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 CellKey^TM 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 CellKey^TM 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.

Keywords: G-protein-biased agonist; analgesic; opioid peptide; rubiscolins; δ opioid receptor.

Figure 1

Molecular structures of rubiscolins.

Figure 2

Effect of rubiscolins on MOR, DOR, and KOR, observed using the CellKey^TM system. The cells expressing MOR (A), DOR (B), and KOR (C) were treated with each compound (10⁻¹¹–10⁻⁵ M), and changes in impedance (ΔZiec) were measured using the CellKey^TM system. Concentration–response curves were prepared by calculating ΔZiec relative to the data obtained for each positive control: 10⁻⁵ M DAMGO for MOR (A), 10⁻⁵ M SNC-80 for DOR (B), and 10⁻⁵ M U-50488H for KOR (C). All data points are presented as means ± S.E.M. for three independent experiments (n = 3–5).

Figure 3

Evaluation of antagonistic effects induced by rubiscolins combined with positive control for MOR or KOR, observed using the CellKey^TM system. The cells expressing MOR (A) and KOR (B) were treated with each positive control alone or in combination with rubiscolin-5, rubiscolin-6, or 10⁻⁵ concentration of each negative control (10⁻¹¹–10⁻⁵ M), and changes in impedance (ΔZiec) were measured using the CellKey^TM system. Concentration–response curves were prepared by calculating ΔZiec relative to the data obtained for each positive control: 10⁻⁵ M DAMGO for MOR (A) and 10⁻⁵ M U-50488H for KOR (B). All data points are presented as means ± S.E.M. for three independent experiments (n = 3–4).

Figure 4

Changes in intracellular cAMP levels induced by rubiscolin-5, rubiscolin-6, and opioid compounds. Cells expressing MOR (A), DOR (B), or KOR (C) were treated with the listed compounds (10⁻¹¹–10⁻⁵ M), and intracellular cAMP levels were measured with the cADDis^® cAMP assay. Concentration–response curves were prepared by calculating cAMP levels relative to the data obtained with 10⁻⁵ M DAMGO for MOR (A), 10⁻⁵ M SNC-80 for DOR (B), and 10⁻⁵ M U-50488H for KOR (C). Data are presented as means ± S.E.M. for three independent experiments (n = 3–5).

Figure 5

Levels of β-arrestin recruitment through OR induced by rubiscolin-5, rubiscolin-6, and opioid compounds. PathHunter^® β-arrestin assay was performed in cells expressing MOR (A), DOR (B), and KOR (C) by treating with each compound (10⁻¹¹–10⁻⁵ M). Concentration–response curves were prepared by calculating intracellular β-arrestin levels relative to the data obtained for each positive control: 10⁻⁵ M DAMGO for MOR (A), 10⁻⁵ M SNC-80 for DOR (B), and 10⁻⁶ M of U-50488H for KOR (C). All data points are presented as means ± S.E.M. for three independent experiments (n = 3–6).

Figure 6

Changes in intracellular cAMP levels induced by rubiscolin-5, rubiscolin-6, and opioid compounds. Cells expressing MOR (A), DOR (B), or MOR/DOR (C) were treated with the listed compounds (10⁻¹¹–10⁻⁵ M), and the intracellular cAMP levels were measured with the cADDis^® cAMP assay. Concentration–response curves were prepared by calculating cAMP levels relative to the data obtained with 10⁻⁵ M DAMGO for MOR (A), 10⁻⁵ M SNC-80 for DOR (B), and 10⁻⁵ M ML335 for MOR/DOR (C). Data are presented as means ± S.E.M. for three independent experiments (n = 6–8).