MECHANISM-BASED ENZYME INHIBITORS: NOVEL ANTIFOLATES

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The proposed project is part of a long range research effort aimed at the development of new approaches to the chemotherapy of proliferative diseases. The main objective of this research is to apply mechanistic considerations to the design and subsquent study of novel antifolates. The study focuses on the role of the poly-gamma-glutamyl chain of folates and antifolates in their cofactor activity and/or cytotoxicity, as a basis for drug design. As specific targets, enzymes of two metabolic cycles, one responsible for the formation and breakdown of polygultamates and the other for the folate polyglutatmate dependent biosynthesis of thymidylate have been chosen. The chemical part of the project involves the rational design, synthesis and characterization of new antifolates. The use of computerized molecular modelling techniques is planned to gain insight into the active sites of target enzymes and their interaction with drug candidates utilizing past and future structure-activity correlations and x-ray crystallographic structural information. The chemical studies are coordinated with the biochemical studies involving the metabolism and enzymology of folyl and antifolyl polyglutamates as well as the evaluation of the enzyme inhibitory activities of the newly synthesized compounds. These studies employ intact and reversibly permeabilized L1210 murine leukemia cells and isolated enzyme systems. The permeabilized cell system premits the direct study of the cellular metabolism of polyglutamates of folates and folate analogues and their interaction with enzymes of intact metabolic pathways. The cytotocicity of the compounds is determined in vitro by measuring the extent of growth inhibition of L1210 leukemia cells in suspension cultures. It is an important goal of the project to correlate cellular and cell-free enzyme inhibitory activity with in vitro cytotoxicity data and to use these correlations to predict in vivo biological activity. The results of this study will help to formulate future research plans including the possibility of therapeutic applications. In addition to their potential therapeutic utility, the proposed analogues may serve as useful tools in the study of the essential role of polyglutamylation in cancer cells. The study of drug- enzyme interactions may further our knowledge of the catalytic mechansms 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.