Tuning the endocannabinoid system: allosteric modulators of the CB₁ receptor

Abstract

Cannabinoid CB₁ receptor antagonists are novel therapeutics with potential for the treatment of a number of conditions including obesity, nicotine addition and metabolic syndrome. In 2005, Price et al. demonstrated that the cannabinoid CB₁ receptor contains an allosteric-binding site which binds synthetic small molecules. In this issue of the British Journal of Pharmacology, Horswill et al. have extended these observations. They demonstrate that a structurally similar small molecule allosterically modulates the cannabinoid CB₁ receptor and reduces body weight and food intake in an acute feeding model. Allosteric modulation now contends as a new strategy in the therapeutic exploitation of cannabinoid receptors that may offer certain advantages over the more familiar small molecules targeting the orthosteric site.

The most studied endocannabinoids are arachidonoylethanolamide (anandamide) and 2-arachidonoyl glycerol (2-AG). These eicosanoids have a neuromodulatory role and are key signalling molecules involved in a range of physiological processes including appetite regulation, pain and memory processing, and addiction. 2-AG is a retrograde signalling molecule and modulates synaptic transmission by interaction with presynaptic cannabinoid CB₁ receptors to inhibit neurotransmitter release. Within the brain, the distribution of CB₁ receptors is heterogeneous; the cerebral cortex, hippocampus, lateral caudate-putamen, substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus and the molecular layer of the cerebellum are all highly populated with CB₁ receptors (Christopoulos and Kenakin, 2002). CB₁ receptor antagonists are being developed directed towards the treatment of obesity (Horvath, 2003; Duffy and Rader, 2007), osteoporosis (Idris et al., 2005), nicotine addiction (Le Foll and Goldberg, 2004) and mental illness (Muccioli and Lambert, 2005).

A few years ago, Price et al. (2005) identified three novel allosteric modulators of the cannabinoid CB₁ receptor. These compounds, synthesized at Organon research, display a number of features that are characteristic of allosteric modulators. Firstly, enhancement of the equilibrium binding of the orthosteric ligand, [³H]CP55950, to mouse brain membranes. Secondly, a ligand-dependent effect, as demonstrated by distinct effects on the equilibrium binding of the different orthosteric radioligands to mouse brain membranes. Thirdly, a slowing of the dissociation rate constant(s) for [³H]CP55940 from the occupied CB₁ receptor. Finally, non-competitive inhibition of orthosteric agonist efficacy: as demonstrated by the effect of the compounds on the E_max of CB₁ receptor agonists in stimulation of [³⁵S]GTPγS binding to mouse brain membranes, inhibition of electrically evoked contractions of the mouse vas deferens and in a hCB₁ receptor luciferase reporter assay. In this issue of the British Journal of Pharmacology, Horswill et al. (2007) report a patently similar profile for PSNCBAM-1; in HEK293-hCB₁ membranes, this compound increased the equilibrium binding of [³H]CP55940, but produced a noncompetitive functional antagonism of agonist-stimulated [³⁵S]GTPγS binding and agonist-induced inhibition of cAMP production. The prosidion (PSN) and the organon (Org) compounds display a ligand-dependent effect, enhancing the specific binding of the CB₁/CB₂ receptor agonist [³H]CP55940 but inhibiting the binding of the inverse agonist [³H]SR141716A.

Clearly, these compounds display an intriguing dichotomy of effect on orthosteric ligand affinity versus efficacy; they are allosteric enhancers of agonist-binding affinity and allosteric inhibitors of agonist-signalling efficacy. Both studies converge to demonstrate a complex scenario for the in vitro pharmacology of these compounds. A key finding in the Horswill et al. paper is the first demonstration of in vivo activity of a CB₁ receptor allosteric modulator in an acute food-intake model. The CB₁ receptor inverse agonist, SR141716A (Acomplia) has recently been approved for treatment of obesity in Europe. Furthermore, a number of other therapeutic indications may be targets for compounds of this class, including nicotine addiction (Le Foll and Goldberg, 2004), the metabolic syndrome (Horvath, 2003; Duffy and Rader, 2007) and inflammation (Costa, 2007). The demonstration that a functional allosteric antagonist of the CB₁ receptor has a hypophagic effect in vivo announces a new strategy of drug discovery that may offer advantages over existing approaches for targeted manipulation of the endocannabinoid system.

Numerous CB₁ receptor antagonists have been synthesized that display a significant degree of subtype selectivity versus the CB₂ receptor (Alexander et al., 2007). However, recently, it has been shown that certain well-established cannabinoid receptor ligands, including the inverse agonists, have affinity for an orphan receptor, GPR55 (Baker et al., 2006; Alexander et al., 2007). Furthermore, certain cannabinoid ligands may interact also with TRPV1 receptors (Ross, 2003). As has been previously demonstrated for other GPCRs, synthesis of receptor subtype-selective ligands can be fraught by high sequence homology within the orthosteric-binding domain between receptor subtypes (Rees et al., 2002; Christopoulos and Kenakin, 2002). Invoking a CB₁ receptor allosteric site may provide a higher degree of subtype selectivity.

Currently, the CB₁ receptor antagonists advancing through clinical trials appear to be inverse agonists (Pertwee, 2005). Inverse agonists of G protein coupled receptor (GPCRs) are known to exert long-lasting effects on receptor function (Greasley and Clapham, 2006) that may be of consequence to patients on certain treatment regimes. Prolonged exposure to CB₁ receptor inverse agonists may prove to be detrimental, beneficial or inconsequential as compared to exposure to a neutral antagonist. The Org compounds do not behave as inverse agonists, having no effect on CB₁ receptor constitutive activity (Price et al., 2005). Horswill et al. report that the PSN compound has no effect on constitutive activity in hCB₁ yeast report assays as compared to SR141716A. However, in the [³⁵S]GTPγS-binding assay, the PSN compound produced an inverse effect, albeit displaying significantly lower inverse efficacy than SR141716A.

The discovery of an allosteric modulator of the cannabinoid CB₁ receptor that has a hypophagic effect in vivo represents a new addition to the existing variety of ligands that recognize the cannabinoid receptors. Allosteric modulation now contends as a novel strategy in the therapeutic exploitation of these receptors that may offer certain advantages over the more familiar small molecules targeting the orthosteric-binding site. Future research will undoubtedly address the question of whether allosteric modulators of the CB₁ receptor are advantageous in certain pathophysiological scenarios by comparison with the currently available orthosteric ligands.