Phenothiazine derivatives as inhibitors of ABC transporters in Candida albicans

Abstract

The increasing resistance of Candida albicans to antimicrobials is a growing challenge. The most pronounced drug elimination mechanism in C. albicans cells are active multi-drug resistance (MDR) pumps CaCdr1p and CaCdr2p, belonging to the ABC (ATP-binding cassette) family of membrane transporters. The xenobiotics are removed from cell using energy generated by hydrolysis of ATP. Scientists have to discover new compounds that would effectively inhibit these efflux pumps. It is important that such compounds should be effective against pumps in fungi, but do not damage cells of the host organism. The fluorescent dye rhodamine 6G (R6G), as a substrate for ABC transporters, was used to assay the performance of MDR pumps in C. albicans. Four strains were used in our experiments: wild type C. albicans ATCC 10231 and three isolates - 2939 (fluconazole sensitive), 3212 (fluconazole resistant) and IZ (floconazole resistant). Inhibition of C. albicans MDR pumps by phenothiazine derivatives with three different alkyl chain substituents was monitored in this study. For experiments the cells were preloaded with R6G by glucose starvation and diluted into 96-well microplates. R6G was removed from the cells with active transporters like other xenobiotics. R6G efflux was initiated adding 2 % glucose to the incubation medium. The efflux was monitored following the intensity of fluorescence. R6G efflux kinetics was different when various concentrations of glucose and inhibitors were added. Phenothiazine acted differently than its derivatives. Phenothiazine did not inhibit MDR pumps and acted as cells. Meanwhile its derivatives methyl-phenothiazine, ethyl-phenothiazine and hexyl-phenothiazine - acted as efflux inhibitors and after glucose addition the fluorescence increased only slightly