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

Mitchell-White, James I.; Briggs, Deborah A.; Mistry, Sarah J.; Mbiwan, Hannah A.; Kellam, Barrie; Holliday, Nicholas D.; Briddon, Stephen J.; Kerr, Ian D.

Abstract

The 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.

Citation

Mitchell-White, J. I., Briggs, D. A., Mistry, S. J., Mbiwan, H. A., Kellam, B., Holliday, N. D., …Kerr, I. D. A time-resolved Förster resonance energy transfer assay to investigate inhibitor binding to ABCG2

• Working Paper Type: Working Paper
• Deposit Date: Feb 3, 2024
• Publicly Available Date: Feb 6, 2024
• Keywords: ABC transporter; ABCG2; FRET; pharmacology; multidrug resistance
• Public URL: https://nottingham-repository.worktribe.com/output/26537536
• Publisher URL: https://www.biorxiv.org/content/10.1101/2023.10.27.564323v1