**1. Introduction**

Opioid analgesics are widely used as key medications for relief from pain, including perioperative pain, cancer pain, and nonmalignant chronic pain. However, their use is sometimes hampered in clinical practice owing to unfavorable side effects, such as tolerance, constipation, and respiratory depression [1,2]. Thus, the discovery of safer opioid analgesics is an urgent requirement. Opioid receptors (ORs), which belong to

**Citation:** Karasawa, Y.; Miyano, K.; Fujii, H.; Mizuguchi, T.; Kuroda, Y.; Nonaka, M.; Komatsu, A.; Ohshima, K.; Yamaguchi, M.; Yamaguchi, K.; et al. In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways. *Molecules* **2021**, *26*, 6079. https://doi.org/10.3390/ molecules26196079

Academic Editors: Mariana Spetea and Richard M. van Rijn

Received: 17 September 2021 Accepted: 30 September 2021 Published: 8 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the G-protein-coupled receptor (GPCR) family [3], are classified into three subtypes—µ (MOR), δ (DOR), and κ (KOR)—and opioid analgesics mainly bind to MOR to exert their effects [4]. Internal signals from ORs are transmitted through two major pathways after the ligand conjugates with Gi/o proteins, followed by internalization of membrane receptors; the two pathways are the G-protein-mediated pathway that is required for analgesia, which is induced by decreasing the intracellular cAMP levels, and the β-arrestin-mediated pathway, which is associated with side effects [5,6]. Therefore, a biased analgesic with a pharmacological profile of favoring the activation of the G protein-mediated pathway over that of the β-arrestin-mediated pathway is desirable because it is considered to be effective and has fewer adverse events [7,8]. From this perspective, some molecules have been studied and indicated as G-protein-biased agonists in the past decades [9,10]. Among them, TRV130 (oliceridine) has been evaluated by intravenous administration in clinical studies and was approved as the first G-protein-biased agonist that can be used in clinical practice [11].

Besides MOR-selective agonists, there are several compounds selective for DOR or KOR that have been investigated in the preclinical studies [12,13]. They are expected to become alternatives for MOR agonists, which can cause severe side effects [14]. Compared with MOR agonists, DOR agonists show weaker effects in modulating acute nociception [12] but obvious effects in treating chronic pain under experimental conditions [15–17]. DOR can also be a therapeutic target for treating emotional disorders, such as depression [13,18]. However, none of the DOR agonists have been developed as an analgesic. Among the DOR agonistic compounds, rubiscolins are naturally occurring opioid peptides isolated from spinach leaves, produced by a pepsin digestion of d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant protein on earth [19,20]. Two types of rubiscolin—rubiscolin-5 and rubiscolin-6—exist, which are composed of penta- or hexa-amino acid residues (Tyr-Pro-Leu-Asp-Leu: YPLDL and Tyr-Pro-Leu-Asp-Leu-Phe: YPLDLF), respectively (Figure 1). Interestingly, these peptides showed antinociceptive effects upon p.o. administration in mice [21], which never occurs for endogenous opioid peptides. Moreover, rubiscolins are promising in terms of their unique effects other than analgesia, such as memory consolidation [22], anxiolytic effect [23], stimulation of food intake [24], enhancement of glucose uptake in skeletal muscle [25], and antidepressant-like effect [26]. Although the potency and actions of rubiscolins as G-protein-biased molecules were partially confirmed in a previous study using DOR [27], their in vitro profiles have not been sufficiently revealed.

**Figure 1.** Molecular structures of rubiscolins.

The heterodimerization of ORs is also a noteworthy aspect [28,29]. It was recently revealed that ORs form heterodimers, which play an important role in pain modulation, and the selective ligand for the µ/δ opioid receptor (MOR/DOR) heteromer induced antinociception similar to that induced by morphine, but with less tolerance [30]. MOR/DOR heteromers have been reported to increase in cultured DRG neurons under pathophysiological conditions, such as chronic pain or subsequent exposure to morphine [31], and heterodimerization appears to be related to morphine-mediated antinociception and development of tolerance [32]. Therefore, MOR/DOR heteromers can also be targets for

developing safer and more effective opioid analgesics [33,34]. We believe that it is preferable for opioid compounds to activate MOR/DOR heteromers, in addition to having a G-protein-biased property.

In the present study, we investigated the in vitro properties of rubiscolins in detail, including the agonistic or antagonistic effects for ORs and intracellular activities through the G-protein- and β-arrestin-mediated pathways, using MOR, DOR, KOR, and MOR/DOR heteromer.

#### **2. Results**
