Biochemical interactions between N-(phosphonacetyl)-L-aspartate and 5-fluorouracil

C. M. Liang, R. C. Donehower, B. A. Chabner

Research output: Contribution to journalArticlepeer-review

Abstract

5-fluorouracil (FU) blocks the synthesis of thymidylate by forming an active metabolite, 5-fluoro-2'-deoxyuridylate (FdUMP) which in the presence of the cofactor 5,10-methylenetetrahydrofolate, binds to thymidylate synthetase (EC 2.1.2.45) to form a stable ternary complex. Exposure of logarithmically growing sarcoma 180 cells to FU (3-300 μM for 2 hr) caused a concentration-dependent decrease in cellular thymidylate synthetase available to bind [6-3H]FdUMP (and to release tritium from [5-3H]dUMP) in the assay solution. These results indicate that FU exposure caused the de novo formation of the ternary complexes and thus decreased the free thymidylate synthetase catalytic sites. When the cells were pretreated with N-(phosphonacetyl)-L-aspartate (PALA) (30-100 μM for 12 hr), exposure to the same concentration of FU (3-100 μM for 2 hr) led to a further decrease in free thymidylate synthetase catalytic sites, indicating the formation of increased inhibited ternary complexes. Increased complex formation of the competitive substrate either increased 5-FdUMP formation or decreased formation of the competitive substrate dUMP. The level of dUMP in the PALA-pretreated cells (100 μM for 12 hr) was 68 ± 3 nmoles/109 cells, which was not much different from that in the control cells (79 ± 2 nmoles/109 cells). FU treatment (100 μM for 2 hr) increased the dUMP level more than 3-fold in the control cells but only 15% in the PALA-pretreated cells, indicating that PALA pretreatment prevented the accumulation of dUMP. After FU treatment (100 μM for 2 hr), PALA-pretreated cells contain 2.5-fold higher levels of 5-FdUMP than control cells. These results suggest that PALA, a transition-state analogue inhibitor of L-aspartate transcarbamylase, by blocking the synthesis of cellular pyrimidine nucleotides and increasing the formation of 5-FdUMP enhances FU inhibition of thymidylate synthetase.

Original languageEnglish (US)
Pages (from-to)224-230
Number of pages7
JournalMolecular Pharmacology
Volume21
Issue number1
StatePublished - Jan 1 1982

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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