Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions

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 19F and 31P NMR spectroscopic study. We have identified 5-FU, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19F 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 31P 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.

Recent Updates on Mechanisms of Resistance to 5-Fluorouracil and Reversal Strategies in Colon Cancer Treatment

“…Thymidine phosphorylase, or also known as platelet-derived endothelial cell growth factor (PD-ECGF) is another crucial enzyme in the metabolism of 5-FU, involved in the conversion of 5-FU to FdUR prior to its phosphorylation by thymidine kinase (TK) to FdUMP [ 241 ]. TYMP overexpression may promote increased intracellular concentration of active 5-FU metabolite, thus enhancing its efficacy.…”

A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate

“…19 F nuclear magnetic resonance ( 19 F NMR) is a non-invasive spectroscopic method that is used to identify and quantify all 5-FU anabolites in a single run ( 80 , 81 ). It was applied to several biological samples such as intracellular extracts from cultured cells ( 80 ), tumour tissues ( 82 , 83 ) and RNA ( 82 ).…”

“…To the best of our knowledge, it has not been used to analyse DNA. 19 F NMR is specific to fluorinated compounds and has to be coupled with 31 P NMR to study endogenous nucleotides and nucleosides ( 81 ).…”