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Antimicrobial Agents and Chemotherapy, April 2004, p. 1256-1271, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1256-1271.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Chemosensitization of Fluconazole Resistance in Saccharomyces cerevisiae and Pathogenic Fungi by a D-Octapeptide Derivative

Department of Oral Sciences, University of Otago, Dunedin,¹ The Centre for Separation Science, Massey University, Palmerston North, New Zealand,² Unité de Biochimie Physiologique, Université Catholique de Louvain, Louvain, Belgium,³ Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan⁴

Received 24 February 2003/ Returned for modification 20 May 2003/ Accepted 2 December 2003

Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5⁺) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 µg ml^-1), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 µg ml^-1). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer D-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted D-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC₅₀], 4 µM) which chemosensitized AD/PDR5⁺ to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC₅₀, 30 µM) and Cdr2p (IC₅₀, 2 µM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter Ben^Rp, or the FLC drug target lanosterol 14-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC₅₀, 1 µM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

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