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

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

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
Volume9
Issue number1
DOIs
Publication statusPublished - 1 Dec 2019

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G-Protein-Coupled Receptors
Rate constants
Ligands
Kinetics
Kinetic parameters
Association reactions
Histamine H1 Antagonists
Kinetic theory
Pharmaceutical Preparations
Assays
Experiments

Cite this

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. / Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor. In: Scientific Reports. 2019 ; Vol. 9, No. 1.
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abstract = "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.",
author = "Reggie Bosma and Stoddart, {Leigh A.} and Victoria Georgi and Monica Bouzo-Lorenzo and Nick Bushby and Loretta Inkoom and Waring, {Michael J.} and Briddon, {Stephen J.} and Vischer, {Henry F.} and Sheppard, {Robert J.} and Amaury Fern{\'a}ndez-Montalv{\'a}n and Hill, {Stephen J.} and Rob Leurs",
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Bosma, R, Stoddart, LA, Georgi, V, Bouzo-Lorenzo, M, Bushby, N, Inkoom, L, Waring, MJ, Briddon, SJ, Vischer, HF, Sheppard, RJ, Fernández-Montalván, A, Hill, SJ & Leurs, R 2019, 'Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor' Scientific Reports, vol. 9, no. 1, 7906. https://doi.org/10.1038/s41598-019-44025-5

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.; Fernández-Montalván, Amaury; Hill, Stephen J.; Leurs, Rob.

In: Scientific Reports, Vol. 9, No. 1, 7906, 01.12.2019.

Research output: Contribution to JournalArticleAcademicpeer-review

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AU - Bosma, Reggie

AU - Stoddart, Leigh A.

AU - Georgi, Victoria

AU - Bouzo-Lorenzo, Monica

AU - Bushby, Nick

AU - Inkoom, Loretta

AU - Waring, Michael J.

AU - Briddon, Stephen J.

AU - Vischer, Henry F.

AU - Sheppard, Robert J.

AU - Fernández-Montalván, Amaury

AU - Hill, Stephen J.

AU - Leurs, Rob

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N2 - 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.

AB - 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.

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