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

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

doi:10.1038/s41598-019-44025-5

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.

Highlights

The pharmacodynamics of a drug are often related to the half-maximal modulation of target function (IC50, EC50), which typically depends on the concentration required to obtain half-maximal target binding (Ki, Kd)
To explore how the binding kinetics of a radioligand affects the Motulsky-Mahan analysis of radioligand association in competition with unlabeled G protein-coupled receptors (GPCRs) ligands, three different radioligands and two fluorescence-based probes for the H1 receptor (H1R) were investigated in this study
Equilibrium binding of increasing concentrations of [3H] mepyramine (Fig. 3a), [3H]levocetirizine (Fig. 3b) or [3H]olopatadine (Fig. 3c) to H1R-expressing HEK293T cell homogenates, revealed that all radioligands saturably bind to the H1R with high affinities, resulting in pKd values of 8.6 ± 0.1, 8.1 ± 0.1 and 8.7 ± 0.1, respectively (Table 1)

Summary

Introduction

The pharmacodynamics of a drug are often related to the half-maximal modulation of target function (IC50, EC50), which typically depends on the concentration required to obtain half-maximal target binding (Ki, Kd). Due to the wide range of radioactive and fluorescently labelled ligands available for H1R, we used this GPCR as a model system to investigate if the measured binding rate constants of unlabeled ligands are influenced by the binding kinetics of the employed labelled probe To this end, [3H]mepyramine and [3H]levocetirizine were used to characterize the binding kinetics of a set of unlabeled H1R ligands by the Motulsky-Mahan methodology. This was followed by the determination of the binding kinetics of H1R ligands via competitive association binding using two different non-radioactive H1R binding assays (BRET-based[29] or HTRF based[30] approaches). Both probe-dependent and assay-dependent factors are important contributors to the accurate determination of binding kinetics of unlabeled ligands

Methods

Results

Conclusion