A novel screening for inhibitors of a pleiotropic drug resistant pump, Pdr5, in Saccharomyces cerevisiae.
Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters. Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5. An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (delta syr1/erg3 null mutation). The sensitivity of the indicator strain (delta syr1/erg3 delta pdr5 delta snq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively. The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin. The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (approximately 0.5 microg/ml). In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506. These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.
Hiraga
K
K
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
Wanigasekera
A
A
Sugi
H
H
Hamanaka
N
N
Oda
K
K
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002108:Ceruletide / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D026141:Drug Resistance, Multiple, Fungal; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012235:Rhodamines / Q000493:pharmacokinetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D016559:Tacrolimus / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008565:Membrane Proteins; C092438:PDR5 protein, S cerevisiae; D012235:Rhodamines; D029701:Saccharomyces cerevisiae Proteins; C026188:rhodamine 6G; D016572:Cyclosporine; D002108:Ceruletide; D003513:Cycloheximide; D016559:Tacrolimus