The long-term goal of this program is to improve human cancer treatment by exploiting aspects of folyl- or antifolylpoly(gamma-glutamate) synthesis. Folylpolyglutamates are essential for cell growth, while polyglutamates of classical antifolates are implicated in, and often critical for, their cytotoxic action. Synthesis of polyglutamate metabolites also plays a role in drug resistance. Detailed understanding of the synthesis and function of folyl- and antifolylpolyglutamates may thus allow development of new agents or strategies designed to exploit this critical process. This long term goal will be addressed in this application through the following Specific Aims.
Specific Aim 1 would explore folylpolyglutamate synthetase (FPGS), the enzyme responsible for synthesis of polyglutamates, as a target for drug development. Since mutational deletion of FPGS activity is lethal, FPGS is a potential therapeutic target for cancer chemotherapy. Rational design of FPGS inhibitors will be based on enzyme mechanism and structure-activity data generated in the applicant's laboratory using homogeneous, recombinant human FPGS. Of interest are potential mechanism-based inhibitors and modifications to enhance potency and/or uptake of recognized folate-based ornithine-containing FPGS inhibitors. The biological effects to be expected from FPGS inhibition will be explored in a model system for folylpolyglutamate deficiency. These studies should define whether FPGS is a viable drug target.
Specific Aim 2 would define the relationship between FPGS and methotrexate (MTX) resistance in human leukemia and human solid tumor model systems. Decreased FPGS activity, as first identified in collaborative studies involving the applicant's laboratory, is now established as a mechanism of resistance to antifolates in vitro, in vivo, and in the clinic. This clinically relevant resistance phenotype will be further characterized as to its frequency and evolution in preclinical models of leukemia and solid tumors. A multicellular spheroid system will be developed and characterized as a model for human solid tumor antifolate resistance.
Specific Aim 3 would define the molecular pharmacology of human FPGS, especially in relation to antifolate resistance. A human FPGS cDNA will be used in further studies of the molecular pharmacology of FPGS and to characterize the nature of the defect in MTX-resistant lines deficient in polyglutamylation (Specific Aim 2). An antipeptide antibody to FPGS will be used to study regulation of FPGS at the protein level and its status in MTX-resistant cell lines. A polyclonal antibody to holo human FPGS protein will also be prepared and utilized.
Showing the most recent 10 out of 50 publications
