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. 2019 May 27;9(1):7906.
doi: 10.1038/s41598-019-44025-5.

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

Reggie Bosma  1 , Leigh A Stoddart  2   3 , Victoria Georgi  4 , Monica Bouzo-Lorenzo  2   3 , Nick Bushby  5 , Loretta Inkoom  1 , Michael J Waring  6   7 , Stephen J Briddon  2   3 , Henry F Vischer  1 , Robert J Sheppard  8 , Amaury Fernández-Montalván  4   9 , Stephen J Hill  2   3 , Rob Leurs  10
Affiliations

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

Reggie Bosma et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Synthesis of [3H]olopatadine. Key: (a) 4-toluenesulfonic acid, EtOH, reflux, 2 h, 89%; (b) (i), 1-chloroethyl chloroformate, DCE, reflux, 4 h; (ii) MeOH, reflux, 2 h, 10% over two steps; (c) [3H]methyl nosylate, DMF, 50 °C, 1 h; (d) NaOH, EtOH/H2O, r.t., 2 h.
Figure 2
Figure 2
Structures of the used probes. T indicates a tritium atom.
Figure 3
Figure 3
Binding of [3H]mepyramine, [3H]levocetirizine and [3H]olopatadine to the H1R. Increasing concentrations of [3H]mepyramine (a), [3H]levocetirizine (b) or [3H]olopatadine (c) were incubated with H1R-expressing cell homogenates for 4 h at 25 °C. Indicated concentrations of [3H]mepyramine (d), [3H]levocetirizine (e) or [3H]olopatadine (f) were incubated with cell homogenate for several incubation times at 25 °C. Representative graphs are shown of ≥3 experiments and the depicted data points represent the mean ± SEM of triplicate values (a–c) or depict individual measurements with duplicate values per time point (d–f). Extracted binding constants and statistical information are shown in Table 1 and Supplementary Table 2.
Figure 4
Figure 4
Association binding of [3H]mepyramine and [3H]levocetirizine in the presence of competing unlabeled ligands at the H1R at 25 °C. The kinetic binding of [3H]mepyramine to H1R-expressing cell homogenates was measured with various concentrations of either mepyramine (a) doxepin (b) or levocetirizine (c). Similarly, the kinetic binding of [3H]levocetirizine to H1R-expressing cell homogenates was measured in competition with various concentrations of either mepyramine (d) doxepin (e) or levocetirizine (f). Representative graphs are shown of ≥3 experiments and each condition was measured in duplicate. Extracted binding constants and statistical information are shown in Table 2 and Supplementary Table 2.
Figure 5
Figure 5
Comparison of the binding properties of unlabeled ligands as measured by using two different probes at 25 °C. Binding rate constants of unlabeled ligands were determined in radioligand binding studies. A correlation plot is depicted for the logkon (a) and logkoff (b) as determined from competitive association experiments using either [3H]mepyramine (x-axis) or [3H]levocetirizine (y-axis) as competitive probe. (c) The correlation plot between the affinity calculated from the kinetic binding rate constants (pKd,kin) and the affinity from competition binding experiments (pKi) is depicted. Errors represent SEM values. Dashed lines represent a perfect correlation respective to the X-axis values and solid lines represent the linear regression lines.
Figure 6
Figure 6
Accuracy of the measured binding rate constants depend on the fitted mean koff of unlabeled ligands at the H1R at 25 °C. The accuracy in which the Motulsky-Mahan model fitted the kon (a) and koff (b) by non-linear regression was examined for the different experimental conditions that were employed in this study. To compare the accuracy of the fitted mean kon and koff values over a broad range, the relative magnitude of the error (SE), as derived from non-linear regression, was calculated for each individual replicate experiment and pooled for all ligands. The relative error was calculated by normalizing the SE by the mean (relative error = SE/mean). Subsequently, the relative error for the kon and koff were plotted against the corresponding mean koff determined from the same competitive association curve. Data points derived from competitive association experiments that employed [3H]levocetirizine are depicted in red. The arrows depict the koff of the used probes with [3H]levocetirizine in red and [3H]mepyramine in blue as reported in Table 1. Dashed lines represent a relative error of 1 (mean = SE).
Figure 7
Figure 7
Association binding of AV082 and Gmep in the presence of competing unlabeled ligands at the H1R at 25 °C. The kinetic binding of AV082 to the H1R, stably expressed on adherent cells, was measured in the presence of various concentrations mepyramine (a) doxepin (b) or levocetirizine (c). AV082 binding was measured continuously by NanoBRET for 60 min. The kinetic binding of Gmep to the H1R, stably expressed on freshly thawed cells in suspension, was measured in the presence of various concentrations mepyramine (d) doxepin (e) or levocetirizine (f). Gmep binding was measured continuously by HTRF for 180 min. Representative graphs are shown of ≥3 experiments (a–f).
Figure 8
Figure 8
Comparison of the binding properties of unlabeled ligands as measured by using orthogonal assays at 25 °C. The binding rate constants of unlabeled ligands that were determined using the fluorescent probes AV082 and Gmep were compared with the binding constants obtained with [3H]mepyramine. A comparison of the pKi values (a) (from equilibrium competition binding experiments) is depicted as well as the comparison of kon (b) and koff (c) values (from kinetic competition association experiments). Dashes lines represent a perfect correlation respective to the X-axis values and solid lines represent the linear regression lines.
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