HUMAN THYMIDYLATE SYNTHETASE .3. EFFECTS OF METHOTREXATE AND FOLATE ANALOGS

The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (1) methotrexate (MTX) analogs modified at the glutamate residue and N10; and (2) tetrahydrofolate (H4PteGlu) analogs modified at N5 and N10. With respect to MTX analogs, it was found that: (1) substitution of the glutamate side chain by α-aminoadipic acid. α-aminopimelic acid or β-aminoglutaric acid slightly affects its Ki; (2) a free α-carboxyl group on the amino acid side chain of MTX, or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterification or amidation of the α-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the γ-carboxyl group of the glutamate side chain decreases its Ki to the enzyme by 5- and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N5 and N10 or by substitution at the N5 position were found to be poor inhibitors under our assay conditions. © 1979.