Abstract
Candida auris is a multidrug-resistant fungal pathogen of major concern, associated with high mortality and frontline azole antifungal resistance. Azole antifungals target lanosterol 14α-demethylase (CYP51), a cytochrome P450 enzyme essential for ergosterol biosynthesis. Resistance has been linked to CYP51 mutations Y132F and K143R, reducing drug efficacy and limiting treatment options. Structural insight into C. auris CYP51 (CauCYP51) is critical for understanding resistance and guiding next-generation antifungal development. However, no crystal structure of CauCYP51 is available. This research aimed to investigate wild-type CauCYP51, and mechanisms of resistance associated with mutations Y132F and K143R using structural and ligand binding methods.
Recombinant hexahistidine-tagged wild-type CauCYP51 and Saccharomyces cerevisiae CYP51 were expressed in an S. cerevisiae host and purified using Ni-NTA and size-exclusion chromatography. UV–visible difference spectroscopy determined binding affinities (Kd, µM) by titrating 1 µM enzyme with increasing azole concentrations. 480 crystallisation conditions were screened with itraconazole, ravuconazole, and ravuconazole-tetrazole. Homology modelling (MODELLER) and docking (GOLD) complemented the experimental studies.
Wild-type CauCYP51 bound the experimental azoles ravuconazole (Kd = 0.284 ± 0.03), ravuconazole-tetrazole (Kd = 0.223 ± 0.02), and the clinical azole posaconazole (Kd = 0.298 ± 0.06) more strongly than the clinical fluconazole (Kd = 0.653 ± 0.04). The K143R mutant was purified and characterised for the first time. It showed type II binding of ravuconazole-tetrazole (Kd = 0.172 ± 0.067), the characteristic spectral pattern observed when azoles bind to CYP51, suggesting strong binding that could overcome resistance. The CauCYP51 Y132F mutant could not be purified. Small wild-type CauCYP51 crystals were sent to the Australian Synchrotron for diffraction testing. Computational studies identified mutation sites homologous to those in other fungal CYP51 enzymes and revealed ravuconazole maintains favourable binding interactions in resistant variants.
These findings advance molecular understanding of azole–CYP51 interactions and support rational design of next-generation antifungals targeting CauCYP51.