Rational Drug Design Based on Chemical Mechanisms

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DESCRIPTION (provided by applicant): During the period of support requested in this application, we plan to investigate further the action of two enzymes, folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) and gamma-glutamyl hydrolase (GH, EC 3.4.19.9), involved in the biosynthesis and hydrolysis of folylpoly-gamma.glutamates the folate "conjugates." A series of phosphapeptides and fluoroglutamate-containing peptides are proposed for investigation as mechanistic probes and/or inhibitors of FPGS and GH. The kinetics of enyzyme-catalyzed gamma-glutamyl ligation (FPGS) and hydrolysis (GH) of the natural substrates and our mechanism-based analogs will be investigated in detail. In a series of collaborations with molecular biologists, crystallographers, and pharmacologists, we will use these newly synthesized molecules to study the role of intracellular poly-gamma-glutamate formation in folate one-carbon biochemistry and antifolate chemotherapy. The specific aims are as follows. 1. Complete the stereoselective synthesis of a phosphinic acid-containing analog of Glu-gamma-Glu. the racemic form of which is a potent FPGS inhibitor when incorporated in folate or antifolate analogs. Complete the synthesis of prodrug forms of these inhibitors for cell culture studies. 2. Complete the synthesis of isotopically labeled DDATHF-based substrates for use in kinetics experiments. Determine the detailed kinetic mechanism of the reaction catalyzed by FPGS especially as it pertains to possible processivity in the ligation of multiple glutamates. 3. In collaboration with x-ray crystallographers use folates and antifolates containing phosphoamino acids to investigate the structural basis of FPGS catalysis, especially in terms of the hypothesized processive mechanism of multiple ligations. 4. Determine the kinetic mechanism of the reaction catalyzed by GH. Of special interest is probing for possible stabilization of reaction intermediates with several gamma-glutamyl peptides containing fluoroamino acids. Complete the synthesis of gammaglutamyl peptides containing glutamate semialdehyde at the C-terminus as potential GH inhibitors. Initiate the synthesis of epoxide-containing peptidomimetics as cysteine orotease inhibitors. 5. Complete the stereoselective synthesis of (2S) 3.3-difluoroglutamic acid and its incorporation into appropriate gammaglutamyl peptides for use in mechanistic studies of GH and FPGS catalysis.