Substrate specificity of mammalian folylpolyglutamate synthetase for 5,10-dideazatetrahydrofolate analogs

Abstract

The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) to polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (FPGS) plays a central role in the activity of this compound as an antineoplastic agent. The availability of a series of DDATHF derivatives differing in structure throughout the molecule has allowed a study of the 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 growth of human CEM leukemic cells. The structure-activity relationships for enzyme from both sources were nearly identical. FPGS from both species showed a broad acceptance for structural changes in the pyridopyrimidine ring, in the phenyl group, and in the intermediate bridge region, with structural changes in these regions being reflected in changes in Km for FPGS but much more modest alterations in Vmax. The data suggested that the phenyl ring was not contributing to any pi-pi hydrophobic interactions. It appeared to function primarily in maintaining a favorable distance between the pyridopyrimidine ring and the glutamate side chain. The lowest Km values were found for DDATHF analogs in which there were small alterations at the 10 position, e.g., 5-deazatetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofolate; the first-order rate constants for these substrates were the highest in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic inhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to be predictive of the potency of tumor cell growth inhibition for most of the compounds in this structural series.