Peptides for opioid tolerance by preventing receptor dimerization

Abstract

The object of the present invention is to provide a novel method for preventing and / or treating opioid tolerance or opioid dependence. Specifically, the object of the present invention is to develop a peptide-based blocker for preventing MOR-DOR and KOR-DOR heterodimerization. Furthermore, the object of the present invention is to develop a homodimer blocker for MOR, DOR, and KOR that modulates downstream signaling without affecting internalization. 【Means for Solving the Problem】 The present invention is a prophylactic agent and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence, comprising a peptide that inhibits dimer formation of an opioid receptor, wherein the opioid receptor dimer is a heterodimer or homodimer formed from one or two opioid receptors selected from the group consisting of a μ type (MOR), a κ type (KOR), and a δ type (DOR).

Description

Technical Field

[0001] This patent application claims priority to Japanese Patent Application No. 2022-096238, filed on June 15, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a peptide drug for suppressing the development of tolerance by blocking the homo- and hetero-dimerization of opioid receptors.

Background Art

[0003] Opioids are important for clinical pain management, but repeated use reduces their analgesic effect, which is called "tolerance". Therefore, long-term opioid therapy is associated with an increased risk of abuse, fatal overdose, dependence, and addiction, causing serious clinical and social problems. Thus, there is an urgent need for a method to suppress the development of tolerance to opioids.

[0004] The three classical opioid receptors (OPRs), μ-, κ-, and δ-OPRs (MOR, KOR, and DOR), are distributed throughout the central and peripheral nervous systems and play important roles in the regulation of pain perception, hedonic homeostasis, mood, and well-being (Non-Patent Documents 1, 2). OPRs are prototype class A G protein-coupled receptors (GPCRs) and are important receptors for various endogenous and synthetic analgesics. Among these classical OPRs, they are one of the most important druggable GPCRs. These OPR subtypes play a crucial role in the development of tolerance and dependence through signal transduction via inhibitory G proteins and arrestins, causing various levels of desirable and harmful drug responses (Non-Patent Documents 3, 4).

[0005] One approach to address issues such as opioid misuse / abuse, tolerance, intoxication, and dependence is to increase the diversity of treatments, including the regulation of OPR heterodimerization (Ong and Cahill, 2014; Non-Patent Document 27; Gaborit and Massotte BJP 2021; Non-Patent Document 28) (Schmid et al., cell 2017; Non-Patent Document 23), but the characteristics of OPR heterodimers remain elusive.

[0006] DOR and MOR (DM) heterodimers (George et al., JBC 2000; Non-Patent Document 29; Gomes and Devi, PNAS 2004; Non-Patent Document 30; Xie and Wang, JN 2009; Non-Patent Document 31; Chefer and Shippenberg, Neuropsychopharm 2009; Non-Patent Document 32; Wang et al., Neuron 2018; Non-Patent Document 33), as well as DOR and KOR (DK) heterodimers (Jordan and Devi, Nature 1999; Non-Patent Document 34; Waldhoer et al., PNAS 2005; Non-Patent Document 35; Ansonoff et al., Psychopharmacology 2010; Non-Patent Document 39; Jacobs et al., Mol Pharm. 2018; Non-Patent Document 37; Jacobs et al., Neuropharm. 2019; Non-Patent Document 38) have been found both in vitro and in vivo, while MOR and KOR (MK) heterodimers have not been detected (Jordan and Devi, Nature 1999; Non-Patent Document 34). DOR and MOR are co-expressed in lamina I projection neurons and lamina II (Wang et al., Neuron 2018; Non-Patent Document 33). The DOR-KOR heteromer was detected by co-immunoprecipitation assays in rat pain-sensing neurons (Berg et al., Mol.Pharm. 2012; Non-Patent Document 36).

[0007] The pharmacological importance of DM and DK heterodimers has been demonstrated. In the mouse central nervous system, chronic morphine treatment increased DM heteromers in pain processing (Gupta et al., 2010; Non-Patent Document 40). Furthermore, inhibition of DM heteromer formation by delivering the first transmembrane domain of MOR (MOR-TM1) reduced morphine antinociceptive tolerance, probably due to preventing DM co-degradation (He et al., Neuron 2011; Non-Patent Document 41). MOR-mediated spinal analgesia is negatively regulated by DOR activation, and opioid tolerance is reduced by pharmacological blockade or genetic deletion of DOR (Gomes and Devi, PNAS 2004; Non-Patent Document 30; Xie and Wang, JN 2009; Non-Patent Document 31; Chefer and Shippenberg, Neuropsychopharm 2009; Non-Patent Document 32). In particular, DOR and KOR signals were affected by DOR-KOR heterodimer interactions (Berg et al., Mol. Pharm. 2012; Non-Patent Document 36; Jacobs et al., Mol Pharm. 2018; Non-Patent Document 37; Jacobs et al., Neuropharm. 2019; Non-Patent Document 38).

[0008] Despite these extensive studies, the basic characteristics of DM and DK heterodimers, including lifespan, sites involved in heterodimerization, and effects on signaling and trafficking, remain unclear.

[0009] Another approach to solving problems such as opioid misuse / abuse, tolerance, toxicity, and dependence is to increase the diversity of treatments, including regulation of OPR homodimerization (Non-Patent Documents 5-8).

[0010] Monomeric OPR may be able to simply activate the signaling cascade, but it has been proposed that, for example, in the case of MOR, changes in the monomer-homodimer equilibrium induced by various agonists play an important role in regulating the relative strength of downstream signals (Non-Patent Document 9).

[0011] On the other hand, the homodimerization of OPR (and class A GPCRs) remains controversial, and the function of the homodimer is almost unknown. The first report on OPR homodimerization using biochemical assays was conducted by Cvejic and Devi in 1997 regarding DOL (Non-Patent Document 10). Numerous reports followed using biochemical assays showing the homodimerization of DOR (Non-Patent Document 11), MOR (Non-Patent Documents 7, 11, 13, 14), and KOR (Non-Patent Documents 7, 15). Furthermore, optical measurements of cells overexpressing OPR found homodimerization (Non-Patent Documents 7, 12, 16, 17), with one report (Non-Patent Document 18) being an exception.

[0012] These conclusions were challenged by five single-molecule imaging studies of OPR expressed at lower physiological concentrations in the plasma membrane (PM) of live cells. These studies found that MOR (Non-Patent Documents 9, 19, 20, 21), DOR (Non-Patent Document 21), and KOR (Non-Patent Document 22) are monomers or have very low homodimer affinity. For example, KOR is monomeric at a density of less than 10 copies / μm 2 and dimers are detectable at a density above 25 copies / μm 2 (the authors claim that this expression level is within the physiological range) (Non-Patent Document 21). These studies strongly question the interpretation of previous OPR homodimerization data obtained by biochemical methods in vitro or under overexpression conditions in the cell PM.

Prior Art Documents

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

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Non-Patent Document 17

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Non-Patent Document 36

Non-Patent Document 37

Non-Patent Document 38

Non-Patent Document 39

Non-Patent Document 40

Non-Patent Document 41

Summary of the Invention

Problems to be Solved by the Invention

[0014] Opioids are important for clinical pain management, but physiological tolerance and dependence can occur with repeated use. Clinical findings mainly show that the analgesic effect is reduced, and after continuous administration of opioids, the analgesic effect is gradually weakened or even disappears, and the same analgesic effect can only be obtained by increasing the dose of opioids. On the other hand, long-term opioid therapy is associated with an increased risk of abuse, dependence, and dose-related fatal overdose.

[0015] The first object of the present invention is to suppress the development of tolerance to morphine. Currently, morphine stands out among all analgesics, and its receptor MOR is the most important in opioid-induced analgesia and reward processing. On the other hand, it is well known that MOR-mediated analgesia is negatively regulated by heterodimerization with DOR and subsequent co-internalization of MOR and DOR. Therefore, the first object of the present invention is to develop a peptide-based blocker to prevent MOR-DOR heterodimerization.

[0016] The second object of the present invention is to develop a blocker for KOR-DOR heterodimerization. Although it is known that KOR and DOR form heterodimers, it is unknown how this affects the analgesic effects of various chemicals that bind to KOR and DOR. Considering the strong effects of MOR-DOR heterodimer blockers on the signals and internalization of MOR and DOR, KOR-DOR heterodimer blockers are potentially very useful.

[0017] The third object of the present invention is to develop homodimer blockers for MOR, DOR, and KOR that regulate downstream signal transduction without affecting internalization.

Means for Solving the Problems

[0018] The inventors of the present invention have invented peptide drugs for enhancing analgesia and suppressing the development of tolerance to morphine, which is a representative of opioid-based analgesics, and optionally other analgesics. These peptides have not been known heretofore. They function by regulating the function of opioid receptors. One of the peptides was found to suppress the development of tolerance to morphine in mice. Opioids function by binding to opioid receptors (OPRs) located on the surface of neurons in the neuronal circuits that regulate pain perception, hedonic homeostasis, mood, and well-being. There are three classical OPRs present in the body, called μ-, κ-, and δ-OPRs (MOR, KOR, and DOR, respectively).

[0019] The inventors of the present invention have discovered the homodimerization and heterodimerization of OPRs, a method of using peptide drugs to block such dimerization, and that such inhibition regulates the downstream signals of OPRs and the internal translocation of OPRs, leading to the present invention.

[0020] The present invention encompasses the following embodiments:

[0021] (1) A prophylactic and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence, comprising a peptide that inhibits the formation of a dimer, wherein the dimer is a heterodimer or homodimer formed from one or two opioid receptors selected from the group consisting of the μ type (MOR), κ type (KOR), and δ type (DOR).

[0022] (2) The preventive and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence according to (1), wherein the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 40, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 1 to 40, the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, and the amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

[0023]

Table 1

[0024] (3) The preventive and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence according to (1), wherein the dimer is a MOR and DOR heterodimer or a KOR and DOR heterodimer.

[0025] (4) The preventive and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence according to (3), wherein the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 30, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences represented by SEQ ID NOs: 1 to 30, the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, and the amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

[0026] (5) The preventive and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence according to (1), wherein the dimer is a homodimer of MOR, KOR or DOR.

[0027] (6) The prophylactic and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence according to (5), wherein the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 40, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 31 to 40, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

[0028] (7) A method for preventing and / or treating opioid tolerance or opioid dependence, comprising the step of administering a peptide that inhibits the formation of heterodimers or homodimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR) to a subject.

[0029] (8) The method for preventing and / or treating opioid tolerance or opioid dependence according to (7), wherein the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 40, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 1 to 40, the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

[0030] (9) The method for preventing and / or treating opioid tolerance or opioid dependence according to (7), wherein the peptide inhibits the formation of MOR and DOR heterodimers or KOR and DOR heterodimers.

[0031] (10) The method for preventing and / or treating opioid tolerance or opioid dependence according to (9), wherein the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 30, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences represented by SEQ ID NOs: 1 to 30, the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

[0032] (11) The method for preventing and / or treating opioid tolerance or opioid dependence according to (7), wherein the peptide inhibits the formation of homodimers of MOR, KOR or DOR.

[0033] (12) The method for preventing and / or treating opioid tolerance or opioid dependence according to (11), wherein the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 40, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 31 to 40, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

[0034] (13) A peptide comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 40, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 40, the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences, which inhibits the formation of heterodimers or homodimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR).

[0035] (14) A peptide comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 30, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 30, the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences, which inhibits the formation of MOR and DOR heterodimers, or KOR and DOR heterodimers.

[0036] (15) A peptide comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 31 to SEQ ID NO: 40, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 31 to SEQ ID NO: 40, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences, which inhibits the formation of homodimers of MOR, KOR, or DOR.

[0037] An agent for enhancing opioid analgesia, comprising a peptide that inhibits dimer formation of opioid receptors, wherein the dimer is a heterodimer or homodimer formed from one or two opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR).

[0038] (17) A method for enhancing opioid analgesia, comprising combining a peptide that inhibits the formation of a heterodimer or homodimer formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR) with an opioid, and administering the combination to a subject who needs to use an opioid, is using an opioid, or is planning to use an opioid.

Advantages of the Invention

[0039] The present invention provides a method for treating opioid tolerance by using a soluble peptide-based inhibitor against dimerization of OPR. Unlike ligand- or kinase-based inhibitors, these peptide-based inhibitors suppress morphine tolerance.

Brief Description of the Drawings

[0040]

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Mode for Carrying Out the Invention

[0041] Unless otherwise noted, all terms in the present invention have the same meaning as those commonly understood by those skilled in the art to which this disclosure pertains. The singular terms "a," "an," and "the" include plural referents unless the context indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context indicates otherwise. In this specification, molecular biological techniques can be carried out by methods described in general experimental manuals known to those skilled in the art or by methods similar thereto, unless otherwise noted.

[0042] Opioids are important for clinical pain management, but repeated use reduces their analgesic effect, which is called "tolerance." Therefore, long-term opioid therapy is associated with an increased risk of abuse, fatal overdose, dependence, and addiction, causing serious clinical and social problems. Thus, there is an urgent need for a method to suppress the development of tolerance to opioids.

[0043] The present invention is to solve these problems. The inventors have invented peptide drugs for suppressing the development of tolerance to morphine, which is representative of opioid-based analgesia, and optionally other analgesics. These peptides have not been known heretofore. They function by regulating the function of opioid receptors. One of the peptides has been found to suppress the development of tolerance to morphine in mice.

[0044] Opioids function by binding to opioid receptors (OPRs) located on the surface of neurons in the neuronal circuits that regulate pain perception, hedonic homeostasis, mood, and well-being. Three classical OPRs, called μ-type, κ-type, and δ-type-OPRs (MOR, KOR, and DOR, respectively), are present in the body.

[0045] The inventors of the present invention discovered the homodimerization and heterodimerization of OPR, a method for inhibiting such dimerization using peptide drugs, and that such inhibition regulates downstream signals and internal translocation of OPR, leading to the present invention.

[0046] (Prophylactic and / or therapeutic agents for preventing and / or treating opioid tolerance or opioid dependence) In one embodiment, the present application includes a prophylactic and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence, comprising a peptide that inhibits the formation of dimers, wherein the dimer is a heterodimer or homodimer formed from one or two opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR).

[0047] The peptide preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 40 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. More preferably, the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 40 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence consisting of an amino acid sequence having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 40 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence consisting of an amino acid sequence having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 to SEQ ID NO: 40 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences.

[0048] [Table 2]

[0049] [Table 3]

[0050] In the present application, "conservative amino acid substitution" means as follows; (i) Replacing a positively charged amino acid with another positively charged amino acid (K, R, H), and an amino acid having a polar uncharged amino acid (S, T, N, Q), and vice versa. (ii) Replacing a negatively charged amino acid with another negatively charged amino acid (D, E), and an amino acid having a polar uncharged amino acid (S, T, N, Q), and vice versa. (iii) Replacing an amino acid, among others, with a polar uncharged amino acid (S, T, N, Q), and replacing them with C, G, and P (containing a side chain with slight hydrophilicity), and vice versa. (iv) Replacing an amino acid, among others, with amino acids C, G, and P. (v) Replacing a nonpolar (hydrophobic) amino acid with another nonpolar amino acid (A, V, I, L, M, F, Y, W), and an amino acid containing a side chain with slight hydrophilicity (C, G, P), and vice versa.

[0051] In the present application, the amino acid can be either natural L-form or artificial D-form. D-form amino acids can be used at the N-terminus and C-terminus of the peptide to reduce the degradation rate of the peptide in the body. In the present application, the amino acid sequence of the chain peptide is described with the N-terminal side on the left and the C-terminal side on the right according to the conventional format of peptide representation. Further, each amino acid symbol with the symbol [D] attached immediately after the amino acid sequence indicates the D-form of the amino acid, and each amino acid symbol without the symbol [D] attached immediately after the amino acid sequence indicates the L-form of the amino acid unless contrary to the context.

[0052] In the present invention, the peptide may be modified by some or all of the amino acid residues in its amino acid sequence. Such modified peptides can be prepared by any method known in the art. For example, the modified peptide can be prepared by modification such as esterification, alkylation, halogenation, phosphorylation, sulfonation, or amidation of the functional group of the side chain of the amino acid residue(s) constituting the peptide.

[0053] In the present invention, the peptide can be fused with, conjugated to, or added to another substance. The peptide can be conjugated to or bound to a specific substance at the N-terminus and / or C-terminus of the peptide via a chemical substance such as a cross-linking agent, or via an agent suitable for linking to the side chain of an amino acid, or by synthetic chemical or genetic engineering techniques. Examples of such substances for improving the blood half-life include polyalkylene glycol molecules such as polyethylene glycol (PEG); fatty acid molecules such as hydroxyethyl starch or palmitic acid; the Fc region of an immunoglobulin; the CH3 domain of an immunoglobulin; the CH4 domain of an immunoglobulin; albumin or a fragment thereof; an albumin-binding peptide; an albumin-binding protein such as streptococcal protein G; and transferrin. Such substances can regulate the solubility of the peptide, improve the stability of the peptide such as protease resistance, or deliver the peptide to a specific tissue or organ.

[0054] In the present invention, the delivery of peptides to the brain can be accomplished by several methods, such as, among others, neurosurgical implantation into the brain, disruption of the blood-brain barrier, lipid-mediated transport, carrier-mediated influx or efflux, plasma protein-mediated transport, receptor-mediated transcytosis, absorptive-mediated transcytosis, neuropeptide transport at the blood-brain barrier, and genetic manipulation of the "Trojan horse" for drug targeting. The above methods are carried out, for example, as described in "Brain Drug Targeting: the future of brain drug development", W.M. Pardridge, Cambridge University Press, Cambridge, UK (2001).

[0055] Known techniques for enabling peptides and other products to cross the blood-brain barrier involve conjugating peptides of a well-known type. These well-known peptides are also known as cell-penetrating peptides, protein transduction domains, brain shuttles, or cell-permeable peptides, and can have, for example, 5 to 30 amino acids. Such peptides typically have a cationic charge derived from the normal representation (generally with respect to proteins) of arginine and / or lysine residues that are thought to facilitate their passage across membranes. Some such peptides have at least 5, 6, 7, or 8 arginine and / or lysine residues. Examples include the antennapedia protein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variants thereof), the TAT protein of human immunodeficiency virus, the protein VP22, the product of the UL49 gene of herpes simplex virus type 1, penetratin, SynBl and 3, transportan, amphipathic, gp41NLS, polyArg, and several plant and bacterial protein toxins such as lysine, abrin, modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat-labile toxin, and Pseudomonas aeruginosa exotoxin A (ETA).Other examples are described in the following references (Temsamani, Drug Discovery Today, 9(23):1012-1019, 2004; De Coupade, Biochem J., 390:407-418, 2005; Saalik Bioconjugate Chem. 15:1246-1253, 2004; Zhao, Medicinal Research Reviews 24(1):1-12, 2004; Deshayes, Cellular and Molecular Life Sciences 62:1839-49, 2005; Gao, ACS Chem. Biol. 2011, 6, 484-491, SG3, Stalmans PLoS ONE 2013, 8(8) e71752, 1-11 and supplement; Figueiredo et al., IUBMB Life 66, 182-194(2014); Copolovici et al., ACS Nano, 8, 1972-94(2014); Lukanowski, Biotech J. 8, 918-930(2013); Stockwell, Chem. Biol. Drug Des. 83, 507-520(2014); Stanzl et al., Accounts. Chem. Res / 46, 2944-2954(2013); Oiler-Salvia et al., Chemical Society Reviews 45:10.1039 / c6cs00076b(2016); Behzad Jafari et al., (2019) Expert Opinion on Drug Delivery, 16:6, 583-605(2019)) (all are incorporated by reference). Still other strategies use additional methods or compositions for enhancing the delivery of cargo molecules, such as PSD-95 inhibitors, to the brain (Dong, Theranostics 8(6):1481-1493(2018)).

[0056] In one embodiment, the present application includes a prophylactic and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence, comprising a peptide that inhibits the dimer formation of opioid receptors, wherein the dimer is a heterodimer formed from two opioid receptors selected from the group consisting of a μ type (MOR), a κ type (KOR), and a δ type (DOR), and wherein the dimer is an MOR and DOR heterodimer or a KOR and DOR heterodimer.

[0057] The peptide preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 30 and the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. More preferably, the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2 to 8, SEQ ID NOs: 11 to 13, SEQ ID NO: 15, SEQ ID NOs: 17 to 30 and the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. The peptide may comprise any one amino acid sequence consisting of an amino acid sequence having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 1 to 30 and in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. The peptide may comprise any one amino acid sequence consisting of an amino acid sequence having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 2 to 8, SEQ ID NOs: 11 to 13, SEQ ID NO: 15, SEQ ID NOs: 17 to 30 and in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. The peptide may comprise any one amino acid sequence having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences.

[0058] In one embodiment, the present application includes a prophylactic and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence, comprising a peptide that inhibits the dimer formation of opioid receptors, wherein the dimer is a homodimer of MOR, KOR or DOR.

[0059] The peptide preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 40, an amino acid sequence having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 31 to 40, or an amino acid sequence having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences.

[0060] Examples of the form of the prophylactic and / or therapeutic agent for preventing and / or treating opioid tolerance or opioid dependence include injections (including intravenous preparations and lyophilized preparations), sublingual preparations, nasal absorbents, transdermal absorbents, capsules, tablets, suppositories, ointments, granules, aerosols, rounds, dispersions, suspensions, emulsions, and bio-embedded preparations.

[0061] The dosage of the preparation containing the peptide of the present invention is not limited to a pharmacologically effective amount and can be determined according to the species of the individual, the type of disease, symptoms, sex, age, medical history, and other factors. Usually, it is 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. The dosage can be administered once a day, twice a day, or more than three times a day.

[0062] (Agent for enhancing opioid analgesia) In one embodiment, the present application includes an agent for enhancing opioid analgesia comprising a peptide that inhibits the dimer formation of opioid receptors, wherein the dimer is a heterodimer or homodimer formed from one or two opioid receptors selected from the group consisting of the μ type (MOR), κ type (KOR), and δ type (DOR). The above dimer is preferably a heterodimer, and more preferably a heterodimer of MOR and DOR. Morphine-induced analgesia can be enhanced by continuous administration of a peptide that inhibits the dimer formation of opioid receptors. The peptide that inhibits the dimer formation of opioid receptors can be used as an enhancer of opioid analgesia.

[0063] The use of an agent that enhances opioid analgesia can reduce the dose of opioid administered. This can prevent, ameliorate, or inhibit the development of opioid dependence and opioid tolerance. In one embodiment, the dose of opioid can be reduced by 10 wt%, 20 wt%, 30 wt%, 50 wt%, or 80 wt% compared to the standard amount administered to a patient.

[0064] The agent that enhances opioid analgesia of the present invention is used in combination with an opioid. The agent that enhances opioid analgesia of the present invention can be administered before, simultaneously with, or after the administration of the opioid.

[0065] The agent that enhances opioid analgesia can be administered to a subject in need of using an opioid, a subject using an opioid, a subject planning to use an opioid, a subject at risk of acquiring opioid tolerance, a subject having opioid tolerance, a subject at risk of opioid dependence, or a subject with opioid dependence.

[0066] The peptides, related matters, and other descriptions in the previous sections regarding prophylactic and / or therapeutic agents for the prevention and / or treatment of opioid tolerance or opioid dependence are directly applicable.

[0067] (Combined drug of opioid and the peptide of the present invention) The present invention includes a combination drug of an opioid and a peptide of the present invention. This combination drug can be a pharmaceutical composition containing the peptide of the present invention and an opioid. The pharmaceutical composition may be for sublingual administration. The combination drug of the present invention can prevent and / or treat opioid tolerance and opioid dependence in addition to the analgesic effect obtained when using an opioid alone, and can also enhance the effect of the opioid. The use of the combination drug of an opioid and a peptide of the present invention can reduce the dose of the opioid to be ingested. This can prevent, improve, or suppress the development of opioid dependence and opioid tolerance. In one embodiment, the dose of the opioid can be reduced by 10 wt%, 20 wt%, 30 wt%, 50 wt%, or 80 wt% compared to the standard amount administered to a patient.

[0068] The peptide of the present invention can be administered before, simultaneously with, or after the administration of an opioid in the combination drug of the present invention.

[0069] The combination drug of the present invention can be administered to a subject suffering from pain and a subject at risk of acquiring opioid tolerance, a subject having opioid tolerance, a subject at risk of opioid dependence, or a subject with opioid dependence.

[0070] (Method for preventing and / or treating opioid tolerance or opioid dependence) In one embodiment, the present application includes a step of administering to a subject a peptide that inhibits the formation of a heterodimer or homodimer formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR), and includes a method for preventing and / or treating opioid tolerance or opioid dependence.

[0071] The peptide preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 40 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. More preferably, the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 40 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 40 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 to SEQ ID NO: 40 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences.

[0072] In one embodiment, the present application includes a step of administering to a subject a peptide that inhibits the formation of heterodimers or homodimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR), wherein the peptide inhibits the formation of MOR and DOR heterodimers or KOR and DOR heterodimers. The present application includes a method for preventing and / or treating opioid tolerance or opioid dependence.

[0073] The peptide preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 30 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide more preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 30 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 30 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 30 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences.

[0074] In one embodiment, the present application includes a step of administering to a subject a peptide that inhibits the formation of a heterodimer or homodimer formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR), wherein the peptide inhibits the formation of a homodimer of MOR, KOR, or DOR. A method for preventing and / or treating opioid tolerance or opioid dependence is provided.

[0075] The peptide preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 40, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 31 to 40, and amino acid sequences having at least 80%, 90%, 95%, or 99% amino acid sequence identity to any of these amino acid sequences.

[0076] (Method for enhancing the analgesic effect of opioids) In one embodiment, the present application includes a step of administering to a subject a peptide that inhibits the formation of a heterodimer or homodimer formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR) opioid receptors, and includes a method for enhancing the analgesic effect of opioids.

[0077] The method for enhancing opioid analgesia can reduce the dose of opioid to be ingested. This can prevent, improve, or suppress the development of opioid dependence and opioid tolerance. In one embodiment, the dose of opioid can be reduced by 10 wt%, 20 wt%, 30 wt%, 50 wt%, or 80 wt% compared to the standard amount administered to the patient.

[0078] The peptide that inhibits the formation of a heterodimer or homodimer formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR) opioid receptors can be administered before, simultaneously with, or after the administration of the opioid in the method for enhancing the analgesic effect of opioids.

[0079] A peptide that inhibits the formation of heterodimers or homodimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR) opioid receptors can be administered to a subject in need of using an opioid, a subject using an opioid, a subject planning to use an opioid, a subject at risk of acquiring opioid tolerance, a subject having opioid tolerance, a subject at risk of opioid dependence, or a subject with opioid dependence in a method of enhancing the analgesic effect of an opioid.

[0080] The description of the peptide in the previous section regarding methods of preventing and / or treating opioid tolerance or opioid dependence is directly applicable.

[0081] (Peptide) In one embodiment, the present application includes a peptide comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 40 and the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. More preferably, the peptide comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2 to 8, SEQ ID NOs: 11 to 13, SEQ ID NO: 15, SEQ ID NOs: 17 to 40 and the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 1 to 40 and in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs: 2 to 8, SEQ ID NOs: 11 to 13, SEQ ID NO: 15 and SEQ ID NOs: 17 to 40 and in the amino acid sequences of SEQ ID NOs: 41 to 67 listed in Table B. The peptide may comprise any one amino acid sequence having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences. The peptide of the present invention inhibits the formation of hetero- or homo-dimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR), and the peptide of the present invention is effective in the prevention and / or treatment of opioid tolerance or opioid dependence. Furthermore, the peptide of the present invention is effective in enhancing the analgesic effect of opioids.

[0082] In one embodiment, this application preferably includes a peptide comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 30 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide more preferably comprises any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 30 and the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 30 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence selected from the group consisting of amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8, SEQ ID NO: 11 to SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 30 and in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 67 listed in Table B. The peptide may comprise any one amino acid sequence having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences. The peptide of the present invention inhibits the formation of MOR and DOR heterodimers or KOR and DOR heterodimers.

[0083] In one embodiment, this application preferably includes a peptide comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 31 to SEQ ID NO: 40, amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NO: 31 to SEQ ID NO: 40, and amino acid sequences having at least 80%, 90%, 95%, or 99% amino acid sequence identity with any of these amino acid sequences. The peptide of the present invention inhibits the formation of MOR, KOR, or DOR homodimers.

Example

[0084] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by these examples.

[0085] <Example 1> Heterodimer blocker DM and DK heterodimers, not MK heterodimers The fluorescently tagged OPR expressed in the PM of CHO-K1 cells used by the inventors throughout this study was functional (expansion data figure 1, labeling efficiency greater than 75%) and was observed at the single molecule level in two colors at 37 °C at video rate (30 Hz) (conditions used throughout this study). Almost all OPR fluorescence spots showed diffusion in the PM, and when DOR and MOR (and DOR and KOR) were expressed in the same cell, they showed frequent transient co-localization and co-diffusion, consistent with transient heterodimer formation (Figure 1-1a). For a simple quantification of the tendency of OPR heterodimer formation, the inventors used a co-localization index, a parameter based on the pair cross-correlation function (PCCF of green and magenta spots), which is a strategy to find molecule B (e.g., green spot) within a circle of radius 100 nm around molecule A (e.g., magenta spot) at a greater distance of 400 - 500 nm (as a negative control, rotate the video frame in the green channel by 180° and then overlay; expansion data figure 1-2). The co-localization index showed DM and DK heterodimerization, but not KM dimerization (Figure 1-1b).

[0086] The heterodimer lifetime was determined by the following method. Each time the inventors detected a co-localization event of spots having different colors, they measured the duration thereof, and after a sufficient number of co-localization durations were obtained (events exceeding 3,000 times in 20 or more cells), the inventors created a distribution of the co-localization durations (histogram; Fig. 1-1c). The inventors found that, as predicted from the theory described in the relevant papers, the histogram could be fitted by the sum of two exponential decay functions (shorter decay time constant and longer decay time constant; τ1 and τ2, respectively), which indicated that τ1 represents the lifetime that non-related molecules accidentally follow together, and τ2 provides the heterodimer lifetime (the lifetime of the actual binding of the two molecules, according to the theory developed by the inventors): 260 ± 11 milliseconds for the DM heterodimer and 240 ± 10 milliseconds for the DK heterodimer after correction for the photobleaching lifetime (Fig. 1-1c; Extended Data 3). The histogram for the MK co-localization duration showed no τ2 component, which was consistent with the lack of detectable heterodimers for this pair (only accidental co-localizations with the lifetime of τ1. Fig. 1-1c). These results are summarized in Fig. 1-1d.

[0087] Since MK heterodimers were undetectable under the observation conditions of the inventors, these results suggest that the amino acid (aa) sequences involved in heterodimerization may be mainly located in specific OPR domains having lower sequence identity / homology among the three OPRs. The candidate sites are summarized in Fig. 1-1e. Although the aa sequence homology in the cytoplasmic C-terminal domain is lower, since they are involved in OPR homodimerization, they are not included in this figure.

[0088] The EL3 domain is involved in DK dimerization As shown in Fig. 1-1e, the aa homology in the N-terminal and C-terminal domains of OPR is low, suggesting that these domains may be involved in DK heterodimerization. Therefore, the inventors generated N-terminal and C-terminal deletion mutants of DOR and KOR and examined their heterodimerization with wild-type (WT) KOR and DOR, respectively (Fig. 1-2a). They were unable to affect DK heterodimerization, indicating that the N-terminal and C-terminal domains are not involved in this process.

[0089] Since MK heterodimerization did not occur, the inventors replaced various MOR domains with the corresponding DOR domains (Fig. 1-1e) and examined which DOR domains in MOR could induce dimers with KOR (Fig. 1-2b left). As previously suggested (Filizola and Weinstein, Protein Eng 2002; Liu and Wang, R.J.Computer-Aided Mol Design 2009), replacing the fourth transmembrane domain (TM4) slightly increased the co-localization index, while replacing extracellular loop 2 (EL2) did not increase the index. However, replacing the extracellular loop 3 (EL3) of MOR with that of DOR increased the co-localization index with KOR to a level close to that for DK heterodimerization (Fig. 1-2b and Fig. 1-1b), indicating that EL3 of DOR plays an important role in the interaction with KOR. Furthermore, the inventors replaced various DOR domains with the corresponding MOR domains and examined which MOR domain(s) in DOR could block the interaction with KOR (Fig. 1-2b right). DK heterodimerization was significantly reduced only when EL3 of DOR was replaced with EL3 of MOR, indicating that EL3 of DOR plays an important role in DK dimerization.

[0090] Furthermore, dimerization could not be induced by replacing the N-terminal domain of MOR with that of DOR (Figure 1-2b, lower left), and DM heterodimerization could not be blocked by replacing the N-terminal domain of DOR with that of MOR (Figure 1-2b, lower right). These results indicate that the N-termini of DOR and KOR are not involved in DK heterodimerization, which is consistent with the N-terminal deletion data.

[0091] The decay histogram for MOR(EL3-DOR)-KOR showed a substantial proportion of the τ2 component when compared to the decay histogram for WT-MOR-WT-KOR, indicating a crucial involvement of DOR's EL3 in DK heterodimerization (Figure 1-2c). On the other hand, the τ2 value for MOR(EL3-DOR)-KOR was not extended to that of WT-DOR-WT-KOR, suggesting the existence of other interaction sites for the DK pair in addition to EL3 (Figure 1-2c).

[0092] EL3-based peptides block DK heterodimers To further investigate the involvement of DOR's EL3 in DK dimerization and to determine whether KOR's EL3 is also involved, the inventors produced soluble peptides containing their EL3 aa sequences (Figure 1-1e) and added them to cells stably co-expressing DOR and KOR (1 μM, final concentration). Both peptides significantly reduced DK dimerization, while the peptide containing the MOR EL3 sequence did not (Figure 1-2d). These results indicate that EL3 is highly involved in DK dimerization.

[0093] However, since the colocalization index did not reach 1 (no colocalization except for accidental colocalization), these results suggest that other domains, such as transmembrane domains, may also be involved in DK dimerization, as previously suggested (Jacobs et al., Mol. Pharm. 2018). To test this possibility, the inventors examined the dimer formation of KOR with TM1 and TM4 of DOR (Figure 1-2e). These DOR TM domains did show a slight tendency to form heterodimers with KOR.

[0094] The N-terminal domain is involved in DM dimerization The first transmembrane domain (TM1) of MOR has been reported to be involved in DM heterodimerization (He et al., Neuron 2011). However, in cortical membranes prepared from wild-type mice, monoclonal antibodies specifically recognized DM heterodimers bound to the cell surface and blocked the DOR antagonist-mediated increase in MOR agonist-mediated signaling (Gupta et al., Sci. Signal. 2010), suggesting that the extracellular domains of MOR and DOR may be involved in DM heterodimerization. Since the aa homology in the N-terminal domain of OPR is low (Figure 1-1e) and the N-terminal domain is not involved in DK heterodimerization, the inventors examined the involvement of the N-terminal domains of both DOR and MOR in DM heterodimerization. First, the inventors systematically deleted partial sequences from the N-terminal domains of DOR and MOR. The inventors found that aa22-27 of DOR and aa32-61 of MOR play important roles in DM heterodimerization, i.e., these domains bind to each other to form DM dimers (Figure 1-3a). Consistently, the histograms of the colocalization duration for DORΔ22-42 with WT-MOR and for MORΔ32-61 with WT-DOR both showed the presence of only a small percentage of the τ2 component, and the dimer lifetime was much shorter (88 ± 12 milliseconds and 91 ± 8 milliseconds, respectively; Figure 1-3b).

[0095] However, the inventors noted that the inhibition by the deletion mutants was incomplete in all cases. The co-localization index decreased to 1.1 - 1.2, but did not completely decrease to 1.0. Therefore, the inventors further utilized the fact that MK heterodimerization did not occur. Thus, the inventors replaced various DOR domains with the corresponding KOR domains (Figure 1-1e) and examined which KOR domains in DOR could induce dimers with KOR (Figure 1-3c). By replacing the N-terminal domain of DOR with the N-terminal domain of KOR, the co-localization index with MOR was significantly reduced (Figures 1-3c and 1-1b), which was consistent with the result that DM heterodimerization was mediated by the N-terminal domains of both DOR and MOR (Figures 1-3a and b). By replacing TM1 or TM4 of DOR with the corresponding KOR TM, the co-localization index when using WT-MOR decreased slightly, but did not decrease with TM6. Replacing the TM1 domain of MOR with the TM1 domain of KOR also slightly reduced the co-localization index when using WT-DOR (the main interaction site of the DK pair is the EL3 domain; Figures 1-3c and 1-2). These results indicate that, despite their very high homology, TM1 and TM4 of DOR interact with MOR, and TM1 of MOR interacts with DOR, as predicted by computational modeling (Filizola and Weinstein, Protein Eng 2002; Liu and Wang, R.J.Computer-Aided Mol Design 2009) and physiological assays (Jacobs et al., Mol.Pharm.2018), respectively. However, the effectiveness of TM-TM interactions for dimerization seems to be weaker than that of N-terminal interactions and EL3 interactions, respectively.

[0096] In previous co-immunoprecipitation assays, the aa53-99 sequence of MOR, which contains a part of the N-terminal region (aa53-70), TM1 of MOR (aa71-94), and five additional amino acids following TM1 (95-99) (the authors refer to this as the TM1 region of MOR, but the present inventors will refer to it as N-term+TM1 to avoid confusion), was found to be involved in DM heterodimerization (He et al., Neuron 2011). This result is consistent with the present inventors' observation that replacement of the TM1 domain of MOR with the TM1 domain of KOR slightly reduced DM heterodimerization (DK interacts mainly through their EL3 domains).

[0097] Peptide-based DM heterodimer blocker The aa sequences deleted from or replaced in the wild-type (WT) OPRs used to obtain the results shown in Figures 1-2a-c may not represent the true interaction sites for heterodimerization. Instead, inhibition of heterodimerization by deletion (N-terminal) or replacement (TM) mutations may be induced by conformational changes in the true interaction sites caused by these deletions and replacements.

[0098] To investigate these possibilities, first, the present inventors produced soluble peptides containing the same aa sequences as the deleted parts of the mutants and added them to cells stably co-expressing WT-DOR and MOR. DOR (MOR) peptides with aa sequences that may be involved in DM dimerization were called Dpep(m-n)DM (Mpep(m-n)MD) (where m and n indicate the amino acid numbers in the original OPR sequence) (Figure 1-3d). Various DpepDM and MpepMD reduced the co-localization index, and the greatest reduction was seen when using Dpep(20-42)DM and Mpep(32-61)MD, which was consistent with the results obtained using deletion mutants (Figure 1-3a). The dependence of the co-localization index shown in Figure 1-3d (upper right) on the Dpep(20-42)DM concentration is the affinity of the D-M heterodimer (K D) shows that it is less than 0.1 μM in 3D. However, this could not be easily converted into a number in 2D where the actual DM interaction mainly occurs. The addition of Dpep(20 - 42)DM reduced the heterodimer lifetime to 124 milliseconds (from 260 milliseconds in its absence) without varying the proportion of τ2 (41%) (Figure 1 - 3d, lower right), supporting DM dimerization via aa20 - 42 in DOR.

[0099] Next, the inventors expressed the N - term + TM1 of MOR and the TM1 of MOR (TM1, 4, and 5 of DOR) conjugated with the SNAPf tag, as previously suggested (Filizola and Weinstein, Protein Eng 2002; Liu and Wang, R.J.Computer - Aided Mol Design 2009), and investigated whether they could interact with WT - DOR (WT - MOR) and compete with the DM interaction (Figure 1 - 3e - g). Both the N - term + TM1 of MOR and the TM1 of MOR co - localized with DOR and WT - MOR. On the other hand, TM1, TM4, and TM5 of DOR showed co - localization with MOR, but to a much lower extent than WT - DOR (Figure 1 - 3f). Both the N - term + TM1 of MOR and the TM1 of MOR decreased DM co - localization, but N - term + TM1 competed more effectively with the DM dimer (Figure 1 - 3g). TM4 of DOR competed with DOR for DM binding, but with lower efficiency (Figure 1 - 3g). These results indicate that for DM heterodimerization, both the N - terminal domains of DOR and MOR are important, but the TM1 domain of MOR that can interact with the TM1, 4, and 5 domains of DOR also plays a role.

[0100] Agonists variously affect DM heterodimerization Next, the inventors examined how inhibition of DM heterodimerization by a peptide blocker affects agonist-induced intracellular signaling and internalization of DOR and MOR (Figure 1-4a). As representative agonists, the inventors used the MOR-agonist DAMGO and morphine (0.5 μM (Yekkirala et al., ACS Chemneuro. 2009)), and the DOR agonist SNC-80 (0.5 μM (Metcalf et al., ACS Chemneuro. 2012)), which bind specifically and rapidly to MOR and DOR on the cell surface. The inventors focused on the initial agonist effects without the excessive complications due to subsequent signaling events and thus observed DM heterodimerization within 5 minutes after agonist addition. DAMGO (morphine)-bound MOR showed more (less) heterodimerization with DOR, i.e., a higher (lower) co-localization index (Figure 1-4b), along with an extended (shortened) heterodimer lifetime (Figure 1-4c). In the presence of Dpep(20-42)DM, the co-localization index and lifetime of heterodimers regarding DOR and agonist-bound MOR (both for DAMGO and morphine) were significantly reduced (Figure 1-4b, c). On the other hand, after addition of 0.5 μM SNC-80, DOR internalization occurred very rapidly (Figure 1-4d), thereby preventing the inventors from determining the co-localization index and heterodimer lifetime of MOR and SNC-80-bound DOR.

[0101] Peptide blockers suppress MD co-internalization In cells stably expressing only MOR, only DOR, and both MOR and DOR (referred to as M cells, D cells, and MD cells, respectively), OPR internalization at a low physiological expression level of approximately 1 copy / μm was monitored by using a membrane-impermeable fluorescent quencher and evaluating the percentage of OPR molecules remaining in the PM (see methods). 2

[0102] The time course of the number of OPRs remaining in the PM was examined both in the presence and absence of 0.5 μM agonist and 10 μM DpepDM in the medium (Figure 1-4d). The time course of MOR / DOR internalization could be operationally fitted by a single exponential function, providing the percentage of internalized MOR / DOR detectable by 60-minute observation and their residence lifetime in the PM.

[0103] MOR internalization was hardly detectable without agonist addition in M cells and MD cells. Addition of the MOR agonist DAMGO induced MOR internalization more effectively in MD cells (51.1%) than in M cells (31.8%). DOR internalization was induced by the MOR agonist DAMGO in MD cells (13.3% vs. 4.3% without DAMGO addition) (Figure 1-4d, upper panel; Supplementary Tables 1 and 2), suggesting co-internalization of DOR and MOR. However, morphine (another MOR agonist) had little effect on any of these internalization processes (Figure 1-4d, middle panel). These results are consistent with previous observations (Derouiche et al., Molecules 2020). Addition of Dpep(20-42)DM to MD cells significantly reduced DAMGO-induced internalization of both MOR and DOR (both percentage and rate), probably due to suppression of DM co-internalization (He et al., Neuron 2011).

[0104] Addition of the DOR agonist SNC-80 induced dramatic DOR internalization in D cells and MD cells, as well as MOR internalization in MD cells, similarly suggesting the occurrence of MD co-internalization (Figure 1-4d, lower panel; Supplementary Tables 1 and 2). Addition of Dpep(20-42)DM to MD cells significantly reduced SNC-80-induced internalization of both MOR and DOR (both percentage and rate), probably due to suppression of DM co-internalization.

[0105] The DM heterodimer enhances MOR signaling Agonist-induced cytoplasmic signals are Ca 2+Evaluated by monitoring mobilization (Fig. 1-4e, f; see methods). Addition of DAMGO and morphine induced intracellular Ca 2+ mobilization in M cells, but not in D cells and K cells, and Ca 2+ mobilization was significantly enhanced in MD cells. The presence of Dpep(20-42)DM in the medium of MD cells alleviated the enhancement of Ca 2+ signals in the presence of DOR in MD cells. These results indicate that DM heterodimerization enhanced MOR-agonist-induced MOR signals.

[0106] Soluble heterodimer blockers suppress morphine tolerance The soluble DM heterodimer blocker peptide Dpep(20-42)DM reduced the expression of antinociceptive tolerance to morphine in mice when examined by the tail-flick test. The peptide dissolved in artificial cerebrospinal fluid (aCSF) was continuously applied to the lateral ventricle for 2 days (10 μg / day) prior to the daily subcutaneous administration of morphine (10 mg / kg / injection; 1 injection / day) (Fig. 1-4g). The analgesic effect of morphine was promoted by Dpep(20-42)DM on all days (days 1, 5, 8, and 11; Extended Data Fig. 6) between 60 and 120 minutes after morphine application. The difference from the peptide group became approximately three-fold at 120 minutes after morphine application on day 11 compared with the decrease in the analgesic effect of morphine in the aCSF group during the experiment (Fig. 1-4h). Soluble heterodimer blockers suppress morphine tolerance.

[0107] Furthermore, application of Dpep(20-42)DM reduced tolerance to morphine. Eight days after morphine treatment, the analgesic effect of morphine was reduced in control aCSF-treated mice. In contrast, the antinociceptive effect of morphine in peptide-treated mice was maintained at approximately 70% of the initial efficacy for 8 - 11 days (Fig. 1-4i). These results suggest that peptide-induced disruption of DM dimers prevented the expression of antinociceptive tolerance to morphine in the brain.

[0108]

Table 4

[0109] The toxicity of DpepDM and DpepDM-TAT was evaluated in T24 cells, a human bladder cancer cell line, after 48-hour exposure at the concentrations administered to the cells (50 μM, 100 μM, and 200 μM) according to the manufacturer's specifications using Abcam's LDH assay kit. Cytotoxicity was measured by subtracting the LDH content in the remaining viable cells from the total LDH in the untreated control. DpepDM and DpepDM-TAT showed no toxicity to T24 cells even at a very high concentration of 200 μM (Figure 1-4j).

[0110] The toxicity of DpepDM and DpepDM-TAT was evaluated in primary cultures of hippocampal neurons (DIV8, DIV12, and DIV14) after 48-, 96-, and 144-hour exposure at a concentration of 100 μM administered to the cells according to the manufacturer's specifications using Abcam's LDH assay kit. Treatment of neurons with Trition-100 (1%) and DMSO (10%) for 48 hours was used as a positive control. DpepDM and DpepDM-TAT showed little toxicity to neurons even at a very high concentration of 100 μM (Figure 1-4k).

[0111] Surprisingly, the peptides of the present invention showed no toxicity even at very high concentrations of 100 μM and 200 μM, whereas general synthetic peptides usually show strong toxicity at concentrations of 10 - 50 μM (Front. Microbiol. 11:1146. doi:10.3389 / fmicb.2020.01146; Scientific Reports, (2018) 8:1763, DOI:10.1038 / s41598-018-19434-7).

[0112] Discussion Here, using advanced single-molecule imaging and analysis, we have approximately 1 copy / μm 2The formation of DM and DK heterodimers and the absence of KM dimers were confirmed at the physiological expression levels. Quite unexpectedly, both DM and DK heterodimers are transiently metastable dimers of approximately 250 milliseconds each, and are continuously formed and dispersed as seen in homodimers of other GPCRs and OPRs. As important interaction sites, in addition to various TM domain interactions discovered / proposed so far by the inventors, for the DM dimer, aa20 - 42 of DOR and aa32 - 61 of MOR in the N - terminal domain, and for the DK dimer, the short EL3 of DOR and KOR were identified. These results provide sites for both stronger specific interactions and weaker basic interactions, as suggested in the "rolling interface" model (Dijkman et al. Nat. Commun. 2018; Johnston et al. Biochem. 2011), and can support heterodimerization (He et al. Neuron, 2011).

[0113] These dimerization sites located in the extracellular domain of OPR were confirmed by peptides having the aa sequences identified as binding sites. These results are consistent with the development of heterodimer - selective antibodies that suppress DM heterodimerization by binding to the extracellular domain (Gomes and Devi, PNAS 2013). The Dpep(20 - 42)DM peptide blocker reduced DM co - internalization. The agonist - induced MOR signal was significantly enhanced upon co - expression with DOR as expected from previous reports (Gomes et al., JN 2000; Yekkirala et al., ACS Chem. Neurosci. 2010). The inventors found that this DOR - induced signal enhancement was suppressed by the addition of Dpep(20 - 42)DM.

[0114] Dpep(20 - 42)DM suppressed the development of morphine tolerance in mice. In previous studies where the N-term+TM1 of MOR linked to the TAT peptide was applied systemically by very acute intraperitoneal injection (three injections within 2.5 hours, 10 mg / kg / injection), it was revealed that the injected membrane-inserting peptide disrupted the DM interaction in the mouse spinal cord and reduced the antinociceptive tolerance to morphine (He et al. Neuron 2011). The DM dimer-blocking peptide Dpep(20 - 42)DM developed in the present invention is much smaller and soluble, and thus can be used complementarily with the previous N-term + TM1 linked to the TAT peptide.

[0115] Previously, the development of antinociceptive tolerance to morphine was thought to be due solely to the downregulation of MOR by internalization enhanced by the DM interaction. However, morphine-induced MOR internalization can be very limited as shown herein (Figure 1 - 4d) and in previous studies (Moller, J. et al. Nat. Chem. Biol. 2020). On the other hand, morphine-induced cytoplasmic signals are enhanced by the co-expression of MOR and DOR in cells (Figure 1 - 4f). Therefore, the development of antinociceptive tolerance to morphine may also involve the downstream signals of MOR. These results support an OPR drug administration strategy to reduce the development of tolerance, toxicity, and dependence to morphine and other analgesics by reducing OPR heterodimer formation using heterodimerization-blocking drugs containing blocking peptides (Gupta et al., Sci. Signal. 2010; He et al. Neuron 2011).

[0116] <Example 2> Homodimer blocker Method cDNA construction All external sources, including newly generated cDNA constructs and constructs from donor and commercial sources, were examined and confirmed by DNA sequencing. The cDNAs encoding rat KOR and DOR were a gift from Professor H. Takeshima of Kyoto University. The cDNA encoding rat MOR tagged with GFP was a gift from Dr. R. Schulz of the University of Munich, Germany. 54 mCherry was a gift from Professor R. Y. Tsien of the University of California, San Diego. 55 The cDNAs encoding SNAPf and mGFP(A206K) were obtained from Promega and Clontech, respectively. When the tag protein SNAPf was attached to the N-terminus of OPR, an additional signal sequence of interleukin 6 was attached to the N-terminus of the tag protein, and a 21-amino acid linker (SGGGSGG×3) was inserted between OPR and the tag protein. A cDNA encoding an OPR point mutant was generated using a site-directed mutagenesis kit (New England Biolabs). For details of the cDNA constructs containing the linker sequence, see Figure 2-s10.

[0117] Cell culture, transfection, and microscopy CHO-K1 cells (Dainippon Pharma) and T24 cells (a gift from Professor M. Sokabe of Nagoya University) were confirmed to be free of mycoplasma contamination by MycoAlert (Lonza). 56was routinely cultured in Ham’s Nutrient Mixture F12 (Sigma-Aldrich) supplemented with 10% (v / v) fetal bovine serum (FBS, Life Technologies), 100 units / ml penicillin, and 0.1 mg / ml streptomycin in an incubator at 37 °C in a 5% CO2 atmosphere. Transfection of CHO-K1 cells with the cDNA of interest was performed by electroporation according to the manufacturer's instructions (4D-Nucleofector, Lonza; SF Cell Line solution and program CHO-K1 for CHO-K1 cells). The transfected cells were seeded in glass-based dishes (35 mm in diameter with a 12 mm diameter glass window, 0.15 mm thick glass; Iwaki, Tokyo; 2×10 5 cells / dish), cultured for 24 - 48 hours, and then observed under a fluorescence microscope. All microscopic observations were performed at 37 °C by placing the entire microscope, except for the distal ends of the excitation and detection arms, in a custom-made microscope environmental chamber made of a heat and electric field insulating plastic sheet and equipped with four heating circulators (SKH0-112-OT, Kokensya, Tokyo, Japan). The Ham’s F12 medium used for microscopic observation did not contain sodium bicarbonate and phenol red and was buffered with 2 mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES, Sigma-Aldrich) at pH 7.4 (referred to as Ham’s F12 observation medium).

[0118] Fluorescent labeling of OPR expressed on the cell surface (Figure 2-s2) Wild-type and mutant OPR with SNAPf tag expressed in PM (the SNAPf tag is located at the extracellular N-terminus) were covalently conjugated with SNAP-Surface 549 (New England Biolabs) and SNAP-CF660R (Shinsei Kagaku) by incubating the cells simultaneously with these fluorescent SNAP ligands at 300 nM in growth medium in a CO2 incubator at 37 °C for 30 minutes. The cells were washed three times with fresh medium (5-minute incubation each time), followed by the addition of Ham’s F12 observation medium. The labeling efficiency was determined by using SNAPf-CD47-mGFP protein expressed in PM. CD47 is a monomeric five-transmembrane protein expressed in PM 37 。Since the inventors could not use OPRs for this purpose because they form transient dimers, monomeric CD47 was used instead. Under the conditions used in the present invention, the inventors achieved labeling of more than 70%: 74 ± 9% for SNAP-Surface 549 and 70 ± 8% for SNAP-CF660R.

[0119] Single fluorescent molecule imaging and tracking in living cells 1 μm 2 Fluorescently labeled OPR expressed in the bottom PM (PM facing the coverslip) with a fluorescence spot number density of 0.5 - 1.5 per μm (total number of two-color spots) was observed at the single molecule level at 37 °C using a custom-built objective lens type TIRF microscope constructed on an inverted microscope (Olympus IX-83) equipped with a 100x 1.49 numerical aperture (NA) objective lens optimized for this study, based on the equipment used so far 57、58 。OPR tagged with a fluorescent probe was excited by TIR illumination using the following power densities: 0.35 μW / μm 2 of 561 nm (Coherent OBIS 561-100 LS) for SNAP-Surface 549; and 0.52 μW / μm 2SNAP-CF660R at 642 nm (Omicron LuxXPlus 640-140). The two-color images were separated by a dichroic mirror and projected onto two detection arms equipped with band-pass filters of 500.0 - 550.0 nm (ET525 / 50m; Chroma) for mGFP, 572.5 - 647.5 nm (ET600 / 50m; Chroma) for the SNAP-Surface 549 dye, and 662.5 - 737.5 nm (ET700 / 75m; Chroma) for the NAP-CF660R dye. Under these conditions, the localization precision (standard deviation assuming a Gaussian distribution) for single dye molecules in living cells was 60 nm when estimated from the PCCF. The fluorescence signals in each channel were first detected and amplified by a two-stage microchannel plate image intensifier (C9016-02MLP24; Hamamatsu Photonics), and the intensified images were projected onto a scientific CMOS camera (C11440-22CU; Hamamatsu Photonics) operating at 30 Hz, and they were synchronized with the same intensifier-camera set placed in the other detection arm. The image sequences in each channel were overlaid after correction of spatial distortion as described previously 57 . The positions (x and y coordinates) of all observed single fluorescent molecules were determined by a in-house computer program as described previously 59 .

[0120] Evaluation of the duration of co-localization Co-localization of two fluorescent molecules was defined as the event that two fluorescent spots representing these molecules become localized within 200 nm of each other as described previously 23、57 . Briefly, in the single-color experiment using SNAP-Surface 549 (Figure 2-s3), cross-correlation analysis was used 23Using this method, the inventors found that when two fluorescent spots, each representing a single molecule of SNAP-Surface 549, are located in close proximity, the threshold distance for identifying one or two spots occurs at 200 nm in this experimental setup. Using this definition, co-localized trajectories were obtained and the co-localization duration was estimated. In two-color single fluorescent molecule tracking simultaneous experiments using dye pairs, the distance between the two molecules was directly measured from the positions (x, y positions) of each molecule (having different colors). Even when examining pairs of molecules of different colors known to be truly associated, the probability of scoring that two molecules are associated is limited by the localization accuracy of each molecule and the accuracy of overlaying the two images. The methods developed so far 57 Based on the accuracy determined by the present invention, the inventors found that for truly associated molecules, when using the criterion that the molecules are within 200 nm of each other, the probability of scoring that two molecules are associated increases to 99%. Therefore, the inventors used this criterion as the definition of co-localization in two-color single molecule simultaneous observation. This distance of 200 nm was consistent with the definition of co-localization in monochromatic experiments. Due to this consistency, in this study, the inventors defined the co-localization of two fluorescent molecules as an event in which two fluorescent spots representing these molecules become localized within 200 nm of each other.

[0121] Each time the inventors found a green-magenta pair (co-localized) located within 200 nm, they measured the duration (co-localized duration) for which their distance remained within 200 nm. After obtaining the co-localization duration for all co-localization events, the inventors created a histogram (distribution) of the co-localization duration. First, by overlaying the magenta image sequence and the green image sequence rotated 180 degrees (double flipped), the distribution of accidental co-localization durations was obtained (Figure 2-s4b). The inventors found that the distribution could be fitted by a single exponential function, and the decay time constant was the accidental co-localization lifetime τ inciIt was found to represent. Next, for the accurately superimposed magenta and green image sequences, the distribution of the co-localization duration was obtained. This distribution can be fitted by the sum of two exponential decay functions (τ1 and τ2, τ1 < τ2). These results are consistent with the theory developed in the present invention that predicts the distribution of the co-localization duration based on the diffusion equation. Since τ1 was almost the same as τ inci (Fig. 2-1d), τ2 provided the co-localization lifetime or the homodimer lifetime (after correction for the photobleaching lifetimes of the two fluorescent probes; Fig. 2-s6). The photobleaching lifetimes (τ bleach ) of the fluorescent probes for 30 Hz observation adsorbed on the cover glass of the glass-based dish were 16.3 ± 1.2 s (n = 500) for SNAP-Surface 549 and 7.8 ± 0.6 s (n = 400) for SNAP-CF660R (Fig. 2-s6), and the correction was performed by using the equation τ(corrected)=[τ(observed) -1 - τ bleach (dye 1) -1 - τ bleach (dye 2) -1 -1 .

[0122] Evaluation of the co-localization index: Quantitative measurement of co-localization For the quantitative evaluation of the degree of co-localization (representing both the frequency and lifetime of co-localization events) in two-color single molecule simultaneous imaging videos, the inventors defined a parameter called the co-localization index (for the results obtained by using two dye molecular species with different excitation / emission wavelengths, the inventors refer to them as green and magenta probes / videos for convenience in this report). This analysis method is essentially based on pair cross-correlation analysis 39 , and the detailed method used in this study is explained in Fig. 2-s4.

[0123] Monte Carlo simulations to examine the dependence of the co-localization index and the pair cross-correlation function (PCCF) on the number density of fluorescent spots (expression level in PM; Fig. 2-s5) ​The co-localization index depends on the number density of fluorescent spots in the PM. In this study, the inventors selected cells showing the number density of fluorescent spots in the PM of 0.5 - 1.5 spots / μm 2 Thus, the inventors used Monte Carlo simulation to examine the extent to which the co-localization index depends on the number density of fluorescent spots (Fig. 2-s5). At the same time, the inventors developed a theory for evaluating the dimer - monomer dissociation equilibrium constant K D from PCCF and the total number of spots in the image.

[0124] Treatment of cells with agonists and FAM-Xpep-TAT For the agonists U-50488, DAMGO, and SNC-80 (Sigma-Aldrich), which are agonists for KOR, MOR, and DOR, respectively, they were dissolved in DMSO (2 mM) and then diluted to a final concentration of 200 μM with Hank's balanced salt solution (HBSS, Nissui) buffered with 2 mM TES at pH 7.4 (T-HBSS). The agonist solution (1 μl) was added to the cells in 1 ml of Ham's F12 observation medium (final concentration 200 nM).

[0125] For the intracellular uptake of FAM-Xpep-TAT (custom synthesized by Cosmo-Bio), the cells were first incubated with 150 μM pyrenebutyric acid (Sigma-Aldrich) in T-HBSS at 37 °C for 5 minutes, and then 2 mM FAM-Xpep-TAT in T-HBSS was added at a final peptide concentration of 20 μM. After incubation at 37 °C for 10 minutes, the cells were washed 3 times with T-HBSS and then fresh Ham's F12 observation medium was added to the cells. The presence of FAM-Xpep-TAT in the cytoplasm was confirmed by the addition of the membrane-impermeable quencher trypan blue 60 .

[0126] Confocal imaging of live cells expressing mGFP-Xpep and live cells that have taken up FAM-Xpep-TAT CHO-K1 cells stably expressing OPR were transfected with mGFP-Xpep. To identify both cells expressing mGFP-Xpep and those not expressing it, cells growing in glass-based dishes were incubated with NucSpot Live 650 Nuclear Stain (Biotium) according to the protocol recommended by the manufacturer. After washing three times with complete medium, it was replaced with Ham’s F12 observation medium. Uptake of FAM-Xpep-TAT in the cells was performed as described in the previous subsection. Confocal fluorescence images were acquired on the same microscope station (using a Plan-Apochromat 100x oil immersion objective lens; NA = 1.49) used for single molecule imaging at 37 °C equipped with an Olympus SR10 spinning disk confocal super-resolution unit.

[0127] GFP and FAM were excited at 488 nm and detected with a 505 - 530 nm bandpass filter. NucSpot Live 650 was excited at 642 nm and detected with a 662.5 - 737.5 nm long pass filter. Concentrations of cytoplasmic mGFP-Xpep and FAM-Xpep-TAT were evaluated using calibration curves obtained by observing purified EGFP protein (BioVision) and FAM-Xpep-TAT dissolved in Ham’s F12 observation medium at various concentrations with the focus on 5 μm above the cover glass surface (Figure 2-s7).

[0128] Agonist-induced Ca mobilization in live cells 2+ Observation of mobilization (Supplementary Figure 2-s1d and Figure 2-6) Since OPR expression levels vary from cell to cell, signal transduction processes must be observed at the level of individual cells. OPR is coupled to the inhibitory trimeric G protein Gi, which induces a decrease in cytoplasmic cAMP concentration by inhibiting adenylyl cyclase. However, cells with low OPR expression levels (0.5 - 1.5 fluorescence spots / μm used for single molecule observation 2Using [it], it was very difficult to measure the decrease in cytoplasmic cAMP levels in individual cells. On the other hand, the increase in cytoplasmic Ca 2+ concentration by PLCβ activated by the Gq signaling pathway could be measured in individual cells. Therefore, in this assay, Gαq was modified so that OPR could be conjugated with Gαq. That is, since the Gα protein binds to a specific GPCR via its short C-terminal sequence 61 , the C-terminal 5-amino acid sequence ECGLY of Gαq was replaced with the C-terminal 5-amino acid sequence DCGLF of Gαi2 (this chimeric protein is called Gqi5). Therefore, the present inventors generated a CHO-K1 cell line stably expressing Gqi5, expressed OPR, and subsequently observed the mobilization of cytoplasmic Ca 2+ using a Ca 2+ -sensitive dye upon agonist addition (Supplementary Figure 2-s1d; Figures 2-6a-c). This method using Gqi5 is widely used in the research of OPR 50 and other Gi-coupled GPCRs 51、52 .

[0129] Briefly stated, CHO-K1 cells stably expressing Gqi5 (Gαq with the C-terminal 5-amino acid sequence [ECGLY] replaced by the 5-amino acid sequence [DCGLF] of the C-terminal Gαi2) were generated and transfected with cDNA encoding wild-type and SNAPf-linked OPR. Ca 2+The sensitive dyes Fluo4-AM (Dojindo) and Rhod3-AM (Thermo Fisher Scientific) were used. Rhod3 was used in the experiments with mGFP-Xpeps and FAM-Xpep-TAT, and Fluo4 was used in the experiments without using these homodimerization inhibitors. These AM dyes were incorporated into cells using the following solutions: 4.6 μM Fluo4-AM in T-HBSS containing 1.25 mM Probenecid (Dojindo) and 0.04% (w / v) Pluronic F127 (Dojindo), and 10 μM Rhod3-AM in T-HBSS containing 2.5 mM Probenecid and 1×PowerLoad™ (Thermo Fisher Scientific) according to the manufacturer's recommendations. These loading solutions (2 ml) were added to the cells, incubated at 37 °C for 30 min in the dark, and then the cells were washed three times with T-HBSS.

[0130] Ca downstream of OPR tagged with SNAPf 2+ For the observation of mobilization, the inventors used single molecule detection (TIRF illumination) at the 561 nm channel (642 nm channel when the inventors used Rhod3) to select cells expressing SNAPf-tagged OPR bound by SNAP-Surface 549 (or SNAP-CF660R for experiments using Rhod3) at a number density of 0.5 - 1.5 fluorescence spots / μm in the basal PM. Next, these cells were observed by epifluorescence illumination using the 488 nm channel to monitor the Fluo4 signal (561 nm channel to observe the Rhod3 signal, and 488 nm channel for mGFP-Xpeps and FAM-Xpep-TAT). Agonist stimulation was performed by adding a DMSO solution of the agonist at a final concentration of 200 nM. To determine the saturation level of the fluorescence signal intensity at higher Ca 2 levels, 1 μM (final concentration) ionomycin (Wako) was added (thereby increasing intracellular Ca 2+ ). 2+Increase the concentration to the extracellular concentration of the cells [1.3 mM). The image sequences of Fluo-4 and Rhod-3 were analyzed using ImageJ software.

[0131] For comparison of the function of SNAPf-OPR with that of wild-type OPR, since cells expressing wild-type OPR should function as a positive control, the present inventors determined that cells expressing SNAPf-OPR at a level of 0.5 - 1.5 spots / μm 2 in the basal PM were to be compared with cells expressing wild-type SNAPf-OPR at a level comparable to or higher than the level of SNAPf-OPR. For this purpose, the expression level of wild-type OPR was monitored by using cells transfected with a cDNA (mCherry-2A-OPR) in which the wild-type OPR sequence was linked to the mCherry sequence via a self-cleavable 2A linker sequence. In this way, mCherry is released from OPR to the cytoplasm in the ER, and subsequently wild-type OPR is transported to the PM. The expression of wild-type OPR was detected by the presence of mCherry in the cytoplasm using epifluorescence illumination at 561 nm (the sensitivity is much lower than single-molecule imaging and indicates the presence of a fairly high concentration of mCherry), showing a higher expression of wild-type OPR.

[0132] Quantification of the internal trafficking of SNAPf-OPR SNAPf-OPR was imaged at the single-molecule level using TIRF microscopy. The signal intensity of each individual spot was measured and the sum of their intensities was calculated. However, the SNAPf-OPR spots found in this manner should include those formed by SNAPf-OPR molecules that have already been internally trafficked and are still located near the PM and detected by TIRF microscopy. These molecules were selectively detected by adding the membrane-impermeable fluorescent quencher Mn 3+ -TSP (Frontier Scientific). Briefly, Mn 3+-TSP was dissolved in T-HBSS containing 1% BSA to a final concentration of 10 mM, and this solution was added to the cells in Ham’s F12 observation medium to a final concentration of 3.3 mM Mn 3+ TSP. The sum of the intensities of the individual fluorescent spots after the addition of the quencher was obtained and subtracted from the sum of the signal intensities before the addition of the quencher.

[0133] Software and statistical analysis The microscope station combining a single-molecule imaging system and a super-resolution confocal microscope was controlled by in-house software based on LabVIEW 2018, and single-molecule video acquisition was performed using MCR software for Windows (Hamamatsu Photonics). Overlay of the single-molecule imaging sequences obtained in two colors and tracking of the spots in the video were performed using a C++-based computer program produced in-house as described previously 57、59、62、63 . The TIRF images of FAM-Xpep-TAT were processed and analyzed using Image J for Windows or MATLAB® 2019a for Windows. Curve fitting was performed by OriginPro 2019b for Windows. Statistical analysis was performed by Welch's two-sided t-test using OriginPro 2019b for Windows and RStudio 1.2.1335 for Windows, except for the co-localization duration data analyzed by the Brunner-Munzel test. A p-value of less than 0.05 was considered statistically significant. The simulation study was performed using in-house software based on MATLAB® 2019a for Windows.

[0134] <Results> Experimental strategy (1) Prediction of the OPR site involved in homodimerization based on amino acid homology Most studies investigating the GPCR homodimerization mechanism of class A have assumed the involvement of transmembrane (TM) domains because a dimerization mechanism common to all dimer-forming GPCRs was mainly sought. 7、28、29 . One of the common features of GPCRs is that, in addition to the presence of the seven transmembrane domains and coupling to G proteins, they may form constitutively transient homodimers, which is understandable.

[0135] However, in the case of the three classical OPRs, KOR, MOR, and DOR, the idea that the TM domain is mainly involved in homodimerization has major problems (although TM interactions can enhance homodimerization). Since their TM domains show approximately 75% amino acid identity and approximately 90% similarity (for rat OPRs; Figure 2-s1a), if they were involved in homodimerization, they would cause heterodimerization at nearly equal levels. However, MOR and KOR showed very limited heterodimerization 15、30 (However, see reference 31), while KOR-DOR and DOR-MOR heteromers have been found to be functionally important 15、32~35 .

[0136] In contrast, the N-terminal extracellular domain (51-70 amino acid sequence) is completely different among the three OPRs, and the C-terminal cytoplasmic domain (47-59 amino acid sequence) is also almost completely different, except for the 9 amino acids in the first part of the C-terminal cytoplasmic domain that are identical among the three OPRs (for rats, only 5 amino acids are identical at the last 38-50 C-terminal residues of KOR, MOR, and DOR). Furthermore, the C-terminal cytoplasmic 15-residue domain of DOR has already been reported to be involved in DOR homodimerization quite some time ago using biochemical approaches. 10 . Therefore, in this study, the inventors hypothesized that the N-terminal and / or C-terminal cytoplasmic domains contain important regions involved in the homodimerization of the three classical OPRs.

[0137] (2) Development of theories and methods for evaluating (A) the homodimer dissociation (dimer - monomer) equilibrium constant K based on single - molecule imaging data D and (B) the homodimer lifetime In the literature, often the argument is whether an OPR (or GPCR) forms a homodimer or not. In reality, however, most proteins form dimers and clusters at very high expression levels, and the physiological expression levels vary greatly from cell to cell. Therefore, the present inventors recognized the need to quantitatively evaluate the affinity of homodimers, that is, the homodimer dissociation (dimer - monomer) equilibrium constant K D Accordingly, the present inventors developed theories and methods for evaluating K D from the PCCF of OPR fluorescently labeled with two colors.

[0138] Furthermore, the present inventors developed a theory based on a diffusion equation for predicting the distribution of co - localization durations. Until now, the present inventors (see also references 23 and 24, 38) and others (references 9, 19, 22, 26, and 27) have relied on an intuitive method for obtaining dimer lifetimes from optical co - localization data. However, due to the theory developed in the present invention, the present inventors now have a solid basis for obtaining dimer lifetimes from single - molecule co - localization data. The final result from this theory is simple, and the distribution of co - localization durations can be fit by the sum of two exponential functions under the experimental conditions of the present inventors, with the longer lifetime providing the dimer lifetime.

[0139] All three OPRs continuously interconvert between transient homodimers and monomers SNAPf - tagged proteins and OPRs conjugated at their N - termini (SNAPf - OPR) were expressed in the PM of CHO - K1 cells (these molecules were functional as shown in FIGS. 2s1b, c), with an efficiency of more than 70% 37 with a fluorescent membrane - impermeable SNAP ligand 9, labeled with SNAP-Surface 549 and SNAP-CF660R (Figure 2-s2). For experiments examining the homodimerization of OPR, the inventors simultaneously labeled SNAPf-OPR (e.g., SNAPf-KOR) with both SNAP-Surface 549 and SNAP-CF660R. As a result, the number density of the two probes on the PM became similar at a spot density of 0.25 - 0.75 spots / μm for each color image. Subsequently, a custom-built total internal reflection fluorescence (TIRF) microscope was used to perform two-color single molecule simultaneous observation at 37 °C at a normal video rate (30 Hz). 2 Nearly all SNAPf-OPR fluorescence spots showed diffusion in the PM. Furthermore, they showed frequent transient co-localization and co-diffusion, consistent with the transient homodimer formation found for other GPCRs (Figure 2-1a). The 200 nm co-localization distance (method) used in the present invention is much larger than the molecular scale (< 10 nm), but co-localization analysis is still useful for detecting molecular interactions: Unrelated molecules can accidentally come together for a short distance over a short period, but the probability of this occurring in multiple frames is small.

[0140] 23、24 Thus, a longer co-localization duration implies the presence of a molecular interaction between two molecules or homodimers, rather than a chance encounter (although the molecular interaction is initiated by a chance encounter). Due to the limited accuracy of single molecule localization when diffusing molecules are observed at video rate, it was necessary to use a long distance such as 200 nm to detect true molecular binding with a probability of over 99%. 38 23、24、26

[0141] Hereinafter, the inventors refer to homoco-localization as homodimerization for clarity and brevity of presentation. The inventors focused on the co-localization of spots with different colors in the simultaneous two-color experiment due to the ease with which the resulting images can be analyzed. See Figure 2-s3 for the monochromatic imaging data. This is consistent with the observations made when using other GPCRs 23、24、26 ​An example of repeated homodimerization of KOR molecules with different partner KOR molecules is shown.

[0142] The degree of OPR homodimerization was analyzed, as previously reported, 39 by the co-localization index based on the pair cross-correlation function (PCCF) of SNAP-Surface 549 (green) and SNAP-CF660R (magenta) spots (Figure 1b, Figures 2-s4a - c). KOR, MOR, and DOR showed significantly higher co-localization indices than the negative control (green image overlaid on the 180°-rotated magenta image; see Figures 2-s4b, 2-1c). Under the expression conditions of 0.5 - 1.5 OPR spots / μm 2 used in this study, the co-localization index was almost independent of the expression level (Figures 2-s5a - f). KOR showed a higher tendency to form homodimers than MOR and DOR (Figure 2-1c; the main results are summarized in Table D and the results of its statistical tests are in Supplementary Table 1). Biochemical studies have, consistent with the results of the present inventors, found that DOR forms homodimers 10 .

[0143] The dissociation (dimer - monomer) equilibrium constant K D of the homodimer was evaluated from the PCCF and the total number of spots in the SNAP-Surface 549 and SNAP-CF660R images. K D was 2.68 ± 0.28, 7.31 ± 0.76, and 7.91 ± 0.26 copies / μm 2 for KOR, MOR, and DOR, respectively (at 37°C; Figure 2-s5c). These values are approximately consistent with those of K D for β2AR and FPR (1.6 and 3.6 copies / μm 2 , respectively), but the K D of the MOR homodimer is approximately 3.5-fold smaller than that previously reported (27.43 ± 11.75 copies / μm 2 ) 19 using a fluorescent antagonist analog.

[0144] Previous single-molecule examinations9、22 According to this, MOR and KOR, when their expression levels were 0.1 - 0.3 copies / μm 2 it was demonstrated that, contrary to the results of the present inventors, they tended to exist as monomers even at 20°C. However, the proportion of homodimers should depend on the copy number density of the protein in the PM, and at the number density of 0.5 - 1.5 OPR spots / μm 2 used in the analysis of the present inventors, MOR and KOR formed homodimers, which was clearly consistent with the MOR data using a fluorescently conjugated MOR antagonist 19 as is obvious.

[0145] The homodimer lifetimes of the three OPRs are on a time scale of approximately 130 milliseconds The durations of all co-localization events of spots having different colors were measured (events exceeding 1,500 times in more than 17 cells). Thereby, the present inventors obtained the distribution of co-localization durations (histogram; Fig. 2-1d). The present inventors developed a theory based on a diffusion equation for predicting the distribution of co-localization durations. According to the theory, the histogram was fitted by the sum of two exponential decay functions (a shorter decay time constant and a longer decay time constant; τ1 and τ2, respectively). On the other hand, the distribution of accidental co-localization durations found by overlaying the green video and the 180°-rotated magenta video (Fig. 2-1d; Fig. 2-s4b) could be fitted by a single exponential function having a decay constant τ inci Since τ1 was almost the same as τ inci (Fig. 2-1d), according to the theory, τ2 provides the co-localization (co-homodimer) lifetime (after correction for the photobleaching lifetimes of the two fluorescent probes; Fig. 2-s6): 149 ± 26, 118 ± 12, and 125 ± 15 milliseconds for KOR, MOR, and DOR, respectively (Fig. 2-1d).

[0146] The amino acid sequences 9 - 26 in the C-terminal cytoplasmic domain play a crucial role in OPR homodimerization In accordance with the strategy of the inventors described at the beginning of the Results section, the inventors first used N-terminal deletion mutants (KOR(Δ1-53), MOR(Δ1-51), and DOR(Δ1-35)) to examine the involvement of the extracellular N-terminal domain in homodimerization. The partial removal of the entire N-terminal extracellular domain blocked OPR expression in the PM, probably due to the deletion of the endogenous signal peptide sequence, and these deletion mutants were used in X-ray crystallographic studies, so these ranges were selected. In these studies, in addition to the deletion of the N-terminal domain, the C-terminal domain was further deleted, the T4-lysozyme sequence was inserted into the third intracellular loop, or specific nanobodies were added to enhance crystallization. 40~43 . All of these N-terminal deletion mutants showed diffusion behavior similar to that of the wild type (without immobilization or clustering) and co-localization indices almost the same as those of the wild type (Figure 2-1c). These results clearly indicate that the N-terminal extracellular domain is not involved in homodimerization.

[0147] Next, the inventors examined C-terminal mutants with systematically varied deletions (Figure 2-2a, b). They showed little immobilization or clustering, and their co-localization indices indicated that the 365-380 amino acid sequence of KOR, the 358-382 sequence of MOR, and the 357-372 sequence of DOR are critical for homodimerization (Figure 2-2b; Supplementary Table 2). The DOR amino acid sequence for homo-oligomerization is consistent with previous biochemical data. 10 .

[0148] The distribution (histogram) of the co-localization duration for the deletion mutants could be fitted by a single exponential function rather than the sum of two exponential functions, and the decay time constant was only slightly longer than the accidental co-localization lifetime (Figure 2-2c; Supplementary Table 3), supporting the co-localization index data, i.e., these deletion mutants rarely form homodimers. On the other hand, the interaction of the transmembrane (TM) domains has traditionally been considered important for GPCR dimerization. 7、28、29、44Since the inventors were unable to completely prevent OPR homodimerization by deletion of the C-terminal region, TM interaction may also be involved in OPR homodimerization, but the C-terminal domain is likely to be the major and crucial interaction site.

[0149] By introducing point mutations in these crucial regions and their surrounding regions (Figure 2a), the inventors found that a few to several basic / acidic residues, as well as proline residues, are important for OPR homodimerization (for specific amino acids, see Figure 2 - 2d; refer to the co-localization index shown in Supplementary Table 2), suggesting that the electrostatic interaction and overall structure of the cytoplasmic C-terminal domain may be important for homodimerization. Indeed, the IUPred2 score (energy-estimation-based prediction for denatured and non-denatured residues; http: / / iupred2a.elte.hu) 45 suggests that the C-terminal region can be intrinsically disordered, and the score decreased with these point mutations (Figure 2 - s1d, e). This result, in addition to specific amino acid interactions, suggests that multiple weak interactions of the intrinsically disordered domain of the C-terminal region of OPR, as found for the dimerization of the transcription factor PU.1 46 can promote OPR dimerization.

[0150] Peptides having amino acid sequences involved in homodimerization prevent homodimer formation. Subsequently, the inventors examined whether peptides having the same amino acid sequence as the deleted portion of the deletion mutants could prevent homodimerization. Since the deletions and point mutations used by the inventors (Figure 2 - 2) may have induced conformational changes in the true homodimer interaction site, leading to inhibition of homodimerization, the use of these peptides was crucial to clearly demonstrate that specific amino acid sequences in the C-terminal regions of KOR, MOR, and DOR are involved in homodimerization (this also applies to the DOR homo-oligomerization amino acid sequence determined so far). 10)。Furthermore, if the inventors were able to develop such homodimerization blockers, they could be used as extremely useful tools for investigating the functions of OPR monomers and homodimers.

[0151] The inventors used two approaches. The first approach used peptides (named mGFP-Kpep, Mpep, and Dpep, collectively called mGFP-Xpep, and the numbers in parentheses following mGFP-Xpep indicate the amino acid residue ranges in wild-type OPR) that had the same amino acid sequence as the deleted portion of the deletion mutants conjugated to the C-terminus of mGFP. These peptide-based molecules were expressed in CHO-K1 cells stably expressing SNAPf-KOR, MOR, or DOL (wild-type) respectively (Figure 2-3a, b), and their effects on OPR homodimerization were examined (Figure 2-3c, d; Supplementary Table 4). The concentration of cytoplasmic mGFP-Xpep was measured by confocal fluorescence microscopy based on calibration using various concentrations of purified mGFP protein dissolved in Ham’s F12 observation medium in a glass-based dish and observed 5 μm above the glass surface (Methods; Figure 2-s7a).

[0152] The OPR homocolocalization index in cells expressing mGFP-Xpep in the cytoplasm (the protein was diffusely and uniformly distributed throughout the cytoplasm; Figure 2-3b) was K DConsistent with the values (2.7 - 7.9 copies / μm2), all three OPRs showed a clear tendency to decrease with the increase in the specific mGFP-Xpep concentration in the cytoplasm from 0 to 7.8 μM (statistical data shown in Fig. 2-3c, Fig. 2-s8a). On the other hand, the control mGFP-peptides (mGFP-Dpep for KOR and MOR, and mGFP-Kpep for DOR) had no effect (Fig. 2-3c; Fig. 2-s8a). Furthermore, the comparison between the co-localization index in the absence of (0 μM) mGFP-Xpep and the average co-localization index for mGFP-Xpep concentrations in the range of 3.8 - 7.8 μM showed a significant reduction in the co-localization index in the presence of mGFP-Xpep for all three OPRs, but not in the presence of the control mGFP-peptides (Fig. 2-3c; Table D). Consistently, the homodimer lifetime was reduced to a level comparable to that for accidental co-localization or C-terminal deletion mutants in cells expressing 3.8 - 7.8 μM mGFP-Xpep (Fig. 2-3d; Table D).

[0153] As another approach, K-, M-, and D-peps were conjugated with the fluorescent dye 5-FAM at their N-termini for visualization and with the TAT sequence (YGRKKRRQRRR) using a G10 linker at their C-termini for membrane permeabilization treatment (FAM-Xpep-TAT; method), and then added to cells pre-incubated with 150 μM pyrenebutyric acid for 5 minutes 47(Figs. 2-4a, b). FAM-peptide-TAT showed a diffuse distribution throughout the cytosol (Fig. 2-4b). Cells containing 2.9 - 3.4 μM FAM-Xpep-TAT (see Fig. 2-s7b for concentration calibration; hereinafter, instead of repeating 2.9 - 3.4 μM, for brevity, the inventors describe this concentration range as approximately 3 μM) were selected, and the effect of these cytoplasmic peptides on OPR homodimerization was examined. The results, consistent with the data obtained using mGFP-Xpep (Fig. 2-3c), showed that approximately 3 μM FAM-peptide-TAT significantly blocked homodimerization (Fig. 2-4c; statistical data shown in Fig. 2-s8b; Table D). Control FAM-Xpep-TAT (FAM-Dpep-TAT for KOR and MOR and FAM-Kpep-TAT for DOR) did not affect the co-localization index.

[0154] The presence of approximately 3 μM FAM-Xpep-TAT in the cytosol significantly reduced the homodimer lifetime to the level of mutants with deletions in the dimer-inducing domain (Fig. 2-4d; Table D). These results are consistent with those obtained by expressing mGFP-Xpep in cells.

[0155] Agonists regulate monomer-dimer interconversion, and FAM-Xpep-TAT peptides block this regulation For the representative agonist KOR, U-50488, for MOR, [D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-enkephalin acetate (DAMGO), and for DOR, SNC-80, added at 0.2 μM (a concentration sufficient to ligate almost all OPR molecules) affected OPR homodimerization differently for the three OPRs (within 5 minutes after addition). Agonist-bound KOR showed less homodimerization (lower co-localization index), while agonist-bound MOR and DOR showed more homodimerization (Fig. 2-4c; Table D), although these results can vary depending on the specific agonist. 9、10. For example, Devi's group has previously found that the DOR agonists DADLE, DSLET, and DPDPE induce fewer homodimers. 10 However, the inventors have found that the DOR agonist SNC-80 increases the proportion of homodimers, suggesting that the dimerization tendency depends on each agonist. Since a comprehensive examination of the agonist effect on OPR homodimerization is beyond the scope of this study, the inventors examined only one agonist for each OPR subtype.

[0156] Furthermore, the effect of the agonist on the homodimer lifetime was consistent with the results of the co-localization index. The homodimer lifetime of agonist-bound KOR became shorter, while the homodimer lifetimes of agonist-bound MOR and DOR became longer (Figure 2-4d).

[0157] In the presence of FAM-Xpep-TAT, the co-localization index of agonist-bound OPR was reduced in different ways depending on the OPR subtype for all OPRs. The co-localization index of agonist-bound KOR, which showed a smaller index when compared with the co-localization index of non-ligated KOR, was further reduced by the presence of FAM-Kpep-TAT. The co-localization index of agonist-bound MOR was reduced to the level of the deletion mutant MOR (Δ358-382) in the presence of FAM-Kpep-TAT (Figure 2-2b). On the other hand, the co-localization index of agonist-bound DOR was reduced in the presence of FAM-Dpep-TAT, but the co-localization index was still higher than that of wild-type DOR in the absence of the agonist.

[0158] The effect of the presence of FAM-Xpep-TAT on the homodimer lifetime of agonist-bound DOR was almost identical to its effect on the co-localization index. The homodimer lifetime of agonist-bound KOR was already comparable to that of the deletion mutant KOR(Δ365-380), and no further reduction was observed with FAM-Kpep-TAT. The homodimer lifetime of agonist-bound MOR was reduced to a level similar to that of the deletion mutant MOR(Δ358-382) in the presence of FAM-Mpep-TAT (Figure 2-2b). The homodimer lifetime of agonist-bound DOR was reduced by the presence of FAM-Dpep-TAT, but was approximately 1.5-fold longer than the homodimer lifetime of wild-type DOR in the absence of agonist.

[0159] Three fundamental constants for describing the monomer-dimer equilibrium for all three OPRs The dimer dissociation equilibrium constant (K D ) was evaluated as done for the results before agonist stimulation (Figure 2-4f). These values support the results of the direct readout of the co-localization index described in the previous subsections.

[0160] The reciprocal of the dimer lifetime (Figure 2-1d, 2-4d) provides the dimer dissociation rate constant (k off ). The k off values before and after agonist binding are summarized in Figure 2-4f. Using the experimentally evaluated k off and K D , the dimer formation rate constant k on was calculated, providing all three fundamental constants for describing the monomer-dimer equilibrium for all three OPRs (Figure 2-4f; see Supplementary Table 5 for SEM). These fundamental constants clearly indicate the presence of homodimers for all three OPRs.

[0161] The ratio of molecules present as dimer to monomer (D / M ratio) with respect to the number of protomers was determined at various expression levels (number density of molecules) using K DCalculated from (FIGS. 2-4e, f). Their overall variation, including agonist-bound and unbound states, is 0.3 to 10 copies / μm 2 in the predicted physiological expression range of OPR of 1 8~20 is in the range of 0.06 to 2.28 (5.7 to 70.0% of protomers in the dimer) in. Thus, a significant amount of dimers are expected to be present in various tissues at any time (the predicted average copy numbers of monomers and dimers per cell at various expression levels are shown in Supplementary Table 6), but importantly, all of these dimers are constantly forming and dissociating with a lifetime of less than 0.3 seconds. When these dimers dissociate into monomers, they form dimers again, but the rate of dimer formation depends on the number density of monomers (and thus the expression level).

[0162] Effect of homodimer-blocking peptide on OPR internalization: OPR monomers and homodimers are internalized at equal rates before and after agonist addition OPR internalization was monitored by using the membrane-impermeable fluorescent quencher Mn(III) meso-tetra(4-sulfonatophenyl) porphyrin (Mn 3+ -TSP). This quencher only suppresses the fluorescence emission from the SNAP-Surface 549 dye on SNAPf-OPR in the PM and does not suppress the fluorescence emission in the cytoplasm. Thus, by subtracting the signal intensity after quencher addition from the signal intensity before addition, we evaluated the percentage of OPR molecules remaining in the PM (FIG. 2-s9; FIG. 2-5a). The time course of the number of OPR remaining in the PM after time 0 was examined both in the presence and absence of 0.2 μM agonist and approximately 3 μM FAM-Xpep-TAT in the cytoplasm (FIG. 2-5b). Observations were made every 5 minutes (each observation was made for a single frame, i.e., 33 milliseconds, and thus the photobleaching of the fluorescent probe can be ignored: the photobleaching lifetime is [16.3 ± 1.2] × [300 / 0.033] seconds or approximately 41 hours). We, according to FIG. 2-4f, approximately 1 copy / μm 2Since the OPR expression was used, before (after) agonist stimulation, 33%, 18%, and 17% (18%, 15%, 29%) of the KOR, MOR, and DOR copies were present as dimers, and most of the OPR molecules were monomers in the presence of FAM-Xpep-TAT.

[0163] The time course of OPR internalization can be operationally fit by a single exponential function (Figure 2-5b), providing the percentage of OPRs with detectable internalization over 35 minutes of observation, their residence lifetime in the PM, and the percentage of molecules with a much longer residence time that cannot be measured in this experimental design. The expression level of β2-arrestin that may be involved in OPR internalization 48 was reported to be higher in T24 cells than in CHO-K1 cells, which is the standard cell line of the present inventors. 49 Therefore, in addition to CHO-K1 cells, which are the standard cell lines of the present inventors, T24 cells 4 were also used (Table D).

[0164] All three OPRs showed a percentage of molecules with a longer unmeasurable residence time in the range of 91 - 97%, while the shorter measurable residence time (related to about 3 - 9% of the molecules) was 24 - 45 minutes. The homodimer-blocking peptide FAM-Xpep-TAT did not significantly affect OPR internalization, indicating that the OPR internalization rate was the same regardless of whether they formed monomers and homodimers (Figure 2-5b; Table D; Supplementary Tables 7 and 8). In the absence of FAM-Xpep-TAT, the percentage of OPR molecules present as dimers was 15 - 33%, but considering the accuracy of these measurements, if the internalization rates of monomers and dimers were at least 2-fold different, the present inventors would have been able to detect the difference.

[0165] Agonist addition significantly increased the detectable internalization rate and internalization speed of all three OPRs, although the effect was small for MOR (agonists were U-50488, DAMGO, and SNC-80 for KOR, MOR, and DOR, respectively). The homodimerization-blocking peptides (both FAM-Xpep-TAT and mGFP-Xpep) had little effect on the internalization of associated OPRs, suggesting that the internalization speed of associated OPRs is the same regardless of whether they form monomers and homodimers (Figure 2-5b; Table D; Supplementary Tables 7 and 8).

[0166] Homodimer-blocking peptides modulate agonist-induced signals in various ways Agonist-induced signals downstream of OPR were examined by a widely used method for Gi-coupled GPCRs using the artificial protein Gqi5 50~52 See the caption and methods of Figure 2-s1b (Figure 2-6) for the method. The inventors used cells expressing OPR at approximately 1 copy / μm 2 and thus the percentage of OPR copies present as dimers was in the range of 15 - 33% in the absence of FAM-Xpep-TA.

[0167] Addition of homodimer-blocking FAM-peptide-TAT alone (thus monomerization of OPR alone) did not induce detectable Ca 2+ mobilization (Figure 2-6d; Table D). On the other hand, addition of each agonist induced Ca 2+ mobilization. The effect of the homodimer-blocking peptides on agonist-induced Ca 2+ mobilization was complex (both FAM-Xpep-TAT and mGFP-Xpep had similar effects for all three OPRs). The homodimer-blocking peptides (OPR monomerization) each induced agonist-induced Ca 2+Mobilization was enhanced, unaffected, or reduced (Figure 2-6d; Table D). These results show that KOR and DOR monomers induce higher and lower signals, respectively, than their respective homodimers (without affecting agonist-induced internalization [Figure 2-5b]), while MOR monomers and homodimers are shown to induce downstream signals at similar levels. This result suggests that homodimer-blocking peptide-TAT can be used as a drug to enhance or suppress agonist-induced cellular responses for KOR and DOR, respectively, without affecting their internalization (see Figure 2-5b).

[0168]

Table 5

[0169] Table D. Co-localization index, homodimer lifetime, percentage of molecules with detectable internalization by this method, residence time in the PM, and Ca 2+ mobilization parameters (F Max -F b ) / F b summary in the presence and absence of various modulators (0.2 μM agonist, 3 μM FAM-Xpep-TAT in the cytoplasm, and approximately 6 μM mGFP-Xpep in the cytoplasm). The agonists used in the present invention were U-50488 for KOR, DAMGO for MOR, and SNC-80 for DOR.

[0170] Discussion Based on single-molecule imaging tracking data to evaluate the K D and lifetime of OPR homodimers, the inventors clearly demonstrated that all three OPRs form transient homodimers even at lower expression levels. The K D values obtained for KOR, MOR, and DOR, respectively, were 2.68 ± 0.28, 7.31 ± 0.76, and 7.91 ± 0.26 copies / μm 2(Figure 2-s5b-d, f, g) shows the values obtained so far for GPCRs (1.6 and 3.6 copies / μm for β2AR and formyl peptide receptor (FPR), respectively) 2 ) 23、25 which are generally consistent. On the other hand, the DAMGO-induced enhancement of homodimerization reported so far 9 was confirmed in this study. Since the inventors used only one agonist for each OPR subtype, further tests using other agonists are important.

[0171] 0.3 - 10 copies / μm 2 of the predicted physiological expression range of OPR 18~20 assuming that, for the overall variation of K including agonist-bound and unbound states D 5.7 - 70.0% of the protomers exist as dimers at any time in various tissues (Figure 2-4e, f). Importantly, all of these dimers have a lifetime of less than 0.3 seconds (Figure 2-1d) and are constantly forming and dissociating at 37°C. When homodimers dissociate into monomers, they form homodimers again with the same and other partner molecules. Longer homodimer lifetimes of about 0.5 - 1 second have been reported, but they were observed at lower temperatures such as room temperature and 20°C 9、26,27 .

[0172] Based on the sequence comparison of the three classical OPRs and the highly likely assumption that the three OPRs form distinct homodimers and heterodimers 10、15、30、33、34、35 the inventors inferred that the amino acid sequences involved in homodimerization have lower amino acid sequence identity and homology. Based on this presumption, the inventors found that a specific amino acid sequence of 9 - 26 residues in the cytoplasmic domain near the C-terminus is greatly involved in the distinct homodimerization of all three classical OPRs (Figure 2-2b, c and Figure 2-3). The TM domain may be involved in homodimerization as previously suggested by the "rolling interface" model 7、28、29Therefore, TM domain interactions can potentially be regulated by interactions in the C-terminal cytoplasmic domain. The N-terminal extracellular domain is not involved in the homodimerization of any of the three OPRs (Figure 2-1c).

[0173] In addition to charged amino acid residues, proline also seemed to be involved in homodimerization (Figure 2-2d). Furthermore, all three OPRs showed IUPred2 scores close to or greater than 0.5 (Figure 2-s1d, e), suggesting a native disordered domain-like conformation / property in the C-terminal region. Point mutations of residues involved in homodimerization reduced the IUPred2 score (Figure 2-s1e). Therefore, in addition to localized electrostatic interactions, many weak interactions can be important for inducing homodimerization through interactions in the cytoplasmic C-terminal domain.

[0174] By developing mGFP-Xpep (Figure 2-3) and FAM-Xpep-TAT (Figure 2-4), the inventors confirmed that a 9- to 26-amino acid sequence near the C-terminal cytoplasmic domain with no sequence similarity is involved in all three OPRs. As far as the inventors know, these Xpeps were the first peptide-based drugs / reagents capable of blocking GPCR homodimerization.

[0175] These homodimer-blocking peptides / proteins have become important tools for investigating the functions of OPR monomers and homodimers. Somewhat surprisingly, Xpep-induced blockade of homodimerization was unable to affect OPR internalization (desensitization), whether before or after agonist addition (Figure 2-5b), indicating that the OPR internalization rate is the same regardless of whether they form monomers or homodimers. On the other hand, it was revealed that by using Xpep, DOR homodimers and KOR homodimers signal differently from their respective monomers, while MOR homodimers do not (Figure 2-6d). This result implies that the interaction mechanisms between receptors and G proteins and / or the competition between G proteins and GPCR-kinases / β-arrestin on the receptor may vary significantly among the three OPRs 53 in a way that involves the homodimer interface.

[0176] Since Xpep-TAT is membrane-permeable, Xpep-TAT can be used as a drug to modulate downstream signals by enhancing the OPR monomer population. Therefore, the results reported herein suggest a new GPCR drug development strategy for regulating downstream GPCR signals by modulating homodimer formation.

[0177]

Table 6

[0178]

Table 7

[0179]

Table 8

[0180]

Table 9

[0181] [Table 10]

[0182] [Table 11]

[0183] [Table 12]

[0184] [Table 13]

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Claims

1. A prophylactic and / or therapeutic agent for the prevention and / or treatment of opioid resistance or opioid dependence, comprising a peptide that inhibits dimer formation, wherein the dimer is a heterodimer or homodimer formed from one or two opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR).

2. The peptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-40, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-40, amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences, as described in claim 1. Table 15

3. The antiprosthetic and / or therapeutic agent for opioid resistance or opioid dependence according to claim 1, wherein the dimer is a MOR and DOR heterodimer, or a KOR and DOR heterodimer.

4. The peptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-30, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences represented by SEQ ID NOs: 2-8, 10-15, 17-30, amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences, as described in claim 3.

5. The antiprosthetic and / or therapeutic agent for opioid resistance or opioid dependence according to claim 1, wherein the dimer is a homodimer of MOR, KOR, or DOR.

6. The preventive and / or therapeutic agent for opioid tolerance or opioid dependence according to claim 5, wherein the peptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 31 to 40, an amino acid sequence having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs. 31 to 40, and an amino acid sequence having at least 80% amino acid sequence identity with any of these amino acid sequences.

7. A method for inhibiting the formation of heterodimers or homodimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR), comprising providing a peptide comprising one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-40, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-40, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

8. The method according to claim 7, wherein the peptide inhibits the formation of a MOR-DOR heterodimer or a KOR-DOR heterodimer.

9. The method according to claim 7 or 8, wherein the peptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-30, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences represented by SEQ ID NOs: 2-8, 10-15, 17-30, amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

10. The method according to claim 7, wherein the peptide inhibits the formation of homodimers of MOR, KOR, or DOR.

11. The method according to claim 10, wherein the peptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 31 to 40, the amino acid sequences having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs. 31 to 40, and the amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences.

12. A peptide comprising one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-40, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-40, amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences, wherein the peptide inhibits the formation of heterodimers or homodimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR).

13. A peptide comprising one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-30, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 2-8, 10-15, 17-30, amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, amino acid sequences having one or more conserved amino acid substitutions in the amino acid sequences of SEQ ID NOs: 41-67 listed in Table B, and amino acid sequences having at least 80% amino acid sequence identity with any of these amino acid sequences, wherein the peptide inhibits the formation of MOR and DOR heterodimers, or KOR and DOR heterodimers.

14. A peptide comprising one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 31 to 40, an amino acid sequence having one or more conservative amino acid substitutions in the amino acid sequences of SEQ ID NOs. 31 to 40, and an amino acid sequence having at least 80% amino acid sequence identity with any of these amino acid sequences, wherein the peptide inhibits the formation of homodimers of MOR, KOR, or DOR.

15. A drug for enhancing opioid analgesia, comprising a peptide that inhibits the formation of heterodimers or homodimers from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR) opioid receptors in the target.

16. A combination of an opioid and a peptide used to enhance opioid analgesia in a subject who needs, is using, or is scheduled to use opioids, which inhibits the formation of heterodimers or homodimers formed from one or two types of opioid receptors selected from the group consisting of μ-type (MOR), κ-type (KOR), and δ-type (DOR).

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