*2.2. Effects of Rubiscolins on the Intracellular cAMP Levels Evaluated Using the cADDis® cAMP Assay*

The activities of compounds through the G-protein-mediated pathway were evaluated by measuring the intracellular cAMP levels for each OR using HEK293 cells stably expressing Halotag®-MOR, T7-tag®-DOR, or Halotag®-KOR (Figure 4). After obtaining results for rubiscolins, SNC-80 (a positive control for DOR), and KNT-127 (an existing selective DOR agonist used as a competitor) [35], we compared the effects of rubiscolins with those of other compounds, including each of the positive controls for the three types of ORs. The Emax and EC<sup>50</sup> values (pEC<sup>50</sup> defined as the negative logarithm of the EC50) for each OR were calculated (Table 1). As was observed for SNC-80 or KNT-127, rubiscolin-6 demonstrated a robust effect on DOR at 10−<sup>5</sup> concentration. On the contrary, they had little effect on MOR and KOR; in contrast, KNT-127 showed full agonistic effects on both MOR and KOR. These results indicate that rubiscolins selectively activate the G-protein-mediated pathway of DOR to exert their pharmacological effects.

**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−<sup>5</sup> 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−<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**). Data are presented as means ± S.E.M. for three independent experiments (*n* = 3–5).


**Table 1.** Emax and pEC<sup>50</sup> values for rubiscolins and opioid compounds obtained in the cAMP assay for MOR, DOR, and KOR.

Emax (means ± S.E.M.) and pEC<sup>50</sup> (-LogEC50, means ± S.E.M.) were calculated according to the results shown in Figure 4. \* *p* < 0.05 versus DAMGO, # *p* < 0.05 versus SNC-80, <sup>+</sup> *p* < 0.05 versus U-50488H. n.d.; not detected.
