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Lookup NU author(s): Professor Herbie Newell, Professor Alan Calvert
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The formation, retention and biological activity of the polyglutamate metabolites of the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717) has been investigated in L1210 murine leukaemia cells grown in vitro. CB3717 polyglutamates were measured by HPLC using high specific activity 3H-CB3717. Following the exposure of cells to 50 μM CB3717 for 6, 12 and 24 hr total cellular radioactivity corresponded to 4.5 ± 1.5, 6.8 ± 3.6 and 5.9 ± 3.4 μM drug derived material, respectively. Of this material, > 70%, 57 ± 3% and 51 ± 5% was in the form of unchanged CB3717 at 6, 12 and 24 hr respectively. The remaining radioactivity was associated with polyglutamate metabolites of CB3717, predominantly the tetra and pentaglutamate forms. Following the removal of extracellular drug after incubation for 24 hr and resuspension in drug free medium, unchanged CB3717 was lost rapidly from the cells such that after 6 hr it accounted for only 5% of total cellular radioactivity. In contrast, levels of CB3717 tetra and pentaglutamates declined solely due to dilution during cell division. Measurement of the whole cell TS activity by 3H-deoxyuridine incorporation into DNA indicated that, despite the loss of unchanged CB3717 from the cell, enzyme activity remained suppressed (< 10% of control) for at least 24 hr after resuspension in drug free medium. The TS inhibitory activity of the polyglutamated metabolites of CB3717 was investigated using enzyme purified from L1210 cells. As inhibitors, the metabolites were 26-, 87-, 119- and 114-fold more potent than CB3717 as the di-, tri-, tetra- and pentaglutamate forms, respectively. However, as inhibitors of dihydrofolate reductase prepared from rat liver, CB3717 polyglutamates were no more than 5-fold more potent than the parent compound. This study has shown that CB3717 can undergo polyglutamation in tumour cells and that the metabolites are preferentially retained giving rise to prolonged TS inhibition. By virtue of their potent TS inhibitory activity these metabolites are, therefore, most probably the intracellular effectors of CB3717 cytotoxicity. Abbreviations: MTX, methotrexate; CB3717,N10-propargyl-5,8-dideazafolic acid; TS, thymidylate synthase; DHFR, dihydrofolate reductase
Author(s): Sikora E, Jackman AL, Newell DR, Calvert AH
Publication type: Article
Publication status: Published
Journal: Biochemical Pharmacology
Year: 1988
Volume: 37
Issue: 21
Pages: 4047-4054
Print publication date: 01/11/1988
ISSN (print): 0006-2952
ISSN (electronic): 1873-2968
URL: http://dx.doi.org/10.1016/0006-2952(88)90094-9
DOI: 10.1016/0006-2952(88)90094-9
PubMed id: 2461200
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