*4.6. β-Arrestin Recruitment Assay with Pathhunter®*

This was performed as described previously [54]. In brief, U2OS OPRM1, CHO-K1 OPRD1, or U2OS OPRK1 cells were seeded at a density of 1.0 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well in 96-well clear-bottom white plates and incubated for 48 h. The cells were stimulated for 90 min (in the case of MOR and DOR) or 180 min (in the case of KOR) in a dilution series for each receptor at 37 ◦C under 5% CO<sup>2</sup> and the PathHunter® working detection solution was added. The luminescence intensity was measured using FlexStation 3 (BioTek Instruments Inc., Winooski, VT, USA) for 1 h at room temperature. Data are expressed as the maximum signal intensity of each test compound as a percentage of the maximum signal intensity of the positive control.

#### *4.7. Statistical Analysis and Approval for the Study*

Data are presented as means ± SEM for at least three independent experiments. Data from cADDis cAMP assays were analyzed using one-way ANOVA followed by Tukey's multiple comparison tests. A value of *p* < 0.05 was considered statistically significant. All analyses and concentration–response curve fitting were performed using Prism 8 (GraphPad Software, San Diego, CA, USA). All experiments were approved and performed in accordance with the Guide for Genetic Modification Safety Committee, National Cancer Center, Japan.

#### **5. Conclusions**

In the present study, we showed that rubiscolins are G-protein-biased full agonists for DOR, as well as partial agonists for the MOR/DOR heteromers, with limited effects on endogenous ligands or opioid analgesics that activate MOR or KOR. Considering the evidence obtained, we believe that rubiscolins could serve as promising seeds for the development of novel, safer opioids and selective DOR agonists that can be orally used for treating pain.

**Supplementary Materials:** The following are available online, Synthesis of rubiscolin-5 and -6.

**Author Contributions:** Conceptualization, Y.U. and K.M.; validation, Yusuke Karasawa and K.M.; investigation, Y.K. (Yusuke Karasawa), T.M., Y.K. (Yui Kuroda), M.N., A.K., K.O., and M.Y.; data curation, Y.U. and K.M.; resources, H.F. and T.M.; writing—original draft preparation, Y.K. (Yusuke Karasawa); writing—review and editing, Y.U. and K.M.; supervision, H.F., K.Y., M.I., and M.H.; project administration, Y.U.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by JSPS KAKENHI Grant Numbers JP18K07404, JP18K08858, JP21K06584, and The National Cancer Center Research and Development Fund 29-A-48.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Guide for Genetic Modification Safety Committee of National Cancer Center, Japan (approval no. B85M1-17, 29 March 2017) and the Recombinant Gene Research Safety Committee of the Jikei University (approval no. D2020-050, 13 January 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The principal author and one coauthor (M.Y.) are employees of a pharmaceutical company (Viatris Pharmaceuticals Japan Inc. and Pfizer Japan Inc., respectively). However, the present study has no financial or other relationships with these companies, as it was entirely sponsored by and performed at the Jikei University School of Medicine, National Cancer Center Research Institute, and Juntendo University Graduate School of Medicine.

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**

