Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions. Issue 12 (10th December 2019)

• Record Type: Journal Article
• Authors: Pawłowski, Piotr H.; Szczęsny, Paweł; Rempoła, Bożenna; Poznańska, Anna; Poznański, Jarosław
• Is Part Of: Bioscience reports. Volume 39:Issue 12(2019)
• Publication Date: 2019-12-10
• Subjects: 5 fluorouracil -- anticancer drug -- metabolic pathways; Molecular biology -- Periodicals; Cytology -- Periodicals; 572.8
• DOI: 10.1042/BSR20192847
• Languages: English
• ISSNs: 0144-8463

Abstract

Abstract: The cytotoxic effect of 5-fluorouracil (5-FU) on yeast cells is thought to be mainly via a misincorporation of fluoropyrimidines into both RNA and DNA, not only DNA damage via inhibition of thymidylate synthase (TYMS) by fluorodeoxyuridine monophosphate (FdUMP). However, some studies on Saccharomyces cerevisiae show a drastic decrease in ATP concentration under oxidative stress, together with a decrease in concentration of other tri- and diphosphates. This raises a question if hydrolysis of 5-fluoro-2-deoxyuridine diphosphate (FdUDP) under oxidative stress could not lead to the presence of FdUMP and the activation of so-called 'thymine-less death' route. We attempted to answer this question with in silico modeling of 5-FU metabolic pathways, based on new experimental results, where the stages of intracellular metabolism of 5-FU in Saccharomyces cerevisiae were tracked by a combination of 19 F and 31 P NMR spectroscopic study. We have identified 5-FU, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19 F signal, assigned to fluorinated unstructured short RNA, has been also identified in the spectra. The concentration of individual metabolites was found to vary substantially within hours, however, the initial steady-state was preserved only for an hour, until the ATP concentration dropped by a half, which was monitored independently via 31 P NMR spectra. After that, the catabolic process leading from triphosphates through monophosphates and nucleosides back to 5-FU was observed. These results imply careful design and interpretation of studies in 5-FU metabolism in yeast.