Estimation of the affinities of allosteric ligands using radioligand binding and pharmacological null methods

The theoretical basis for using radioligand binding and pharmacological techniques to estimate the dissociation constants of drugs which interact allosterically with receptors is described. This theory predicts that an allosteric ligand changes the affinity of another ligand which binds at the primary recognition site on the receptor complex without affecting the binding capacity of the primary ligand. The magnitude of this effect depends on the amount of cooperativity (positive or negative) between the binding of ligands at the allosteric and primary recognition sites. It is possible to estimate the dissociation constant of an allosteric ligand by measuring its effect on the binding of a radioligand at a fixed concentration. In this situation, the dissociation constant of the allosteric ligand can be calculated from the concentration of ligand which causes half of its maximal effect on radioligand binding. The effects of an allosteric ligand on the pharmacological responses to an agonist can be attributed to changes in affinity and intrinsic efficacy of the agonist-receptor complex. If the agonist used in the pharmacological experiments has a large receptor reserve, or if the allosteric ligand only influences the affinity of the agonist, then the dissociation constant of the allosteric ligand can be calculated from the results of experiments in which the concentration of agonist required for half-maximal response (EC50) is measured in the presence of various concentrations of the allosteric ligand. If the agonist used in the pharmacological experiments is a partial agonist with little receptor reserve, then the shift in the EC50 of the agonist caused by the allosteric ligand may be dependent on the affinity of the agonist.