Reference: Parker JE, et al. (2008) Differential azole antifungal efficacies contrasted using a Saccharomyces cerevisiae strain humanized for sterol 14 alpha-demethylase at the homologous locus. Antimicrob Agents Chemother 52(10):3597-603

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Abstract


Inhibition of sterol-14alpha-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs and with high frequencies of fungal infection new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51 although selective inhibitors of the human target are also of interest as anti-cholesterol agents. A strain of Saccharomyces cerevisiae was produced that was humanised with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51). The strain was validated with respect to gene expression, protein localisation, growth characteristics and sterol content. The MIC was determined and compared to the wild type parental strain (BY4741) using clotrimazole, econazole, fluconazole itraconazole, ketoconazole, miconazole and voriconazole. The humanised strain showed reduced susceptibility to the orally active azole drugs up to >1000 fold while the topical agents showed no difference. Data from growth kinetic measurements substantiated this but also revealed reduced effectiveness against the humanised strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14alpha-methyl-ergosta-8, 24(28)-dien-3beta-6alpha-diol, than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and differentiates azole antifungals clearly in a side-by-side comparison.

Reference Type
Journal Article
Authors
Parker JE, Merkamm M, Manning NJ, Pompon D, Kelly SL, Kelly DE
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