The Allosteric Ternary Complex Model Radioligand Binding And Affinity

In order to design and critique methods for screening for allosteric modulators, it is necessary to understand the macromolecular basis of the interaction of allosteric ligands with a GPCR. The simplest model of a three-way interaction between a GPCR, an orthosteric ligand, and an allosteric modulator is the allosteric ternary complex model (ATCM) proposed by Stockton et al. [13] , which is illustrated in Fig. 12.1. Both an orthosteric ligand (A) and an allosteric ligand (B) can interact with a receptor (R), forming either binary species (AR, BR) or a ternary complex (ARB). KA and KB denote the equilibrium dissociation constants of AR and BR, respectively. The symbol a denotes the coopera-tivity factor and is a quantitative measure of the maximal, reciprocal alteration of affinity of A and B for their respective binding sites when both ligands bind simultaneously to form the ternary complex, ARB.

In short, the value of a dictates whether the allosteric ligand (B) has a positive or negative effect on the binding of the orthosteric ligand (A) and vice versa. Values of a > 1 denote positive allosteric modulation of affinity, whereas values of a < 1 denote negative allosteric modulation. A value of a = 1 represents a special situation whereby the binding of the allosteric modulator does not alter the affinity of the orthosteric probe and vice versa.

Ke/a

KA/a

KA/a

Figure 12.1 Orthosteric (a) and allosteric ternary complex (b) models. Both schemes describe the interaction of two compounds, A and B, with a receptor, R. These ligands interact with R with equilibrium dissociation constants, KA and KB, respectively. Where both ligands compete for the orthosteric site, the binding of each ligand is mutually exclusive. However, where the second ligand, B, is an allosteric modulator, both ligands can interact with the receptor to form an allosteric ternary complex (ARB). The magnitude and direction of the allosteric effect on ligand binding affinity is quantified by a. Values of a > 1 denote positive cooperativity (positive modulation), whereas values of a < 1 (but >0) denote negative cooperativity (negative modulation). Neutral binding cooperativity at equilibrium occurs when a = 1.

The effect of allosteric modulators on the binding of a radioligand is shown in Fig. 12.2. A positive allosteric modulator produces a concentration-dependent, saturable increase in the affinity (Fig. 12.2a) of the radioligand, which is more readily visualized on a semilogarithmic plot (Fig. 12.2c). Conversely, a negative allosteric modulator produces a concentration-dependent,

120

g in

100

di

in bi

80

o

60

o

.

40

S?

20

0

(c)

120

g in

100

di

80

in bi

ic

60

ci

.

40

S?

20

0

(e)

g in

200

di

in

bi

150

ic

Radioligand (nM)

Radioligand (nM)

Radioligand (nM)

ing200 ndi bin150

rol 50

Radioligand (nM)

-10 -9 -8 Log [radioligand] (M)

Figure 12.2 (a) Effect of a range of concentrations of positive allosteric modulator (Kb = 1 |M; a = 3)on the saturation binding of a radioligand (KD = 1 nM) according to the ATCM; (c) transformed into a semilog plot. (b) and (d) show the same for a negative allosteric modulator (KB = 1 |M; a = 0.3).Titration curves for the positive (e) and negative (f) allosteric modulators on the equilibrium binding of a single concentration (1 x Kd) of the same radioligand.

saturable decrease in the affinity of the radioligand, similarly represented in Fig. 12.2b,d. It is possible to analyze such data sets according to the ATCM to generate estimates of the affinity of the allosteric modulator (KB) and the coop-erativity (a) between the modulator and the orthosteric radioligand probe.

However, to run what is in effect a Schild analysis using a radioligand binding assay is unusual and not particularly cost-effective in a drug discovery setting. It is much more common to use a single concentration of a radioligand and run a titration curve of a test compound (often referred to as "competition binding" as, historically, such assays were used to screen analogues of endogenous ligands). The effects of both positive and negative allosteric modulators in such an assay design are shown in Fig. 12.2e,f. Note that a positive modulator yields an enhancement of the level of a bound radioligand, whereas the negative modulator inhibits the binding of the radioligand. These curves can also be analyzed to quantify the affinity and cooperativity of the modulator. It is important to note that the negative modulator is unable to fully inhibit the specific binding of the radioligand. This effect (and the definition of the maximum asymptote for the positive allosteric modulator) is key to determining the degree of cooperativity between the modulator and the radioligand. If the negative modulator was to fully inhibit the specific binding of the radioli-gand, it would be impossible to distinguish such a compound from a simple competitive antagonist (as the degree of negative cooperativity, a ^ 0). If a compound is truly a negative allosteric modulator, this can be resolved by evaluating the test compound against multiple, higher concentrations of radio-ligand (e.g., 1 x KD, 10 x KD), but typically, this method is less suitable for higher throughput assessment of compounds.

However, there are a number of circumstances whereby an allosteric modulator may be missed using a radioligand binding protocol as described above. First, an allosteric modulator of neutral affinity cooperativity would neither inhibit nor potentiate the binding of a radioligand and would appear as an inactive compound. An example of such a compound is tetra-W84, which does not alter equilibrium [3H]-N-methyl scopolamine (NMS) binding at muscarinic M2 receptors [14]. However, tetra-W84 significantly retards the rate of both association and dissociation of the radioligand, revealing its allosteric mechanism of action (the effect is equal on both association and dissociation; hence, there is no net effect on equilibrium binding). Measuring effects of compounds on orthosteric radioligand dissociation is a very useful way of unmasking an allosteric mechanism at GPCRs as the only way that a compound can alter the rate of dissociation of a preformed receptor-ligand complex is via a topographically distinct site on the receptor [14]. This is partially amenable to a higher throughput screening assay using a two-point kinetic format [14] but is limited to merely confirming an allosteric mechanism rather than providing information on whether a compound is a positive, negative, or neutral modulator.

It is also possible that a compound that appears inactive in an equilibrium radioligand binding assay may possess effects that could be detected in functional assays (e.g., effects on signaling efficacy or direct agonism/inverse agonism—see Section 12.3), and indeed, this was one of the main drivers for the switch from radioligand binding to functional assay formats. For example, a number of allosteric modulators of mGlu receptors do not perturb equilibrium binding of orthosteric radiolabels, for example, CPCCOEt (vs. [3H]-glutamate binding at mGluR1; [15]) and CDPPB (vs. [3H]-quisqualic acid binding at mGluR5; [16]), yet are well-described allosteric modulators in functional assays. Their mechanism of action is discussed further in Section 12.3.

The second possibility concerns the choice of orthosteric radioligand used. For GPCRs where the endogenous ligand is of relatively low affinity or labile, for example, acetylcholine at muscarinic receptors, it is common for the radio-ligand of choice to be a high-affinity competitive antagonist. However, one of the hallmarks of allosteric interactions is that although the affinity of an allosteric modulator for a receptor is an intrinsic property of the molecule, its cooperativity is a property unique to the orthosteric/allosteric ligand pair. Invariably, drug discovery efforts aimed toward identifying allosteric modulators are seeking compounds that perturb the binding or function of the endogenous agonist for the receptor in question. In practice, the phenomenon of "probe-dependence" can result in misleading results if the orthosteric radiolabel is not the endogenous ligand—for example, staurosporine is a positive allosteric modulator of [3H]-NMS binding at the M1 muscarinic receptor but a negative modulator of acetylcholine binding [17] . Thus, wherever possible, the endogenous ligand for the receptor should be used when screening for allosteric modulators.

0 0

Post a comment