A time-resolved Förster resonance energy transfer assay to investigate inhibitor binding to ABCG2

Abstract

AbstractThe human ATP-binding cassette (ABC) transporter, ABCG2 is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Förster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps.HighlightsABCG2 is a multidrug pump which moves between states having low or high affinity for substrates and inhibitorsWe introduce a time-resolved Förster resonance energy transfer assay to measure interaction of substrates and inhibitors to ABCG2We confirm that NBD dimerization is associated with a switch from a high to a low affinity site for an ABCG2 inhibitor