Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor

Bosma, Reggie; Stoddart, Leigh A.; Georgi, Victoria; Bouzo-Lorenzo, Monica; Bushby, Nick; Inkoom, Loretta; Waring, Michael J.; Briddon, Stephen J.; Vischer, Henry F.; Sheppard, Robert J.; Fern�ndez-Montalv�n, Amaury; Hill, Stephen J.; Leurs, Rob

Abstract

© 2019, The Author(s). Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H1 receptor (H1R) antagonists were determined using radioligands with either slow (low koff) or fast (high koff) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.

Citation

Bosma, R., Stoddart, L. A., Georgi, V., Bouzo-Lorenzo, M., Bushby, N., Inkoom, L., …Leurs, R. (2019). Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor. Scientific Reports, 9, Article 7906. https://doi.org/10.1038/s41598-019-44025-5

• Journal Article Type: Article
• Acceptance Date: Apr 25, 2019
• Online Publication Date: May 27, 2019
• Publication Date: Dec 1, 2019
• Deposit Date: Jan 13, 2020
• Publicly Available Date: Jan 13, 2020
• Journal: Scientific Reports
• Print ISSN: 2045-2322
• Electronic ISSN: 2045-2322
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 9
• Article Number: 7906
• DOI: https://doi.org/10.1038/s41598-019-44025-5
• Public URL: https://nottingham-repository.worktribe.com/output/3110142
• Publisher URL: https://www.nature.com/articles/s41598-019-44025-5
• Additional Information: Received: 30 January 2019; Accepted: 25 April 2019; First Online: 27 May 2019; : The authors declare no competing interests.