Radioligand Binding Assays

Equilibrium Binding Assays Detection of allosteric interactions using an equilibrium binding assay relies on the ability of a putative allosteric ligand to alter the binding affinity of a radiolabeled orthosteric ligand to either increase or decrease the specific binding of the orthosteric probe. The most common assay in this regard is to perform a modulator titration curve against a single fixed concentration of orthosteric radioligand. According to the simple ATCM, the maximum fractional binding (FB) of the orthosteric ligand is a function of the cooperativity of the interaction, and is given by [30]:

From this relationship, it can be seen that an allosteric ligand exhibiting very high negative cooperativity (a ^ 0) can completely inhibit the specific binding of the radioligand and therefore be misinterpreted as a competitive orthosteric ligand. If the negative cooperativity is not too high, then the interaction will deviate from the expectations of competition due to the fact that appreciable ternary complex (ARB, Fig. 3.1a) will still be detected at high concentrations of modulator, resulting in a nonzero degree of specific radioligand binding (Fig. 3.3). Thus, one means of differentiating allosteric antagonism from competitive antagonism is to utilize large concentrations (>KA) of orthosteric radioligand as a probe.

A second issue that needs to be considered in the conduct and interpretation of equilibrium binding assays is that an allosteric modulator with a weak

Ligand Binding Assay

centrations of an allosteric modulator (KB = 10-7M) under conditions of different degrees of positive or negative cooperativity (a values indicated in the figure) on the percent specific binding of an orthosteric radioligand, denoted A*. Note that for high degrees of negative cooperativity (boxed region, top panel), the interaction becomes difficult to distinguish from competitive antagonism. However, increasing the concentration of radioligand (bottom panel) can unmask the allosteric effect.

centrations of an allosteric modulator (KB = 10-7M) under conditions of different degrees of positive or negative cooperativity (a values indicated in the figure) on the percent specific binding of an orthosteric radioligand, denoted A*. Note that for high degrees of negative cooperativity (boxed region, top panel), the interaction becomes difficult to distinguish from competitive antagonism. However, increasing the concentration of radioligand (bottom panel) can unmask the allosteric effect.

cooperativity factor (a ~ 1) may not be detected due to a lack of sensitivity of the method in this instance; this is common for allosteric modulators that exert their effects predominantly on receptor signaling rather than binding. Utilizing another orthosteric ligand belonging to a separate chemical class may overcome this, assuming that the allosteric modulator exhibits a different degree of binding cooperativity with the new probe. In general, however, radioligand binding assays are most useful if the modulator behaves according to the simple ATCM with a reasonable degree of either positive or negative cooperativity. There are a few instances where high-affinity allosteric radioligands have been developed, for example, for M2 muscarinic receptors [31] or metabo-tropic glutamate receptors (mGluRs) [32] , but by and large, the equilibrium binding assay using a radioactive allosteric ligand to screen for other allosteric ligands that act at the same site is not yet a routine method of detection of allosteric modulators.

Dissociation/Association Kinetic Binding Assays An alternative application of radioligand binding assays to detect and quantify allosteric interactions involves the analysis of allosteric effects on the rates of dissociation or association of the orthosteric radioligand with the GPCR of interest. Kinetic binding assays can be used individually or in tandem with equilibrium binding assays; if an allosteric modulator has the ability to change the affinity of an orthosteric ligand, then this will be manifested either as a change in the association and/or dissociation rates of the orthosteric probe on a modulator-occupied receptor. In general, the measurement of association kinetics is problematic with respect to interpretation because competitive (orthosteric) ligands will also alter the apparent association rate of the radioligand simply by delaying the time taken for the radiolabeled orthosteric probe to reach equilibrium. In contrast, the only way that the dissociation rate of a pre-equilibrated radioligand-receptor complex can be modified is if the interacting ligand binds to another site on this complex to change the receptor conformation prior to the radioligand dissociating. Thus, radioligand dissociation kinetic assays represent a useful means of detecting and validating an allosteric mode of action. Furthermore, under certain conditions, these assays can also be used to quantify the allosteric effect in terms of the ATCM [33, 34]. An additional advantage of kinetic binding assays is that they have the potential to detect allosteric ligands that possess neutral binding cooperativity (a = 1) at equilibrium, if the mechanistic basis of the neutral cooperativity is due to the modulator altering orthosteric ligand association and dissociation rates to the same extent [ 34]. However, there are a number of situations where the utility of dissociation kinetic assays is limited. First, when the allosteric interaction is primarily manifested by an alteration in the association, rather than dissociation, rate of the orthosteric ligand. Second, under conditions of very high negative cooperativity, the affinity of the modulator for the radioligand-bound receptor may be sufficiently low such that, unless impractically high concentrations of modulator are utilized, there is no perturbation of dissociation kinetics. Third, a dissociation kinetic assay will fail to detect allosterism if the effect of the modulator is primarily on orthosteric ligand efficacy rather than affinity.

Another consequence of the effects of some allosteric modulators on orthosteric ligand binding kinetics is that the interaction between the modulator and radiolabeled orthosteric probe may not reach equilibrium over the time course of the assay, leading to complex behaviors that are not accommodated by the simple equilibrium ATCM [33, 35, 36].

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