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

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.Candida yeasts are the fourth most common pathogens responsible for systemic bloodstream infections. Candida albicans is the most frequently isolated species, contributing to Ͼ50% of cases (41). It generally shows little permeability to a large variety of toxic compounds, which is believed to result to a large degree from the existence of an active permeability barrier (42). Cdr1p and Cdr2p are two homologous ATPbinding cassette (ABC) multidrug resistance (MDR) transporters of broad specificity that confer resistance to the most widely used azole antifungals as well as to terbinafine, amorolfine, and many other metabolic inhibitors (43,48). CDR1 is constitutively expressed in azole-sensitive isolates, where it modifies the intrinsic level of susceptibility to antifungals, as its inactivation by deletion increases sensitivity (47). The effectiveness of the small number of antifungals available for the treatment of life-threatening systemic mycoses is further reduced by overexpression of both CDR1 and CDR2 in many azole-resistant clinical isolates (46, 59).Cdr1p and Cdr2p are structural and functional homologues of the Pdr5p and Snq2p MDR transporters of the model yeast Saccharomyces cerevisiae (25,29,50). A large-scale screening of Pdr5p substrate specificity identified it as the most important MDR ABC transporter, conferring resistance to most classes of currently available antifungals and other xenobiotics, with some overlap in sp...

Microbial Efflux Pump Inhibition: Tactics and Strategies

“…(ii) Phenothiazines potentiate ketoconazole in strains overexpressing Pdr5p, Snq2p and Yor1p while also exhibiting intrinsic antimicrobial activity [138]. iii) Propafenones potentiate azoles and terbinafine in strains lacking Cdr1p and Cdr2p [139].…”

The Quorum-Sensing Molecule Farnesol Is a Modulator of Drug Efflux Mediated by ABC Multidrug Transporters and Synergizes with Drugs in Candida albicans

“…C. albicans cells at a concentration of 10 3 CFU/ml were inoculated in RPMI 1640 medium containing either FAR or antioxidant ascorbic acid (AA) alone or a combination of both FAR and AA as detailed previously (26). At predetermined time points (0, 4,8,12,16,20, and 24 h) (at 30°C incubation; agitation, 200 rpm), a 100-l aliquot was removed, serially diluted (10-fold) in 0.9% saline, and plated on YEPD agar plates. Colony counts were determined after incubation at 30°C for 48 h (26).…”

The Monoamine Oxidase A Inhibitor Clorgyline Is a Broad-Spectrum Inhibitor of Fungal ABC and MFS Transporter Efflux Pump Activities Which Reverses the Azole Resistance of Candida albicans and Candida glabrata Clinical Isolates

“…As there are several fluorescent substrates of efflux pumps, fluorescence-based assays of activity are applicable to measuring the activity of ABC transporters (16,21,44). A dif-ferential signal can be obtained between cells which are actively effluxing a previously loaded fluorescent substrate (low signal at endpoint) and cells for which efflux is inhibited by a test compound (high signal at endpoint).…”