The proposed studies are directed at a deeper understanding and novel approaches to the antifolates. We plan to evaluate several determinants of their preclinical therapeutics which includes 1) the relationship of glutamylation of antifolates to tumor cytotoxicity and host toxicity 2) the biochemical mechanisms by which antifolates exert their cytotoxicity through perturbations in the cell folylpolyglutamates 3) the biochemical and pharmacological processes which regulate glutamylation and hydrolysis of folyl and antifolylpolyglutamates and 4) the interaction of the folate- analog inhibitors of thymidylate synthase with the target enzyme. In approaching the first goal a series of folates and antifolates have been synthesized containing a fluorine in the 4 carbon of glutamate which severely regards glutamylation but does not alter other properties of the molecule. 4-Fluoromethotrexate has been used to show changes in the activity of methotrexate when it can't be glutamylated. 4-Fluorofolinic will be used to determine the rescue capacity with tumor cells and host tissue. This experimental design is intended to determine if selective host rescue can be achieved more effectively by preventing glutamylation of the rescue agent. A highly sensitive assay for folate coenzymes developed by Priest and coworkers has been adapted and modified in our laboratory to give essentially quantitative recoveries of 5,10CH2H4PteGlun, H4PteGlun, H2PteGlun, 10-HCO-H4PteGlun, and 10-HCOH2PteGlun which are the key folates involved in purine and pyrimidine biosynthesis. We will measure these species following treatment of cells with methotrexate and 4- fluoromethotrexate and rescue with folinic and 4-fluorofolinic acid. Combinations of DHFR inhibitors with propargylquinazoline inhibitors of thymidylate synthase and the GAR transformylase inhibitor 5,10 dideazatetrahydrofolate will be evaluated with cells cultured in folinic acid or 5 methyltetrahydrofolate to determine if greater than additive drug activity occurs. Although we have already shown synergistic drug activity with combinations in folic acid containing medium, the use of the reduced folate substrates will suggest the potential for in vivo activity. If positive drug interactions are found, mechanistic evaluations will be undertaken including a thorough analysis of folate perturbations as described above. In addition a detailed mechanistic analysis of the activity of propargylquinazolines will be undertaken with wild type cells and those amplified 70 fold for thymidylate synthase. These investigations are directed determining in detail the cellular response to this new class of inhibitors. A theme throughout all these studies is understanding the mechanisms which regulate the glutamylation and hydrolysis of folyl and antifolyl polyglutamates. This will be evaluated at the enzymatic level by investigation of folylpolyglutamate synthetase and gamma-glutamylhydrolase and at the cellular level by examining agents which perturb the rate of polyglutamate formation and breakdown.
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