Evidence that G-quadruplexes form in pathogenic fungi and represent promising antifungal targets.

Fungi are estimated to cause the death of almost 4 million people annually, and we urgently need new drug targets to overcome antifungal resistance. We found that four-stranded nucleic acid structures called G-quadruplexes (G4s) could form within the critical priority fungal pathogen Aspergillus fumigatus. Sequences with the potential to form G4s could be found in genes involved in fungal growth, virulence, and drug resistance. This included cyp51A, which encodes the target of azoles. Notably, we observed the formation of both canonical and unusual acid-stabilised G4s in these sequences. We found that PhenDC3 (a G4-stabilising ligand) could refold DNA into antiparallel G4 structures in cyp51A that were associated with decreased transcription. PhenDC3 also had potent fungistatic activity, prevented germination, synergised with the antifungal amphotericin B in vitro and in vivo, and displayed low genotoxicity and cytotoxicity towards human cells. Interestingly, PhenDC3 had greater antifungal activity towards the pan-azole-resistant A. fumigatus TR34/L98H isolate, and another G4-stabiliser, pyridostatin, killed multi-drug-resistant Candida auris. Taken together, G4s represent a promising target for the development of antifungals with novel mechanisms of action.