A time-resolved Förster resonance energy transfer assay to investigate drug and 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.

• Journal Article Type: Article
• Acceptance Date: Jan 17, 2024
• Online Publication Date: Feb 6, 2024
• Publication Date: 2024-03
• Deposit Date: Feb 3, 2024
• Publicly Available Date: Feb 6, 2024
• Journal: Archives of Biochemistry and Biophysics
• Print ISSN: 0003-9861
• Electronic ISSN: 1096-0384
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 753
• Article Number: 109915
• DOI: https://doi.org/10.1016/j.abb.2024.109915
• Keywords: ABC1 transporter; ABCG2; FRET; Pharmacology; Multidrug resistance
• Public URL: https://nottingham-repository.worktribe.com/output/30664847
• Additional Information: This article is maintained by: Elsevier; Article Title: A time-resolved Förster resonance energy transfer assay to investigate drug and inhibitor binding to ABCG2; Journal Title: Archives of Biochemistry and Biophysics; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.abb.2024.109915; Content Type: article; Copyright: © 2024 The Author(s). Published by Elsevier Inc.