Recommended Opioid Receptor Tool Compounds: Comparative In Vitro for Receptor Selectivity Profiles and In Vivo for...

Opioid agonist ligands bind opioid receptors and stimulate downstream signaling cascades for various biological processes including pain and reward. Historically, before cloning the receptors, muscle contraction assays using isolated organ tissues were used followed by radiolabel ligand binding assays on native tissues. Upon cloning of the opioid G protein-coupled receptors (GPCRs), cell assays using transfected opioid receptor DNA plasmids became the standard practice including ³⁵S-GTPγS functional and cAMP based assays. A number of research laboratories have studied key "tool" reference opioid receptor ligands for decades and used them as control reference compounds. Some, but not all, of these commonly used tool compounds have been characterized and compared side by side in parallel assays for selectivity profiles at the different human opioid receptors isoforms. Herein, we performed the standard FLIPR calcium mobilization assay using HEK293 cells engineered to stably express the Gα_Δ6qi4myr in parallel, at human MOR, KOR, DOR, and NOP opioid receptors. The following tool compounds: morphine, fentanyl, oxycodone, DAMGO, DPDPE, U69593, deltorphin II, and nociceptin, were examined herein. These included the substance use disorder (SUD) compounds morphine, fentanyl, and oxycodone. Additionally, the antagonist tool compounds naloxone, NTI, norBNI, and β-FNA were assayed in parallel at the human MOR, KOR, DOR, and NOP opioid receptors. Furthermore, the agonist tool compounds were tested in the same in vivo tail-flick antinociception assays via intrathecal injection for ED₅₀ potencies. These data provide both in vitro comparative pharmacology as a reference for cellular activities and in vivo antinociception profiles for these tool compounds.