*2.3. Effects of Rubiscolins on β-Arrestin Recruitment Measured Using the PathHunter® Assay*

To determine the activities of rubiscolins through the β-arrestin-mediated pathway, we performed the PathHunter® β-arrestin recruitment assay using CHO-K1 cells stably expressing MOR and DOR (DiscoverX, Fremont, CA, USA), and U2OS cells stably expressing KOR (DiscoverX). We also evaluated the effects of SNC-80 and KNT-127. Compared with SNC-80, rubiscolins displayed little effect on DOR, as shown in Figure 5, whereas KNT-127 moderately recruited β-arrestin in DOR. Given these results, among the DOR-selective compounds used in the experiment, rubiscolin-5 and rubiscolin-6 were considered the most irrelevant with regard to the activity through the β-arrestin-mediated pathway. In contrast, all the DOR-selective compounds showed little effect on MOR and KOR, compared with each positive control.

**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−<sup>5</sup> M). Concentration–response curves were prepared by calculating intracellular β-arrestin levels 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>6</sup> M of U-50488H for KOR (**C**). All data points are presented as means ± S.E.M. for three independent experiments (*n* = 3–6).

#### *2.4. Effects of Rubiscolins on the MOR/DOR Heteromer*

Finally, we examined the effects of rubiscolins on the MOR/DOR heteromer through the G-protein-mediated pathway using the cADDis® cAMP assay. As shown in Figure 6 and Table 2, rubiscolins acted as partial agonists, similarly to SNC-80, compared with ML335 [30], a specific agonist for MOR/DOR.

**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−<sup>5</sup> 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−<sup>5</sup> M DAMGO for MOR (**A**), 10−<sup>5</sup> M SNC-80 for DOR (**B**), and <sup>10</sup>−<sup>5</sup> M ML335 for MOR/DOR (**C**). Data are presented as means <sup>±</sup> S.E.M. for three independent experiments (*<sup>n</sup>* = 6–8).

**Table 2.** Emax and pEC<sup>50</sup> values for rubiscolins and opioid compounds obtained in the cAMP assay for MOR, DOR, and MOR/DOR.


Emax (means ± S.E.M.) and pEC<sup>50</sup> (-LogEC50, means ± S.E.M.) were calculated according to the results shown in Figure 6. \* *p* < 0.05 versus DAMGO, # *p* < 0.05 versus SNC-80, <sup>+</sup> *p* < 0.05 versus ML335. n.d.; not detected.

#### **3. Discussion**

In the present study, both rubiscolin-5 and -6 were indicated as G-protein-biased DOR full agonists without affecting MOR and KOR. The limited antagonistic effects of rubiscolins on MOR and KOR (Figure 3) confirmed using the three types of ORs, for the first time in this study, indicate that they rarely interfere with the cellular signaling mediated by endogenous or exogenous opioid ligands. The endogenous opioid system plays a critical role in modulating stress [36,37], anxiety [38,39], and the immune system [40]; hence, other than its role in analgesia, it is preferable that opioid agonists do not exert antagonistic effects on untargeted ORs, as these can lead to unexpected side effects that occur by attenuating the activities of endogenous ligands, such as enkephalins, β-endorphin, or dynorphin A. In addition, rubiscolins can potentially be administered in combination with exogenous ligands, such as MOR and KOR agonists and antagonists, without modulating their expected effects, which means that they are unique and attractive seeds that exhibit DOR selectivity, considering that the existing opioids can affect untargeted ORs to varying degrees [4]. As for the combination therapy of analgesics including opioids, opioid-sparing effects of non-opioid analgesics combined with opioids can reduce opioid consumption and its related side effects, especially in the perioperative pain management in terms of avoiding the toxicity and chronic use of opioids [41–43]. Rubiscolins can be novel candidates for use in combination with opioids. Therefore, further research is needed to

investigate the efficacy and safety of DOR agonists, including rubiscolins, in combination with MOR agonists.

In view of the results of our cAMP assay, rubiscolins can be considered DOR-biased agonists, consistent with previous reports. However, compared with the findings in a previous study on the bias factor of rubiscolins, which found that rubiscolin-5 was more G-protein-biased than rubiscolin-6 [27], our results indicate that rubiscolin-6 is relatively stronger than rubiscolin-5 in activating the G-protein-mediated intracellular pathway, and the β-arrestin recruitment levels induced by rubiscolin-5 or rubiscolin-6 are equivalently negligible (Table 1 and Figure 5). Indeed, there is a structural difference between the two peptides, as rubiscolin-6 has Phe, an additional aromatic residue, at the sixth position. Although its function is not clear, rubiscloin-6 has been shown to have a higher receptor affinity and is about twice as potent in analgesia as rubiscolin-5 [21], as was observed in our study. Rubiscolin-6 has also been reported to have broad beneficial effects related to the central nervous system, other than analgesia [22–26]. As an example, for the development of DOR selective agonists, considering such effects, NC-2800 is under Phase 1 clinical study to determine the indication of major depressive disorder (https://jrct.niph.go.jp/ en-latest-detail/jRCT2071210033 accessed on 30 September 2021). The development of DOR-selective agonists as alternative antidepressants is expected to offer a solution for the unmet need related to the patient's adherence to the current treatment of depression, since their efficacy is independent of representative side effects of selective serotonin reuptake inhibitors, such as digestive symptoms [13]. Their antidepressant-like or anxiolytic-like activities are also desirable in the context of treating pain, considering psychological factors, such as depression and anxiety, are intimately associated with pain behavior, especially in chronic pain conditions [44–46]. Therefore, based on our results, we believe that rubiscolin-6 has more potential to be developed as a G-protein-biased DOR agonist than rubiscolin-5, not only as an analgesic but also as a medicine for treating other indications that are significantly different for DOR and MOR agonists, despite their mild analgesic properties compared with that of MOR agonists.

In OR signaling pathways, β-arrestin-mediated pathway is involved in unfavorable side effects, such as tolerance through the intracellular pathway in MOR or dysphoria through that of KOR. Interestingly, β-arrestin recruitment by rubiscolins on any type of OR was low, although moderate changes were observed even with KNT-127 for DOR (Emax (%): 35.4 ± 1.3), an existing selective DOR agonist [35], when compared with SNC-80 (Emax (%): 100.0 ± 2.6) (Figure 5B). Given these results, rubiscolins can be considered the safest among selective DOR agonists, possibly with fewer side effects, such as convulsion that sometimes occurs upon administration of DOR agonists [18], or increase in alcohol intake correlated with β-arrestin recruitment induced by DOR agonists [47].

Here, we report the effects of rubiscolins on MOR/DOR heteromers for the first time. Rubiscolins showed partial agonistic effects on the MOR/DOR heteromer (Figure 6C and Table 2). Moreover, the finding that rubiscolins have unique profiles in exerting their effects, mainly through the activation of the G-protein-mediated pathway in DOR, and in part through the MOR/DOR heteromer, is novel. In contrast, ML335 was reproduced as a full agonist of MOR/DOR, consistent with the findings in a previous study [30]. However, ML335 also acted as a full agonist for both MOR and DOR, and also partially recruited β-arrestin through both MOR and DOR. This suggests that there is still an unmet need to develop biased agonists that have more specific selectivity for MOR/DOR heteromers. A limitation of the present study is that we do not have data for the induction of β-arrestin recruitment on the MOR/DOR heteromer by rubiscolins, because it is not commercially available to investigate using the PathHunter® β-arrestin assay. In addition, although little evidence has been obtained on how DOR-selective agonists affect the MOR/DOR heteromer, interestingly, recent research has suggested that simultaneous treatment with MOR agonists and DOR antagonists can modulate tolerance induced by MOR agonists [48,49]. Therefore, further research is required to decipher how rubiscolins act as G-protein-biased

molecules for MOR/DOR heteromers, how they can contribute to analgesia and other effects, and to elucidate the utility of G-protein-biased MOR/DOR agonists.

Rubiscolins have the advantage of oral availability, although their absorption is not well known. In general, oligopeptides are thought to be metabolized by digestive enzymes (peptidases) and are then taken up in the form of dipeptides or tripeptides by the digestive organs, such as the stomach and small intestine. Whereas other opioid peptides are easily degraded, rubiscolins may not be disassembled and captured, and then pass through the blood–brain barrier to exhibit their antinociception [20]. They have been hypothesized to be resistant to proteolytic enzymes because of the Pro residue in the second position of their molecular structure, although it is not a characteristic feature of rubiscolins because the Tyr-Pro sequence at the N-terminus is generally present in the YP-type opioid peptides and is thought to be essential for opioid activity [21]. Thus, for the development of novel G-protein-biased DOR analgesics, further research is needed to decipher the mechanism of their uptake and to know whether some kind of active transporter is involved.

From a clinical perspective, the opioid crisis is currently a global challenge [50]. In general, opioid analgesics targeting MOR are shuffled to provide a "switching therapy", so as to balance the benefits and risks of individual opioids [51]; sometimes, a rescue dose with immediate effect is added for breakthrough pain in cancer patients. However, the kinds of opioid analgesics are limited, and their dosages often tend to increase owing to the loss in efficacy, opioid-induced hyperalgesia, or tolerance. Therefore, analgesics with safer and more effective profiles with new mechanisms of action, such as rubiscolins, appear to be promising alternatives. Hopefully, they could be used for reducing the dosage of current opioids and for resolving the opioid crisis, as part of the opioid rotation strategy, if their efficacy is proven to be on par with that of the existing opioids.

#### **4. Materials and Methods**

#### *4.1. Chemicals*

The following reagents were used: D-Ala(2)-*N*-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), (+)-4-[(a*R*)-a-((2*S*,5*R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-*N*,*N*diethylbenzamide (SNC-80), trans-3,4-dichloro-*N*-methyl-*N*-(2-(1-pyrrolidinyl)-cyclohexyl) benzeneacetamide (U-50488H), naloxone, norbinaltorphimine, forskolin, KNT-127, ML335 (Sigma-Aldrich, St. Louis, MO, USA); rubiscolin-5 (H-Tyr-Pro-Leu-Asp-Leu-OH) and rubiscolin-6 (H-Tyr-Pro-Leu-Asp-Leu-Phe-OH) were chemically synthesized by standard solid-phase peptide synthesis as described in Supplementary Materials. Forskolin was diluted with dimethyl sulfoxide (DMSO) and other chemicals were diluted with water.

#### *4.2. Cell Line*

Human embryonic kidney 293 (HEK293) cells were obtained from American Type Culture Collection (ATCC®, Manassas, VA, USA), and HEK293 cells stably expressing Halotag®-MOR, T7-tag®-DOR, Halotag®-KOR, or Halotag®-MOR/T7-tag®-DOR were generated by transfection of the constructed plasmids using Lipofectamine reagent (Life Technologies, Carlsbad, CA, USA).

#### *4.3. Cell Culture*

HEK293 cells (stably expressing Halotag®-MOR, T7-tag®-DOR, Halotag®-KOR, or Halotag®-MOR/T7-tag®-DOR) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 5 µg/mL puromycin (InvivoGen, San Diego, CA, USA) for Halotag®-MOR, 250 µg/mL hygromycin B solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for T7-tag®-DOR, or 700 µg/mL genistein (Glico, Palo Alto, CA, USA) and 100 µg/mL hygromycin for Halotag®-KOR and Halotag®-MOR/T7-tag®-DOR. The incubation was done in a humidified atmosphere with 5% CO<sup>2</sup> at 37 ◦C.
