New High-Throughput Screening Assay to Reveal Similarities and Differences in Inhibitory Sensitivities of Multidrug ATP-binding Cassette Transporters

Antimicrob Agents Chemother. 2009 Apr;53(4):1516-27. doi: 10.1128/AAC.00956-08. Epub 2009 Feb 2.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / antagonists & inhibitors*
  • ATP-Binding Cassette Transporters / physiology
  • Antifungal Agents / pharmacology*
  • Fluphenazine / pharmacology
  • Fungal Proteins / antagonists & inhibitors*
  • Ketoconazole / pharmacology
  • Membrane Transport Proteins
  • Rhodamines / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / antagonists & inhibitors*
  • Saccharomyces cerevisiae Proteins / physiology

Substances

  • ATP-Binding Cassette Transporters
  • Antifungal Agents
  • CDR1 protein, Candida albicans
  • Fungal Proteins
  • Membrane Transport Proteins
  • PDR5 protein, S cerevisiae
  • Rhodamines
  • SNQ2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • rhodamine 6G
  • Ketoconazole
  • Fluphenazine