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Antimicrobial Agents and Chemotherapy, October 2008, p. 3597-3603, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00517-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Differential Azole Antifungal Efficacies Contrasted Using a Saccharomyces cerevisiae Strain Humanized for Sterol 14{alpha}-Demethylase at the Homologous Locus{triangledown}

J. E. Parker,1 M. Merkamm,2 N. J. Manning,3 D. Pompon,2 S. L. Kelly,1 and D. E. Kelly1*

Institute of Life Science and School of Medicine, Swansea University, Swansea, Wales SA2 8PP, United Kingdom,1 Laboratoire d'Ingénierie des Protéines Membranaires, CNRS-Centre de Génétique Moléculaire, 91198 Gif-sur-Yvette Cedex, France,2 Clinical Chemistry, Sheffield Children's Hospital, Sheffield S10 2TH, United Kingdom3

Received 22 April 2008/ Returned for modification 29 May 2008/ Accepted 28 July 2008

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-3β-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.

* Corresponding author. Mailing address: Institute of Life Science and School of Medicine, Swansea University, Swansea, Wales SA2 8PP, United Kingdom. Phone: 44 1792 602184. Fax: 44 1792 602280. E-mail: D.Kelly{at}swansea.ac.uk

{triangledown} Published ahead of print on 11 August 2008.

Antimicrobial Agents and Chemotherapy, October 2008, p. 3597-3603, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00517-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Warrilow, A. G. S., Jackson, C. J., Parker, J. E., Marczylo, T. H., Kelly, D. E., Lamb, D. C., Kelly, S. L. (2009). Identification, Characterization, and Azole-Binding Properties of Mycobacterium smegmatis CYP164A2, a Homolog of ML2088, the Sole Cytochrome P450 Gene of Mycobacterium leprae. Antimicrob. Agents Chemother. 53: 1157-1164 [Abstract] [Full Text]  


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