Antifungal activity of the nucleoside mimetic 5-fluorouridine against <i>Candidozyma auris</i> and <i>Candida</i> species

ABSTRACT The emergence of multidrug-resistant Candidozyma auris has created an urgent need for antifungals with novel mechanisms of action. We screened 121 nucleoside and nucleobase derivatives against C. auris and other clinically relevant Candida species to identify effective antimetabolite scaffolds. Among all compounds tested, only the C-5-fluorinated nucleosides 5-fluorouridine (5-FU) and 5-fluorocytidine exhibited potent antifungal activity. 5-FU demonstrated consistently low minimum inhibitory concentrations (0.5–10 µg/mL) across multiple Candida species, including fluconazole-resistant strains, and displayed fungicidal activity against C. auris at concentrations lower than several frontline antifungals. Uridine rescue assays confirmed competitive interference with endogenous uridine metabolism. Checkerboard assays revealed synergistic or additive interactions between 5-FU and clinically used azoles and echinocandins. Toxicity evaluation using plant, nematode, and human cell models showed minimal cytotoxicity even at concentrations far exceeding antifungal minimum inhibitory concentrations. In silico docking analyses revealed dual inhibition of thymidylate synthase and RNA polymerase III, explaining the fungicidal phenotype and broad-spectrum efficacy. These findings identify 5-fluorouridine as a promising antifungal candidate and establish C-5-halogenated nucleosides as a privileged scaffold for antifungal drug development.