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

• Published in: Scientific reports Vol. 9; no. 1; p. 7906
• Main Authors: 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., Fernández-Montalván, Amaury, Hill, Stephen J., Leurs, Rob
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 27-05-2019; Nature Publishing Group
• Subjects: 14/5; 631/154/436/2387; 631/1647/2196/2197; 631/45/612/194; 96/10; 96/33; Binding, Competitive; Cetirizine - chemistry; Cetirizine - pharmacokinetics; Datasets as Topic; Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes - chemistry; Fluorescent Dyes - pharmacokinetics; G protein-coupled receptors; Gold; HEK293 Cells; Histamine H1 Antagonists - chemistry; Histamine H1 Antagonists - pharmacokinetics; Histamine H1 receptors; Humanities and Social Sciences; Humans; Kinetics; Ligands; Molecular Probes - chemistry; Molecular Probes - pharmacokinetics; multidisciplinary; Olopatadine Hydrochloride - chemistry; Olopatadine Hydrochloride - pharmacokinetics; Protein Binding; Pyrilamine - chemistry; Pyrilamine - pharmacokinetics; Radioisotopes; Radioligand Assay - methods; Receptors, Histamine H1 - metabolism; Science; Science (multidisciplinary); Tritium
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-44025-5

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 H
1
receptor (H
1
R) antagonists were determined using radioligands with either slow (low k
off
) or fast (high k
off
) 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.