CB₁ Receptor Allosteric Modulators Display Both Agonist and Signaling Pathway Specificity

Abstract

We have previously identified allosteric modulators of the cannabinoid CB 1 receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB 1 receptor agonist [ 3 H]CP55940 but producing a decrease in CB 1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB 1 receptor activation. We assessed the effect of these compounds on CB 1 receptor agonist-induced [35 S]GTPgS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and b-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB 1 agonist binding kinetics. -mediated), and inhibition (Ga i -mediated) of cAMP production and b-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kineticbinding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB 1 agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor

“…3) to determine the cooperativity between Org27569 and the cannabinoids ( Table 4). As in previous findings (Baillie et al, 2013) Org276529 Displays Pathway-Dependent Allosteric Modulation at CB 1 Rs, Depending on the Probe. Similar to binding studies, the allosteric activity of Org27569 on functional measures of CB 1 R activity has been shown previously to depend on the orthosteric probe used, such that it increases CP55940-induced pERK1/2, without affecting the WIN55,212-2-mediated response (Baillie et al, 2013).…”

“…As in previous findings (Baillie et al, 2013) Org276529 Displays Pathway-Dependent Allosteric Modulation at CB 1 Rs, Depending on the Probe. Similar to binding studies, the allosteric activity of Org27569 on functional measures of CB 1 R activity has been shown previously to depend on the orthosteric probe used, such that it increases CP55940-induced pERK1/2, without affecting the WIN55,212-2-mediated response (Baillie et al, 2013). Org27569 has also previously been shown to display pathway-specific, or biased, allosteric modulation at CB 1 Rs (Ahn et al, 2012;Baillie et al, 2013).…”

“…The bias factors again highlight that, whereas WIN55,212-2 demonstrates a similar profile to 2-AG with a bias factor not dissimilar from 1, HU-210 and methanandamide exhibit strong bias toward cAMP inhibition. CP55940, D 9 -THC, and anandamide also displayed a preference toward cAMP inhibition, although it did not reach statistical significance (Tables 2 and 3 (Price et al, 2005;Baillie et al, 2013). In binding assays, Org27569 had no effect on [ 3 H]WIN55,212-2 binding, although in functional assays there was evidence that it potentiated WIN55,212-2 binding (Price et al, 2005;Baillie et al, 2013).…”

“…CP55940, D 9 -THC, and anandamide also displayed a preference toward cAMP inhibition, although it did not reach statistical significance (Tables 2 and 3 (Price et al, 2005;Baillie et al, 2013). In binding assays, Org27569 had no effect on [ 3 H]WIN55,212-2 binding, although in functional assays there was evidence that it potentiated WIN55,212-2 binding (Price et al, 2005;Baillie et al, 2013). Therefore, we further sought to evaluate the potential for Org276529 to display probe dependence with both endogenous and exogenous cannabinoids.…”

Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB 1

“…Similarly, our evidence that Org 27569 traps CB 1 in a G proteininactive intermediate state (R′) could explain why Org 27569 acts as classical antagonist in regards to G-protein activation, while at the same time, eliciting varying degrees of MAPK signaling (23,43), presumably by acting as an arrestin-biased allosteric agonist (23,24). The reduced TM6 movement would inhibit productive G-protein coupling, whereas the enhanced H8/TM7 movements could change the accessibility of the C-terminal tail, enabling the receptor to preferentially engage with β-arrestin.…”