RATIONAL DRUG DESIGN BASED ON CHEMICAL MECHANISMS

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During the period of support requested in this application, we plan to continue our efforts on the synthesis of potent and specific enzyme inhibitors based on enzyme mechanism data obtained in our laboratory and from the literature. The proposed studies involve a combination of synthetic organic chemistry, mechanistic enzymology, and biochemical pharmacology. The overall goal of our research is to investigate the role of specific biochemical pathways in the control of cell growth. More specifically, in the first portion of the research proposed herein, we will continue our use of fluoroglutamate-containing folates and antifols to assess the role of polyglutamate biosynthesis and hydrolysis in mammalian cells. For example, we will complete the synthesis of gamma-fluoroleucovorin and use it to define the role of polyglutamates in leucovorin "rescue" of cells following high-dose methotrexate chemotherapy. We will also exploit our recent finding that 3,3-difluoroglutamate (F2Glu) stimulates the formation of polyglutamates in cell-free systems. Following improvement of the synthetic route to F2Glu, we will synthesize a series of F2Glu-containing folates and antifols in order to investigate the biochemical pharmacology of compounds with enhanced ability to form polyglutamate derivatives. In the second portion of the proposed research we will employ our new synthetic methodology for the synthesis of complex acetylenic nucleosides. We will complete the synthesis of our proposed specific "multisubstrate adduct inhibitors" of methylases with which we have many years of experience (catechol O-methyltransferase, phenethanolamine N-methyltransferase) and study their effect on isolated enzymes. If these proposed new methylase inhibitors are as specific and potent as predicted, we will use them to study catecholamine biosynthesis in neuroblastoma cells. As with the folate work, our ultimate goal in this work is to develop potent and specific inhibitors of each methyltransferase in order to study their role in cell growth and/or cell function.