RATIONAL DRUG DESIGN BASED ON CHEMICAL MECHANISMS

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DESCRIPTION: During the period of support requested in this application, we plan to investigate further the mechanism of action of two enzymes, folypoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) and gamma-glutamyl hydrolase (GH, EC 3.4.22.17), involved in the biosynthesis and hydrolysis of folypoly-gamma-glutamates; i.e., the folate "conjugates". Recently synthesized substrates and inhibitors, together with selected phosphapeptides and fluoroamino acid containing peptides to be synthesized during this period of support, will be used as mechanistic probes and/or inhibitors of FGPS and GH. In collaboration with Drs. John McGuire and John Galivan, we will use these newly synthesized molecules to study the role of intracellular poly-gamma-glutamate formation in folate one-carbon biochemistry and in antifolate chemotherapy. The specific aims are as follows: 1. Using recombinant human FPGS, evaluate the kinetics of the reaction under pre-steady-state conditions in order to distinguish between a processive and non-processive kinetic mechanism. 2. Determine the mechanistic basis for the "position-dependent" enhancement or termination of FPGS-catalyzed ligation using 3,3-difluoroglutamate-containing oligopeptides. 3. Using purified GH isolated rat hepatoma cells, determine the metal content and mechanism of GH-catalyzed hydrolysis of olgi-gamma-glutamyl peptides. Using newly synthesized fluoroglutamate-containing dipeptides, determine if tetrahedral intermediates accumulate during catalysis. 4. Complete the synthesis of phosphapeptide analogs of two similar tetrahedral intermediates postulated to form during the FPGS- and GH-catalyzed reaction. Evaluate these new compounds as FPGS and/or GH inhibitors and, if found to be effective in cell-free assays, investigate the action of these compounds as modulators of polyglutamate biosynthesis and/or polyglutamate hydrolysis in intact mammalian cells.