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

Abstract

Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.

Fig 1

Effect of clorgyline on R6G efflux from S. cerevisiae AD/CaCDR1 and control AD/pABC3 yeast cells. The efflux of R6G from either AD/CaCDR1 (filled circles) or AD/pABC3 (open circles) cells in the presence of 0.4% glucose was measured 6 min following glucose addition in the presence of clorgyline at the concentrations indicated, as described in Materials and Methods. Glucose-dependent efflux is expressed relative to the amount of R6G effluxed from controls with no added clorgyline (100% values). Each data point represents the mean of quadruplicate determinations in a representative experiment repeated once. Variation from the mean did not exceed 15%.

Fig 2

Effect of clorgyline on the growth of S. cerevisiae strains expressing individual fungal efflux pumps in the absence or presence of FLC. Growth of the control S. cerevisiae strain AD/pABC3 or recombinant strains expressing either C. albicans Cdr1p, Cdr2p, or Mdr1p, C. glabrata Cdr1p, or C. krusei Abcb1p were analyzed by agarose diffusion chemosensitization assays as described in Materials and Methods, using CSM medium (pH 7.0) containing either no added FLC (top rows) or FLC (bottom rows) at the relevant concentrations (0.25 × MIC values). Paper disks containing clorgyline at the concentrations indicated were placed on the surface of agarose plates seeded with yeast. Plates were incubated at 30°C for 24 h.

Fig 3

Representative checkerboard susceptibility assays showing chemosensitization to FLC by clorgyline of S. cerevisiae strains AD/CaCDR1 and AD/CaMDR1 and C. albicans azole-resistant clinical isolate MML611. Yeast cells were grown in CSM medium pH 7.0 containing the indicated concentrations of FLC and clorgyline at 30°C for 24 h. Growth was measured spectrophotometrically as described in Materials and Methods, and values are given as a percentage of growth in control wells without added compounds.

Fig 4

Effect of clorgyline on the growth of C. albicans or C. glabrata clinical isolates with decreased FLC susceptibilities in the absence or presence of FLC. (A) Inhibition of C. albicans MML605, MML609, or MML611 or C. glabrata MML99 growth in the absence (top row) or presence (bottom row) of FLC at the relevant concentrations (0.25 × MIC values) as demonstrated by agarose diffusion chemosensitization assays using CSM medium (pH 7.0). Clorgyline (100 μg) was added to each disk. FLC MIC values are reported as μg ml⁻¹. (B) Growth of C. albicans isolates MML604 and MML610 in the absence or presence of FLC, without or with added clorgyline, at the indicated concentrations (0.25 × MIC values for FLC; 15 μg ml⁻¹ for clorgyline) as demonstrated by agarose dilution assays.