Estimation of the dissociation rate of unlabelled ligand-receptor complexes by a “two-step” competition binding approach

Ann Packeu, Marie Wennerberg, A. Balendran, Georges Vauquelin

Onderzoeksoutput: Articlepeer review

30 Citaten (Scopus)

Samenvatting

Background: Owing to the increasing awareness that the in vivo effectiveness of ligands may also be dictated by the rate by which they dissociate form their target receptors, drug candidates are getting more and more screened for this kinetic property.
Purpose and approach: The dissociation rate of unlabelled ligand-receptor complexes can be estimated indirectly from their ability to slow-down the association of subsequently added radioligand molecules. The presently explored "two-step competition" binding approach consists of preincubating the receptor-preparation with a wide range of ligand concentrations, washing off free ligand molecules, adding radioligand and monitoring its receptor binding after a fixed time.
Key results: Based on the rationale that binding of both ligands is mutually exclusive and that they bind according to the law of mass-action to a single class of sites, the unlabelled ligand's disociation rate can be estimated from the upward shift that the competition curve experiences after washing. The relevance of the "two-step competition" approach is explored by simulations and by comparing the dissociation behaviour of unlabelled D2 dopamine- and CB1 cannabinoid receptor antagonists in this and alternative approaches. Besides providing satisfactory estimations of dissociation rates, the method also detects the ability of the unlabelled ligand molecules to be released from "sinks" such as the cell membrane.
Conclusions and implications: Due to the requirement of rapid intermediate wash steps and the ability to perform radioligand binding at only one time point, this approach is particularly suited for binding studies on intact plated cells.
Originele taal-2English
Pagina's (van-tot)1311-1328
Aantal pagina's18
TijdschriftBritish Journal of Pharmacology
Volume161
Nummer van het tijdschrift6
StatusPublished - nov 2010

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