Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51).
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Warrilow AG, Martel CM, Parker JE, Melo N, Lamb DC, Nes WD, Kelly DE, Kelly SL
Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51).
Antimicrob Agents Chemother. 2010 Oct;54(10):4235-45. doi: 10.1128/AAC.00587-10. Epub 2010 Jul 12.
- PubMed ID
- 20625155 [ View in PubMed]
- Abstract
Purified Candida albicans sterol 14-alpha demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K(s) values of 11 and 25 muM, respectively, and a K(m) value of 6 muM for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (DeltaA(max)) and the azole concentration required to obtain a half-DeltaA(max) being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K(d) values of 10 to 26 muM under oxidative conditions, compared with 47 muM for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min(-1)), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.
DrugBank Data that Cites this Article
- Drug Targets
Drug Target Kind Organism Pharmacological Action Actions Clotrimazole Cytochrome P450 51 Protein Yeast YesAntagonistInhibitorDetails Econazole Cytochrome P450 51 Protein Yeast YesAntagonistDetails Ketoconazole Lanosterol 14-alpha demethylase Protein YesInhibitorDetails