Assessment of high-efficacy agonism in synthetic cannabinoid receptor agonists containing l- tert -leucinate

Synthetic cannabinoid receptor agonists (SCRAs) represent a class of new psychoactive substances that pose great health risks attributed to their wide-ranging and severe adverse effects. Recent evidence has shown that SCRAs with key moieties can confer superagonism, yet this phenomenon is still not well understood. In this study, we developed a structure-activity relationship (SAR) for SCRA superagonism by comparing eight compounds differing by their head moiety (l-valinate vs. l- tert -leucinate), core moiety (indole vs. indazole), and tail moiety (5-fluoropentyl vs. 4-fluorobenzyl) through different modes of bioluminescence resonance energy transfer (BRET). We found that l- tert -leucinate head moiety and indazole core moiety conferred superagonism across multiple Gα _i/o proteins and β-arrestin 2. Finally, after generating CB1R mutant constructs, we found that transmembrane 2 (TM2) interactions to the head moiety of tested SCRAs at F170, F177, and H178 are key to eliciting activity.