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 pumpmediated 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.

The roles of CDR1, CDR2, and MDR1 in kaempferol-induced suppression with fluconazole-resistant Candida albicans

“…Interestingly, KAE in combination with FLC could synergistically inhibit all the strains after 48 h of incubation in the T-K test (Figure 1) including C. albicans z2003, indicating that it was useful for T-K test to further assess the interpretations acquired in a checkerboard assay when FICI was a little higher than 0.5. The upregulations of multidrug efflux pump controlled by Cdr1p, Cdr2p belonging to ATP-binding cassette superfamily (APC transporter) and Mdr1p, a member of major facilitator superfamily (MFS) were implicated in most fluconazole-resistant C. albicans strains (Niimi et al 2004;Holmes et al 2012;Prasad and Rawal 2014) as FLC was a substrate for CDR1, CDR2, and MDR1 (Kohli et al 2001;Nakamura et al 2001). Rhodamine 6G was the most common used fluorescent dye of ABC pump substrate requiring ATP as energy to observe transporter-mediated FLC resistance (Lamping et al 2007;Peralta et al 2012).…”

The Influence of Biodegradable Gemini Surfactants, N,N’-bis(1-Decyloxy-1-Oxopronan-2-yl)-N,N,N’,N’ Tetramethylpropane-1,3-Diammonium Dibromide and N,N’-bis(1-Dodecyloxy-1-Oxopronan-2-yl) N,N,N’,N’-Tetramethylethane-1,2-Diammonium Dibromide, on Fungal Biofilm and Adhesion

“…There are few classes of drugs used for treatment of fungal infections 33 , however pathogenic mi- croorganisms have developed several mechanisms of resistance to overcome this barrier. An example is being active efflux by ABC and MFS membrane transporters or alteration in plasma membrane lipid composition 34,35 . A combination of gemini surfactants and common fungicides could contribute to overcoming fungal infections at much lower concentrations.…”

Resistance reversal induced by a combination of fluconazole and tacrolimus (FK506) in Candida glabrata

“…The intracellular concentration of rh6G can be used to investigate the drug efflux mechanism in azole-resistant C. glabrata (Moon et al, 2009;Holmes et al, 2012). Rh6G efflux was investigated using an Epics XL-3 flow cytometer (Beckman Coulter) at 525 nm with the exponentialphase C. glabrata cells (4.5610 6 c.f.u.…”

“…These processes lead to the increased production of drug efflux pumps and ergosterol. Efforts have been made to reverse the FLC resistance in C. glabrata by blocking the processes as described above (Holmes et al, 2012). Further investigation is required to determine whether FK506 could enhance the susceptibility of resistant C. glabrata to FLC by inhibiting these processes.…”