The long-term goal of this program is to improve treatment of human cancer by exploiting aspects of folyl- (or antifolyl-) polyglutamate synthesis. Folylpolyglutamates are essential for cell growth, while polyglutamates of classical antifolates are implicated in their cytotoxic action and resistance and may have a role in selectivity. Detailed understanding of the synthesis and function of (anti)folylpolyglutamates may thus allow design of new agents or strategies to exploit this critical process. This long-term goal will be addressed through three specific aims: 1. Exploration of folylpolyglutamate synthetase (FPGS), the enzyme responsible for polyglutamate synthesis, as a drug target. Mutational inactivation of FPGS is lethal, thus FPGS is a potential cancer chemotherapy target. Inhibitor design is based on enzyme mechanism and structure-activity data of the applicant using recombinant human FPGS. After synthesis by expert folate chemists in collaborating laboratories, antifolates will be studied in the applicant's laboratory. Of primary interest will be the optimization of mechanism-based phosphorous-containing inhibitors found during the last grant period. Analogs that inhibit both purified FPGS and polyglutamylation in intact cells (i.e., are transported) will be studied to clarify their specificity and cellular effects. Promising drugs will undergo initial toxicity and therapeutic testing in vivo to define whether FPGS is a useful therapeutic target. 2. Determine the functional significance of the apparent physico-chemical difference between mitochondrial (mFPGS) and cytosolic FPGS (cFPGS). Although encoded by one gene, mFPGS and cFPGS differ in SDS-PAGE mobility, which may reflect a functional difference, particularly as related to antifolate resistance (Aim 3). The applicant will explore the hypothesis that this physico-chemical variance, discovered under this grant, is reflected in kinetic, substrate specificity, and/or regulation alterations. Also, since the structural difference may be reflective of function, he will examine hypotheses that it is caused by mitochondrial leader sequence truncation or by post-translational modification. 3. Elucidate the role of cFPGS and mFPGS in MTX resistance. Clinically, MTX is often given as a bolus or short (24 hr) infusion at high dose. The applicant has shown that this regimen selects for resistance via FPGS deficiency. FPGS is expressed in both cytosol and mitochondria. MTX cannot enter mitochondria, while reduced folate monoglutamates can. It is known (Shane et al.) that expression of mFPGS alone can establish folylpolyglutamate pools in both cytosol and mitochondria and allow normal cell growth. Thus, he hypothesizes that a differential decrease in cFPGS, rather than a parallel decrease in both isoforms, can contribute to resistance to pulse MTX exposure. This may explain why high-level resistance to pulse MTX can occur in the absence of a large decrease in total FPGS activity or a rise in glutamyl hydrolase activity.
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