Mechanism-based inhibition of human folylpolyglutamate synthetase: design, synthesis, and biochemical characterization of a phosphapeptide mimic of the tetrahedral intermediate.
Folylpolyglutamate synthetase (FPGS) catalyzes anATP-dependent ligation reaction that results in the synthesis of poly(gamma-glutamate) metabolites of folates and some antifolates. We have synthesized and characterized the prototype of a new class of mechanism-based FPGS inhibitor in which a phosphonate moiety mimics the tetrahedral intermediate formed 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 substrate for human FPGS, but is a linear, competitive inhibitor (Kis = 46 nM) with respect to methotrexate as the variable substrate. Inhibition is not time-dependent and preincubation of FPGS with this phosphonate does not increase the degree of inhibition, suggesting that it is not a slow, tight-binding inhibitor involving a time-dependent isomerization, EI --> EI*. Substructures containing the phosphonate moiety but lacking the pterin are much less inhibitory to FPGS, indicating that a significant 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 controlling folate homeostasis in cells. This intermediate would arise from direct attack of H2O on the dipeptide, 4-NH2-10-CH3-Pte-L-Glu-gamma-L-Glu. The fact that 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate is not an inhibitor of gamma-GH strongly suggests that hydrolysis of poly-gamma-glutamates catalyzed by gamma-GH does not involve the direct attack of water at the scissile amide bond. Methotrexate, its gamma-glutamyl dipeptide metabolite, and 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate are equipotent as inhibitors of human dihydrofolate reductase (the primary target of methotrexate), but the phosphonate does not significantly inhibit another important folate-dependent enzyme, thymidylate synthase. Thus, the phosphonate moiety in this analog represents an important new lead in the development of FPGS inhibitors.
Tsukamoto
T
T
Departments of Chemistry and Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, 48109-1055, USA.
Haile
W H
WH
McGuire
J J
JJ
Coward
J K
JK
D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D004151:Dipeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D010453:Peptide Synthases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013816:Thermodynamics; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000037:antagonists & inhibitors
D004151:Dipeptides; D004791:Enzyme Inhibitors; D011623:gamma-Glutamyl Hydrolase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase