Innovating Your Drug Discovery

Employing Assay “Volume Control” to Unveil Efficacies in Allosteric Lead Molecules for GPCRs

Drugs interact with living physiology; an in vitro assay of drug activity can be considered a snapshot of the full movie seen in vivo of the drug exposed to a range of tissues of varying sensitivities. An assay measures signaling at one sensitivity, namely that of the particular in vitro assay being utilized, but the more of these snapshots we have, the better will be our understanding of what drugs will do in vivo.


It is well known that physiological tissues control signal magnitude through adjustment of receptor expression levels and the relative stoichiometry of receptors to signaling components. This natural control of signaling is operative for all tissues in vivo. Such adjustment of tissue sensitivity can have a dominating control on the effects of agonists. For example, a low efficacy agonist such as prenalterol can produce nearly full agonism in thyroxine-treated guinea pig atria and no agonism (instead becomes an antagonist) in skeletal muscle such as the digitorum longus (Kenakin, 1985). An in vitro assay of drug activity measures signaling at one defined sensitivity, but if the sensitivity of the in vitro assay differs from that of the relevant therapeutic tissues in vivo, then a possibly costly dissimulation can occur leading to an incorrect choice of drug candidate moving forward. This is why testing drugs in an array of assays of differing sensitivity can be a valuable tool to correctly characterizing hidden efficacies of new molecules.


A case in point are positive allosteric modulators (PAMs) for GPCRs. These unique molecules are becoming important therapeutic entities as they have many beneficial features for agonist signal modification. Specifically, they can augment natural responses and preserve the way in which physiological systems are wired (not alter the complex patterns as they function in Nature). In addition, they produce saturable effects that allow them to change the operational sensitivity of systems without signal overload. PAMs may have quite different patterns of activity in tissues of varying sensitivity and these patterns can be used to quantify their efficacies and predict effects in vivo.

One pattern is sensitization of tissues to agonist response as shown in Figure 1. A. for a tissue of moderate sensitivity. This single assay yields important information such as, in this case, the fact that the PAM sensitizes the tissue to the agonist. However, there are two important questions left unanswered; (1) in a more sensitive tissue, will direct agonism to the PAM be seen?, and (2) does the potentiation of agonist effect occur because of effects on agonist affinity, efficacy, or both? Answers to these questions can be obtained by testing the PAM in two more assays of varying sensitivity.

Figure 1. The effects of a PAM possessing direct efficacy and potentiating activity for efficacy
Figure 1. The effects of a PAM possessing direct efficacy and potentiating activity for efficacy. A. In a tissue of moderate sensitivity, sinistral displacement of the agonist dose response curve is observed. B. In a sensitive tissue, the direct agonism to the PAM is shown by the elevated baseline. C. In a tissue of low sensitivity where the agonist is a partial agonist, the efficacy increase produced by the PAM is shown by the increased maximal response to the agonist.


Figure 1. B. shows the same PAM in an assay of higher sensitivity. There are many chemical series whereby PAM activity transfers to direct agonism in highly sensitive tissues; presumably, this is related to the fact that PAMs stabilize the formation of receptor active states. Therefore, the data this figure predicts that if this PAM encounters a sensitive tissue, it would produce direct agonism. In many cases PAMs are used to avoid direct agonism as these may lead to side effects. Thus, a PAM without agonism would produce effects only when the system is activated naturally and otherwise will produce no effect (thus reducing side effects). The detection of direct efficacy may be a harbinger of the side effects of a therapeutic PAM.


PAMs can potentiate responses through elevation of agonist affinity, efficacy, or both; it can be very important to know which of these activities is operative for a therapeutic PAM. If the PAM is aimed to rejuvenate a failing system, i.e. neurotransmission in Alzheimer’s disease, then an increase only in agonist affinity may not produce increased signaling as the original signal is weak and after PAM treatment would still be weak. Instead, what is needed is an increase in efficacy whereby a low level signal is magnified. Under these circumstances, an inoperative receptor system could be revitalized by the efficacy-based PAM. For example, a D1123.32 mutation of the muscarinic M4 receptor makes it completely refractory to the natural agonist acetylcholine, yet application of efficacy-active PAM LY2033298 can completely restore the receptor sensitivity to the agonist (Leach et al. 2011). In general, efficacy-based PAMs would be predicted to produce more robust sensitization of signaling systems.


It is not possible to differentiate whether sensitization to a full agonist is due to increased affinity or efficacy as both of those will produce a leftward shift of the agonist dose response curve. However, if the agonist is a partial agonist, then increases in efficacy will be shown as an increased maximal response, i.e. increases in affinity will not change the maximal response. Therefore, if the PAM could be tested in an assay where the agonist is a partial agonist, then increased efficacy effects can be unveiled, i.e. see Figure 1. C. Once efficacy involvement has been identified, then any possible role for changes in affinity can be detected through a comparison of dose-response curves to the functional allosteric model (Bdioui et al. 2018). Thus, testing of the PAM in the three systems would fully characterize the activity as PAM agonism with increased efficacy; an example of such a molecule can be found in the muscarinic M2 receptor PAM BQCA (benzylquinolone carboxylic acid ) (Bdioui et al. 2019).


Assay volume control can be attained through various means such as differences in receptor expression levels, alkylation of surface receptors, choice of host cell, and augmentation of signal disposition (i.e., GRK co-expression for β-arrestin). Exploration of these approaches could yield valuable information for the full characterization of candidate efficacies. It has been estimated that 50% of new drug candidate failures occur because of therapeutic efficacy (Arrowsmith, 2011); this is in addition to issues of safety that add to this high rate of attrition. Demonstration of such failure usually occurs in the most complex systems showing pathology-controlled pharmacology at the end stage of discovery and development, a costly point in the process. While complete examination of all of the efficacies of a candidate molecule may not prevent such failures at the outset, it may prevent follow-up failures, i.e. why follow up a failed molecule with one that has the same profile of efficacies? Full candidate characterization also can help identify which collection of efficacies are important therapeutically as an effective treatment may require multiple activities, not a single one. For example, the blockade of β-adrenoceptors has been identified as an important treatment for heart failure. However, the testing of 16 β-blockers in clinical trials shows an unequal spectrum of benefit with the multiple efficacy molecule carvedilol emerging as the best (Metra et al. 2004). In general, investment into determining a full characterization of the efficacies of new candidate molecules may yield dividends in late-stage development; thus, an array of in vitro assays can be a valuable asset in this regard.


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