Mechanism-based inhibitors of folylpoly-gamma-glutamate synthetase and gamma-glutamyl hydrolase: control of folylpoly-gamma-glutamate homeostasis as a drug target.

Intracellular folate pools consist primarily of gamma-glutamyl isopeptide conjugates of reduced forms of the vitamin, folic acid. Biosynthesis of these oligomeric isopeptides is catalyzed by the enzyme folylpoly-gamma-glutamate synthetase (FPGS). The highly anionic character of the oligomers renders them unable to cross the cell membrane and, therefore, these forms of reduced folates (and certain antifolates) accumulate in cells to high concentration. gamma-Glutamyl hydrolase (GH) catalyzes the hydrolysis of the oligo-gamma-glutamates derivatives to monoglutamyl forms, which are substrates for the reduced folate carrier and able to exit the cell. This chapter describes research, primarily from our laboratories, on the design, synthesis, and biochemical evaluation of several novel analogues of glutamic acid, gamma-glutamyl peptides, and derivatives of folic acid as well as of antifolate drugs. These include a series of fluoroglutamic acids, fluoroglutamate-containing isopeptides, phosphorus-containing pseudopeptides, and epoxide-containing peptidomimetics. The fluoroglutamic acids and fluoroglutamate-containing folates and antifolates exhibit position-dependent effects on the reactions catalyzed by FPGS and GH, thus providing insight into the catalytic mechanism and control of these enzymes. The phosphinic acid-containing pseudopeptides are the most potent inhibitors of FPGS identified to date, and were designed based on mechanistic enzymology data from our laboratories and others, prior to the publication of any structural information about the targeted enzymes.