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

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.

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.

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.

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.

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.