Mapping the interaction of the Candida albicans multidrug resistance ABC protein Cdr1 with pump inhibitor clorgyline that stimulates ATPase activity 

M. Niimi1,2, K. Niimi2, S. Saiwan1, S. Assavachin1, A. Chindamporn1, R.D. Cannon2 and E. Lamping2

1Mycology Unit, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand and 2Sir John Walsh Research Institute, University of Otago, Faculty of Dentistry, Dunedin, New Zealand 

Objectives: The over-expression of ATP-binding cassette (ABC) transporters is a major cause of clinical fungal resistance to azole antifungals. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. The monoamine oxidase A inhibitor clorgyline has been shown to be a broad-spectrum inhibitor of fungal multidrug efflux pumps. The objective of this study was to investigate the interaction of clorgyline with fungal ABC transporters. Methods: Naturally acquired suppressor mutants of a Saccharomyces cerevisiae strain over-expressing Candida albicans ABC transporter Cdr1 that were resistant to clorgyline inhibition of rhodamine 6G efflux were isolated on yeast extract, peptone, dextrose (YPD) agar plates containing clorgyline and sub-minimum growth inhibitory concentration (MIC) of rhodamine 6G. Functional analyses, and molecular modeling, of Cdr1 from wild type and suppressor mutants were performed. The analyses comprised determination of mutation sites in Cdr1 suppressor mutants, measurement of the MICs of Cdr1 substrates, and whole cell rhodamine 6G efflux and in vitro pump ATPase assays. Results: Unexpectedly, we found that clorgyline stimulated the ATPase activities of fungal pleiotropic drug resistant (PDR) transporters, including S. cerevisiae Pdr5, Candida albicans Cdr1 and Cdr2, and Candida glabrata Cdr1 and Cdr2, 4- to 5-fold. All clorgyline-resistant mutants over-expressing C. albicans Cdr1 had acquired point mutations in Cdr1 that were of highly conserved amino acids. The point mutations were F556C and F556L, both in the second transmembrane segment (TMS2), F608I and V616F in TMS3, a double mutant T671P/G915C in TMS5 and the second nucleotide binding domain (NBD2) and Q1244E in TMS8. The mutants had altered MICs for several Cdr1 substrates, and their R6G efflux and Cdr1 ATPase activities responded poorly to clorgyline when compared with wildtype Cdr1. Conclusion: Fungal PDR transporters are known to have high, but non-inducible, ATPase activities. This is why our discovery of clorgyline as a strong stimulator of the ATPase activity of fungal PDR transporters was unexpected. Our results suggest that clorgyline interacts directly with Cdr1 and inhibits drug efflux by changing the kinetics of the transporter. Our surprising discovery opens a promising new avenue for drug discovery and the fight against fungal drug resistance.

6 / Mapping the interaction of the Candida albicans multidrug resistance ABC protein Cdr1 with pump inhibitor clorgyline that stimulates ATPase activity