Biochemical and antitumor effects of 5,8-dideazaisopteroylglutamate, a unique quinazoline inhibitor of thymidylate synthase.

The effects of 5,8-dideazaisopteroylglutamate (A/-[p-{[(2amino-4-hydroxy-6-quinazolinyl)amino]methyl}benzoyl]-Lglutamic acid) (IAHQ) on thymidylate synthase and dihydrofolate reducÃ-ase have been examined as a biochemical basis for the anticancer activity of IAHQ in several tumor systems. IAHQ, when administered at 85 mg/kg on Days 2 and 10 after tumor implantation, delayed the growth of methotrexate-resistant Co lon Tumor 38 and resulted in 6 of 20 tumor-free animals at 90 days. IAHQ was also active against human colon adenocarcinoma (HCT-8), murine L1210, P388, L5178Y leukemias, and Walker 256 rat carcinoma in tissue culture, requiring concen trations of 0.5, 5, 5, 5, and 5 JUM,respectively, to inhibit cell growth 50% compared to untreated cells. Several lines of evidence indicate that, unlike methotrexate, IAHQ exerts its effects via inhibition of thymidylate synthase. Thymidine alone protected HCT-8 cells from IAHQ cytotoxicity. CCRF-CEM human leukemic cells 75-fold resistant to IAHQ showed no cross-resistance to methotrexate. Although IAHQ was a more potent inhibitor of dihydrofolate reducÃ-ase[K, slope (K¡s), 0.7 /IM] than Ihymidylale synlhase (K¡s5 ¡UM), Ihe possible poly-y-glutamyl melabolile of IAHQ, 5,8-dideazaisopteroyl-yL-glutamyl-y-L-glulamyl-L-glulamic acid, is a better inhibitor of thymidylate synthase (K¡s0.09 ,UM)than dihydrofolate reduc Ã-ase(KiS 0.7 jtiM). Significant binding of IAHQ and 5,8-dideazaisopteroyl-y-L-glutamyl-y-L-glutamyl-L-glutamic acid to thymidylate synthase required the presence of deoxyuridylate. IAHQ and, to a greater extent, 5,8-dideazaisopteroyl-y-u-glutamyl-y-L-glutamyl-L-glutamic acid stimulated binding of deox yuridylate to one site on thymidylate synthase with dissociation constants for deoxyuridylate in the ternary complexes of 1.2 and 0.041 JUM,respectively. These data indicate that the bio chemical basis for the antitumor effects of IAHQ is probably its intracellular conversion to poly-y-glutamyl metabolites, which inhibit thymidylate synthase via quinazoline-deoxyuridylate-enzyme ternary complex formation.