Biochemical effects of a quinazoline inhibitor of thymidylate synthetase, N-(4-(N-(( 2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino) benzoyl)-L-glutamic acid (CB3717), on human lymphoblastoid cells.
The biochemical effects of the antitumor agent N-(4-(N-(( 2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino) benzoyl)-L -glutamic acid (CB3717) were studied in WI-L2 cultured human lymphoblastoid cells. CB3717 was a potent inhibitor of human thymidylate synthetase; the inhibition was competitive with 5,10-methylenetetrahydrofolate (Ki = 4.9 X 10(-9) M). CB3717 also inhibited human dihydrofolate reductase, competitively with dihydrofolate (Ki = 2.3 X 10(-8) M). The growth-inhibitory effect of CB3717 could be prevented completely by 10 microM thymidine. Administration of thymidine could be delayed for up to 8 hr after CB3717 treatment without cytotoxicity but, if thymidine was delayed for 24 hr, severe toxicity resulted. Incubation for 16 hr in the presence of a growth-inhibitory concentration of CB3717 did not result in the appearance of dihydrofolate in WI-L2 cells. These results indicate that, in the presence of CB3717, thymidylate synthetase, rather than dihydrofolate reductase, became rate-limiting for the cycle of dihydrofolate oxidation and reduction. Treatment of cells for 16 hr at an IC50 concentration of CB3717 caused a decrease of 88% in cellular dTTP and a 2,300% increase in dUMP. The level of dUDP also increased, and traces of dUTP appeared in treated cells. No large changes were seen in ribonucleotide pools. A kinetic analysis was made, by computer simulation, of predicted consequences of metabolic effects of compounds that inhibit both dihydrofolate reductase and thymidylate synthetase. It was concluded that, even if the Ki of the inhibitor for thymidylate synthetase were 3 orders of magnitude higher (weaker) than the Ki for dihydrofolate reductase, thymidylate synthetase could still become rate-limiting.
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D003854:Deoxyribonucleotides / Q000378:metabolism; D003857:Deoxyuridine / Q000494:pharmacology; D005492:Folic Acid / Q000031:analogs & derivatives*; D005493:Folic Acid Antagonists; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D011799:Quinazolines / Q000494:pharmacology*; D012265:Ribonucleotides / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D003854:Deoxyribonucleotides; D005493:Folic Acid Antagonists; D011799:Quinazolines; D012265:Ribonucleotides; C031662:CB 3717; D005492:Folic Acid; D008780:Methyltransferases; D013940:Thymidylate Synthase; D003857:Deoxyuridine; D008727:Methotrexate