Functional Assays

Although radioligand binding assays provide a direct means for detecting and quantifying allosteric interactions at GPCRs, they are limited in that they can only measure allosteric changes in radioligand affinity. In order to fully define the pharmacology of an allosteric ligand, it is necessary to consider its effects in a functional context, which can reveal allosteric effects on signaling efficacy as well as whether or not the modulator possesses agonism (either positive or inverse) in its own right. Furthermore, it is more frequently possible to use the endogenous ligand in a functional assay, relative to a binding assay, to directly probe the influence of the allosteric ligand on the natural agonist response.

As with the radioligand assay, the simplest allosteric effect in the context of a functional assay occurs when only the affinity of an orthosteric ligand for its receptor changes. In this scenario, the orthosteric agonist concentration-response curve is shifted to the left by an allosteric enhancer while an allosteric inhibitor will shift the curve to the right; no effects are expected on either the maximum or basal system responses. According to the ATCM, the translocation of concentration-response curves in this situation will approach a limit as determined by the cooperativity, a . with the orthosteric agonist, beyond which increasing concentrations of modulator will be unable to further shift the agonist curve. In this respect, the ATCM can be readily applied to functional interaction data to determine the affinity of the allosteric modulator and the cooperativity between the interacting ligands. For allosteric inhibitors that have very high negative cooperativity with the orthosteric agonist tested, this limit may not be reached over the concentration range utilized. Such allosteric modulators may be mistaken for competitive antagonists. Indeed, this is predicted in the ATCM (a ^ 0), as described above.

If the presence of an allosteric modulator results in a change in the maximal agonist effect (Emax) or the basal responsiveness of the system, then this is indicative of orthosteric efficacy modulation, in the case of the former, or allosteric agonism, in the case of the latter. The allosteric two-state model (Fig. 3.1b) accommodates efficacy modulation and agonism in addition to affinity modulation. However, the model is not readily amenable to fitting experimental data due to the large number of parameters. An alternative approach has recently been presented, based on combining the operational model of agonism [37] with the ATCM [21, 25] to yield an operational model of allosterism:

([A] Kb + KaKb + Ka[B] + a [A] [B])n + ([A] (Kb + ap [B]) + Tb[B] Ka )n where E is the effect, and A, B, KA . and KB are as defined for the ATCM (Fig. 3.1a) above. As for the simple ATCM, allosteric modulation of binding affinity in the operational model of allosterism is governed by the cooperativ-ity factor a. Allosteric modulation of orthosteric ligand efficacy is incorporated into the model by the introduction of another (empirical) parameter, p, which scales from zero to infinity and denotes the magnitude by which the allosteric modulator modifies the stimulus generated by the orthosteric agonist on the ARB ternary complex. The parameters ta and tb relate to the ability of the orthosteric and allosteric ligands, respectively, to promote receptor activation. Both ta and tb incorporate the intrinsic efficacy of each ligand, the total density of receptors, and the efficiency of stimulus-response coupling. The

Log [Orthosteric Agonist] M

Figure 3.4 Effects of cellular stimulus-response coupling on the manifestation of allosteric modulation and agonism. Simulations based on an operational model of allosterism (see text) for a system with low coupling efficiency or receptor expression (top panel) or high coupling efficiency or receptor expression (low panel). For all simulations, the following parameter values were used: Em = 100, n = 1, KA = 10-6M, Kb = 10-7M, a = 30, P = 10. The allosteric modulator, B, was present at concentrations ranging from 0.3nM to 3 |M. Despite the same pair of ligands being tested at the same receptor, strikingly different apparent behaviors are imposed by the stimulus-response machinery of the host system, which can be modeled by changes in the parameter, t, of the operational model. Data adapted from Reference 21.

Log [Orthosteric Agonist] M

Figure 3.4 Effects of cellular stimulus-response coupling on the manifestation of allosteric modulation and agonism. Simulations based on an operational model of allosterism (see text) for a system with low coupling efficiency or receptor expression (top panel) or high coupling efficiency or receptor expression (low panel). For all simulations, the following parameter values were used: Em = 100, n = 1, KA = 10-6M, Kb = 10-7M, a = 30, P = 10. The allosteric modulator, B, was present at concentrations ranging from 0.3nM to 3 |M. Despite the same pair of ligands being tested at the same receptor, strikingly different apparent behaviors are imposed by the stimulus-response machinery of the host system, which can be modeled by changes in the parameter, t, of the operational model. Data adapted from Reference 21.

parameters Em and n denote the maximal possible system response and the slope factor of the transducer function that links occupancy to response, respectively [21].

In addition to the modulator KB value, the operational model of allosterism describes modulation as being mediated by two parameters, a and p, which can vary for each and every set of interacting ligands at a GPCR. Theoretically, however, these should not change for a given set of ligands and GPCR between different assays of GPCR function. An important caveat to this is the potential for pathway-specific modulation; allosteric modulator-engendered functional selectivity would be indicated by assay-dependent changes in the value of the P parameter.

With respect to the t (agonism) parameters of the model, because these are determined not only by the intrinsic properties of each ligand but also by the biological system under investigation, they can change between different assay systems (Fig. 3.4). Analyzing an effector pathway that has low stimulus-response coupling efficiency or in a cellular background with very low levels of receptor expression can yield a low tb value for an agonist and, as such, its efficacy may not be discernible; if the compound is allosteric, the interaction will manifest primarily as a change in the potency and/or maximal response to orthosteric ligand, with no effect on basal signaling. In the case of receptor overexpression or high stimulus-response coupling efficiency, and subsequently high tb values, the allosteric ligand efficacy will substantially increase the basal responsiveness of the assay system, and may also shift the orthosteric agonist potency. However, enhancement of the maximal orthosteric agonist response may not be evident, as a GPCR that has high coupling efficiency may already be approaching the maximal possible response of the entire cellular system (Em) for any agonist being tested [21]. These are important considerations in terms of designing screening strategies for allosteric ligand- based drug discovery programs, interpreting the pharmacology of putative allosteric ligands and also translating research from recombinant systems to tissues and beyond.

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