The fungal expel of 5-fluorocytosine derived fluoropyrimidines mitigates its antifungal activity and generates a cytotoxic environment

Sastré-Velásquez, Luis Enrique; Dallemulle, Alex; Kühbacher, Alexander; Baldin, Clara; Alcazar-Fuoli, Laura; Niedrig, Anna; Müller, Christoph und Gsaller, Fabio (2022): The fungal expel of 5-fluorocytosine derived fluoropyrimidines mitigates its antifungal activity and generates a cytotoxic environment.
In: PLOS Pathogens 18(12), e1011066

Abstract

Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR. Author summary Aspergillus fumigatus is the most common cause of invasive aspergillosis, a life-threatening fungal infection that is associated with worryingly high mortality rates. An antifungal that is barely used for the treatment of aspergillosis illustrates the nucleobase analog 5-fluorocytosine (5FC). A major drawback of this antimycotic compound constitutes its low in vitro activity against A. fumigatus, which is owed to transcriptional repression of its uptake at neutral pH. In contrast to other clinically administered anti-Aspergillus agents, 5FC has no intrinsic antifungal capacity but has to be activated within the cell through the pyrimidine salvage pathway. In this work, we characterized a new set of pyrimidine salvage enzymes that participate in its metabolic activation and show that during 5FC exposure, A. fumigatus generates and exports significant amounts of its derived fluoropyrimidines such as cytotoxic 5-fluorouracil (5FU) and 5-fluorouridine (5FUR). This extrusion diminishes 5FC antifungal activity and, simultaneously, creates a toxic environment for mammalian cells, which suggests the use of fungal efflux pump inhibitors in combination with 5FC as a promising therapeutic strategy.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Chemie und Pharmazie > Department für Pharmazie - Zentrum für Pharmaforschung
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 1553-7366
• Sprache: Englisch