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

BACKGROUND AND PURPOSE: Because the in vivo effectiveness of ligands may also be determined by the rate by which they dissociate from their target receptors, drug candidates are being increasingly screened for this kinetic property. The dissociation rate of unlabelled ligand-receptor complexes can be estimated indirectly from their ability to slow the association of subsequently added radioligand molecules. EXPERIMENTAL APPROACH: We used the 'two-step competition' binding approach consisting of pre-incubating 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. 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 dissociation rate can be estimated from the upward shift that the competition curve experiences after washing. KEY RESULTS: The relevance of the 'two-step competition' approach was explored by computer simulations and by comparing the dissociation behaviour of unlabelled D(2) dopamine and CB(1) 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: As the 'two-step competition' requires rapid intermediate washing steps and needs radioligand binding to be measured at only one time point, this approach is particularly suited for binding studies on intact plated cells. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

Unravelling the complex dissociation of [(3)H]-rimonabant from plated CB(1) cannabinoid receptor-expressing cells.

The dissociation profile of the antagonist [(3)H]-rimonabant from recombinant CB(1) cannabinoid receptors expressed in plated HEK293 cells followed a complex pattern when measured in medium only. After a rapid decline, the specific binding levelled off at about 20% below the initial value. To unravel the responsible mechanism(s), we examined the relative contribution of binding to cells and walls of the culture wells respectively. Washout was also performed in the presence of an excess of unlabelled ligand and/or bovine serum albumin (BSA). The findings suggest that dissociated [(3)H]-rimonabant molecules not only undergo rebinding to the same or neighbouring receptors but also partition in the cell membranes and fix to the walls. As these non-receptor associations still occur in presence of unlabelled ligand, they can be erroneously regarded to represent 'specific binding'. While the unlabelled ligand was most effective in preventing receptor rebinding, BSA was most effective in preventing non-receptor associations. To measure receptor-dissociation only, washout is best performed in presence of unlabelled ligand and BSA or any other protein that can pick-up free radioligand molecules. Yet, washout in medium only could hint at mechanisms that affect the in vivo residence time of the drug in question.

Molecular mechanisms for the persistent bronchodilatory effect of the beta 2-adrenoceptor agonist salmeterol.

BACKGROUND: Beta(2)-adrenoceptor agonists are effective bronchodilators. In vitro studies demonstrated long-lasting airway smooth muscle relaxation by salmeterol after washout, the quick disappearance of this effect in presence of antagonists and its recovery after antagonist removal. Current explanations invoke salmeterol accumulation in the membrane ('diffusion microkinetic' model) or the existence of salmeterol-binding 'exosites'. An alternative model based on 'rebinding' of a dissociated ligand to the receptor molecules also produces an apparent decrease in the ligand's dissociation rate in the absence of competing ligands. PURPOSE AND APPROACH: Computer-assisted simulations were performed to follow the receptor-occupation by a salmeterol-like ligand and a competing ligand as a function of time. The aptness of the models to describe the above in vitro findings was evaluated. KEY RESULTS: The 'diffusion microkinetic' model is sufficient to explain a long-lasting beta(2)-adrenoceptor stimulation and reassertion as long as the membrane harbors a high concentration of the agonist. At lower concentration, 'rebinding' and, in second place, 'exosite' binding are likely to become operational. CONCLUSIONS AND IMPLICATIONS: The 'rebinding' and 'exosite' binding mechanisms take place at a sub-cellular/molecular scale. Pending their demonstration by experiments on appropriate, simple models such as intact cells or membranes thereof, these mechanisms remain hypothetical in the case of salmeterol. Airway smooth muscle contraction could also be governed by additional mechanisms that are particular to this macroscopic approach.