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

Reggie Bosma, Leigh A. Stoddart, Victoria Georgi, Monica Bouzo-Lorenzo, Nick Bushby, Loretta Inkoom, Michael J. Waring, Stephen J. Briddon, Henry F. Vischer, Robert J. Sheppard, Amaury Fernández-Montalván, Stephen J. Hill, Rob Leurs*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


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.

Original languageEnglish
Article number7906
JournalScientific Reports
Issue number1
Publication statusPublished - 1 Dec 2019


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