MECHANISM-BASED INHIBITION OF HUMAN FOLYLPOLYGLUTAMATE SYNTHETASE - DESIGN, SYNTHESIS, AND BIOCHEMICAL-CHARACTERIZATION OF A PHOSPHAPEPTIDEMIMIC OF THE TETRAHEDRAL INTERMEDIATE

Folylpolyglutamate synthetase (FPGS) catalyzes an ATP-dependent ligati on reaction that results in the synthesis of poly(gamma-glutamate) met abolites of folates and some antifolates, We have synthesized and char acterized the prototype of a new class of mechanism-based FPGS inhibit or in which a phosphonate moiety mimics the tetrahedral intermediate f ormed during the ligation reaction. This phosphonate, 4-amino-4-deoxy- 10-methyl-pteroyl-L- glutamyl-gamma-[Psi{P(O)(OH)-O}]glutarate (4-NH2- 10-CH3-Pte-L-Glu-gamma-[Psi{P(O)(OH)-O}] glutarate), is not a substrat e for human FPGS, but is a linear, competitive inhibitor (K-is = 46 nM ) with respect to methotrexate as the variable substrate, Inhibition i s not time-dependent and preincubation of FPGS with this phosphonate d oes not increase the degree of inhibition, suggesting that it is not a slow, tight-binding inhibitor involving a time-dependent isomerizatio n, EI --> EI. Substructures containing the phosphonate moiety but lac king the pterin are much less inhibitory to FPGS, indicating that a si gnificant portion of the inhibitor binding energy is derived from the pterin moiety, a feature also observed in substrate binding. 4-NH2-10- CH3-Pte-L- Glu-gamma-[Psi{P(O)(OH)-O}]glutarate is also an analog of a proposed tetrahedral intermediate in the reaction catalyzed by gamma- glutamyl hydrolase (gamma-GH), another enzyme of importance in control ling folate homeostasis in cells. This intermediate would arise from d irect attack of H2O on the dipeptide, 4-NH2-10-CH3-Pte-L-Glu-gamma-L-G lu. The fact that NH2-10-CH3-Pte-L-Glu-gamma-[Psi{P(OH)-O}]glutarate i s not an inhibitor of gamma-GH strongly suggests that hydrolysis of po ly-gamma-glutamates catalyzed by gamma-GH does not involve the direct attack of water at the scissile amide bond. Methotrexate, its gamma-gl utamyl dipeptide metabolite, and -10-CH3-Pte-L-Glu-gamma-[Psi{P(O)(OH) -O}]glutarate are equipotent as inhibitors of human dihydrofolate redu ctase (the primary target of methotrexate), but the phosphonate does n ot significantly inhibit another important folate-dependent enzyme, th ymidylate synthase, Thus, the phosphonate moiety in this analog repres ents an important new lead in the development of FPGS inhibitors. (C) 1998 Academic Press.