The overall goal of this project is the synthesis of novel substrates and inhibitors of folylpolyglutamate synthetase (FPGS) with a view to defining the structural requirements for tight binding to the active site and gaining a better understanding of the molecular mechanism of action of this physiologically important enzyme. In addition, these studies will yield new information about the optimal structural features for cellular uptake of folate analogs via the reduced folate carrier (RFC) and membrane folate-binding protein (mFBP) transport pathways. It is hoped that knowledge gained through these studies will contribute to possible development of inhibitors of folate polyglutamylation and/or uptake as a discrete class of antifolates for cancer therapeutics. Specific types of compounds to be synthesized will include: (1) intermediate product and transition state analogs of the FPGS reaction; (2) folate and tetrahydrofolate analogs that retain the glutamic acid moiety but are modified in the `bay region' comprised by the B-ring, bridge, aryl, and CONH moieties; (3) analogs in which the gamma-CO2H group is replaced by NO2, SO2H, and PO2H2 groups; (4) analogs with gamma,gamma-difluoroornithine side chains; and (5) GSH-activated and other prodrug derivatives of the best available ornithine-type analogs. The kinetics of interaction of these compounds with FPGS will be determined as part of an established collaboration with Dr. R.G. Moran (Medical College of Virginia, Richmond), whereas their membrane transport characteristics in different cells will be determined in collaboration with Dr. G. Jansen (Free University Hospital, Amsterdam). In addition, where such studies are indicated, compounds will be co-crystallized with FPGS and the crystals analyzed by X-ray crystallography to obtain critical information about how FPGS inhibitors bind to the active site. The crystallographic studies will be part of an ongoing collaboration with Dr. V. Cody (Hauptman-Wood Medical Research Institute, Buffalo). It is hoped that these interactive studies may ultimately lead to development of a therapeutic agent based on the exploitation of qualitative or quantitative differences in binding specificity which are thought to exist between/among FPGS isoforms in tumor and nontumor tissues, and which may also be present, in principle, at the level of membrane transport. In addition to the binding and uptake assays, the new compounds derived from this project will be tested for the ability to inhibit growth of cultured tumor cells, with the hope of identifying one or more promising candidates for scaled up synthesis and further preclinical development.
