title: Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative.
abstract: 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 microg 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 microg 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 [IC(50)], 4 microM) which chemosensitized AD/PDR5(+) to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC(50), 30 microM) and Cdr2p (IC(50), 2 microM), 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(R)p, or the FLC drug target lanosterol 14 alpha-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC(50), 1 microM), 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.
authors.affiliation: Department of Oral Sciences, University of Otago, Dunedin, New Zealand.
authors.forename: K
authors.initials: K
authors.lastname: Niimi
authors.forename: D R K
authors.initials: DR
authors.lastname: Harding
authors.forename: R
authors.initials: R
authors.lastname: Parshot
authors.forename: A
authors.initials: A
authors.lastname: King
authors.forename: D J
authors.initials: DJ
authors.lastname: Lun
authors.forename: A
authors.initials: A
authors.lastname: Decottignies
authors.forename: M
authors.initials: M
authors.lastname: Niimi
authors.forename: S
authors.initials: S
authors.lastname: Lin
authors.forename: R D
authors.initials: RD
authors.lastname: Cannon
authors.forename: A
authors.initials: A
authors.lastname: Goffeau
authors.forename: B C
authors.initials: BC
authors.lastname: Monk
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000935:Antifungal Agents / Q000494:pharmacology*; D002175:Candida / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004353:Drug Evaluation, Preclinical; D025141:Drug Resistance, Fungal; D004847:Epithelial Cells / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects; D015725:Fluconazole / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006801:Humans; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000494:pharmacology* / Q000633:toxicity; D019151:Peptide Library; D012235:Rhodamines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects*; D012685:Sepharose
chemical_list: D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D005456:Fluorescent Dyes; D009842:Oligopeptides; D019151:Peptide Library; D012235:Rhodamines; D015725:Fluconazole; D012685:Sepharose; D000251:Adenosine Triphosphatases