A two‐state model for the kinetics of competitive radioligand binding

Abstract

Background and PurposeLigand–receptor binding kinetics is receiving increasing attention in the drug research community. The Motulsky and Mahan model, a one‐state model, offers a method for measuring the binding kinetics of an unlabelled ligand, with the assumption that the labelled ligand has no preference while binding to distinct states or conformations of a drug target. As such, the one‐state model is not applicable if the radioligand displays biphasic binding kinetics to the receptor.Experimental ApproachWe extended the Motulsky and Mahan model to a two‐state model, in which the kinetics of the unlabelled competitor binding to different receptor states (R1 and R2) can be measured. With this extended model, we determined the binding kinetics of unlabelled N‐5′‐ethylcarboxamidoadenosine (NECA), a representative agonist for the adenosine A1 receptor. Subsequently, an application of the model was exemplified by measuring the binding kinetics of other A1 receptor ligands. In addition, limitations of the model were investigated as well.Key ResultsThe kinetic rate constants of unlabelled NECA were comparable with the results of kinetic radioligand binding assays in which [3H]‐NECA was used. The model was further validated by good correlation between simulated results and the experimental data.ConclusionThe two‐state model is sufficient to analyse the binding kinetics of an unlabelled ligand, when a radioligand shows biphasic association characteristics. We expect this two‐state model to have general applicability for other targets as well.

Aβ oligomers induce pathophysiological mGluR5 signaling in Alzheimer’s disease model mice in a sex-selective manner

“…Although it needs to be confirmed in future pharmacological studies, we speculate that A preferentially binds to the mGluR5 allosteric site based on the ability of mGluR5 NAM to reverse A-induced changes in neuronal cultures and the incomplete displacement of the radioactive ligand by A. However, the latter can also be attributed to the existence of two populations of receptors that exhibit different MPEP affinities, such that mGluR5 may exist in two conformational states, with A likely selectively binding to the high-affinity site, or that a less accessible MPEP binding site may exist (41).…”

Cancer‐related somatic mutations alter adenosine A 1 receptor pharmacology—A focus on mutations in the loops and C‐terminus

“…Supporting, these mutant receptors N148S EL2 and V152L EL2 showed lower potency and affinity of CPA compared to the wild‐type hA 1 AR. Moreover, mutant receptor A151V EL2 preferred a one‐site CPA binding model with decreased affinity, which showed that the equilibrium was shifted to one certain receptor conformation, most likely G protein uncoupled 43 . Interestingly, mutant receptors N148S EL2 , A151V EL2 , and V152L EL showed a significantly lower affinity of DPCPX.…”

Considerations for improved performance of competition association assays analysed with the Motulsky–Mahan's “kinetics of competitive binding” model

“…Experimentalists are encouraged to use the MC simulations presented here for detailed evaluation of their own procedures (adjustments may be necessary if they differ significantly from the kPCA assay set‐up, Schiele et al, ) and to address the remaining open questions, such as the influence of ligand and target depletion. Along these lines, further studies could include the expansion of these analyses to recent variants of the “kinetics of competitive binding” equation that incorporate alternative scheduling of the labelled and unlabelled ligand addition (Hoare, ; Shimizu, Ogawa, & Nakayama, ) or two‐state binding (Guo et al, ). All in all, this work should enable the optimised design and analysis of competition association assays.…”