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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

Lookup NU author(s): Professor Alan Calvert

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Abstract

The biochemical effects of the antitumor agent N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino)benzoyl)-l-glutam 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 (K_i = 4.9 × 10⁻⁹M). CB3717 also inhibited human dihydrofolate reductase, competitively with dihydrofolate (K_i = 2.3 × 10⁻⁸M). The growth-inhibitory effect of CB3717 could be prevented completely by 10 μM 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 K_i of the inhibitor for thymidylate synthetase were 3 orders of magnitude higher (weaker) than the K_i for dihydrofolate reductase, thymidylate synthetase could still become rate-limiting. Abbreviations: CB3717, N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino)-benzoyl)-l-glutamic acid, DHFR. dihydrofolate reductase; TS. thymidylate synthetase; MTX-methotrexate; FH₂, dihydrofolate

Publication metadata

Author(s): Jackson RC, Jackman AL, Calvert AH

Publication type: Article

Publication status: Published

Journal: Biochemical Pharmacology

Year: 1983

Volume: 32

Issue: 24

Pages: 3783-3790

ISSN (print): 0006-2952

ISSN (electronic): 1873-2968

URL: http://dx.doi.org/10.1016/0006-2952(83)90150-8

DOI: 10.1016/0006-2952(83)90150-8

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