Inhibiting Drug Efflux Pumps Relevant to Fungal Infections and Cancer

The development of multidrug resistance (MDR) following upregulation of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters poses a significant problem in antimicrobial and anticancer drug therapy. If inhibitors of these transporters can be found, combination treatments may overcome efflux-mediated drug resistance, thus enhancing cell sensitivity to drug therapy. Objectives: To develop and optimise a reproducible fluorescence-based assay of pump activity which mimics high throughput screening conditions. Methods: We used the fluorescent dye Nile Red, an intracellular lipid-soluble pump substrate which is only fluorescent when cell-associated. This study was conducted using a previously validated expression system involving recombinant strains of the model non-pathogenic yeast S. cerevisiae, in which individual efflux pumps had been cloned. Yeast cells were pre-loaded with Nile Red before distribution into microtitre plate wells. Nile Red efflux from whole cells in the presence or absence of glucose was determined by measurement of loss of fluorescence using a Synergy 2 microplate reader (Biotek, USA) with excitation and emission wavelengths at 485 and 520 nm, respectively. Results: A cell density of 7x10⁷ cells/mL was selected for an optimal efflux response. Following addition of glucose, there was a clear dose response to glucose in all recombinant strains tested. However, efflux kinetics were complex and initiation of pump activity was faster at lower glucose concentrations (5-10 mM) whereas at 20-50 mM glucose, efflux lasted longer. Efflux kinetics were slower at 25 °C than at 30 °C. Effective inhibition of efflux from Mdr1p pumps by two novel peptide compounds was observed. Conclusions: We have optimised a fluorescence-based assay to measure the activity of fungal and human membrane efflux pumps. Using this optimized assay, further research can be undertaken to screen for clinically safe inhibitors. This summer studentship was supported by the Division of Health Sciences, USA NIH (R01DE016885) and NZFRST (U00X0607).