SUBSTRATE-SPECIFICITY OF MAMMALIAN FOLYLPOLYGLUTAMATE SYNTHETASE FOR 5,10-DIDEAZATETRAHYDROFOLATE ANALOGS

The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) t o polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (F PGS) plays a central role in the activity of this compound as an antin eoplastic agent. The availability of a series of DDATHF derivatives di ffering in structure throughout the molecule has allowed a study of th e structural requirements for substrate activity with mouse liver and hog liver FPGS. Kinetics of the polyglutamation reaction in vitro have been related to the potency of these compounds as inhibitors of the g rowth of human CEM leukemic cells. The structure-activity relationship s for enzyme from both sources were nearly identical. FPGS from both s pecies showed a broad acceptance for structural changes in the pyridop yrimidine ring, in the phenyl group, and in the intermediate bridge re gion, with structural changes in these regions being reflected in chan ges in K-m for FPGS but much more modest alterations in V-max. The dat a suggested that the phenyl ring was not contributing to any pi-pi hyd rophobic interactions. It appeared to function primarily in maintainin g a favorable distance between the pyridopyrimidine ring and the gluta mate side chain. The lowest K-m values were found for DDATHF analogs i n which there were small alterations at the 10 position, e.g., 5-deaza tetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofol ate; the first-order rate constants for these substrates were the high est in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic i nhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to b e predictive of the potency of tumor cell growth inhibition for most o f the compounds in this structural series.