The long-term goal of the proposed research is to develop improved treatment for human cancer by exploiting aspects of folyl- or antifolylpoly(gamma-glutamate) synthesis. Folylpolyglutamates are essential for cell proliferation, while polyglutamates of classical antifolates are implicated in, and often critical for, their cytotoxic action. Synthesis of polyglutamate metabolites plays a role in drug resistance and may have a role in selectivity. Complete biochemical understanding of the synthesis and function of folyl- and antifolylpolyglutamates may thus allow the development of new agents or strategies designed to exploit this critical process. This long term goal will be addressed in this grant period through the following specific aims which incorporate elements unique to this lab: 1. Design, synthesis, and characterization of human folylpolyglutamate synthetase (FPGS) inhibitors. Rational design of inhibitors is based on FPGS substrate specificity data generated in this lab using purified, well- characterized human FPGS. Novel antifolates that could potentially inhibit FPGS (based on structure) or that will add to the FPGS structure-activity data base are also studied. Syntheses are performed by recognized experts in antifol chemistry, principally Drs. M.G. Nair and J.R. Piper. All antifols will be fully characterized in this lab. Of interest are 2nd generation analogs of the first, human FPGS-specific inhibitor, 5,8-dideaza-pteroyl-ornithine (Dr. Piper), which was designed and characterized in this lab. FPGS inhibitors will be used in investigations of polyglutamate metabolism and its regulation in intact human leukemia cell lines. Basic knowledge derived from such studies may allow more effective use of current antifols or suggest new targets for therapeutic intervention. 2. Characterization of the development and properties of human leukemia cell lines that are resistant to methotrexate (MTX) because of decreased MTX polyglutamate synthesis. In the first and only cell lines with acquired MTX resistance having reduced MTX polyglutamate synthesis as the sole source, resistance was traced to decreased FPGS activity. Similar resistance has been identified in the clinic. This clinically relevant resistance phenotype will be characterized. Further verification that this is the sole mechanism of resistance will be obtained, along with cross-resistance data. Comparison of the enzymatic and physical properties of the sensitive and resistant cell FPGS may provide both insight into the nature of the change and information useful for the design of FPGS inhibitors (Specific Aim 1). We also propose to study the kinetics of occurrence of this phenotype and factors affecting the frequency of occurrence in vitro. 3. Development of molecular probes for human FPGS, the enzyme responsible for polyglutamate synthesis. A human FPGS cDNA will be isolated to use in studies of the regulation and molecular pharmacology of this enzyme. This probe would also be used to characterize the nature of the defect in the MTX-resistant lines deficient in polyglutamylation (Specific Aim 2). An expression vector will be prepared to make the enzyme available in large quantity for enzyme characterization and inhibitor design studies. In addition, antibodies to the FPGS protein will be prepared to study regulation at the protein level.
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