In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and beta-Arrestin-Mediated Pathways

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the beta-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective delta opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKey(TM) assay, cADDis(R) cAMP assay, and PathHunter(R) beta-arrestin recruitment assay, using cells stably expressing mu, delta, kappa, or mu/delta heteromer opioid receptors. In the CellKey(TM) assay, rubiscolins showed selective agonistic effects for delta opioid receptor and little agonistic or antagonistic effects for mu and kappa opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased delta opioid receptor agonists based on the results obtained in cADDis(R) cAMP and PathHunter(R) beta-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the mu/delta dimers. In conclusion, rubiscolins could serve as attractive seeds, as delta opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.