This application requests continuing support for work on antifolate-based inhibition of de novo purine biosynthesis. Previous work has resulted in the discovery of deaza analogs of tetrahydrofolate, the first potent folate antimetabolites specifically inhibitory to this critical pathway of intracellular metabolism. These compounds possess broad anti-tumor activity in pre-clinical model systems, including many in which classical antifolates have little or no activity. DDATHF (Lometrexol) is currently in Phase I clinical trials at a number of centers worldwide, including our own. Our work accomplished during the current grant period has focussed largely on the biochemical pharmacology of this new class of compounds. Work proposed in this application is aimed at providing basic information about the interactions of these and other antifolates with their target enzymes, to begin to understand the structure of those targets, to investigate biochemical determinants of drug action at the clinical level, and to explore possible ways in which these drugs might be used in combination with others. Specifically we will: 1. Continue investigations to determine the precise structural requirements for inhibition of each of the two folate-dependent enzyme activities of de novo purine biosynthesis, GAR transformylase (GARTF) and AICAR transformylase (AICARTF). 2. Develop systems for the expression of human GARTF and AICARTF in order to probe the structure of target enzymes. These expression systems will be utilized for the production of site specific mutants, based on information determined through active site labelling. 3. To test the hypothesis that in vitro polyglutamylation is an indicator of response in the context of phase I & II clinical trials of Lometrexol ongoing at our institution. 4. Investigate the basis for the interaction of DDATHF in biochemical modulation of fluoropyrimidines.
