase (GCS) the enzyme catalyzing the first step in GSH synthesis. In such animals, the ongoing reactions of GSH utilization cause GSH depletion and increased sensitivity to alkylating agents and platinum compounds as well as to radiation therapy. Buthionine sulfoximine is currently undergoing clinical trial as an adjuvant to melphalan chemotherapy. Effective depletion of tumor GSH requires that GSH synthesis be potently and consistently inhibited; the potential rate of GSH synthesis is sufficiently high that even a brief lapse in GCS inhibition can result in substantial repletion of tumor GSH and restoration of resistance. The applicant proposes to develop improved agents for inhibition of GSH synthesis by elucidating the enzymology of GCS and by synthesizing and testing novel inhibitors.
Specific aims are as follows (1) The interaction of human GCS with its substrate and known inhibitors will be characterized. (2) Novel analogs of BSO with improved affinity for g-GCS will be synthesized and tested in vitro and in vivo. (3) Novel covalently reactive transition state inhibitors of g-GCS will be synthesized and tested in vitro and in vivo. (4) The ability of novel inhibitors to increase efficacy of melphalan- and cisplatin-based chemotherapy of human ovarian cancer xenografts will be characterized in nude mice.
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| Misra, I; Griffith, O W (1998) Expression and purification of human gamma-glutamylcysteine synthetase. Protein Expr Purif 13:268-76 |
