Cellular and Molecular Basis of Constitutive Activity and Inverse Agonism

The term "inverse agonist'' was first used to describe the actions of certain compounds, such as the b-carbolines, at the ionotropic GABAA receptors, where it was observed that the compounds not only antagonized the actions of ► benzodiazepines at the receptor, but also appeared to evoke an effect opposite to that of the benzodiazepine in the absence of the latter (Costa and Cotecchia 2005). However, given that the mechanism of action of benzo-diazepines is to act as positive ► allosteric modulator of the endogenous agonist, ► GABA, rather than as true agonists in their own right, compounds such the b-car-bolines are more appropriately classed as "negative allosteric modulators'' rather than true inverse agonists; the reversal of "basal" activity in this instance reflects allosteric antagonism of the endogenous agonist. This is an important pharmacological consideration that extends beyond the specific GABAA example. Namely, the in vivo effects of drugs that reduce basal activity may reflect the true inverse agonism of a constitutively active receptor, or they may reflect the antagonism of an endogenous-ly released agonist of that receptor.

Given that the unambiguous demonstration of inverse agonism requires the experimental observation that the effect is not due to antagonism of an endogenous agonist, most observations and assays of inverse agonist behavior are routinely made using a variety of cell-based in vitro bioassays. It was in this context that the earliest report of true inverse agonism occurring at a GPCR was presented by Costa and Herz, who demonstrated the constitutive activation of guanosine triphosphatase (GTPase) activity by the §-► opioids receptor expressed natively in NG108-15 cell membranes and its reversal by compounds that were previously classed as simple opioid receptor antagonists (Bond et al. 2000). Moreover, that same study was the first to differentiate antagonists on the basis of the ability to inhibit constitutive activity (inverse agonists) from the ability of not perturbing constitutive activity but still antagonizing the actions of both agonists and inverse agonists (i.e., act as neutral antagonists). Since the initial report of constitutive GPCR activity and inverse agonism, there has been an explosion in the number of ligands identified to express this type of pharmacology (Bond et al. 2000), including compounds acting at prototypical Family A neurotransmitter receptors, such as the adrenoceptors (a1, a2, and b2), ► muscarinic receptor (M1, M2, M3, and M5), histamine H2 receptors, serotonin receptors (5-HT1A, 5-HT2A, 5-HT2C, 5-HT4, 5-HT6, and

Inverse Agonists. Fig. 2. Agonism and inverse agonism arising due to ligand-induced redistribution of multiple receptor conformational states. The figure shows the distribution of three theoretical receptor states: R0 inactive; R, active conformation that induces functional output 1; R2 active conformation that induces functional output 2. In the absence of ligand (left side), most of the receptors exist in an inactive state, but some constitutive activity may be detected. In the presence of ligand (right side), the conformations of the receptor change such that the behavior of the system is altered; in this example, the abundance of receptors in the R, is reduced, and thus the ligand would be classified as an inverse agonist in an assay measuring functional output 1. However, the R2 state is increased by ligand, and so an assay of functional output 2 would classify the ligand as an agonist.

5-HT7), and dopamine receptors (D2, D3, and D5), as well as Family B and C GPCRs, such as the vasoactive intestinal peptide (Kenakin 2004) and metabotropic glutamate (Bond and Ijzerman 2006) receptors. Importantly, a number of established or potential ► antipsychotics, ► antidepressants, and other psychopharmacological drugs have inverse agonist activity at such targets, e.g., serotonin, dopamine, histamine, opioid, cannabinoid, and muscarinic receptor subtypes (Bond and Ijzerman 2006). As mentioned earlier, GPCRs are highly dynamic proteins, and their ability to fluctuate between various conformational states is a natural property common to most proteins governed at the molecular level by random thermal fluctuations in the energy of the protein in its microenvironment. Physiologically, however, organisms have evolved to keep most constitutive receptor activation at relatively low levels; it would be metabolical-ly wasteful to maintain a permanent state of cellular activation, in addition to reducing the dynamic response range available for signal transduction. For GPCRs, this is generally governed by key microdomains of the receptors that have evolved to minimize the spontaneous isomeri-zation of the proteins into active conformations (Pontier et al. 2008). Thus, at any point in time, only a small fraction of a given population of GPCRs will be in a ligand-independent active state (Fig. 2). An important question, thus, is under what conditions is constitutive activation of a GPCR appreciable such that inverse agonist activity of various ligands is readily detected? Perhaps, a more important consideration is under what circumstances does the phenomenon become therapeutically relevant?

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