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Antimicrobial Agents and Chemotherapy, April 2009, p. 1516-1527, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.00956-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

New High-Throughput Screening Assay To Reveal Similarities and Differences in Inhibitory Sensitivities of Multidrug ATP-Binding Cassette Transporters{triangledown}

Marcin Kolaczkowski,1* Anna Kolaczkowska,2 Noboru Motohashi,3 and Krystyna Michalak1

Department of Biophysics, Wroclaw Medical University, Ul. Chalubinskiego 10, 50-368 Wroclaw, Poland,1 Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Ul. Norwida 31, 50-375 Wroclaw, Poland,2 Meiji Pharmaceutical University, 2-522-1 Noshio, Tokyo 2048588, Japan3

Received 18 July 2008/ Returned for modification 23 October 2008/ Accepted 18 January 2009

Cdr1p is the major ATP-binding cassette multidrug transporter conferring resistance to azoles and other antifungals in Candida albicans. In this study, the identification of new Cdr1p inhibitors by use of a newly developed high-throughput fluorescence-based assay is reported. The assay also allowed monitoring of the activity and inhibition of the related transporters Pdr5p and Snq2p of Saccharomyces cerevisiae, which made it possible to compare its performance with those of previously established procedures. A high sensitivity, resulting from a wide dynamic range, was achieved upon high-level expression of the Cdr1p, Pdr5p, and Snq2p transporters in an 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 for Cdr1p, Pdr5p, and Snq2p. All transporters showed similar sensitivities to M961 inhibition. In contrast, Cdr1p was less sensitive to inhibition by fluphenazine, whereas phenothiazine selectively inhibited Snq2p. The inhibition potencies 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. They also correlated with the 50% inhibitory concentration 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.

* Corresponding author. Mailing address: Department of Biophysics, Wroclaw Medical University, Ul. Chalubinskiego 10, 50-368 Wroclaw, Poland. Phone: (48) 71 7841403. Fax: (48) 71 7840088. E-mail: mkolacz2{at}poczta.onet.pl

{triangledown} Published ahead of print on 2 February 2009.

Antimicrobial Agents and Chemotherapy, April 2009, p. 1516-1527, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.00956-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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