| Title | A new high-throughput screening assay to reveal similarities and differences in inhibitory sensitivity of multidrug ATP-binding cassette transporters. | | Author(s) | Kolaczkowski M, Kolaczkowska A, Motohashi N, Michalak K | | Institution | Department of Biophysics, Wroclaw Medical University, ul. Chalubinskiego 10, 50-368 Wroclaw, Poland; Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, ul. Norwida 31, 50-375 Wroclaw, Poland; Meiji Pharmaceutical University, 2-522-1 Noshio, Tokyo 2048588, Japan. | | Source | Antimicrob Agents Chemother 2009 Feb 2. | | Abstract | Cdr1p is the major ATP-binding cassette (ABC) multidrug transporter conferring resistance to azoles and other antifungals in Candida albicans. In this study the identification of new Cdr1p inhibitors using a newly developed high-throughput, fluorescence-based assay is reported. The assay also allowed monitoring the activity and inhibition of the related transporters Pdr5p and Snq2p of Saccharomyces cerevisiae, which made possible to compare its performance with previously established procedures. The high sensitivity, resulting from a wide dynamic range, was achieved upon high-level expression of the Cdr1p, Pdr5p, and Snq2p transporters in the S. cerevisiae strain in which the endogenous interfering activities were further reduced by genetic manipulation. An analysis of a set of therapeutically used and newly synthesized phenothiazine derivatives revealed different pharmacological profiles of Cdr1p, Pdr5p, and Snq2p. All transporters showed similar sensitivity to M961 inhibition. In contrast Cdr1p was less sensitive to inhibition by fluphenazine, whereas phenothiazine selectively inhibited Snq2p. The inhibition potency measured by the new assay reflected the ability of the compounds to potentiate the antifungal effect of ketoconazole (KTC), which was detoxified by the overproduced transporters. It also correlated with the IC50 values for inhibition of Pdr5p-mediated transport of rhodamine 6G in isolated plasma membranes. The most active derivative, M961, potentiated the activity of (KTC) against an azole-resistant CDR1-overexpressing C. albicans isolate. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19188399 |
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