4.6 Article

Characterisation of Candida parapsilosis CYP51 as a Drug Target Using Saccharomyces cerevisiae as Host

Journal

JOURNAL OF FUNGI
Volume 8, Issue 1, Pages -

Publisher

MDPI
DOI: 10.3390/jof8010069

Keywords

Candida parapsilosis; cytochrome P450; CYP51; lanosterol 14 alpha-demethylase; fungal pathogen; antifungal resistance; azoles; Saccharomyces cerevisiae expression; X-ray crystal structure; VT-1129

Funding

  1. Health Research Council of New Zealand [13/263, 16/232, 19/397]
  2. University of Otago doctoral scholarship

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The study characterized the wild type C. parapsilosis CYP51 and its resistance-causing point mutation Y132F, revealing increased resistance to fluconazole and voriconazole with the Y132F mutation. The research provides insights into the susceptibility of recombinant CYP51 from C. parapsilosis to azole drugs, which may help advance structure-directed antifungal discovery.
The fungal cytochrome P450 lanosterol 14 alpha-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 & ANGS;. Structural analysis of azole-enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.

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