Cells and methods for evaluating binding response of β-arrestin 2 to opioid μ receptor

Abstract

To provide a system for evaluating the binding response of β-arrestin 2 to opioid μ receptor.SOLUTION: Provided is a transformed animal cultured cell into which a gene encoding a fusion protein containing an opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 are introduced, and in which a fused protein which is either one of the fusion protein containing the opioid μ receptor or the fusion protein containing the β-arrestin 2 contains luciferase consisting of an amino acid sequence set forth in SEQ ID NO: 1, and the other fusion protein contains a protein tag consisting of an amino acid sequence set forth in SEQ ID NO: 3. Also provided are a method for evaluating a β-arrestin 2 activation level mediated by a test substance via the opioid μ receptor, and a method for screening for a biased opioid μ receptor agonist, by using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cells and methods for assessing the binding response of β-arrestin 2 to the opioid μ receptor. [Background technology]

[0002] Pain is defined as an "unpleasant sensory and emotional experience," and chronic pain, in particular, is a symptom that must be eliminated because it reduces the patient's quality of life (QOL). Opioid analgesics are commonly used as treatment for cancer pain and postoperative pain, which account for a large proportion of pain patients.

[0003] There are three known major opioid receptors: μ, κ, and δ. While opioid analgesics commonly used as therapeutic drugs exert strong analgesic effects by acting on the opioid μ receptor, they are known to cause serious side effects, such as drug dependence that can lead to abuse and respiratory depression that can be fatal. Therefore, there is a need to develop analgesics with reduced side effects, such as drug dependence and respiratory depression.

[0004] Opioid receptors are G protein-coupled receptors (GPCRs). When bound to opioid receptor agonists, they bind to G proteins, resulting in analgesic effects. However, they also activate G protein-coupled receptor kinases (GRKs), which uncouple the G proteins and stably bind to β-arrestins, resulting in side effects. The signal transduction pathway mediated by the binding of opioid receptors to G proteins is called the G protein pathway, and the signal transduction pathway mediated by the binding of opioid receptors to β-arrestins is called the β-arrestin pathway.

[0005] One method for reducing the side effects specific to opioid μ receptor agonists is the use of biased compounds. Biasing of opioid receptor agonists refers to the tendency of intracellular signaling, activated by binding of an opioid receptor agonist to an opioid receptor, to be biased toward either the G protein pathway or the β-arrestin pathway. It has been reported that the G protein pathway is involved in the analgesic effect of the opioid μ receptor, while the β-arrestin pathway is involved in side effects such as respiratory depression and constipation (Non-Patent Documents 1 and 2). Opioid μ receptor agonists that have a weak ability to activate the β-arrestin pathway (i.e., low β-arrestin activation level) and preferentially activate the G protein pathway (i.e., high G protein activation level), and thus have a bias toward the G protein pathway, can be used as an analgesic with excellent separation of pharmacological effects and side effects, and are highly safe.

[0006] PathHunter is used to evaluate the β-arrestin binding response to GPCRs. (R) β-arrestin GPCR Assay (DiscoverX; hereinafter referred to as "PathHunter Assay"; Patent Document 1) and Tango TM GPCR Assay (Invitrogen; hereinafter referred to as "Tango Assay"; Patent Document 2) is commercially available as a cell-based assay kit and is widely used.

[0007] However, these assay kits are not capable of detecting with high sensitivity the β-arrestin binding reaction (Non-Patent Document 3), which occurs within a short time frame of a few minutes after GPCR activation. When using the PathHunter Assay, two β-galactosidase fragments with mutual binding affinity are fused to a GPCR and a β-arrestin, respectively. Upon binding of β-arrestin to the GPCR, β-galactosidase is reconstituted into an active enzyme. Because this enzymatic reconstitution of β-galactosidase requires time, it is recommended that the reaction be allowed to continue for at least 90 minutes after the addition of the test substance. When using the Tango Assay, in cells expressing a GPCR fused to a transcription factor via a protease cleavage site and a β-arrestin fused to a protease, the binding of β-arrestin to the GPCR cleaves the transcription factor, activating the reporter gene. Because it takes time for the transcription factor to induce reporter gene expression, it is recommended that the Tango Assay be allowed to continue for 5 hours after the addition of the test substance. Therefore, these assay kits are not suitable for the immediate evaluation of β-arrestin binding to opioid receptors induced by opioid receptor agonists; a method that can detect β-arrestin binding within a few minutes of GPCR activation is desirable.

[0008] Another method for assessing β-arrestin binding to GPCRs is based on the detection of bioluminescence resonance energy transfer (BRET). In this method, one of the GPCR and β-arrestin is labeled with a BRET donor, and the other with an acceptor. The binding of β-arrestin to GPCRs is assessed by detecting BRET that occurs between the donor and acceptor upon binding of the GPCR and β-arrestin. Specific examples of methods using BRET include a method using a cell-free composition (a membrane fraction obtained by disrupting cell membranes) to detect BRET between Renilla luciferase and green fluorescent protein 2 (GFP2) (Patent Document 3), and a method using cells transiently expressing a target protein to detect BRET between NanoLuciferase and yellow fluorescent protein (YFP) (Non-Patent Document 4). Non-Patent Document 4 also reports that overexpressing GRK2 in cells increases the sensitivity of β-arrestin binding detection in BRET assays.

[0009] However, for example, in the method of Non-Patent Document 4, the spectral separation between the emission peak of the luminescence signal derived from NanoLuc (registered trademark) luciferase as the energy transfer donor and the emission peak of the fluorescent signal derived from YFP as the acceptor is small, and there remain challenges in highly sensitive detection of β-arrestin binding.

[0010] Thus, there is still a need to develop an assay system suitable for assessing the binding response of β-arrestins to opioid receptors. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2007 / 106456 [Patent Document 2] International Publication No. 2007 / 127538 [Patent Document 3] International Publication No. 2010 / 085844 [Non-patent literature]

[0012] [Non-Patent Document 1] Raehal et al., Journal of Pharmacology and Experimental Therapeutics, 2005, 314(3), 1195-1201 [Non-patent document 2] Manglik et al., Nature, 2016, Vol. 537(7619), pp. 186-190 [Non-patent document 3] Spillmann et al., International Journal of Molecular Sciences, 2020, Vol. 21, No. 4949 [Non-patent document 4] Gillis et al., Science Signaling, 2020, Vol. 13, No. 625, eaaz3140 Summary of the Invention [Problem to be solved by the invention]

[0013] An objective of the present invention is to provide an evaluation system for the binding reaction of β-arrestin 2 to the opioid μ receptor. [Means for solving the problem]

[0014] As a result of extensive research to solve the above problems, the inventors discovered a cell line and method that enables highly accurate and consistent evaluation of the β-arrestin 2 binding reaction to the opioid μ receptor using a combination of NanoLuc (registered trademark) luciferase and HaloTag (registered trademark) by a BRET assay, thereby completing the present invention.

[0015] That is, the present invention includes the following.

[0016] [1] A gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 are introduced into the mouse. A transformed animal cultured cell, wherein one of the fusion proteins, either the fusion protein containing the opioid μ receptor or the fusion protein containing the β-arrestin 2, contains a luciferase having the amino acid sequence shown in SEQ ID NO: 1, and the other fusion protein contains a protein tag having the amino acid sequence shown in SEQ ID NO: 3. [2] The gene encoding the fusion protein containing the opioid μ receptor encodes a fusion protein in which the luciferase is fused to the C-terminus of the opioid μ receptor; The transformed animal cultured cell described in [1] above, wherein the gene encoding the fusion protein containing the β-arrestin 2 encodes a fusion protein in which the protein tag is fused to the C-terminus of β-arrestin 2. [3] In the absence of an opioid μ receptor agonist, the transformed animal cultured cells a) in the presence of a luminescent substrate specific to the luciferase, the luminescence generated by the reaction between the luciferase and the luminescent substrate exhibits a luminescence intensity that is 35 times or more higher than that in the absence of the luminescent substrate; and b) The cultured transformed animal cell according to [1] or [2] above, which exhibits fluorescence from a fluorescently labeled ligand specific to the protein tag, which is generated by irradiation with excitation light after addition of the fluorescently labeled ligand, with a fluorescence intensity that is 8 times or more higher than that in the absence of the fluorescently labeled ligand. [4] The cultured transformed animal cell according to any one of [1] to [3] above, which is a transformant of an HEK293 cell. [5] The cultured transformed animal cell according to any one of [1] to [4] above, which is a cell line HEK-MGA #7 having accession number NITE AP-04097. [6] A method for producing a transformed animal cultured cell that can be used to evaluate the binding response of β-arrestin 2 to the opioid μ receptor, comprising: i) a step of introducing a gene encoding a fusion protein comprising an opioid μ receptor, a gene encoding a fusion protein comprising β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 into a cultured animal cell to obtain a transformed cell, wherein one of the fusion proteins, the opioid μ receptor-containing fusion protein or the β-arrestin 2-containing fusion protein, comprises a luciferase having the amino acid sequence shown in SEQ ID NO: 1, and the other fusion protein comprises a protein tag having the amino acid sequence shown in SEQ ID NO: 3; ii) adding a fluorescently labeled ligand specific to the protein tag to the transformed cells obtained in step i) in the absence of an opioid μ receptor agonist, and irradiating the transformed cells with excitation light to measure the fluorescence derived from the fluorescently labeled ligand; iii) adding a luminescent substrate specific for the luciferase to the transformed cells obtained in step i) in the absence of an opioid μ receptor agonist, and measuring luminescence; iv) from the transformed cells obtained in step i), a) in step ii), the fluorescence generated by irradiation with excitation light exhibits a fluorescence intensity that is 8 times or more higher than that in the absence of the fluorescently labeled ligand; and b) In step iii), the luminescence generated by the reaction between the luciferase and the luminescent substrate exhibits a luminescence intensity 35 times or more compared to that in the absence of the luminescent substrate. selecting the cells; A method comprising: [7] A step of contacting a test substance with the cultured transformed animal cell according to any one of [1] to [5] above; measuring the level of induction of β-arrestin2 binding to the opioid μ receptor by measuring bioluminescence resonance energy transfer (BRET) that occurs in the presence of a fluorescently labeled ligand specific to the protein tag and a luminescent substrate specific to the luciferase in the cultured transformed animal cells contacted with the test substance; Using the measured induction level of β-arrestin 2 binding response to the opioid μ receptor as a level of β-arrestin 2 activation via the opioid μ receptor, and comparing it with a level of complete activation of β-arrestin 2 via the opioid μ receptor; A method for assessing the level of β-arrestin 2 activation mediated by an opioid μ receptor by a test substance, comprising: [8] Measuring the level of β-arrestin 2 activation mediated by the opioid μ receptor by the test substance using the method described in [7] above; comparing the measured β-arrestin 2 activation level with the G protein activation level via the opioid μ receptor and / or the full activation level of β-arrestin 2 via the opioid μ receptor to select a test substance that exhibits bias in intracellular signal transduction via the opioid μ receptor; A method for screening for an opioid μ receptor agonist having bias, comprising: [Effects of the Invention]

[0017] The present invention provides cells and methods that enable stable evaluation of the binding response of β-arrestin 2 to the opioid μ receptor. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram of a method for producing a cell line and a BRET assay using the same in an example of the present application. [Figure 2] Figure 2 is a graph showing the BRET ratios obtained by the BRET assay in the solvent-treated group (open bars) and the positive control substance-treated group (filled bars) before (pre) and after (post) treatment. A: #2, B: #7, C: #15, D: #17, E: #18, F: Representative examples of clonal cell lines in which no change in BRET ratio was observed. [Figure 3]Figure 3 shows the results of evaluating the β-arrestin 2 binding activity of positive control substances to the human opioid μ receptor, using five clonal cell lines in which BRET was detected: A: #2, B: #7, C: #18, D: #15, and E: #17. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail below.

[0020] The present invention relates to cells suitable for use in evaluating the binding response of β-arrestin2 to the opioid μ receptor based on a BRET assay using luciferase and a protein tag, and a method for evaluating the binding response of β-arrestin2 to the opioid μ receptor using the cells. More specifically, the present invention relates to the cells and evaluation method based on a BRET assay using a luciferase (NanoLuc luciferase) consisting of the amino acid sequence shown in SEQ ID NO: 1 and a protein tag (HaloTag) consisting of the amino acid sequence shown in SEQ ID NO: 3. In particular, the present invention provides a method for evaluating the level of opioid μ receptor-mediated β-arrestin2 activation by a test substance using cells suitable for evaluating the binding response of β-arrestin2 to the opioid μ receptor. The present invention also provides a biased screening method for opioid μ receptor agonists using such an evaluation method. In the present invention, an opioid μ receptor agonist refers to a substance that can bind to the opioid μ receptor and activate intracellular signaling (specifically, at least a G protein pathway) mediated by the opioid μ receptor.

[0021] In the present invention, cells that can be used to evaluate the binding response of β-arrestin 2 to the opioid μ receptor are cultured cells transfected with genes expressing a fusion protein containing the opioid μ receptor, a fusion protein containing β-arrestin 2, and G protein-coupled receptor kinase 2 (GRK2). More specifically, the cells used in the present invention are cultured cells transfected with a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 (GRK2). In the cultured cells of the present invention, one of the fusion proteins encoded by the transgenes, either the opioid μ receptor-containing fusion protein or the β-arrestin 2-containing fusion protein, contains a luciferase (NanoLuc luciferase) consisting of the amino acid sequence set forth in SEQ ID NO: 1, and the other fusion protein contains a protein tag (HaloTag) consisting of the amino acid sequence set forth in SEQ ID NO: 3. The gene encoding the fusion protein containing the opioid μ receptor contains a nucleotide sequence encoding the opioid μ receptor protein (opioid μ receptor gene). A gene encoding a fusion protein containing β-arrestin 2 contains a nucleotide sequence encoding β-arrestin 2 protein (β-arrestin 2 gene). A gene encoding G protein-coupled receptor kinase 2 (GRK2) (GRK2 gene) contains a nucleotide sequence encoding GRK2. In the present invention, the term "gene" refers to a nucleic acid (such as DNA or RNA) having a nucleotide sequence encoding the amino acid sequence of a protein, and may or may not contain an initiation codon and / or a termination codon.

[0022] In the present invention, the opioid μ receptor protein, β-arrestin 2 protein, and GRK2 protein, as well as the base sequences (genes) encoding these proteins, may be derived from any animal, but are preferably derived from chordates, more preferably from vertebrates (e.g., mammals, birds, reptiles, amphibians, or fish), and particularly preferably from mammals. Examples of mammals include, but are not limited to, animals belonging to the orders Primates, Rodentia, Dodontia, Artiodactyla, Perissodactyla, or Carnivora. Examples of animals belonging to the order Primates (primates) include humans and non-human primates (e.g., chimpanzees, gorillas, orangutans, Japanese macaques, bonobos, cynomolgus monkeys, rhesus monkeys, and squirrel monkeys), more preferably humans. Examples of animals belonging to the order Rodentia include, but are not limited to, mice, rats, hamsters, and guinea pigs. Examples of animals belonging to the order Dodontia include, but are not limited to, rabbits. Animals belonging to the order Artiodactyla include, but are not limited to, cattle, pigs, sheep, camels, llamas, deer, wild boars, whales, and dolphins. Animals belonging to the order Perissodactyla include, but are not limited to, horses, rhinoceroses, and tapirs. Animals belonging to the order Carnivora include, but are not limited to, dogs, cats, pandas, weasels, otters, and bears. The opioid μ receptor, β-arrestin 2, and GRK2 are preferably derived from the same species.

[0023] The opioid μ receptor protein, β-arrestin 2 protein, and GRK2 protein, as well as the base sequences (genes) encoding these proteins, may be wild-type or mutants (preferably biologically active mutants).

[0024] The "opioid μ receptor" in the present invention is a subtype of opioid receptor, which is a G protein-coupled receptor (GPCR). The "opioid μ receptor" used in the present invention may be a wild-type or a mutant.

[0025] In one embodiment, the opioid μ receptor (preferably a human opioid μ receptor) of the present invention can be a protein of (i) or (ii) below: (i) a protein consisting of an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 98% or more or 99% or more to the amino acid sequence shown in SEQ ID NO: 5; (ii) A protein consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) amino acids in the amino acid sequence shown in SEQ ID NO: 5. The opioid μ receptor of the present invention has G protein binding activity.

[0026] In one embodiment, the base sequence encoding the opioid μ receptor (preferably, the human opioid μ receptor) of the present invention (or the opioid μ receptor gene, preferably the human opioid μ receptor gene) may be a base sequence (gene) selected from the group consisting of the following (i) to (iv): (i) a base sequence (gene) encoding a protein consisting of an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more or 99% or more, for example, 99.5% or more, to the amino acid sequence shown in SEQ ID NO: 5; (ii) a base sequence (gene) encoding a protein consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) amino acids in the amino acid sequence shown in SEQ ID NO: 5; (iii) a base sequence (gene) having a sequence identity of 90% or more, preferably 95% or more, more preferably 99% or more, even more preferably 99.5% or more or 99.7% or more, for example, 99.9% or more, to the base sequence shown in SEQ ID NO: 6; (iv) A nucleotide sequence (gene) having an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) nucleotides in the nucleotide sequence shown in SEQ ID NO: 6. The nucleotide sequence encoding the opioid μ receptor (opioid μ receptor gene) of the present invention encodes a protein having G protein binding activity.

[0027] In one embodiment, the opioid μ receptor of the present invention is a human opioid μ receptor. In a preferred embodiment, the human opioid μ receptor protein consists of the amino acid sequence shown in SEQ ID NO: 5. In a preferred embodiment, the nucleotide sequence encoding the human opioid μ receptor protein is the nucleotide sequence shown in SEQ ID NO: 6. The nucleotide sequence shown in SEQ ID NO: 6 (or an opioid μ receptor gene consisting of it) encodes the opioid μ receptor protein consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0028] "Arrestins" are cytoplasmic adaptor proteins that bind to GPCRs, thereby causing desensitization of the GPCRs and intracellular signal transduction in the arrestin pathway. Arrestins are classified into visual arrestins and non-visual arrestins, and "β-arrestin2" is a non-visual arrestin. "β-arrestin2" in the present invention is a type of arrestin that binds to the opioid μ receptor to cause intracellular signal transduction in the β-arrestin pathway. The "β-arrestin2" used in the present invention may be a wild-type or mutant.

[0029] In one embodiment, the β-arrestin2 (preferably human β-arrestin2) of the present invention can be a protein (i) or (ii) below: (i) a protein consisting of an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more or 99% or more, for example 99.5% or more, to the amino acid sequence shown in SEQ ID NO: 7; (ii) A protein consisting of the amino acid sequence shown in SEQ ID NO: 7, which has 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) amino acid insertions, deletions, substitutions, and / or additions. The β-arrestin 2 of the present invention has opioid μ receptor binding activity.

[0030] In one embodiment, the base sequence encoding β-arrestin2 (preferably, human β-arrestin2) (or β-arrestin2 gene, preferably human β-arrestin2 gene) of the present invention may be a base sequence (gene) selected from the group consisting of the following (i) to (iv): (i) a base sequence (gene) encoding a protein consisting of an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more or 99% or more, for example, 99.5% or more, to the amino acid sequence shown in SEQ ID NO: 7; (ii) a base sequence (gene) encoding a protein consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) amino acids in the amino acid sequence shown in SEQ ID NO: 7; (iii) a base sequence (gene) having a sequence identity of 90% or more, preferably 95% or more, more preferably 99% or more, even more preferably 99.5% or more or 99.7% or more, for example, 99.9% or more, to the base sequence shown in SEQ ID NO: 8; (iv) A base sequence (gene) having an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) bases in the base sequence shown in SEQ ID NO: 8. The base sequence encoding β-arrestin 2 (β-arrestin 2 gene) of the present invention encodes a protein having opioid μ receptor binding activity.

[0031] In one embodiment, the β-arrestin2 of the present invention is human β-arrestin2. In a preferred embodiment, the human β-arrestin2 protein consists of the amino acid sequence shown in SEQ ID NO: 7. In a preferred embodiment, the nucleotide sequence encoding the human β-arrestin2 protein is the nucleotide sequence shown in SEQ ID NO: 8. The nucleotide sequence shown in SEQ ID NO: 8 (a β-arrestin2 gene consisting of) encodes the β-arrestin2 protein consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0032] In the present invention, "G protein-coupled receptor kinase 2 (GRK2)" is a type of GRK, a serine-threonine kinase that phosphorylates GPCRs. When an agonist-bound GPCR, such as the opioid μ receptor, activates GRK2, the activated GRK2 phosphorylates the C-terminus of the GPCR. The phosphorylated GPCR uncouples from the G protein and binds to arrestin, resulting in intracellular signal transduction via the arrestin pathway. In the present invention, GRK2 phosphorylates the opioid μ receptor and promotes the binding of β-arrestin 2 to the opioid μ receptor. The "G protein-coupled receptor kinase 2 (GRK2)" used in the present invention may be a wild-type or mutant.

[0033] In one embodiment, the GRK2 (preferably human GRK2) of the present invention can be a protein of (i) or (ii) below: (i) a protein consisting of an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more or 99% or more, for example 99.5% or more, to the amino acid sequence shown in SEQ ID NO: 9; (ii) A protein consisting of the amino acid sequence shown in SEQ ID NO: 9, which has an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) amino acids. GRK2 in the present invention has serine / threonine kinase activity.

[0034] In one embodiment, the base sequence (or GRK2 gene, preferably human GRK2 gene) encoding GRK2 (preferably human GRK2) in the present invention may be a base sequence (gene) selected from the group consisting of the following (i) to (iv): (i) a base sequence (gene) encoding a protein consisting of an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more or 99% or more, for example, 99.5% or more, to the amino acid sequence shown in SEQ ID NO: 9; (ii) a base sequence (gene) encoding a protein consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 10 amino acids (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) in the amino acid sequence shown in SEQ ID NO: 9; (iii) a base sequence (gene) having a sequence identity of 90% or more, preferably 95% or more, more preferably 99% or more, even more preferably 99.5% or more or 99.7% or more, for example, 99.9% or more, to the base sequence shown in SEQ ID NO: 10; (iv) A base sequence (gene) having an insertion, deletion, substitution, and / or addition of 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) bases in the base sequence shown in SEQ ID NO: 10. The base sequence encoding β-arrestin 2 (β-arrestin 2 gene) of the present invention encodes a protein having opioid μ receptor binding activity.

[0035] In one embodiment, the GRK2 of the present invention is human GRK2. In a preferred embodiment, the human GRK2 protein consists of the amino acid sequence shown in SEQ ID NO: 9. In a preferred embodiment, the nucleotide sequence encoding the human GRK2 protein is the nucleotide sequence shown in SEQ ID NO: 10. The nucleotide sequence shown in SEQ ID NO: 10 (the GRK2 gene consisting of) encodes the GRK2 protein consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0036] In the present invention, "sequence identity" refers to the percentage (%) of residues that match between a reference biological sequence (nucleotide sequence or amino acid sequence) and a target biological sequence when the sequences are aligned (usually the percentage of matching residues relative to the entire length of the reference biological sequence). "Sequence identity" can be calculated, for example, using EMBOSS Needle (Weizhong L et al., Nucleic Acids Res., 2015, 43:W580-W584) with the following default parameters: Gap Open Penalty = 10 Gap Extend Penalty = 0.5 Matrix = EBLOSUM62 End Gap Penalty = false

[0037] In the present invention, a gene encoding a fusion protein containing an opioid μ receptor encodes a fusion protein containing the opioid μ receptor and NanoLuc luciferase or HaloTag. Furthermore, a gene encoding a fusion protein containing β-arrestin2 encodes a fusion protein containing β-arrestin2 and either NanoLuc luciferase or HaloTag, whichever molecule is not fused to the opioid μ receptor. A fusion protein containing an opioid μ receptor and NanoLuc luciferase or HaloTag is preferably a fusion protein in which NanoLuc luciferase or HaloTag is fused to the C-terminus of the opioid μ receptor. A fusion protein containing β-arrestin2 and NanoLuc luciferase or HaloTag is preferably a fusion protein in which NanoLuc luciferase or HaloTag is fused to the C-terminus or N-terminus of β-arrestin2. In the fusion protein of the present invention, NanoLuc luciferase or HaloTag may be fused to the "C-terminus" or "N-terminus" of the opioid μ receptor or β-arrestin 2 via another polypeptide such as a linker between the C-terminus or N-terminus of the opioid μ receptor or β-arrestin 2 and the NanoLuc luciferase or HaloTag. Preferred combinations of proteins to be contained in the fusion protein of the present invention are shown in Table 1.

[0038] [Table 1]

[0039] The configuration of the fusion protein containing the opioid μ receptor and the fusion protein containing β-arrestin 2 may be any combination of Nos. 1 to 4 shown in Table 1, but the combination of No. 2, in which NanoLuc luciferase is fused to the C-terminus of the opioid μ receptor and HaloTag is fused to the C-terminus of β-arrestin 2, is more preferred.

[0040] A luciferase consisting of the amino acid sequence shown in SEQ ID NO: 1 is commercially available from Promega under the name "NanoLuc (registered trademark) luciferase." In the present invention, the luciferase consisting of the amino acid sequence shown in SEQ ID NO: 1 is also referred to as NanoLuc luciferase. NanoLuc luciferase is derived from the deep-sea shrimp Oplophorus gracilirostris, and is a 19 kDa enzyme obtained by modifying a catalytically active subunit. NanoLuc luciferase produces a strong luminescent signal at a wavelength of 460 nm upon reaction with the luminescent substrate furimazine (2-furanylmethyldeoxy-coelenterazine).

[0041] A protein tag consisting of the amino acid sequence shown in SEQ ID NO: 3 is commercially available from Promega under the trademark "HaloTag (registered trademark)." In the present invention, the protein tag consisting of the amino acid sequence shown in SEQ ID NO: 3 is also referred to as HaloTag (or HaloTag). HaloTag is a self-labeling protein tag derived from haloalkane dehalogenase. A fusion protein containing HaloTag is labeled by covalently binding to HaloTag a fluorescently labeled ligand specific to the protein tag (HaloTag) that has a chloroalkane (hereinafter also referred to as a "HaloTag-specific fluorescently labeled ligand" or "HaloTag fluorescently labeled ligand").

[0042] In the present invention, the cells into which the gene encoding a fusion protein containing the opioid μ receptor, the gene encoding a fusion protein containing β-arrestin 2, and the gene encoding GRK2 are introduced are cultured animal cells. The cultured animal cells may be derived from any animal, but are preferably derived from chordates, more preferably from vertebrates (e.g., mammals, birds, reptiles, amphibians, or fish), and particularly preferably from mammals. Examples of mammals include, but are not limited to, animals belonging to the orders Primates, Rodentia, Dodontia, Artiodactyla, Perissodactyla, or Carnivora. Examples of animals belonging to the order Primates (primates) include humans and non-human primates (e.g., chimpanzees, gorillas, orangutans, Japanese macaques, bonobos, cynomolgus monkeys, rhesus monkeys, and squirrel monkeys), more preferably humans. Examples of animals belonging to the order Rodentia include, but are not limited to, mice, rats, hamsters, and guinea pigs. Examples of animals belonging to the order Dodecadenta include, but are not limited to, rabbits. Examples of animals belonging to the order Artiodactyla include, but are not limited to, cattle, pigs, sheep, camels, llamas, deer, wild boars, whales, and dolphins. Examples of animals belonging to the order Perissodactyla include, but are not limited to, horses, rhinoceroses, and tapirs. Examples of animals belonging to the order Carnivora include, but are not limited to, dogs, cats, pandas, weasels, otters, and bears. The cultured cells are preferably derived from the same species as the opioid μ receptor, β-arrestin 2, and / or GRK2 to be introduced. In one embodiment, the cultured cells are human cultured cells. In one embodiment, the human cultured cells are cultured cells derived from a human fetus. In one embodiment, the human cultured cells may be, but are not limited to, HEK293 cells, which are derived from human fetal kidney. HEK293 cells do not contain any exogenously introduced genes and barely express the opioid μ receptor. In the present invention, the term "cultured cells" refers to cells cultured in vitro.

[0043] Introduction (artificial introduction) of a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 into cultured cells can be carried out by conventional methods. For example, gene introduction into cultured cells may be carried out by the calcium phosphate method, lipofection, DEAE-dextran method, electroporation, microinjection, or the like using a gene expression nucleic acid construct such as an expression vector (e.g., a plasmid vector) or expression cassette containing the gene. Alternatively, gene introduction into cultured cells may be carried out by transduction using a viral vector (viral particle) having a viral genome incorporating the gene. The viral vector may be an adeno-associated viral vector, an adenoviral vector, a retroviral vector, a lentiviral vector, or the like. The gene is preferably introduced under the control of a promoter. To facilitate confirmation of gene introduction, a gene expression nucleic acid construct such as an expression vector (e.g., a plasmid vector) or an expression cassette, or a viral vector containing the gene as well as a selection marker gene such as a drug resistance gene, may be used. In one embodiment, an expression vector comprising a gene encoding a fusion protein comprising the opioid μ receptor comprises the nucleotide sequence shown in SEQ ID NO: 11. In one embodiment, an expression vector comprising a gene encoding a fusion protein comprising β-arrestin2 comprises the nucleotide sequence shown in SEQ ID NO: 12. In one embodiment, an expression vector comprising a gene encoding GRK2 comprises the nucleotide sequence shown in SEQ ID NO: 13. After gene introduction into cultured cells, it is preferable to select cultured cells expressing the gene. Cultured cells may be selected using a selection marker.

[0044] The present invention provides transformed cells prepared as described above, into which a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 have been introduced. In the present invention, the term "transformed cell" refers to a cell that harbors an exogenously introduced gene and whose phenotype has been changed due to the cell expressing the introduced gene or being in a state where it can be expressed, and is also referred to as a "transformant."

[0045] In one embodiment, a transformed cell into which a gene encoding a fusion protein comprising the opioid μ receptor, a gene encoding a fusion protein comprising β-arrestin2, and a gene encoding GRK2 has been introduced preferably stably expresses the gene encoding the fusion protein comprising the opioid μ receptor, the gene encoding the fusion protein comprising β-arrestin2, and the gene encoding GRK2. In the present invention, "stable expression" of a transgene (or protein) refers to constitutive (continuous) expression of the transgene in a state where the transgene is integrated into at least one site in the cell genome (chromosome) or in an episomal vector stably maintained in proliferating cells.

[0046] Transformed cells into which a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 have been introduced are preferably monocloned. Monoclonal cloning can be achieved by standard methods such as limiting dilution or colony isolation (seeding cells at low density in a petri dish and then picking the formed cell colonies). Monoclonal transformed cells are also referred to as cell clones, clonal cell strains, or cell strains.

[0047] Transformed cells according to the present invention, into which a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin2, and a gene encoding GRK2 have been introduced, can generate luminescence through the reaction between NanoLuc luciferase and a luminescent substrate specific to NanoLuc luciferase, even in the absence of an opioid μ receptor agonist, and can generate fluorescence derived from a HaloTag-specific fluorescent-labeled ligand upon irradiation with excitation light after the addition of the HaloTag-specific fluorescent-labeled ligand. In the present invention, with regard to the fluorescence derived from the HaloTag-specific fluorescent-labeled ligand, the "excitation light" irradiated onto the transformed cells is light of a wavelength capable of exciting the fluorophore in the HaloTag-specific fluorescent-labeled ligand to generate fluorescence.

[0048] The transformed cells of the present invention, into which a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin2, and a gene encoding GRK2 have been introduced, are preferably capable of: i) exhibiting high luminescence intensity upon reaction of NanoLuc luciferase with a luminescent substrate specific to NanoLuc luciferase, in the absence of an opioid μ receptor agonist; and ii) exhibiting high fluorescence intensity derived from the fluorescently labeled ligand upon irradiation with excitation light after addition of a HaloTag-specific fluorescently labeled ligand. Specifically, the transformed cells of the present invention are capable of: a) In the presence of a luminescent substrate specific to NanoLuc luciferase, the luminescence generated by the reaction between NanoLuc luciferase and the luminescent substrate exhibits a luminescence intensity that is 35 times or more higher than that in the absence of the luminescent substrate; and b) It is preferable that the fluorescence from the fluorescently labeled ligand specific to HaloTag generated by irradiation with excitation light after addition of the fluorescently labeled ligand exhibits a fluorescence intensity that is 8 times or more higher than that in the absence of the fluorescently labeled ligand.

[0049] In a more preferred embodiment, the transformed cell of the present invention is capable of expressing the following in the absence of an opioid μ receptor agonist: a) in the presence of a luminescent substrate specific to NanoLuc luciferase, the luminescence generated by the reaction between NanoLuc luciferase and the luminescent substrate exhibits a luminescence intensity that is 50 times or more higher than that in the absence of the luminescent substrate; and b) The fluorescence from the fluorescently labeled ligand specific to HaloTag, which is generated by irradiation with excitation light after addition of the fluorescently labeled ligand, exhibits a fluorescence intensity 14 times or more compared to when the fluorescently labeled ligand is not added.

[0050] In the present invention, "in the absence of an opioid μ receptor agonist" refers to a condition in which no opioid μ receptor agonist is added to cells. "In the absence of an opioid μ receptor agonist" can typically be a condition in which no test substance, including an opioid μ receptor agonist, is added to cells.

[0051] Here, furimazine (2-furanylmethyldeoxy-coelenterazine) can be used as a luminescent substrate specific to NanoLuc luciferase, and HaloTag 1000 (HaloTag 1000) which exhibits a fluorescence wavelength of 618 nm can be used as a fluorescently labeled ligand specific to HaloTag. (R) NanoBRET TM 618 Ligand (Promega) can be used, but the combination of luminescent substrate and fluorescently labeled ligand is not limited to this. When furimazine is used as the luminescent substrate, it is preferable to measure the luminescence intensity (RLU) at 460 nm to detect the signal from the luminescent substrate. (R) NanoBRET TM When 618 Ligand is used as a fluorescently labeled ligand, it is preferable to detect a signal from the fluorescently labeled ligand alone by irradiating it with excitation light at a wavelength of 540 nm and measuring the fluorescence intensity (RFU) at 620 nm.

[0052] The above luminescence and fluorescence measurements can be performed after culturing the cells for 3-4 hours in a carbon dioxide incubator at 37°C with 5% CO2. This cell culture is performed in 1% GlutaMAX TMIt is preferable to carry out the experiment in a CO2-independent medium (Thermo Fisher Scientific) supplemented with Calcium Supplement (Thermo Fisher Scientific).

[0053] In transformed cells into which a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 according to the present invention has been introduced, the gene expression level of the introduced gene can be measured by standard methods. For example, in the present invention, the gene expression level of the opioid μ receptor can be measured by standard methods such as real-time PCR (quantitative PCR) based on reverse transcription PCR (RT-PCR) or in situ hybridization using a nucleic acid probe or primer that specifically hybridizes with mRNA transcribed from the gene of interest. For example, a nucleic acid probe or primer that specifically hybridizes with mRNA transcribed from the opioid μ receptor gene can be appropriately designed by those skilled in the art based on the nucleotide sequence of the opioid μ receptor gene. Calculation of gene expression level in real-time PCR can be performed by standard methods such as the calibration curve method or the comparative Ct method, but the comparative Ct method is preferred. When measuring the gene expression level of an introduced gene, it is also preferable to measure in parallel the expression level of an endogenous housekeeping gene that is always expressed within a certain range, such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, in order to standardize the measured values.

[0054] For PCR, reaction conditions can be selected that optimize the yield and specificity of the PCR amplification product. Examples of PCR reaction parameters include the length and base sequence of primers, the temperature and reaction time of the annealing and extension steps, and the Mg ion concentration or salt concentration. Those skilled in the art can appropriately set these parameters. In one embodiment, the temperature cycle conditions for real-time PCR used to measure the gene expression level of the opioid μ receptor may be as follows: an initial denaturation step at 95°C for 10 minutes, followed by another denaturation step at 95°C for 15 seconds, an annealing step at 60°C for 1 minute, and an extension step at 95°C for 15 seconds, with the denaturation step and extension step repeated 40 times.

[0055] A preferred example of a transformed cell according to the present invention into which a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 have been introduced is the cell line HEK-MGA #7. Cell line HEK-MGA #7 was deposited on March 11, 2024, with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under accession number NITE AP-04097.

[0056] Transformed cells of the present invention, into which a gene encoding a fusion protein containing the opioid μ receptor, a gene encoding a fusion protein containing β-arrestin2, and a gene encoding GRK2 have been introduced, can be used in an assay utilizing bioluminescence resonance energy transfer (BRET), which occurs when NanoLuc luciferase and a HaloTag-specific fluorescently labeled ligand bound to HaloTag are brought into close proximity due to binding between the opioid μ receptor and β-arrestin2 in the presence of a luminescent substrate specific for NanoLuc luciferase. BRET enables real-time measurement of protein-protein interactions, allowing the binding of β-arrestin2 to the opioid μ receptor to be detected with high temporal resolution. More specifically, the transformed cells of the present invention can be used to evaluate the β-arrestin2 binding response to the opioid μ receptor. For example, the transformed cells of the present invention can be used to evaluate the β-arrestin2 binding response to the opioid μ receptor induced by a test substance. The transformed cells of the present invention can also be used to evaluate the level of β-arrestin 2 activation mediated by the opioid μ receptor by a test substance.

[0057] In one embodiment, the present invention provides a method for assessing the level of β-arrestin2 activation by a test substance via the opioid μ receptor, comprising contacting the test substance with a transformed cell of the present invention and then measuring bioluminescence resonance energy transfer (BRET) in the presence of a fluorescently labeled ligand specific for HaloTag and a luminescent substrate specific for NanoLuc luciferase.

[0058] In one embodiment, the method of the present invention comprises the following steps: A step of contacting a test substance with the transformed cell of the present invention; measuring the level of induction of β-arrestin2 binding to the opioid μ receptor in the transformed cells contacted with the test substance by measuring bioluminescence resonance energy transfer (BRET) in the presence of a HaloTag-specific fluorescently labeled ligand and a NanoLuc luciferase-specific luminescent substrate; Using the measured induction level of β-arrestin 2 binding response to the opioid μ receptor as a level of β-arrestin 2 activation via the opioid μ receptor, and comparing it with a level of complete activation of β-arrestin 2 via the opioid μ receptor; The method may be a method for evaluating the level of β-arrestin 2 activation by a test substance via the opioid μ receptor, comprising:

[0059] The HaloTag-specific fluorescently labeled ligand may be any substance capable of specifically binding to a protein tag consisting of the amino acid sequence set forth in SEQ ID NO: 3 and emitting fluorescence. The HaloTag-specific fluorescently labeled ligand used in the present invention is preferably, but not limited to, HaloTag® NanoBRET® 618 Ligand (Promega), which exhibits a fluorescence wavelength of 618 nm. The HaloTag® NanoBRET® 618 Ligand is a compound represented by the following chemical formula (see Supplementary Figure 2 in Thirukkumaran et al., Frontiers in Chemistry, 2020, Vol. 7, 938).

[0060] [ka]

[0061] The luminescent substrate specific to NanoLuc luciferase may be any substance that emits light upon reaction with NanoLuc luciferase. The luminescent substrate specific to NanoLuc luciferase used in the present invention is preferably, but not limited to, furimazine. The luminescent substrate specific to NanoLuc luciferase is, for example, NanoBRET containing furimazine. TM Nano-Glo (R) It is commercially available as Substrate (Promega).

[0062] The "test substance" of the present invention refers to a substance that is the subject of the method of the present invention, and is used to evaluate its ability to induce the binding reaction of β-arrestin2 to the opioid μ receptor or the level of β-arrestin2 activation mediated by the opioid μ receptor. In one embodiment, the "test substance" of the present invention is an opioid μ receptor agonist or a candidate substance thereof, or at least one substance containing such an agonist. The "test substance" is not particularly limited and may be a natural or non-natural organic or inorganic compound, an in vivo substance, a substance produced by genetic engineering (genetically recombinant substance), or a chemically synthesized substance. The "test substance" may be a protein, an antibody, a peptide, a nucleic acid (such as DNA, RNA, or artificial nucleic acid), a polymeric compound, or a low molecular weight compound.

[0063] The "test substance" may be a concentration-difference-biasing compound or an intensity-difference-biasing compound. In the present invention, a concentration-difference-biasing compound is a compound that exhibits concentration-difference bias in intracellular signaling mediated by the opioid μ receptor. In the present invention, "concentration-difference bias" refers to the property of an opioid μ receptor agonist that more strongly activates the G protein pathway at a lower concentration than the β-arrestin 2 pathway, but can fully activate the β-arrestin 2 pathway (exhibiting full agonism of β-arrestin 2 pathway activation). In the present invention, an intensity-difference-biasing compound is a compound that exhibits intensity-difference bias in intracellular signaling mediated by the opioid μ receptor. In the present invention, "intensity-difference bias" refers to the property of an opioid μ receptor agonist that weakly activates the β-arrestin 2 pathway and is unable to fully activate the β-arrestin 2 pathway, i.e., exhibits partial agonism of β-arrestin 2 pathway activation.

[0064] In the method of the present invention, the test substance may be dissolved or suspended in a solvent and added to the cells. The solvent is not particularly limited as long as it is a pharmaceutically acceptable carrier, and examples thereof include cell culture medium (culture medium) and various buffer solutions (e.g., HEPES buffer and phosphate buffer). The treatment dose (treatment concentration), number of treatments, treatment timing, and treatment period of the test substance can be appropriately determined.

[0065] The transformed cells of the present invention are preferably contacted with a test substance in a culture medium. The medium used to contact the transformed cells with a test substance may be any medium, such as a culture medium containing or not containing serum (e.g., FBS), a culture medium containing or not containing HEPES, a culture medium containing or not containing phenol red, or a CO2-dependent or CO2-independent medium. In one embodiment, 1% GlutaMAX TMThe transformed cells of the present invention may be contacted with a test substance in a CO2-independent medium (Thermo Fisher Scientific) supplemented with 1% GlutaMAX Supplement (Thermo Fisher Scientific). TM Supplement (Thermo Fisher Scientific) may be added before use. The transformed cells of the present invention may be contacted with a test substance after culturing them in the above-mentioned culture medium for a predetermined period of time (e.g., 3 to 4 hours). Such culturing may be carried out, for example, in a carbon dioxide incubator at 37°C and 5% CO2. When the transformed cells of the present invention are contacted with a test substance, if the test substance binds to the opioid μ receptor expressed in the transformed cells and activates the opioid μ receptor, G protein is activated and a binding reaction of β-arrestin2 to the opioid μ receptor is induced, which may result in β-arrestin2 activation.

[0066] In the method of the present invention, a fluorescently labeled ligand specific to HaloTag may be added to the cells before or after, or simultaneously with, contacting the test substance with the transformed cells of the present invention. In one embodiment, the fluorescently labeled ligand specific to HaloTag is preferably added to the cells before contacting the test substance with the transformed cells of the present invention. In the method of the present invention, a luminescent substrate specific to NanoLuc luciferase may be added to the cells before or after, or simultaneously with, contacting the test substance with the transformed cells of the present invention. In one embodiment, the luminescent substrate specific to NanoLuc luciferase is preferably added to the cells after contacting the test substance with the transformed cells of the present invention (e.g., 2 to 5 minutes after the contact). In one embodiment, after adding the fluorescently labeled ligand specific to HaloTag to the cells (e.g., the day after the addition), the test substance may be contacted with the transformed cells of the present invention, and then the luminescent substrate specific to NanoLuc luciferase may be added to the cells after the contact (e.g., 2 to 5 minutes after the contact). When the HaloTag-specific fluorescent-labeled ligand and NanoLuc luciferase are brought into close proximity, BRET can occur between the luminescence derived from NanoLuc luciferase and the fluorescent-labeled ligand in the presence of the HaloTag-specific fluorescent-labeled ligand and a luminescent substrate specific for NanoLuc luciferase. When a binding reaction of β-arrestin2 to the opioid μ receptor is induced via binding of a test substance to the opioid μ receptor expressed in a transformed cell, the HaloTag-specific fluorescent-labeled ligand bound to the HaloTag fused to either the opioid μ receptor or β-arrestin2 comes into close proximity with the NanoLuc luciferase fused to the other (β-arrestin2 or opioid μ receptor), and BRET can occur in the presence of the HaloTag-specific fluorescent-labeled ligand and a luminescent substrate specific for NanoLuc luciferase. In the present invention, "BRET occurring in the presence" of a HaloTag-specific fluorescently labeled ligand and a NanoLuc luciferase-specific luminescent substrate refers to BRET that occurs in a state in which the HaloTag-specific fluorescently labeled ligand and the NanoLuc luciferase-specific luminescent substrate that have been added to cells are present within the cells.In the method of the present invention, BRET as described above may be measured.

[0067] In the method of the present invention, the transformed cells of the present invention may be seeded on a plate or the like at a predetermined cell number and cultured for a certain period of time before contacting the transformed cells of the present invention with a test substance. The plate, cell number, culture time, and the like can be appropriately determined by those skilled in the art. The transformed cells of the present invention may be thawed frozen cells or unfrozen cells that have been passaged a certain number of times. In one embodiment, the transformed cells of the present invention may be seeded on a plate or the like, and a fluorescently labeled ligand specific to HaloTag may be added to the cells during culture prior to contact with the test substance.

[0068] In one embodiment, the method of the present invention may comprise the following steps: i) seeding the transformed cells of the present invention in a culture medium, for example, a medium containing serum (such as FBS), and culturing them in the presence of a fluorescently labeled ligand specific for HaloTag; ii) Replace the serum-containing medium used in step i) with CO2-independent medium (e.g., 1% GlutaMAX) that does not contain serum (e.g., FBS). TM Substituting the medium (CO2-independent medium) with supplements; iii) adding the test substance to the transformed cells after replacing the medium in step ii) to bring the test substance into contact with the luminescent substrate; iv) adding a luminescent substrate specific for NanoLuc luciferase to the transformed cells contacted with the test substance in step iii); and v) After step iv), measuring BRET within 10 minutes, preferably within 5 minutes, of step iii).

[0069] BRET can be measured by measuring the fluorescence (energy transfer acceptor) derived from the HaloTag-specific fluorescent-labeled ligand and the luminescence (energy transfer donor) derived from NanoLuc luciferase in the presence of a HaloTag-specific fluorescent-labeled ligand and a NanoLuc luciferase-specific luminescence substrate using a spectrofluorometer or the like. BRET measurement is preferably performed within 10 minutes, and more preferably within 5 minutes, of contacting the test substance with the transformed cells of the present invention. The BRET ratio can be calculated as follows based on the measured values ​​of the obtained fluorescent and luminescence signals (i.e., fluorescence intensity and luminescence intensity).

[0070]

number

[0071] The BRET ratio is an index of the efficiency of energy transfer between luminescence from NanoLuc luciferase (energy transfer donor) and fluorescence from the HaloTag-specific fluorescently labeled ligand (energy transfer acceptor). When a test substance binds to the opioid μ receptor, the activated opioid μ receptor binds to β-arrestin2, bringing the donor and acceptor systems fused to the opioid μ receptor and β-arrestin2, respectively, into close proximity, resulting in energy transfer. In other words, the greater the binding of β-arrestin2 to the opioid μ receptor, the greater the BRET ratio. Therefore, the BRET ratio can be used as an index to measure (determine / calculate) and evaluate the level of β-arrestin2 binding to the opioid μ receptor induced by a test substance (the induction level of β-arrestin2 binding to the opioid μ receptor) and its time-dependent changes.

[0072] In the BRET assay, it is also preferable to perform a similar experiment (control experiment) in parallel using the same amount of solvent, a solvent containing a negative control substance that does not affect the measurement items, or a positive control substance (a positive control substance known to induce the binding reaction of β-arrestin2 to the opioid μ receptor) instead of the above-mentioned test substance as a control. A control experiment in which the same amount of solvent as the test substance or a solvent containing a negative control substance that does not affect the measurement items is used in the BRET assay is a negative control experiment. A control experiment in which a positive control substance (a positive control substance that is a full agonist of the opioid μ receptor known to induce the binding reaction of β-arrestin2 to the opioid μ receptor) is used in the BRET assay is a positive control experiment. The induction level of the β-arrestin2 binding reaction to the opioid μ receptor can also be measured (determined / calculated), for example, based on the BRET measurement results (e.g., BRET ratio) obtained using the test substance in the BRET assay and the BRET measurement results (e.g., BRET ratio) obtained in the control experiment. For example, based on the BRET ratio obtained using the test substance and the BRET ratio obtained in a control experiment, the difference in the BRET ratio from the control experiment, the BRET induction rate, the BRET change rate, and / or the 50% effective concentration (EC 50 ) and / or maximum response (E max ) can be calculated and used as the induction level of the β-arrestin 2 binding response to the opioid μ receptor. In the method of the present invention, the difference in the BRET ratio from such a control experiment for the β-arrestin 2 pathway, the BRET induction rate, the BRET change rate, and / or the 50% effective concentration (EC 50 ) and / or maximum response (E max ) may be used as an induction level of the β-arrestin 2 binding response to the opioid μ receptor, and the time course of the β-arrestin 2 binding response to the opioid μ receptor or the properties of the test substance may be evaluated.

[0073] The BRET induction rate can be calculated using the following formula.

[0074]

number

[0075] The BRET change rate can be calculated using the following formula.

[0076]

number

[0077] 50% effective concentration (EC 50 ) or maximum response (E max The maximum response (E) can be calculated based on the change in BRET induction rate (e.g., graph) depending on the concentration of the test substance. max ) is the maximum BRET induction rate (%), and the 50% effective concentration (EC 50 ) is the concentration of the test substance at which the BRET induction rate reaches 50% of the maximum response.

[0078] In the methods of the present invention, the level of induction of β-arrestin2 binding to the opioid μ receptor, measured as described above, may be used as the level of opioid μ receptor-mediated β-arrestin2 activation, and the level may be compared with the level of full opioid μ receptor-mediated β-arrestin2 activation to assess the level of opioid μ receptor-mediated β-arrestin2 activation by a test substance. As the "level of full opioid μ receptor-mediated β-arrestin2 activation," it is particularly preferred to use the level of opioid μ receptor-mediated β-arrestin2 activation by DAMGO, an opioid μ receptor full agonist. Because the methods of the present invention can assess the β-arrestin2 binding response to the opioid μ receptor, they are suitable for assessing the level of opioid μ receptor-mediated β-arrestin2 activation by a test substance.

[0079] The present invention provides a method for evaluating the level of β-arrestin2 activation by a test substance via the opioid μ receptor, comprising measuring the level of induction of the β-arrestin2 binding reaction to the opioid μ receptor by measuring BRET in the presence of a HaloTag-specific fluorescently labeled ligand and a NanoLuc luciferase-specific luminescent substrate in the transformed cell of the present invention contacted with the test substance as described above, and comparing the level thus measured as the level of β-arrestin2 activation via the opioid μ receptor with the level of complete activation of β-arrestin2 via the opioid μ receptor.

[0080] The method of the present invention is also useful for evaluating bias in intracellular signaling mediated by the opioid μ receptor by comparing the level of β-arrestin2 activation mediated by a test substance through the opioid μ receptor with the level of G protein activation (G protein pathway activation level) and / or the level of full activation of β-arrestin2 through the opioid μ receptor.

[0081] Bias in intracellular signaling mediated by the opioid μ receptor includes concentration difference bias and intensity difference bias. The method according to the present invention can be used to evaluate such intensity difference bias and / or concentration difference bias of a test substance.

[0082] In the present invention, the 50% effective concentration (EC ) of a test substance at the level of G protein activation via the opioid μ receptor is determined. 50 ) was the 50% effective concentration (EC 50 ), that is, the opioid μ receptor-mediated G protein activation occurs at a lower concentration than the β-arrestin 2 activation, and the maximum response (E max) is 90% or more compared to the full activation level of β-arrestin 2 via the opioid μ receptor, the test substance can be determined to have "concentration difference bias." In addition, in the present invention, the maximum response (E) of the β-arrestin 2 activation level via the opioid μ receptor for the test substance is max ) is less than 90% of the full activation level of β-arrestin 2 mediated by the opioid μ receptor, the test substance can be determined to have "intensity difference bias." In the present invention, "full activation level of β-arrestin 2 mediated by the opioid μ receptor" refers to the activation level of β-arrestin 2 when activation of β-arrestin 2 mediated by the opioid μ receptor is completely induced, and specifically, DAMGO (i.e., [D-Ala 2 ,N-methyl-Phe 4 ,Gly 5 This refers to the level of opioid μ receptor-mediated β-arrestin 2 activation by opioid μ receptor full agonists such as [-ol]-enkephalin. DAMGO, known as an opioid μ receptor full agonist, has the following formula: [ka] DAMGO may be used in the form of a free form, a salt thereof (for example, but not limited to, hydrochloride, methanesulfonate, acetate, etc.), or a hydrate.

[0083] The present invention also provides a screening method for biased opioid μ receptor agonists (e.g., concentration-difference-biasing compounds or intensity-difference-biasing compounds) using the above-described method. In the screening method of the present invention, the above-described method is used to measure the level of β-arrestin 2 activation by a test substance via the opioid μ receptor, and the β-arrestin 2 activation level is used to select test substances that exhibit bias in intracellular signaling via the opioid μ receptor, for example, bias toward the G protein pathway or the β-arrestin 2 pathway, thereby enabling screening for biased opioid μ receptor agonists. The measured β-arrestin 2 activation level is preferably compared with the level of G protein activation via the opioid μ receptor (preferably, the level of G protein activation via the opioid μ receptor by the test substance) and / or the full activation level of β-arrestin 2 via the opioid μ receptor. By this comparison, it is possible to select a test substance that exhibits a bias in intracellular signaling mediated by the opioid μ receptor, specifically, a test substance that has a bias toward the G protein pathway (concentration difference or intensity difference bias) or a test substance that has a bias toward the β-arrestin 2 pathway (concentration difference or intensity difference bias). In the screening method of the present invention, the test substance is preferably an opioid μ receptor agonist. The test substance selected as described above can be used as a biased opioid μ receptor agonist.

[0084] In the screening method of the present invention, the level of β-arrestin 2 activation via the opioid μ receptor can be measured using, for example, the BRET ratio, the difference in the BRET ratio from a control experiment, the BRET induction rate, the BRET change rate, and / or the 50% effective concentration (EC 50 ) and / or maximum response (E max ) may be used, but the 50% effective concentration (EC 50 ) and / or maximum response (E max ) is preferably used.

[0085] In the screening method of the present invention, the 50% effective concentration (EC 50 ) and / or maximum response (E max ) was used as the 50% effective concentration (EC 50 ) and / or maximum response (E max ) (preferably, the EC 50 and / or E max ), and / or the maximum response (E) of the full activation level of β-arrestin 2 via the opioid μ receptor. max ) and / or maximum response (E max ) is particularly preferred. If the test substance exhibits the above-mentioned "concentration difference bias" as a result of the comparison, the test substance can be selected as a concentration difference biasing compound. Furthermore, if the test substance exhibits the above-mentioned "intensity difference bias" as a result of the comparison, the test substance can be selected as a concentration difference biasing compound.

[0086] The screening method of the present invention may include a step of measuring the level of G protein activation via the opioid μ receptor used for comparison. Alternatively, a known value of the level of G protein activation via the opioid μ receptor may be used for comparison. The level of G protein activation via the opioid μ receptor can be measured by measuring the concentration of cAMP, an indicator of G protein pathway activation, using cells stably expressing the opioid μ receptor, as described, for example, in Nakao et al., European Journal of Pharmacology, 2012, Vol. 695, pp. 57-61. Alternatively, the level of G protein activation via the opioid μ receptor can be measured by, for example, using cell membranes overexpressing the opioid μ receptor and measuring the concentration of cAMP, an indicator of G protein pathway activation. 35S]GTPγS (guanosine 5'-O-[gamma-thio]triphosphate) binding is used as an index; cells expressing the opioid μ receptor are used to evaluate the concentration of cAMP produced by stimulating with forskolin or the like as an index; and cells expressing a fusion protein in which a luminescent molecule is fused to the opioid μ receptor, or a fusion protein in which a luminescent molecule is fused to one of the G protein α subunit and the G protein γ subunit and a fluorescent molecule is fused to the other, are used to evaluate BRET as an index (all of these methods are described by Uprety et al., eLife, 2021, Vol. 10, e56519).

[0087] According to the screening method of the present invention, by selecting a test substance that has a bias toward the G protein pathway (concentration difference or intensity difference bias), it is possible to screen for opioid μ receptor agonists with reduced side effects, and by selecting a test substance that has a bias toward the β-arrestin 2 pathway (concentration difference or intensity difference bias), it is possible to screen for opioid μ receptor agonists that may have strong side effects.

[0088] The present invention also provides the use of the transformed cells of the present invention for assessing the β-arrestin 2 binding response to the opioid μ receptor or for assessing the level of β-arrestin 2 activation via the opioid μ receptor by a test substance.

[0089] The present invention also provides a method for producing transformed cultured animal cells that can be used to evaluate the binding response of β-arrestin 2 to the opioid μ receptor, the method comprising the step of introducing into cultured animal cells a gene encoding a fusion protein comprising the opioid μ receptor, a gene encoding a fusion protein comprising β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 to obtain transformed cells, wherein one of the fusion proteins, either the fusion protein comprising the opioid μ receptor or the fusion protein comprising β-arrestin 2, comprises NanoLuc luciferase (a luciferase consisting of the amino acid sequence set forth in SEQ ID NO: 1), and the other fusion protein comprises HaloTag (a protein tag consisting of the amino acid sequence set forth in SEQ ID NO: 3).

[0090] In one embodiment, the present invention provides a method for producing a transformed animal cell line in culture that can be used to assess β-arrestin 2 binding responses to opioid μ receptors, comprising: i) introducing into cultured animal cells a gene encoding a fusion protein comprising an opioid μ receptor, a gene encoding a fusion protein comprising β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 to obtain a transformed cell, wherein one of the fusion proteins, the opioid μ receptor-containing fusion protein or the β-arrestin 2-containing fusion protein, contains NanoLuc luciferase, and the other fusion protein contains a fluorescently labeled ligand specific to HaloTag; ii) adding a HaloTag-specific fluorescently labeled ligand to the transformed cells obtained in step i) in the absence of an opioid μ receptor agonist, irradiating the cells with excitation light, and measuring the fluorescence derived from the fluorescently labeled ligand; iii) adding a luminescent substrate specific for NanoLuc luciferase to the transformed cells obtained in step i) in the absence of an opioid μ receptor agonist, and measuring luminescence; iv) from the transformed cells obtained in step i), a) In step ii), the fluorescence generated by irradiation with excitation light exhibits a fluorescence intensity that is 8 times or more higher than that in the absence of the HaloTag-specific fluorescently labeled ligand; and b) In step iii), the luminescence generated by the reaction of NanoLuc luciferase with the luminescent substrate exhibits a luminescence intensity 35 times or more compared to that in the absence of the luminescent substrate. selecting the cells; In one embodiment, in step iv) of the above method, the luminescence generated by the reaction of NanoLuc luciferase with the luminescent substrate exhibits a luminescence intensity that is 50 times or more higher than that in the absence of the luminescent substrate, and b) Preferably, cells are selected that exhibit fluorescence from the fluorescent-labeled ligand specific to HaloTag, which is generated upon irradiation with excitation light after addition of the fluorescent-labeled ligand, at a fluorescence intensity 14-fold or greater compared to that observed in the absence of the fluorescent-labeled ligand. This cell selection step allows for the acquisition of cultured transformed animal cells that are more suitable for evaluating the β-arrestin2 binding reaction to the opioid μ receptor. Cultured transformed animal cells prepared by this method can be suitably used in the above-mentioned method for evaluating the level of β-arrestin2 activation via the opioid μ receptor by a test substance. [Example]

[0091] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the descriptions of these examples.

[0092] Example 1: Creation and cloning of cells stably expressing human opioid μ receptor, human β-arrestin 2, and human GRK2 In conventional evaluation systems using the BRET assay, it was difficult to detect the binding of β-arrestin2 to the opioid μ receptor with high accuracy or to obtain stable measurement results with little variance. Furthermore, because intracellular signaling accompanying activation of the opioid μ receptor is a phenomenon that occurs in vivo, evaluation systems using cultured cells that are closer to the living body are more suitable for analyzing and screening the mechanism of action of opioid μ receptor agonists with the aim of creating analgesics with reduced side effects. Therefore, evaluation systems using cell-free compositions, such as those described in Patent Document 3, are not appropriate.

[0093] To establish an appropriate evaluation system for the binding of β-arrestin2 to the opioid μ receptor, we considered using a combination of NanoLuc luciferase and HaloTag in a BRET assay and attempted to generate cells for this purpose. First, we generated cells stably expressing human opioid μ receptor (SEQ ID NO: 5) fused with NanoLuc luciferase (SEQ ID NO: 1) at its C-terminus, human β-arrestin2 (SEQ ID NO: 7) fused with HaloTag (SEQ ID NO: 3) at its C-terminus, and human G protein-coupled receptor kinase 2 (human GRK2; SEQ ID NO: 9) (Figure 1).

[0094] Specifically, first, HEK293 cells (Category CRL-1573, ATCC) were transfected with the gene expression plasmid vectors (Vector Builder) shown in Table 2 using Lipofectamine 3000 Reagent (Invitrogen).

[0095] [Table 2]

[0096] After transfection, the cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and antibiotics (500 μg / mL G418, 200 μg / mL hygromycin B, or 10 μg / mL puromycin) for approximately two weeks, and cells were selected for each antibiotic. Cells that exhibited resistance to the three antibiotics were selected as cells that had been introduced with all three expression vectors listed in Table 2 and stably expressed the three introduced genes (hereinafter referred to as "HEK-MGA cells"). When untransformed HEK293 cells were cultured in the same medium, the cells died within two weeks.

[0097] A single clonal cell line was generated from the obtained HEK-MGA cells. 5,000 to 10,000 HEK-MGA cells were seeded onto a 10-cm culture dish and cultured in culture medium for approximately two weeks. Cell colonies were then isolated and cultured to obtain a clonal cell line of HEK-MGA cells. The culture medium for the clonal cell line was DMEM containing 10% FBS, 500 μg / mL G418, 200 μg / mL hygromycin B, and 10 μg / mL puromycin (this medium was used as the culture medium in the following examples). The clonal cell line was cultured and subcultured as follows: After washing the cells with D-PBS(-), they were detached by trypsin / EDTA treatment. Culture medium was added to recover the detached cells, which were then centrifuged (150 × g, 5 minutes, room temperature). The supernatant was removed, and the resulting cell pellet was resuspended in culture medium. The cell suspension diluted to an appropriate dilution ratio was seeded onto a culture dish and cultured in a carbon dioxide incubator at 37°C and 5% CO. The resulting multiple clonal cell lines were used in the following examples.

[0098] Example 2: Evaluation of HEK-MGA cell clones Using each of the clonal cell lines obtained in Example 1, a BRET assay was performed in the following four test groups: (i) Treatment with solvent without adding HaloTag fluorescent-labeled ligand or NanoLuc luciferase luminescent substrate (n = 1); (ii) No HaloTag fluorescent-labeled ligand was added, NanoLuc luciferase luminescent substrate was added, and the vehicle was treated (n = 1); (iii) HaloTag fluorescently labeled ligand was added, NanoLuc luciferase luminescent substrate was added, and then the vehicle was treated (n=2). (iv) HaloTag fluorescently labeled ligand added, NanoLuc luciferase luminescent substrate added, and treatment with a positive control substance (n=2).

[0099] For BRET assays, use NanoBRET TM Nano-Glo (R) A BRET assay was performed using a BRET Detection System (Promega). The day before the BRET assay, cells cultured in a 37°C, 5% CO2 carbon dioxide incubator were washed with D-PBS(-) and detached by trypsin / EDTA treatment. The detached cells were collected by adding culture medium and centrifuged (150 × g, 5 minutes, room temperature). The supernatant was removed, and 2 × 10 cells were seeded using Opti-MEM I Reduced Serum Medium, no phenol red (Thermo Fisher Scientific) containing 4% FBS (in the following examples, unless otherwise specified, this medium will be referred to as "seeding medium"). 5 The concentration of the cell suspension was adjusted to 1 / 1000 of the amount of DMSO for the cells in test groups (i) and (ii), and 1 / 1000 of the amount of HaloTag fluorescent-labeled ligand, HaloTag, for the cells in test groups (iii) and (iv). (R) NanoBRET TM 618 Ligand (Promega) was added. 4 Cells were seeded onto a 96-well white plate pre-coated with poly-D-lysine at 100 μL / well and cultured overnight in a 5% CO2 incubator at 37°C to prepare a cell plate.

[0100] On the day of the BRET assay, a test substance solution was prepared. The test substance used was a positive control substance (DAMGO, a known standard opioid μ receptor agonist, final concentration 10 μM). As a control, a solvent (DMSO, final concentration 0.1%) was used instead of the test substance. The solvent was dimethyl sulfoxide (DMSO) (Sigma-Aldrich) diluted with 1% GlutaMAX TM The positive control was prepared at 100 μM (10 times the final concentration) by diluting a 10 mM stock solution of DAMGO (Sigma-Aldrich) dissolved in DMSO with the measurement medium.

[0101] On the day of the BRET assay, the seeding medium was removed from the cell plate prepared above and replaced with 80 μL / well of measurement medium. The cells were then cultured for 3-4 hours in a carbon dioxide incubator at 37°C with 5% CO2. TM Nano-Glo (R) Substrate (Promega) was diluted 100-fold with assay medium to prepare a 5x Nano-Glo substrate solution (5x the final concentration). The 5x Nano-Glo substrate solution was added to cells in test groups (ii) to (iv) at 20 μL / well, and BRET was measured before treatment with solvent or test substance. For cells in test group (i) without luminescent substrate, assay medium was added at 20 μL / well, and BRET was measured before treatment with solvent. Subsequently, the cells were treated with the above test substance or control solvent diluted with assay medium to 10x the final concentration, and added at 11 μL / well. BRET was measured again 5 minutes after treatment with solvent or test substance.

[0102] BRET measurements were performed using the EnVision microplate reader.TM This was performed using 2103 Multilabel Plate Readers (PerkinElmer) by measuring the light intensities (1) and (2) at the following two wavelengths: (1) the fluorescence intensity (cps) of the energy transfer acceptor with a peak wavelength of 618 nm measured using a Cy5 FP 620 filter, and (2) the luminescence intensity (cps) of the energy transfer donor with a peak wavelength of 460 nm measured using an Umbelliferone 460 filter.

[0103] The BRET ratio was calculated using the following formula based on the BRET measurement results.

[0104]

number

[0105] The results of the BRET assay are shown in Figure 2. The vertical axis of the graph in Figure 2 represents the BRET ratio, and the # numbers above the graph indicate the HEK-MGA clonal cell line number. "Pre" on the horizontal axis of the graph indicates cells before treatment with the test substance or vehicle control, while "Post" indicates cells after treatment with the test substance or vehicle control. The bars on the graph represent the average and individual values ​​for duplicate wells of the same treatment. Figures 2A–E show the results for all clonal cell lines (HEK-MGA #2, #7, #15, #17, and #18, respectively) in which the BRET ratio increased after treatment (post) compared to before treatment (pre). This increase in BRET ratio indicates that BRET occurred due to the binding of β-arrestin 2 to the opioid μ receptor. Figure 2F shows a representative example of the results for a clonal cell line in which no change in the BRET ratio was observed in the positive control substance-treated group after treatment (post) compared to both groups before treatment (pre) and the vehicle-treated group after treatment (post). BRET measurements showed that fluorescence from the HaloTag fluorescent-labeled ligand was detected in all clone cell lines tested, while luminescence from NanoLuc luciferase was detected in all clone cell lines tested, but not in clone cell lines HEK-MGA #5, #8, #10, and #22.

[0106] Furthermore, to confirm the expression of the introduced gene in the cloned cell lines, the relative luminescence intensity of each cloned cell line was calculated based on the BRET measurement results using the following formula: (2) The luminescence intensity (cps) of the energy transfer donor with a peak wavelength of 460 nm measured using an Umbelliferone 460 filter after the addition of the measurement medium or 5x Nano-Glo substrate solution (before solvent treatment) in test groups (i) and (ii) without the addition of the HaloTag fluorescent-labeled ligand.

[0107]

number

[0108] The relative value of the luminescence intensity at a wavelength of 460 nm indicates the cellular expression level of the human opioid μ receptor-NanoLuc luciferase fusion protein.

[0109] Furthermore, for test groups (i) to (iv), on the day of the BRET assay, the seeding medium in the cell plates was replaced with the measurement medium, and after 3 to 4 hours of incubation in a 37°C, 5% CO2 carbon dioxide incubator (before adding the luminescent substrate for NanoLuc luciferase), the fluorescence intensity was measured using a microplate reader, SpectraMax® Paradigm (Molecular Devices). The cell plates were irradiated with 540 nm light, which is the excitation light for the HaloTag NanoBRET 618 Ligand, and the fluorescence intensity (RFU) at 620 nm was measured. The relative fluorescence intensity of each clone cell line was calculated using the following formula:

[0110]

number

[0111] The relative value of the fluorescence intensity at a wavelength of 620 nm indicates the cellular expression level of the human β-arrestin2-HaloTag fusion protein.

[0112] Table 3 shows the relative values ​​of luminescence intensity and fluorescence intensity for HEK-MGA cell clones (a total of 22 types of cell lines #1 to #22).

[0113] [Table 3]

[0114] The results in Figure 2 and Table 3 indicate that, even among cells stably expressing the human opioid μ receptor-NanoLuc luciferase fusion protein and the human β-arrestin2-HaloTag fusion protein, there were clonal cell lines (HEK-MGA #2, #7, #15, #17, and #18) in which an increase in the BRET ratio could be detected, i.e., the binding reaction of β-arrestin2 to the opioid μ receptor induced by an opioid μ receptor agonist could be detected, and clonal cell lines (HEK-MGA #1, #3, #4, #5, #6, #8, #9, #10, #11, #12, #13, #14, #16, #19, #20, #21, and #22) in which this reaction could not be detected.

[0115] Example 3: Examination of the suitability of HEK-MGA cell clones for evaluating β-arrestin 2 binding to human opioid μ receptors Using all of the clonal cell lines (HEK-MGA #2, #7, #15, #17, and #18) in which an increase in the BRET ratio was detected in Example 2, the β-arrestin 2 binding response to the human opioid μ receptor was evaluated by BRET assay.

[0116] A positive control substance (DAMGO, final concentration 10 μM) was used as the test substance. The test substance was dissolved in DMSO to prepare a 10 mM stock solution and stored at −20°C or below until the day of use. As a control, a solvent (DMSO, final concentration 0.1%) was used instead of the test substance.

[0117] For BRET assays, use NanoBRET TM Nano-Glo (R) A BRET assay was performed using a BRET Detection System (Promega). The day before the BRET assay, cells cultured in a carbon dioxide incubator at 37°C and 5% CO2 were washed with D-PBS(-) and detached by trypsin / EDTA treatment. The detached cells were collected by adding culture medium and centrifuged (150 × g, 5 minutes, room temperature). The supernatant was removed, and 2 × 10 cells were plated using the seeding medium described in Example 2. 5The concentration of the cell suspension was adjusted to 1 / 1000 of the amount of HaloTag fluorescently labeled ligand, HaloTag (R) NanoBRET TM 618 Ligand (Promega) was added. 4 Cells were seeded onto a 96-well white plate pre-coated with poly-D-lysine at 100 μL / well and cultured overnight in a 5% CO2 incubator at 37°C to prepare a cell plate.

[0118] On the day of the BRET assay, the stock solution of the test substance was diluted with the measurement medium of Example 2 to adjust to the target concentration (10 times the final concentration). The final concentration of DMSO in all test substance solutions was adjusted to 0.1%.

[0119] On the day of the BRET assay, the seeding medium was removed from the cell plate prepared above and replaced with 90 μL / well of measurement medium, and the cells were cultured in a 37°C, 5% CO2 incubator for 3-4 hours. TM Nano-Glo (R) Substrate (Promega) was diluted 100-fold with assay medium to prepare a 5x Nano-Glo substrate solution (5x the final concentration). The test substance or solvent prepared above was added at 10 μL / well to treat the cells. Five minutes after treatment with the test substance or solvent, 5x Nano-Glo substrate solution was added at 25 μL / well, and BRET was measured immediately thereafter. BRET measurements were performed using EnVision 2103 Multilabel Plate Readers (PerkinElmer) in the same manner as in Example 2, and the BRET ratio was calculated in the same manner as in Example 2.

[0120] Furthermore, based on the BRET ratio, the BRET induction rate was calculated using the following formula.

[0121]

number

[0122] This BRET induction rate is expressed as the percentage of the BRET ratio of each test substance treatment group, with the average BRET ratio in the solvent-treated group being defined as a BRET induction rate of 0% and the average BRET ratio in the positive control substance (DAMGO)-treated group being defined as a BRET induction rate of 100%.

[0123] Data were processed using Microsoft® Excel® for Microsoft 365 (Microsoft Corporation). Regression to a four-parameter sigmoid curve and the 50% effective concentration (EC 50 ) and maximum response (E max ) was calculated and graphs were created using GraphPad Prism 9.3.1 (GraphPad Software).

[0124] The results of the BRET assay for each clone cell line are shown in Figure 3. The horizontal axis of the graph indicates the concentration of the test substance (log (mol / L)), and the vertical axis indicates the BRET induction rate (%). The # numbers at the top of the graph indicate the HEK-MGA clone cell line number. Each plot shows the mean ± standard deviation of duplicate wells treated with the same treatment. As a result, as shown in Figure 3, HEK-MGA #2, #7, and #18 exhibited characteristics suitable for evaluation in terms of uniformity within the same treatment and regression to a sigmoid curve. On the other hand, HEK-MGA #15 and #17 demonstrated poor uniformity within the same treatment and poor regression to a sigmoid curve.

[0125] As shown in Table 3, HEK-MGA #2, #7, and #18, which are cell lines suitable for evaluation, had relative fluorescence intensity values ​​of 8 or greater and relative luminescence intensity values ​​of 35 or greater. On the other hand, HEK-MGA #15 and #17 had relative fluorescence intensity values ​​of 8 or greater, but relative luminescence intensity values ​​of less than 35. This indicates that in order to have characteristics suitable for evaluation in the present invention, the relative fluorescence intensity derived from the HaloTag fluorescent-labeled ligand must be 8 or greater and the relative luminescence intensity derived from NanoLuc luciferase must be 35 or greater.

[0126] To select the most suitable clone from HEK-MGA #2, #7, and #18, which were deemed suitable for evaluation, the BRET change rate was calculated for each clone cell line. The BRET change rate was calculated based on the BRET ratio using the following formula:

[0127]

number

[0128] The results are shown in Table 4. The BRET change rate of HEK-MGA #7 was 1.45, the highest.

[0129] [Table 4]

[0130] EC of cell line HEK-MGA #7 50 and E max The calculation results are shown in Table 5.

[0131] [Table 5]

[0132] Based on these results, HEK-MGA #7 was selected as the most suitable cell line for evaluating the β-arrestin2 binding response to the opioid μ receptor agonist. HEK-MGA #7 was deposited at the National Institute of Technology and Evaluation (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818, Japan) on March 11, 2024 (Accession No. NITE AP-04097). [Industrial Applicability]

[0133] The cells and methods of the present invention can detect the β-arrestin 2 binding reaction to the opioid μ receptor induced by a test substance with high accuracy and little variability, and are therefore useful for screening opioid μ receptor agonists that have weak β-arrestin 2 pathway activation ability and reduced side effects.

[0134] <Sequence list> SEQ ID NO: 1: NanoLuc luciferase SEQ ID NO: 2: NanoLuc luciferase gene SEQ ID NO: 3: HaloTag SEQ ID NO: 4: Nucleotide sequence encoding HaloTag SEQ ID NO: 5: Human opioid μ receptor protein SEQ ID NO: 6: Human opioid μ receptor gene SEQ ID NO: 7: Human β-arrestin 2 protein SEQ ID NO: 8: Human β-arrestin 2 gene SEQ ID NO: 9: Human G protein-coupled receptor kinase 2 (GRK2) protein SEQ ID NO: 10: Human G protein-coupled receptor kinase 2 (GRK2) gene SEQ ID NO: 11: Nucleotide sequence of expression vector VB211001-1033vys SEQ ID NO: 12: Nucleotide sequence of expression vector VB210518-1378<em>f SEQ ID NO: 13: Nucleotide sequence of expression vector VB210518-1365eqk

Claims

1. a gene encoding a fusion protein containing an opioid μ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 are introduced; A cultured transformed animal cell, wherein one of the fusion proteins, either the fusion protein containing the opioid μ receptor or the fusion protein containing β-arrestin 2, contains a luciferase consisting of the amino acid sequence shown in SEQ ID NO: 1, and the other fusion protein contains a protein tag consisting of the amino acid sequence shown in SEQ ID NO:

3.

2. the gene encoding the fusion protein containing the opioid μ receptor encodes a fusion protein in which the luciferase is fused to the C-terminus of the opioid μ receptor; The transformed animal cultured cell according to claim 1, wherein the gene encoding the fusion protein containing β-arrestin 2 encodes a fusion protein in which the protein tag is fused to the C-terminus of β-arrestin 2.

3. In the absence of an opioid μ receptor agonist, the transformed animal cultured cells a) in the presence of a luminescent substrate specific to the luciferase, the luminescence generated by the reaction between the luciferase and the luminescent substrate exhibits a luminescence intensity that is 35 times or more higher than that in the absence of the luminescent substrate; and b) the fluorescence generated by the fluorescent-labeled ligand specific to the protein tag upon irradiation with excitation light after addition of the fluorescent-labeled ligand exhibits a fluorescence intensity that is 8 times or more higher than that in the absence of the fluorescent-labeled ligand; The transformed animal cultured cell according to claim 1.

4. The transformed animal cultured cell according to claim 1, which is a transformant of HEK293 cells.

5. 2. The cultured transformed animal cell according to claim 1, which is a cell line HEK-MGA #7 having accession number NITE AP-04097.

6. A method for producing a transformed animal cultured cell that can be used to evaluate the binding response of β-arrestin 2 to the opioid μ receptor, comprising: i) introducing into cultured animal cells a gene encoding a fusion protein comprising an opioid μ receptor, a gene encoding a fusion protein comprising β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 to obtain a transformed cell, wherein one of the fusion proteins, i.e., the fusion protein comprising the opioid μ receptor or the fusion protein comprising β-arrestin 2, comprises a luciferase consisting of the amino acid sequence shown in SEQ ID NO: 1, and the other fusion protein comprises a protein tag consisting of the amino acid sequence shown in SEQ ID NO: 3; ii) adding a fluorescently labeled ligand specific to the protein tag to the transformed cells obtained in step i) in the absence of an opioid μ receptor agonist, and irradiating the transformed cells with excitation light to measure the fluorescence derived from the fluorescently labeled ligand; iii) adding a luminescent substrate specific for the luciferase to the transformed cells obtained in step i) in the absence of an opioid μ receptor agonist, and measuring luminescence; iv) from the transformed cells obtained in step i), a) in step ii), the fluorescence generated by irradiation with excitation light exhibits a fluorescence intensity that is 8 times or more higher than that in the absence of the fluorescently labeled ligand; and b) In step iii), the luminescence generated by the reaction between the luciferase and the luminescent substrate exhibits a luminescence intensity that is 35 times or more higher than that in the absence of the luminescent substrate. selecting the cells; A method comprising:

7. A step of contacting a test substance with the cultured transformed animal cell according to any one of claims 1 to 5; measuring the level of induction of β-arrestin 2 binding to the opioid μ receptor by measuring bioluminescence resonance energy transfer (BRET) that occurs in the cultured transformed animal cells contacted with the test substance in the presence of a fluorescently labeled ligand specific to the protein tag and a luminescent substrate specific to the luciferase; Using the measured induction level of β-arrestin 2 binding response to the opioid μ receptor as a level of β-arrestin 2 activation via the opioid μ receptor, and comparing it with a level of complete activation of β-arrestin 2 via the opioid μ receptor; A method for evaluating the level of β-arrestin 2 activation mediated by an opioid μ receptor by a test substance, comprising:

8. Measuring the level of β-arrestin 2 activation mediated by the opioid μ receptor by a test substance using the method of claim 7; comparing the measured β-arrestin 2 activation level with the G protein activation level via the opioid μ receptor and / or the full activation level of β-arrestin 2 via the opioid μ receptor to select a test substance that exhibits bias in intracellular signal transduction via the opioid μ receptor; A method for screening for an opioid μ receptor agonist having bias, comprising:

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