title: Differential azole antifungal efficacies contrasted using a Saccharomyces cerevisiae strain humanized for sterol 14 alpha-demethylase at the homologous locus.
abstract: Inhibition of sterol-14 alpha-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections 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 anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized 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 14 alpha-methyl-ergosta-8, 24(28)-dien-3beta-6 alpha-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 clearly differentiates azole antifungals in a side-by-side comparison.
authors.affiliation: Institute of Life Science and School of Medicine, Swansea University, Swansea, Wales SA2 8PP, United Kingdom.
authors.forename: J E
authors.initials: JE
authors.lastname: Parker
authors.forename: M
authors.initials: M
authors.lastname: Merkamm
authors.forename: N J
authors.initials: NJ
authors.lastname: Manning
authors.forename: D
authors.initials: D
authors.lastname: Pompon
authors.forename: S L
authors.initials: SL
authors.lastname: Kelly
authors.forename: D E
authors.initials: DE
authors.lastname: Kelly
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D001483:Base Sequence; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics / Q000502:physiology; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D058886:Sterol 14-Demethylase
chemical_list: D000935:Antifungal Agents; D001393:Azoles; C548418:CYP51A1 protein, human; D065607:Cytochrome P-450 Enzyme Inhibitors; D004271:DNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003577:Cytochrome P-450 Enzyme System; D058886:Sterol 14-Demethylase