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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
Assessment of high-efficacy agonism in synthetic cannabinoid receptor agonists containing l-tert-leucinate
Christopher Lucaj1, Soo Jung Oh1, Charlotte Pitha1
1Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Bouvé College of Health Sciences, Center for Drug Discovery, Northeastern University, Boston, MA, 02115, United States.
Abstract:
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. Here we report structure-activity relationships (SARs) of modular SCRAs contributing to 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) assays. We found that the l-tert-leucinate head moiety and indazole core moiety conferred superagonism across multiple Gαi/o proteins and β-arrestin-2. After generating the cannabinoid type 1 receptor (CB1R) mutant constructs, we found that transmembrane 2 (TM2) interactions to the head moiety of tested SCRAs at F170, F174, F177, and H178 are key to eliciting activity. Finally, we found that l-tert-leucinate SCRAs confer a high-efficacy response in ex vivo slice electrophysiology.
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