Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae

The Pdr5p multidrug ABC ("ATP-binding cassette") transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified PdrSp displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effecters. The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer. It also bound a series of known drug substrates and modulators. Interestingly, yeast PdrSp interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter. The extent of high-affinity binding of prenyl-flavonoids to purified PdrSp was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3,3-dimethylallyl)galangin was the most efficient, with a K-i of 0.18 mu M for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive, Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast PdrSp ABC transporter.