Misuse of synthetic cannabinoid receptor agonists (SCRAs) has risen worldwide and is often associated with serious adverse effects including hallucinations, cardiovascular abnormalities, coma, or death. Many SCRAs are aminoalkylindole molecules that are distinct from the naturally occurring psychoactive phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC), found in cannabis. It is established that Δ9-THC is a partial agonist at cannabinoid CB1 receptors, whereas SCRAs demonstrate much higher efficacy and potency at this site, and this likely relates to a much higher incidence of medically adverse effects. Moreover, some SCRAs exhibit a maximal efficacy at CB1 receptors exceeding typical full agonists, and this has been termed “superagonism.” Additionally, superagonism is usually associated with increased potency at CB1 receptors. At present, it is unknown whether these unique pharmacological characteristics at CB1 receptors directly relate to the adverse effects of some SCRAs, or whether their actions at non-CB1 sites may also be involved. In the current study, we examined molecular mechanisms of superagonism of SCRAs at CB1 receptors.

We first tested a series of SCRAs in our bioluminescence resonance energy transfer (BRET) assay in a CB1-Gi protein engagement configuration. Unlike other receptor function assays, this approach avoids caveats associated with signal amplification that can lead to false-positives for superagonism. Based on the structure-activity relationship for superagonism established by this BRET assay, we then performed molecular dynamics simulations of the CB1 receptor complexed with selected SCRAs to investigate the molecular determinants of SCRA binding efficacy, and to characterize superagonism conformational states of the CB1 receptor-SCRA complex. Finally, to evaluate superagonism of selected SCRAs at CB1 receptors in the mouse brain, we performed electrophysiological experiments in hippocampal brain slices to determine effects of these SCRAs on glutamate release inhibition mediated by CB1 receptors. In short, our findings delineate molecular mechanisms of superagonism at CB1 receptors and further our understanding of the mechanisms through which SCRAs may cause serious adverse health effe