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

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 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.

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…In fact, one of the first applications of this method used a fluorescent peptide ligand to determine the binding kinetics of ligands to the formyl peptide receptor endogenously expressed on human neutrophils using flow cytometry (Sklar, Sayre, McNeil, & Finney, ). The Motulsky and Mahan method requires the kinetic ( k on and k off ) parameters of a labelled ligand (radiolabelled or fluorescent‐labelled) to be predetermined, and then by measuring the effect of an unlabelled ligand on these rates, the binding kinetics of the unlabelled ligand can be determined (Figure ; see Bosma et al, , for the Motulsky–Mahan equation used to simulate these data). Due to the ease of collecting larger datasets with fluorescence techniques, there have been several recent papers comparing the binding kinetics of unlabelled ligands measured using radioligand‐ and fluorescence‐based methods (Bosma et al, ; Bouzo‐Lorenzo et al, ; Nederpelt et al, ).…”

“…The Motulsky and Mahan method requires the kinetic ( k on and k off ) parameters of a labelled ligand (radiolabelled or fluorescent‐labelled) to be predetermined, and then by measuring the effect of an unlabelled ligand on these rates, the binding kinetics of the unlabelled ligand can be determined (Figure ; see Bosma et al, , for the Motulsky–Mahan equation used to simulate these data). Due to the ease of collecting larger datasets with fluorescence techniques, there have been several recent papers comparing the binding kinetics of unlabelled ligands measured using radioligand‐ and fluorescence‐based methods (Bosma et al, ; Bouzo‐Lorenzo et al, ; Nederpelt et al, ). Using the gonadotropin‐releasing hormone receptor, Nederpelt et al () compared the binding kinetics of a number of agonists measured in radioligand and TR‐FRET assays.…”

“…For the adenosine A 3 receptor, a comparison of radioligand and NanoBRET (BRET with the small bright luciferease Nanoluc as the donor) assays using labelled ligands with markedly different binding kinetics suggested that it is difficult to determine the kinetics of fast binding unlabelled ligands when using a probe with a very slow off rate (Bouzo‐Lorenzo et al, ). Using the histamine H 1 receptor as a model system, Bosma et al () compared the binding kinetics of a number of unlabelled antagonists using two radioligands and both NanoBRET and TR‐FRET fluorescence‐based assays. This large dataset allowed multiple comparisons to be made and found that for the H 1 receptor, the association rates ( k on ) compared well across all three assay set‐ups, and there was good correlation between the dissociation rates measured in the radioligand and TR‐FRET assays but not with the NanoBRET assays.…”

“…Simulated competition association binding curves with increasing k off values of the labelled ligand simulations were generated in GraphPad Prism using the Motulsky and Mahan equation. k on (both k 1 and k 3 ) was set to 1 × 10 6 M −1 ·min −1 for all simulated data (see Bosma et al, , for full equation on the Motulsky–Mahan model). For (a, d), k off (M −1 ) of the labelled ligand ( k 2 ) was set to 0.1, (b, e) to 0.01, and (c, f) to 0.001, simulating a 10‐fold increase in k off each time and a “fast,” “slow,” and “very slow” labelled ligand.…”

“…As the Motulsky and Mahan method is the most commonly used method to measure binding kinetics of unlabelled ligands, the above examples emphasise the importance of selecting a labelled probe with the appropriate kinetics for accurate determination of test compounds. It is also becoming clear that a probe with fast binding kinetics may be more generally applicable for measuring the kinetics of an unlabelled ligand (Bosma et al, ; Sykes, Jain, & Charlton, ). As radioligands are only subtly different from the parent pharmacophore, differences between radioligand probe binding kinetics are often less pronounced.…”