Styrylquinolines inhibit the ABC transporters of Candida albicans Joanna Szczepaniak1, Robert Musioł2, Wioleta Cieślik2, Anna Romanowicz1, Anna Krasowska1 1Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland 2Department of Organic Chemistry, University of Silesia, Katowice, Poland Corresponding author e-mail: joanna.szczepaniak@uwr.edu.pl ATP ADP + Pi H+ ABC MFS Cdr1p Cdr2p Mdr1p HO N Styrylquionoline molecule may interact with ergosterol in similar manner to amphotericin B, cytotoxic antifungal drug. OH Amphotericin B Styryloquinoline Overexpression of ABC pumps is one of the major resistance mechanisms of Candida albicans. Here we present styrylquinolines, a novel group of quinoline drugs we evaluated for the antifungal activity of these drugs more deeply, particularly their activity modulation towards Cdr1p, the main multidrug transporter of C. albicans. Styrylquinolines have antifungal properties. MIC values fo tested styrylquinolines [µg/ml]:   WT cdr1Δ cdr2Δ cdr1Δ cdr2Δ WK14B >122.11 34.74 WK15B 122.11 30.53 WK15 26.31 3.29 52.62 13.15 WK14 Rhodamine 6G is ABC transporters substrate. WK14B and WK14 show inhibition of R6G efflux. WK15 and WK15B have no effect on R6G export from cells. WK14B and WK15B are characterized by synergistic action with fluconazole against both wild type strain as well as cdr2Δ strain. No synergistic effect was observed in the case of the cdr1Δ strain. This presumably occurs because styrylquinolines have only a minor effect on Cdr2p, as the cdr1Δ strain MIC profile is comparable with that of the wild-type strain. Styrylquinolines block ABC transporters action. FIC index for tested styrylquinolines and fluconazole (≤0.5 synergistic action; 0.5 – 4.0 indifferent; ≥ 4.0 anatgonistic) Styrylquinolines block early biofilm formation. Both compounds with a hydroxyl group in the phenyl ring, WK14 and WK15, show the lowest MIC among the drugs tested. These same two compounds affected the early biofilm formation of C. albicans cells, phase that shows the highest levels of ABC transporters expression in biofilm formation. Substitution of an acetoxy group in the same positions (WK14B, WK15B) decreases drug activity, especially for cdr2Δ and wild-type strains. The cells with CDR1 deletion show a significant increase in sensitivity to styrylquinolines, suggesting that these compounds are Cdr1p substrates. This indicates a possible mode of action as competitive inhibitors of this transporter.   WT cdr1Δ cdr2Δ WK14B 0.26 1 0.03 WK15B 1.2 0.02 WK15 2 WK14 The Cdr1p-GFP protein level in crude extract is also higher in treated cells, same effect as observed with epifluorescent microscopy. Styrylquinolines (SQ) are novel antifungal drugs. SQs work against both C. albicans biofilm and planktonic cells. Tested compounds blocked rhodamine 6G efflux and work synergistically with fluconazole suggesting a possible mode of action as competitive inhibitors of this transporter. SQs induce production of Cdr1p and cause this protein delocalisation from cell membrane. The treatment of C. albicans cells with ABC transporters substrates increases their expression. Fluorescent microscopy shows a stronger signal from GFP-tagged Cdr1p in the case of cells incubated with styrylquinolines than the control or fluphenazine – known inductor of Cdr1p expression. A. WK15B, WK14, and WK15 show partial delocalization of the signal to the inside of the cell. B. At concentrations higher than the MIC concentration of WK14 and WK15, the signal comes from the whole cells. This study is supported by Polish National Center for Science NCN grant 2013/09/B/NZ7/00423 and by Wroclaw Centre of Biotechnology, programme: The Leading National Research Centre (KNOW) for years 2014-2018. Szczepaniak et al., 2016 in review