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

Abstract

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-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 δ 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 CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

Opioids and Their Receptors: Present and Emerging Concepts in Opioid Drug Discovery II

“…However, modeling and competitive functional assays suggest that the molecule may possess some negative modulatory capabilities. The study by Karasawa et al confirmed previous work by Cassell et al showing rubiscolin-5 (Tyr-Pro-Leu-Asp-Leu) and rubiscolin-6 (Tyr-Pro-Leu-Asp-Leu-Phe) to selectively bind and activate δORs without recruiting β-arrestin 2 [ 2 , 6 ]. The authors noted significant changes in the efficacy of rubiscolin peptides to inhibit intracellular cAMP in cells co-expressing δOR and µOR, potentially indicating an affinity for putative δOR-µOR heteromers; however, this type of assay comes with multiple limitations in terms of controlling receptor expression and dissecting the cAMP signal that originates from the monomers, this could be better resolved in a model system that eliminates monomer signaling [ 7 ].…”

“…Three research articles cover the discovery of novel δ-opioid receptor (δOR) ligands with distinct pharmacological profiles [ 2 , 3 , 4 , 5 ]. Meqbil et al identified a novel δOR agonist with a unique scaffold lacking basic nitrogen from a high-throughput screen [ 4 ].…”

Validation of an Analytical Methods of 3',4',5-Trihydroxy-3-Methoxy-6,7- Methylenedioxyflavone 4'-Glucuronide for Standardization of Spinacia oleracea

“…The healthy bene ts of spinach have been noted to involve its speci c phytochemicals, such as carotenoids, avonoids, terpenoids, polyketides, lignans, saponins, and peptides (Riso et al 2004;Park et al 2011; Karasawa et al 2021). One of the key components responsible for these bene ts is avonoids, a type of phytochemical in spinach (Bergqust et al 2005;Tao et al 2023).…”

The Therapeutic Potential of Naturally Occurring Peptides in Counteracting SH-SY5Y Cells Injury

“…[ 8 ]). In turn, rubiscolins are peptides isolated from spinach protein D-ribulose-1,5-bisphosphate carboxylase (RuBisCo); they act as G-protein-biased full agonists for δ-opioid receptor, as well as partial agonists for the µ/δ-opioid receptor heteromers [ 9 , 10 , 11 ]. Their chemical structure is quite distinct from other traditional opioid peptide structures; however, the presence of the protonated Tyr at the N-terminus, Pro in position 2 (as in endomorphins), and an aliphatic amino acid (Leu) in position 3 seems to be the minimal requirement for the opioid activity, as was soon confirmed in the structure–activity relationship studies [ 9 , 10 ].…”

“…Orally administered R-6 showed an antinociceptive, anxiolytic, and antidepressant effect, influencing learning and memory. Moreover, rubiscolins are promising in terms of their unique effects, such as stimulation of food intake or enhancement of glucose uptake in skeletal muscle [ 10 , 11 ].…”