The proposed project is part of a long range research effort aimed at the development of new approaches to the chemotherapy of neoplastic diseases. The main objective of this research is to design novel target-oriented antifolates based on biochemical and mechanistic rationales derived from structural and functional information and to study their effects at the cellular and molecular level. As specific targets, the proposed study focuses on enzymes of two interrelated metabolic cycles, one responsible for the formation and breakdown of the poly-gamma-glutamates of folates and antifolates and the other the folylpolyglutamate dependent biosynthesis of thymidylate (the TS cycle). The proposed research sets the following specific aims: To design and synthesize potential inhibitors of folylpolyglutamate synthetase, gamma-glutamyl hydrolase (conjugase), thymidylate synthase, dihydrofolate reductase and serine hydroxymethyl transferase; to evaluate the effects of the inhibitors on their respective targets in cellular enzyme systems; to determine the growth inhibitory effects of the target compounds on L1210 mouse and CCRF-CEM human leukemia cells in culture; to study the mechanism of selected inhibitor-enzyme interactions at the molecular level using techniques of enzymology, X-ray crystallography, molecular mechanics and computer graphics; to study the polyglutamylation of active antifolates and its importance for biological activity; to correlate cellular and cell-free enzyme inhibitory activity with in vitro cytotoxicity data and based on these correlations select candidates for in vivo biological testing. The study of drug-enzyme interactions may further our knowledge of the mechanisms of folate dependent enzymes and furnish new rationales for inhibitor design. Since folate metabolism is intimately linked to nucleic acid biosynthesis and cellular proliferation, this research may lead to important observations in tumor biology and new approaches to cancer treatment.
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