*2.1. Effects of Rubiscolins on the Functions of ORs Evaluated Using the CellKeyTM System*

The effects of rubiscolins on the three types of ORs (MOR, DOR, and KOR) were evaluated using the CellKeyTM system (MDS Sciex, Foster City, CA, USA) in HEK293 cells stably expressing Halo-tag®-MOR, T7-tag®-DOR, or Halotag®-KOR. Changes in cellular impedance were detected as activities of OR using this system. The changes in impedance induced by rubiscolins in positive controls of MOR (DAMGO), DOR (SNC-80), and KOR (U-50488H) were compared to confirm their agonistic effects on each OR. Rubiscolins showed dose-dependent effects only on DOR, whereas little effect was observed on MOR and KOR (Figure 2).

**Figure 2.** Effect of rubiscolins on MOR, DOR, and KOR, observed using the CellKeyTM system. The cells expressing MOR (**A**), DOR (**B**), and KOR (**C**) were treated with each compound (10−11–10−<sup>5</sup> M), and changes in impedance (∆Ziec) were measured using the CellKeyTM system. Concentration–response curves were prepared by calculating ∆Ziec relative to the data obtained for each positive control: 10−<sup>5</sup> M DAMGO for MOR (**A**), 10−<sup>5</sup> M SNC-80 for DOR (**B**), and 10−<sup>5</sup> M U-50488H for KOR (**C**). All data points are presented as means ± S.E.M. for three independent experiments (*n* = 3–5).

Moreover, we examined the antagonistic effects induced by a combination of rubiscolins with the positive control of MOR (DAMGO) or KOR (U-50488H), by comparing with the effect of a combination of each positive control with 10−<sup>5</sup> concentration of a negative control for MOR (naloxone) or KOR (norbinaltorphimine: norBNI), respectively. Unlike for the combination with 10−<sup>5</sup> concentration of negative control that completely suppressed the agonistic effects of the positive control for both MOR and KOR, rubiscolins had little antagonistic effects on MOR and KOR (Figure 3). These results suggest that rubiscolins act as selective DOR agonists without affecting the other subtypes (MOR and KOR) of ORs.

**Figure 3.** Evaluation of antagonistic effects induced by rubiscolins combined with positive control for MOR or KOR, observed using the CellKeyTM 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−<sup>5</sup> concentration of each negative control (10−11–10−<sup>5</sup> M), and changes in impedance (∆Ziec) were measured using the CellKeyTM system. Concentration–response curves were prepared by calculating ∆Ziec relative to the data obtained for each positive control: 10−<sup>5</sup> M DAMGO for MOR (**A**) and 10−<sup>5</sup> M U-50488H for KOR (**B**). All data points are presented as means ± S.E.M. for three independent experiments (*n* = 3–4).
