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

Daniel J. Fernandes, Joseph R. Bertino, John B. Hyne

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The effects of 5,8-dideazaisopteroylglutamate (N-[p-{[(2-amino-4-hydroxy-6-quinazolinyl)amino]methyl}benzoyl]-L- glutamic acid) (IAHQ) on thymidylate synthase and dihydrofolate reductase 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 Colon 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 concentrations of 0.5, 5, 5, 5, and 5 μm, 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 reductase [Kislope (KiS), 0.7μm] than thymidylate synthase (Kis 5 m), the possible poly-γ-glutamyl metabolite of IAHQ, 5,8-dideazaisopteroyl-γ-L-glutamyl-γ-L-glutamyl-L-glutamic acid, is a better inhibitor of thymidylate synthase (KiS 0.09 m) than dihydrofolate reductase (KiS 0.7 m). Significant binding of IAHQ and 5,8-di-deazaisopteroyl-γ-L-glutamyl-γ-L-glutamyl-L-glutamic acid to thymidylate synthase required the presence of deoxyuridylate. IAHQ and, to a greater extent, 5,8-dideazaisopteroyl-γ-L-glu-tamyl-γ-L-glutamyl-L-glutamic acid stimulated binding of deoxyuridylate to one site on thymidylate synthase with dissociation constants for deoxyuridylate in the ternary complexes of 1.2 and 0.041 µm, respectively. These data indicate that the biochemical basis for the antitumor effects of IAHQ is probably its intracellular conversion to poly-γ-glutamyl metabolites, which inhibit thymidylate synthase via quinazoline-deoxyuridylate-enzyme ternary complex formation.

Original languageEnglish (US)
Pages (from-to)1117-1123
Number of pages7
JournalCancer Research
Issue number3
StatePublished - Mar 1 1983
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research


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