New high-throughput screening assay to reveal similarities and differences in inhibitory sensitivities of multidrug ATP-binding cassette transporters
Marcin Kolaczkowski , Anna Kołaczkowska , Noboru Motohashi , K. Michalak
AbstractCdr1p is the major ATP-blnding cassette multidrug transporter conferring resistance to azoles and other antifungals in Candida albicans. In this study, the identification of new Cdrlp 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 Cdrlp, 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 Cdrlp, Pdr5p, and Snq2p. All transporters showed similar sensitivities to M961 inhibition. In contrast, Cdrlp 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 CßRi-overexpressing C. albicans isolate.
|Journal series||Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, e-ISSN 1098-6596, (A 40 pkt)|
|Publication size in sheets||0.55|
|ASJC Classification||; ;|
|Publication indicators||= 20; = 18; : 2009 = 1.653; : 2009 = 4.802 (2) - 2009=4.819 (5)|
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