Inverse agonism

In contrast to agonists that produce receptor activation and neutral antagonists that do not affect basal receptor activity, inverse agonists are able to decrease basal receptor

Figure 177 The cubic ternary complex model (Weiss et al., 1996) allows the inactive receptor state to interact with a G protein (circled). Reprinted from Journal of Theoretical Biology, 181, Weiss, J. M., Morgan, P. H., Lutz, M. W. & Kenakin, T. P., The cubic ternary complex receptor-occupancy model III. Resurrecting efficacy, 381-397. Copyright (1997), with permission from Elsevier.

Figure 177 The cubic ternary complex model (Weiss et al., 1996) allows the inactive receptor state to interact with a G protein (circled). Reprinted from Journal of Theoretical Biology, 181, Weiss, J. M., Morgan, P. H., Lutz, M. W. & Kenakin, T. P., The cubic ternary complex receptor-occupancy model III. Resurrecting efficacy, 381-397. Copyright (1997), with permission from Elsevier.

Figure 178 Increase in basal receptor activity to detect inverse agonism: A) increased basal a2D-adrenergic receptor activity in recombinant PC12 cells by reducing the Na+ concentration, B) effect of different ligands on constitutively active p-adrenergic receptor mutant, C) effect of different ligands on Sf9 cells with over-expressed p-adrenergic receptor. This figure has been adapted from Tian, W. N., Duzic, E., Lanier, S. M. and Deth, R. C. (1994) Molecular Pharmacology, 45, 524-531; Samama, P., Pei, G., Costa, T., Cotecchia, S. and Lefkowitz, R. J. (1994) Molecular Pharmacology, 45, 390-394; and Chidiac, P., Hebert, T., Valiquette, M., Dennis, M. and Bouvier, M. (1994) Molecular Pharmacology, 45, 490-499, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 178 Increase in basal receptor activity to detect inverse agonism: A) increased basal a2D-adrenergic receptor activity in recombinant PC12 cells by reducing the Na+ concentration, B) effect of different ligands on constitutively active p-adrenergic receptor mutant, C) effect of different ligands on Sf9 cells with over-expressed p-adrenergic receptor. This figure has been adapted from Tian, W. N., Duzic, E., Lanier, S. M. and Deth, R. C. (1994) Molecular Pharmacology, 45, 524-531; Samama, P., Pei, G., Costa, T., Cotecchia, S. and Lefkowitz, R. J. (1994) Molecular Pharmacology, 45, 390-394; and Chidiac, P., Hebert, T., Valiquette, M., Dennis, M. and Bouvier, M. (1994) Molecular Pharmacology, 45, 490-499, with permission from the American Society for Pharmacology and Experimental Therapeutics.

activity. This implies that the receptor possesses some constitutive activity (i.e. that it activity is not completely zero in the resting state). Inverse agonism is well known for benzodiazepine receptors, but this phenomenon has also been shown to take place for many GPCRs (Figure 178). To observe inverse agonism, constitutive GPCR activity can be obtained by:

• Over-expressing the receptor in recombinant systems (Table 21).

• Altering the ionic milieu of the assay system. This can often be achieved by substituting Na+ by K+.

• Using constitutively active receptor mutants. Yet, one criticism to this approach is that it generally requires mutagenesis of the GPCR and that this might alter the

Table 21 Inverse agonists for over-expressed wild-type adrenergic receptors. Reproduced from Kenakin, T. (1996) Pharmacological Reviews, 48, 413-463, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Receptor

System

Drug

ß2-Adrenergic

Sf9 membranes

DCI

pindolol

labetolol

timolol

CHW membranes

labetolol

pindolol

alprenolol

propranolol

timolol

turkey erythrocytes

propranolol

pindolol

TG-4 murine atria

ICI 118,551

ßj-Adrenergic

cardiomyocytes

atenolol

propranolol

a2-Adrenergic

PC-12 cells

rauwolscine

yohimbine

WB 4101

idazoxan

phentolamine

yohimbine

bovine aorta

rauwolscine

PC-12 cells

rauwolscine

details of receptor pharmacology. A second feature of many GPCR mutations with enhanced constitutive activity is their reduced stability in the absence of a ligand.

The physiological relevance of inverse agonists has been questioned because it is often only observed in artificial systems. In this respect, overexpression of wild-type receptors is thought to provide the most reliable information. Whereas experiments with constitutively active receptor mutants are most subject to caution, there is also evidence that GPCR mutation may sometimes lead to pathologically relevant constitutive activity. For example, transgenic mice that express a constitutively active mutant of the P2-adrenergic receptor have been shown to display cardiac abnormalities and only inverse agonists were shown to correct these abnormal responses. Hence, the therapeutic potential of inverse agonists is proposed in human diseases ascribed to constitutively active mutant receptors as well as in diseases involving non-mutated receptors, which either have high basal activity or have constitutive activity due to over-expression.

Partial Full Invert Agonists
Figure 179 Difference in Ligand efficacy in a quiescent (i.e. with no basal activity) and a constitutively active GPCR signalling system (with respect to the full agonist = 1).

Inverse agonists may also contribute to the pharmacological characterization of orphan GPCRs. Traditionally, drug candidates are tested for their ability to mimic or inhibit ligand binding to the targeted receptor. However, as there are no previously known ligands for orphan GPCRs, competition ligand-binding studies cannot be performed and thus functional assays are at the core of all such screening programmes. High levels of activity can be obtained by over-expressing or mutating the orphan GPCR

Figure 180 Simulations according to the cubic ternary complex model. Upon increasing L, partial agonists may become (neutral) antagonists and even inverse agonists. The number of active receptors is defined by [AR*G] + [R*G]. Reprinted from Trends in Pharmacological Science, 16, Kenakin, T., Pharmacological proteus?, 256-258. Copyright (1995), with permission from Elsevier.

Figure 180 Simulations according to the cubic ternary complex model. Upon increasing L, partial agonists may become (neutral) antagonists and even inverse agonists. The number of active receptors is defined by [AR*G] + [R*G]. Reprinted from Trends in Pharmacological Science, 16, Kenakin, T., Pharmacological proteus?, 256-258. Copyright (1995), with permission from Elsevier.

of interest. This allows the discovery of 'inverse agonists' and provides a more sensitive assay for the discovery of receptor agonists (since constitutively activated GPCRs often exhibit high affinity for agonists). It should be noted that neutral antagonists and inverse agonists behave in an identical manner in the absence of constitutive receptor activity and, in fact, it is only with the advent of constitutive active receptor systems that many ligands thought to be neutral antagonists were found to be inverse agonists (Figure 179).

The P-adrenergic receptor ligand dichloroisoproterenol is a well-known partial agonist in many systems, but it exhibits inverse agonism in p2-receptors over-expressing Sf9 cells. Due to this duality in behaviour, such ligands have been termed protean ligands. The cubic ternary complex model permits weak partial agonists in systems with low basal activity to become inverse agonists in constitutively active systems (Figure 180). However, this is only possible if the receptor is permitted to adopt more than one activated state (see also Section 4.13).

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