OBJECTIVE: Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation.
METHODS: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA.
RESULTS: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed.
CONCLUSIONS: Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including β-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB1) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB1 activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB1. Six putative allosteric binding sites were identified in silico, including novel sites previously associated with cholesterol binding, and key residues within each site were mutated to alanine. The recently determined ZCZ011 binding site was found to be essential for allosteric agonism, as GAT228, GAT229 and ZCZ011 all increased wild-type G protein dissociation in the absence of an orthosteric ligand; activity that was abolished in mutants F191A3.27 and I169A2.56. PAM activity was demonstrated for ZCZ011 in the presence of the orthosteric ligand CP55940, which was only abolished in I169A2.56. In contrast, the PAM activity of GAT229 was reduced for mutants R220A3.56, L404A8.50, F191A3.27 and I169A2.56. This indicates that allosteric modulation may represent the net effect of binding at multiple sites, and that allosteric agonism is likely to be mediated via the ZCZ011 site. This study underlines the need for detailed understanding of ligand receptor interactions in the search for pure CB1 allosteric modulators.

Chemotherapy-induced peripheral neuropathy (CIPN) is one of chemotherapies\' most often documented side effects. Patients with CIPN experience spontaneous burning, numbness, tingling, and neuropathic pain in their feet and hands. Currently, there is no effective pharmacological treatment to prevent or treat CIPN. Activating the cannabinoid receptor type 1 (CB1) by orthosteric agonists has shown promising results in alleviating the pain and neuroinflammation associated with CIPN. However, the use of CB1 orthosteric agonists is linked to undesirable side effects. Unlike the CB1 orthosteric agonists, CB1 positive allosteric modulators (PAMs) don\'t produce any psychoactive effects, tolerance, or dependence. Previous studies have shown that CB1 PAMs exhibit antinociceptive effects in inflammatory and neuropathic rodent models. This study aimed to investigate the potential benefits of the newly synthesized GAT229, a pure CB1 PAM, in alleviating neuropathic pain and slowing the progression of CIPN. GAT229 was evaluated in a cisplatin-induced (CIS) mouse model of peripheral neuropathic pain (3 mg/kg/d, 28 d, i.p.). GAT229 attenuated and slowed the progression of thermal hyperalgesia and mechanical allodynia induced by CIS, as evaluated by the hotplate test and von Frey filament test. GAT229 reduced the expression of proinflammatory cytokines in the dorsal root ganglia (DRG) neurons. Furthermore, GAT229 attenuated nerve injuries by normalizing the brain-derived neurotrophic factor and the nerve growth factor mRNA expression levels in the DRG neurons. The CB1 receptor antagonist/inverse agonist AM251 blocked GAT229-mediated beneficial effects. According to our data, we suggest that CB1 PAMs might be beneficial in alleviating neuropathic pain and slowing the progression of CIPN.