Cannabinoid pharmacology has proven nettlesome with issues of promiscuity a common theme among both agonists and antagonists. One recourse is to develop allosteric ligands to modulate cannabinoid receptor signaling. Cannabinoids have come late to the allosteric table. The ‘first-generation’ negative and positive allosteric modulators (NAMs and PAMs) represent an important first effort. However, most studies have relied on synthetic agonists, often tested in over-expression systems rather than a defined neuronal model system that utilizes endogenously synthesized and released cannabinoids. We have systematically examined first-generation NAMs and a PAM on endocannabinoid modulation of synaptic transmission in cultured autaptic hippocampal neurons. These neurons exhibit CB1 and 2-arachidonoyl glycerol (2-AG)-mediated depolarization induced suppression of excitation (DSE) and therefore serve as a model to test CB1 modulators in a neuronal model of endogenous cannabinoid signaling. We find ORG27569, PSNCBAM-1, and PEPCAN12 attenuate DSE and do not directly inhibit CB1 receptors. Of these PSNCBAM-1 is the most efficacious while PEPCAN12 has the distinction of being an endogenous NAM. The reported NAMs pregnenolone and hemopressin as well as the reported PAM lipoxin A4 are without effect in this model of endocannabinoid signaling. In summary, three of the allosteric modulators evaluated function in a manner consistent with allosterism in a neuronal 2-AG-based model of endogenous cannabinoid signaling.

2-arachidonoylglycerol (2-AG) is the most abundant endogenous cannabinoid in the brain and an agonist at two cannabinoid receptors (CB1 and CB2). The synthesis, degradation and signaling of 2-AG have been investigated in detail but its relationship to other endogenous monoacylglycerols has not been fully explored. Three congeners that have been isolated from the CNS are 2-linoleoylglycerol (2-LG), 2-oleoylglycerol (2-OG), and 2-palmitoylglycerol (2-PG). These lipids do not orthosterically bind to cannabinoid receptors but are reported to potentiate the activity of 2-AG, possibly through inhibition of 2-AG degradation. This phenomenon has been dubbed the ‘entourage effect’ and has been proposed to regulate synaptic activity of 2-AG. To clarify the activity of these congeners of 2-AG we tested them in neuronal and cell-based signaling assays. The signaling profile for these compounds is inconsistent with an entourage effect. None of the compounds inhibited neurotransmission via CB1 in autaptic neurons. Interestingly, each failed to potentiate 2-AG-mediated depolarization-induced suppression of excitation (DSE), behaving instead as antagonists. Examining other signaling pathways we found that 2-OG interferes with agonist-induced CB1 internalization while 2-PG modestly internalizes CB1 receptors. However in tests of pERK, cAMP and arrestin recruitment, none of the acylglycerols altered CB1 signaling. Our results suggest 1) that these compounds do not serve as entourage compounds under the conditions examined, and 2) that they may instead serve as functional antagonists. Our results suggest that the relationship between 2-AG and its congeners is more nuanced than previously appreciated.

The cannabinoid signaling system is found throughout the CNS and its involvement in several pathological processes makes it an attractive therapeutic target. Because orthosteric CB1 cannabinoid receptor ligands have undesirable adverse effects there has been great interest in the development of allosteric modulators - both negative (NAMs) and positive (PAMs) - of these receptors. NAMs of CB appeared first on the scene, followed more recently by PAMs. Because allosteric modulation can vary depending on the orthosteric ligand it is important to study their function in a system that employs endogenous cannabinoids. We have recently surveyed first generation NAMs using cultured autaptic hippocampal neurons. These neurons express depolarization induced suppression of excitation (DSE), a form of synaptic plasticity that is mediated by CB and 2-arachidonoyl glycerol (2-AG); they are therefore an excellent neuronal model of endogenous cannabinoid signaling in which to test CB modulators. In this study we find that while two related compounds, GAT211 and ZCZ011, each show PAM-like responses in autaptic hippocampal neurons, they also exhibit complex pharmacology. Notably we were able to separate the PAM- and agonist-like responses of GAT211 by examining the enantiomers of this racemic compound: GAT228 and GAT229. We find that GAT229 exhibits PAM-like behavior while GAT228 appears to directly activate the CB receptor. Both GAT229 and ZCZ011 represent the first PAMs that we have found to be effective in using this 2-AG utilizing neuronal model system. Because these compounds may exhibit both probe selectivity and biased signaling it will be important to test them with anandamide as well as other signaling pathways.

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