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

Abstract

To provide a system for evaluating the binding response of β-arrestin 2 to an opioid κ receptor.SOLUTION: Provided is a transformed animal cultured cell into which a gene encoding a fusion protein containing an opioid κ 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 κ 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 κ receptor, and a method for screening for a biased opioid κ 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 κ 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 κ receptor agonists (agonists) are expected to be highly safe analgesics because they do not cause side effects such as drug dependence or respiratory depression mediated by the opioid μ receptor. However, side effects such as sedation and aversion mediated by the opioid κ receptor are known, and separating such side effects from pharmacological efficacy remains a challenge (Non-Patent Document 1).

[0005] 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.

[0006] One method for reducing the side effects specific to opioid κ receptor agonists is the use of biased compounds. The bias 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 κ receptor, and that opioid κ receptor agonists with a bias toward the G protein pathway, which activates the pathway more strongly, are less likely to cause sedation (Non-Patent Document 2). Opioid κ receptor agonists with a bias toward the G protein pathway, which 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), can be highly safe analgesics with excellent separation of pharmacological effects and side effects.

[0007] 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.

[0008] 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 proceed 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 proceed 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.

[0009] 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.

[0010] 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.

[0011] 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]

[0012] [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]

[0013] [Non-Patent Document 1] Dalefield et al., Frontiers in Pharmacology, 2022, Vol. 13, pp. 837671 [Non-patent document 2] Mores et al., Frontiers in Pharmacology, 2019, Vol. 10, pp. 407 [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]

[0014] An objective of the present invention is to provide an evaluation system for the binding reaction of β-arrestin 2 to the opioid κ receptor. [Means for solving the problem]

[0015] 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 κ receptor by a BRET assay using a combination of NanoLuc (registered trademark) luciferase and HaloTag (registered trademark), thereby completing the present invention.

[0016] That is, the present invention includes the following.

[0017] [1] A gene encoding a fusion protein containing an opioid κ 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 κ receptor or the fusion protein containing the 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 κ receptor encodes a fusion protein in which the luciferase is fused to the C-terminus of the opioid κ 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] The transformed animal cultured cell according to [1] or [2] above, wherein the gene expression level of the opioid κ receptor is 1,500 times or more compared to the gene expression level of the opioid κ receptor in a corresponding non-transformed animal cultured cell. [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-KGA #35 having accession number NITE AP-04098. [6] 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 κ 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 κ receptor as a level of β-arrestin 2 activation via the opioid κ receptor, and comparing it with a level of complete activation of β-arrestin 2 via the opioid κ receptor; A method for evaluating the level of β-arrestin 2 activation mediated by an opioid κ receptor by a test substance, comprising: [7] The method according to [6] above, wherein the test substance is contacted with the cultured transformed animal cells in a serum-free, CO2-independent medium. [8] Measuring the level of β-arrestin 2 activation mediated by the opioid κ receptor by a test substance using the method described in [6] or [7] above; comparing the measured β-arrestin 2 activation level with the G protein activation level via the opioid κ receptor and / or the full activation level of β-arrestin 2 via the opioid κ receptor to select a test substance that exhibits bias in intracellular signal transduction via the opioid κ receptor; A method for screening for an opioid κ receptor agonist having bias, comprising: [9] 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 κ receptor, comprising: i) a step of introducing a gene encoding a fusion protein comprising an opioid κ receptor, a gene encoding a fusion protein comprising β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 into cultured animal cells to obtain transformed cells, wherein one of the fusion proteins, i.e., the fusion protein comprising the opioid κ 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) selecting from the obtained transformed cells, transformed cells in which the expression level of the opioid κ receptor gene is 1500 times or more compared to the expression level of the opioid κ receptor gene in the corresponding non-transformed animal cultured cells; A method comprising: [Effects of the Invention]

[0018] The present invention provides cells and methods that enable stable evaluation of the binding response of β-arrestin 2 to the opioid κ receptor. [Brief explanation of the drawings]

[0019] [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 shows representative examples of the results of measuring fluorescence intensity and luminescence intensity performed using the obtained clone cell lines. A shows the results of fluorescence intensity measurement (representative example), and B and C show the results of luminescence intensity measurement (representative example). B shows a representative example of a measurement result in which luminescence was confirmed, and C shows a representative example of a measurement result in which luminescence was not confirmed. [Figure 3] Figure 3 is a graph showing the BRET ratios obtained by 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: #3, B: #6, C: #18, D: #19, E: #29, F: #35, G: #7, H: #12, I: #13, J: #38. K shows the result for #40 as a representative example of a clonal cell line in which no luminescence was detected. [Figure 4] Figure 4 shows the results of evaluating the β-arrestin 2 binding reactions of concentration-difference biasing compounds and intensity-difference biasing compounds, performed using six types of clone cell lines in which BRET was detected. Black circles: concentration-difference biasing compounds, open triangles: intensity-difference biasing compounds. A: #3, B: #6, C: #29, D: #35, E: #18, F: #19. [Figure 5] FIG. 5 shows the results of measuring the expression level of the human opioid κ receptor gene in 10 clones in which luminescence derived from NanoLuc luciferase was detected. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be described in detail below.

[0021] The present invention relates to cells suitable for use in evaluating the binding reaction of β-arrestin2 to the opioid κ receptor, based on a BRET assay using luciferase and a protein tag, and a method for evaluating the binding reaction of β-arrestin2 to the opioid κ 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 β-arrestin2 activation via the opioid κ receptor by a test substance, using cells suitable for evaluating the binding reaction of β-arrestin2 to the opioid κ receptor. The present invention also provides a biased screening method for opioid κ receptor agonists using such an evaluation method. In the present invention, an opioid κ receptor agonist refers to a substance that can bind to the opioid κ receptor and activate intracellular signaling (specifically, at least a G protein pathway) via the opioid κ receptor.

[0022] In the present invention, cells that can be used to evaluate the binding response of β-arrestin 2 to the opioid κ receptor are cultured cells expressing a transgene containing a fusion protein containing the opioid κ 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 into which a gene encoding a fusion protein containing the opioid κ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 (GRK2) have been introduced. In the cultured cells of the present invention, one of the fusion proteins encoded by the transgenes, either the opioid κ 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 κ receptor contains a nucleotide sequence encoding the opioid κ receptor protein (opioid κ 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.

[0023] In the present invention, the opioid κ 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), with humans being more preferred. 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 κ receptor, β-arrestin 2, and GRK2 are preferably derived from the same species.

[0024] The opioid κ 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).

[0025] The "opioid κ receptor" in the present invention is one subtype of opioid receptor, which is a G protein-coupled receptor (GPCR). The "opioid κ receptor" used in the present invention may be a wild-type or a mutant.

[0026] In one embodiment, the opioid kappa receptor (preferably, a human opioid kappa receptor) of the present invention can be a protein of the following (i) or (ii): (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 κ receptor of the present invention has G protein binding activity.

[0027] In one embodiment, the base sequence encoding the opioid κ receptor (preferably, the human opioid κ receptor) of the present invention (or the opioid κ receptor gene, preferably the human opioid κ 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 κ receptor (opioid κ receptor gene) of the present invention encodes a protein having G protein binding activity.

[0028] In one embodiment, the opioid κ receptor of the present invention is a human opioid κ receptor. In a preferred embodiment, the human opioid κ 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 κ receptor protein is the nucleotide sequence shown in SEQ ID NO: 6. The nucleotide sequence shown in SEQ ID NO: 6 (or an opioid κ receptor gene consisting of) encodes the opioid κ receptor protein consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0029] "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 κ 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.

[0030] 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 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. The β-arrestin 2 of the present invention has opioid κ receptor binding activity.

[0031] 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 κ receptor binding activity.

[0032] 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.

[0033] 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 κ 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 κ receptor and promotes the binding of β-arrestin 2 to the opioid κ receptor. The "G protein-coupled receptor kinase 2 (GRK2)" used in the present invention may be a wild-type or mutant.

[0034] 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.

[0035] 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 κ receptor binding activity.

[0036] 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.

[0037] 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

[0038] In the present invention, a gene encoding a fusion protein containing an opioid κ receptor encodes a fusion protein containing the opioid κ receptor and NanoLuc luciferase or HaloTag. Furthermore, a gene encoding a fusion protein containing β-arrestin 2 encodes a fusion protein containing β-arrestin 2 and either NanoLuc luciferase or HaloTag, whichever molecule is not fused to the opioid κ receptor. A fusion protein containing an opioid κ 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 κ receptor. A fusion protein containing β-arrestin 2 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-arrestin 2. In the fusion protein of the present invention, NanoLuc luciferase or HaloTag may be fused to the "C-terminus" or "N-terminus" of opioid κ receptor or β-arrestin 2 via another polypeptide such as a linker between the C-terminus or N-terminus of opioid κ receptor or β-arrestin 2 and NanoLuc luciferase or HaloTag. Preferred protein combinations contained in the fusion protein of the present invention are shown in Table 1.

[0039] [Table 1]

[0040] The configuration of the fusion protein containing the opioid κ receptor and the fusion protein containing β-arrestin 2 may be any combination of Nos. 1 to 4 shown in Table 1, but combination No. 2, in which NanoLuc luciferase is fused to the C-terminus of the opioid κ receptor and HaloTag is fused to the C-terminus of β-arrestin 2, is more preferred.

[0041] 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).

[0042] 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").

[0043] In the present invention, the cells into which the gene encoding the fusion protein containing the opioid κ receptor, the gene encoding the 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. Mammals include, but are not limited to, animals belonging to the orders Primates, Rodentia, Dodontia, Artiodactyla, Perissodactyla, or Carnivora. 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. 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 Dodontidae 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 κ 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 cells derived from a human fetal kidney. HEK293 cells do not contain any exogenously introduced genes and barely express the opioid κ receptor. In the present invention, the term "cultured cells" refers to cells cultured in vitro.

[0044] Introduction (artificial introduction) of a gene encoding a fusion protein containing the opioid κ 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 κ 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.

[0045] The present invention provides transformed cells prepared as described above, into which a gene encoding a fusion protein containing the opioid κ 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 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."

[0046] In one embodiment, a transformed cell into which a gene encoding a fusion protein comprising the opioid κ receptor, a gene encoding a fusion protein comprising β-arrestin 2, and a gene encoding GRK2 have been introduced preferably stably expresses the gene encoding the fusion protein comprising the opioid κ receptor, the gene encoding the fusion protein comprising β-arrestin 2, 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 that is stably maintained in proliferating cells.

[0047] Transformed cells into which a gene encoding a fusion protein containing the opioid κ 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.

[0048] Transformed cells according to the present invention, into which a gene encoding a fusion protein containing the opioid κ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 have been introduced, can generate luminescence through a reaction between NanoLuc luciferase and a luminescent substrate specific to NanoLuc luciferase, even in the absence of an opioid κ 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.

[0049] Transformed cells according to the present invention into which a gene encoding a fusion protein containing the opioid κ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 have been introduced preferably exhibit an opioid κ receptor gene expression level that is at least 1500-fold, more preferably at least 1600-fold, and even more preferably at least 1850-fold higher than that of corresponding non-transformed cells (e.g., untransformed HEK293 cells when the transformed cells are HEK293 cell transformants) (i.e., when the opioid κ receptor gene expression level in corresponding non-transformed cells is set to 1, the relative value of the opioid κ receptor gene expression level in the transformed cells is preferably at least 1500, more preferably at least 1600, and even more preferably at least 1850). In the present invention, the term "opioid κ receptor gene expression level" refers to the expression level of mRNA encoding the opioid κ receptor protein in the cells. The mRNA encoding the opioid κ receptor protein encompasses both mRNA transcribed from an endogenous opioid κ receptor gene and mRNA transcribed from a gene encoding a fusion protein containing the opioid κ receptor. In a transformed cell into which a gene encoding a fusion protein containing the opioid κ 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 κ 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 opioid κ receptor gene. A nucleic acid probe or primer that specifically hybridizes with mRNA transcribed from the opioid κ receptor gene can be appropriately designed by one skilled in the art based on the nucleotide sequence of the opioid κ receptor gene.An example of a suitable primer capable of specifically hybridizing with and amplifying mRNA transcribed from the opioid κ receptor gene is a primer set comprising a primer (forward primer) containing or consisting of the nucleotide sequence shown in SEQ ID NO: 14 and a primer (reverse primer) containing or consisting of the nucleotide sequence shown in SEQ ID NO: 15. Calculation of gene expression levels in real-time PCR can be performed by standard methods such as the calibration curve method and the comparative Ct method, with the comparative Ct method being preferred. When measuring the gene expression level of the opioid κ receptor, it is also preferable to measure in parallel the expression level of an endogenous housekeeping gene that is always expressed at a constant level, such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, in order to standardize the measured values.

[0050] 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 nucleotide 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 κ receptor may be as follows: an initial denaturation step at 95°C for 10 minutes, followed by a 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.

[0051] A preferred example of a transformed cell according to the present invention into which a gene encoding a fusion protein containing the opioid κ receptor, a gene encoding a fusion protein containing β-arrestin 2, and a gene encoding GRK2 have been introduced is the cell line HEK-KGA #35. Cell line HEK-KGA #35 was deposited on March 11, 2024, with the National Institute of Technology and Evaluation (NPMD), Patent Microorganisms Depositary (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under accession number NITE AP-04098.

[0052] Transformed cells of the present invention, into which a gene encoding a fusion protein containing the opioid κ 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 κ 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 κ 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 κ receptor. For example, the transformed cells of the present invention can be used to evaluate the β-arrestin2 binding response to the opioid κ 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 κ receptor by a test substance.

[0053] In one embodiment, the present invention provides a method for assessing the level of β-arrestin2 activation by a test substance via the opioid κ 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 HaloTag-specific fluorescently labeled ligand and a NanoLuc luciferase-specific luminescent substrate.

[0054] 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-arrestin 2 binding to the opioid κ 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 κ receptor as a level of β-arrestin 2 activation via the opioid κ receptor, and comparing it with a level of complete activation of β-arrestin 2 via the opioid κ receptor; The method may be a method for evaluating the level of β-arrestin 2 activation by a test substance via the opioid κ receptor, comprising:

[0055] 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).

[0056] [ka]

[0057] 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).

[0058] Furimazine (2-furanylmethyldeoxy-coelenterazine) can be used as a luminescent substrate specific to NanoLuc luciferase, and HaloTag 1000 (HaloTag 1000) (2-furanylmethyldeoxy-coelenterazine) (HaloTag 10 ... (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.

[0059] A "test substance" in the present invention is a substance that is the subject of evaluation in the method of the present invention for its ability to induce the binding reaction of β-arrestin 2 to the opioid κ receptor or the level of β-arrestin 2 activation mediated by the opioid κ receptor. In one embodiment, a "test substance" in the present invention is an opioid κ 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 compound or inorganic compound, an in vivo substance, a substance produced by genetic engineering (genetically engineered substance), a chemically synthesized substance, or the like. 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.

[0060] 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 κ receptor. In the present invention, "concentration-difference bias" refers to the property of an opioid κ 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, a intensity-difference-biasing compound is a compound that exhibits intensity-difference bias in intracellular signaling mediated by the opioid κ receptor. In the present invention, "intensity-difference bias" refers to the property of an opioid κ receptor agonist that weakly activates the β-arrestin pathway 2 and is unable to fully activate the β-arrestin 2 pathway, i.e., exhibits partial agonism of β-arrestin 2 pathway activation.

[0061] 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.

[0062] 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 independent medium. In particular, to distinguish between concentration-difference-biasing compounds and intensity-difference-biasing compounds, it is preferable to contact the transformed cells of the present invention with a test substance in a CO2-independent medium that does not contain serum (e.g., FBS), such as CO2-independent medium (Thermo Fisher Scientific). The CO2-independent medium that does not contain serum (e.g., FBS), such as CO2-independent medium (Thermo Fisher Scientific), contains 1% GlutaMAX TM It is also preferable to use the transformant cells of the present invention with the addition of Supplement (Thermo Fisher Scientific). The contact of a test substance with the transformed cells of the present invention may be carried out 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 transformant cells of the present invention are contacted with the test substance, if the test substance binds to the opioid κ receptor expressed in the transformed cells and activates the opioid κ receptor, G protein activation will occur and a binding reaction of β-arrestin2 to the opioid κ receptor will be induced, resulting in β-arrestin2 activation.

[0063] 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 a 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 κ receptor is induced via binding of a test substance to the opioid κ receptor expressed in a transformed cell, the HaloTag-specific fluorescent-labeled ligand bound to HaloTag fused to either the opioid κ receptor or β-arrestin2 comes into close proximity with NanoLuc luciferase fused to the other (β-arrestin2 or opioid κ 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.

[0064] 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.

[0065] 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 transformed cells; 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).

[0066] 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).

[0067]

number

[0068] The BRET ratio is an index of the efficiency of energy transfer between luminescence from NanoLuc luciferase (energy transfer donor) and fluorescence from a HaloTag-specific fluorescently labeled ligand (energy transfer acceptor). When a test substance binds to the opioid κ receptor, the activated opioid κ receptor binds to β-arrestin2, bringing the donor and acceptor systems fused to the opioid κ 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 κ receptor, the greater the BRET ratio. Therefore, the level of β-arrestin2 binding to the opioid κ receptor induced by a test substance (the induction level of β-arrestin2 binding to the opioid κ receptor) and its time-dependent changes can be measured (determined / calculated) and evaluated using the BRET ratio as an index.

[0069] In the BRET assay, it is also preferable to perform a similar experiment (control experiment) in parallel using, as a control, 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-arrestin 2 to the opioid κ receptor) instead of the above-mentioned test substance. 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 an opioid κ receptor full agonist known to induce the binding reaction of β-arrestin 2 to the opioid κ receptor) is used in the BRET assay is a positive control experiment. The induction level of the β-arrestin 2 binding reaction to the opioid κ 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 κ 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 κ receptor to evaluate time-dependent changes in the β-arrestin 2 binding response to the opioid κ receptor, as well as the properties of the test substance.

[0070] The BRET induction rate can be calculated using the following formula.

[0071]

number

[0072] The BRET change rate can be calculated using the following formula.

[0073]

number

[0074] 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.

[0075] In the methods of the present invention, the level of induction of β-arrestin2 binding to the opioid κ receptor, measured as described above, may be used as the level of β-arrestin2 activation mediated by the opioid κ receptor, and the level may be compared with the level of full activation of β-arrestin2 mediated by the opioid κ receptor to assess the level of opioid κ receptor-mediated β-arrestin2 activation by a test substance. As the "level of full activation of β-arrestin2 mediated by the opioid κ receptor," it is particularly preferred to use the level of β-arrestin2 activation mediated by U-69593 or U-50488, which are full agonists of the opioid κ receptor. Because the methods of the present invention can assess the β-arrestin2 binding to the opioid κ receptor, they are suitable for assessing the level of opioid κ receptor-mediated β-arrestin2 activation by a test substance.

[0076] The present invention provides a method for evaluating the level of β-arrestin2 activation by a test substance via the opioid κ receptor, comprising measuring the level of induction of the β-arrestin2 binding reaction to the opioid κ 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 that has been contacted with the test substance as described above, and comparing the level of induction of the β-arrestin2 binding reaction to the opioid κ receptor with the complete activation level of β-arrestin2 via the opioid κ receptor, using the level as the activation level of β-arrestin2 via the opioid κ receptor.

[0077] The method of the present invention is also useful for evaluating bias in intracellular signaling mediated by the opioid κ receptor by comparing the level of β-arrestin2 activation mediated by the opioid κ receptor by a test substance with the level of G protein activation (G protein pathway activation level) mediated by the opioid κ receptor and / or the level of complete activation of β-arrestin2 mediated by the opioid κ receptor.

[0078] Bias in intracellular signaling mediated by the opioid κ receptor includes concentration difference bias and intensity difference bias. The method of the present invention can be used to evaluate such intensity difference bias and / or concentration difference bias of a test substance, and is particularly suitable for evaluating concentration difference bias.

[0079] In the present invention, the 50% effective concentration (EC ) of a test substance at the level of G protein activation via the opioid κ receptor is determined. 50 ) was the 50% effective concentration (EC 50 ), that is, the opioid κ 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 κ 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 κ receptor for the test substance is max ) is less than 90% compared to the level of full activation of β-arrestin2 via the opioid κ receptor, the test substance can be determined to have "intensity difference bias." In the present invention, "full activation level of β-arrestin2 via the opioid κ receptor" refers to the level of β-arrestin2 activation when β-arrestin2 activation via the opioid κ receptor is fully induced, and specifically refers to the level of β-arrestin2 activation via the opioid κ receptor by a full opioid κ receptor agonist such as U-50488 or U-69593. U-50488, which is known as an opioid κ receptor full agonist, may be (−)-U-50488 (i.e., trans-(−)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide), (±)-U-50488 (i.e., trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide), or (+)-U-50488 (i.e., trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide), and is not particularly limited thereto, but (−)-U-50488 is more preferably used. U-50488 may be used in the form of a free form, a salt thereof (e.g., but not limited to, hydrochloride, methanesulfonate, acetate, etc.), or a hydrate. U-69593, known as an opioid κ receptor full agonist, is (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide. U-69593 may be used in the form of a free form, a salt thereof (e.g., but not limited to, hydrochloride, methanesulfonate, acetate, etc.), or a hydrate.

[0080] The present invention also provides a screening method for biased opioid κ receptor agonists (e.g., concentration-difference-biasing compounds or intensity-difference-biasing compounds) using the above-mentioned method. In the screening method of the present invention, the above-mentioned method is used to measure the level of β-arrestin 2 activation by a test substance via the opioid κ receptor, and the β-arrestin 2 activation level is used to select a test substance that exhibits bias in intracellular signaling via the opioid κ receptor, for example, a bias toward the G protein pathway or the β-arrestin 2 pathway, thereby enabling screening for biased opioid κ receptor agonists. The measured β-arrestin 2 activation level is preferably compared with the level of G protein activation via the opioid κ receptor (preferably, the level of G protein activation via the opioid κ receptor by the test substance) and / or the full activation level of β-arrestin 2 via the opioid κ receptor. By this comparison, it is possible to select a test substance that exhibits a bias in intracellular signal transduction mediated by the opioid κ 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 κ receptor agonist. The test substance selected as described above can be used as an opioid κ receptor agonist with a bias.

[0081] In the screening method of the present invention, the level of β-arrestin 2 activation via the opioid κ 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.

[0082] 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 κ 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.

[0083] The screening method of the present invention may include a step of measuring the level of G protein activation via the opioid κ receptor used for comparison. Alternatively, a known value of the level of G protein activation via the opioid κ receptor may be used for comparison. The level of G protein activation via the opioid κ receptor can be measured by measuring the concentration of cAMP, an indicator of G protein pathway activation, using cells stably expressing the opioid κ receptor, as described in, for example, Nakao et al., European Journal of Pharmacology, 2012, Vol. 695, pp. 57-61. Alternatively, the level of G protein activation via the opioid κ receptor can be measured by, for example, using cell membranes overexpressing the opioid κ receptor and measuring the concentration of cAMP, an indicator of G protein pathway activation. 35S]GTPγS (guanosine 5'-O-[gamma-thio]triphosphate) binding level as an index (Cao et al., ACS Chemical Neuroscience, 2020, Vol. 11, pp. 3036-3050); a method using cells expressing opioid κ receptors and evaluating the concentration of cAMP produced by stimulation with forskolin or the like as an index (Kaski et al., Journal of Pharmacology and Experimental Therapeutics, 2019, Vol. 371, pp. 487-499); a method using cells expressing opioid κ receptors and evaluating the phosphorylation of ERK (extracellular signal-regulated kinase) as an index (Schattauer et al., The Journal of Biological Chemistry, 2012, Vol. 287, pp. 41594-41607), or a method in which BRET is used as an index to evaluate the activity of G protein α subunits and G protein γ subunits (Han et al., Nature, 2023, Vol. 617, pp. 417-425) using cells expressing a fusion protein in which a luminescent molecule is fused to one of the G protein α subunits and a fluorescent molecule is fused to the other.

[0084] 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 κ 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 κ receptor agonists that may have strong side effects.

[0085] The present invention also provides the use of the transformed cells of the present invention for evaluating the β-arrestin 2 binding response to the opioid κ receptor or for evaluating the level of β-arrestin 2 activation via the opioid κ receptor by a test substance.

[0086] 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 κ receptor, the method comprising the step of introducing into cultured animal cells a gene encoding a fusion protein comprising the opioid κ 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 κ 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).

[0087] In one embodiment, the present invention provides a method for producing a transformed animal cultured cell that can be used to assess β-arrestin 2 binding response to an opioid κ receptor, comprising the steps of: i) a step of introducing a gene encoding a fusion protein comprising an opioid κ receptor, a gene encoding a fusion protein comprising β-arrestin 2, and a gene encoding G protein-coupled receptor kinase 2 into cultured animal cells to obtain a transformed cell, wherein one of the fusion proteins, the opioid κ receptor-containing fusion protein or the β-arrestin 2-containing fusion protein, comprises NanoLuc luciferase (a luciferase consisting of the amino acid sequence shown in SEQ ID NO: 1), and the other fusion protein comprises HaloTag (a protein tag consisting of the amino acid sequence shown in SEQ ID NO: 3); ii) selecting from the obtained transformed cells a transformed cell in which the gene expression level of the opioid κ receptor is 1500 times or more, more preferably 1600 times or more, and even more preferably 1850 times or more, compared to the gene expression level of the opioid κ receptor in a corresponding non-transformed cultured animal cell; The present invention also provides a method for evaluating the β-arrestin 2 binding response to the opioid κ receptor. This method may include measuring the gene expression level of the opioid κ receptor in the transformed cells obtained in step i) and comparing it with the gene expression level of the opioid κ receptor in corresponding non-transformed cultured animal cells. By selecting transformed cells having such an expression level of the opioid κ receptor gene, cells more suitable for evaluating the β-arrestin 2 binding response to the opioid κ receptor can be obtained. The transformed cultured animal cells prepared by this method can be suitably used in the above-mentioned method for evaluating the level of β-arrestin 2 activation via the opioid κ receptor by a test substance. [Example]

[0088] 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.

[0089] Example 1: Creation and cloning of cells stably expressing human opioid κ 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-arrestin 2 to the opioid κ receptor with high accuracy or to obtain stable measurement results with little variance. Furthermore, because intracellular signaling accompanying activation of the opioid κ 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 κ 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.

[0090] To establish an appropriate evaluation system for the binding of β-arrestin2 to the opioid κ 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 κ 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).

[0091] 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).

[0092] [Table 2]

[0093] 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-KGA cells"). When untransformed HEK293 cells were cultured in the same medium, the cells died within two weeks.

[0094] A single clonal cell line was generated from the obtained HEK-KGA cells. 5,000 to 20,000 HEK-KGA 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-KGA 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). Cell culture and cell passage of the clonal cell line were performed as follows: After washing the cells with D-PBS(-), the cells 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.

[0095] Example 2: Evaluation of HEK-KGA cell clones A BRET assay was performed using each of the clonal cell lines obtained in Example 1.

[0096] 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"). 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.

[0097] On the day of the BRET assay, a test substance solution was prepared. The test substance used was a positive control substance (U-69593, a known standard opioid κ 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 a concentration of 100 μM (10 times the final concentration) by diluting a 10 mM stock solution of U-69593 (Sigma-Aldrich) dissolved in DMSO with the measurement medium.

[0098] 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 37°C, 5% CO2 incubator. 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). 20 μL of the 5x Nano-Glo substrate solution was added to the cells at a concentration of 20 μL / well, and bioluminescence resonance energy transfer (BRET) was measured before treatment with the test substance or vehicle. Subsequently, 11 μL / well of the test substance or control vehicle, diluted with assay medium to a concentration 10x the final concentration, was added to the cells, and BRET was measured again 5 minutes after treatment with the test substance or vehicle.

[0099] 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.

[0100] The BRET ratio was calculated using the following formula based on the BRET measurement results.

[0101]

number

[0102] To confirm the expression of the transgene in the clonal cell lines, the fluorescence intensity from the HaloTag fluorescent-labeled ligand and the luminescence intensity from NanoLuc luciferase were measured using the cell plates prepared above with a microplate reader, SpectraMax® Paradigm (Molecular Devices). Fluorescence intensity measurements were performed after replacing the seeding medium of the cell plates with the measurement medium used in the BRET assay described above and culturing the cells for 3–4 hours in a carbon dioxide incubator at 37°C and 5% CO2. The cell plates were irradiated with 540 nm light, the excitation light for the HaloTag NanoBRET 618 Ligand, and the fluorescence intensity (RFU) at specific wavelengths (580–700 nm in 10 nm increments) was measured. Expression of the human β-arrestin2-HaloTag fusion protein in each of the obtained clonal cell lines was confirmed by examining whether or not fluorescence from the HaloTag NanoBRET 618 Ligand with a peak around 618 nm was observed. Luminescence intensity was measured in the BRET assay described above after adding 20 μL / well of 5x Nano-Glo substrate solution to the cells. Luminescence intensity (RLU) was measured at specific wavelengths (400–550 nm, in 10 nm increments). Expression of the human opioid kappa receptor-NanoLuc luciferase fusion protein was confirmed in each clone cell line by examining whether luminescence, peaking around 460 nm, was observed due to the reaction between NanoLuc luciferase and the luminescent substrate.

[0103] Representative examples of fluorescence and luminescence intensity measurements are shown in Figure 2. Figure 2A shows representative fluorescence intensity measurements. The horizontal axis of the graph indicates the measurement wavelength (nm), the vertical axis indicates the fluorescence intensity (RFU) derived from the HaloTag fluorescent-labeled ligand, and the plots on the graph show the average fluorescence intensity at each wavelength (n = 4). Figures 2B and 2C show representative luminescence intensity measurements. The horizontal axis of the graph indicates the measurement wavelength (nm), the vertical axis indicates the luminescence intensity (RLU) derived from NanoLuc luciferase, and the plots on the graph show the luminescence intensity at each wavelength (n = 1). Figure 2B shows representative results for a clone cell line in which luminescence was detected, and Figure 2C shows representative results for a clone cell line in which luminescence was not detected. Measurements of fluorescence and luminescence intensities revealed that fluorescence from the HaloTag fluorescent-labeled ligand was detected in all clones obtained. However, luminescence from NanoLuc luciferase was detected in some clones (HEK-KGA #3, #6, #7, #12, #13, #18, #19, #29, #35, and #38) and not in others (HEK-KGA #1, #2, #4, #5, #8, #9, #10, #11, #14, #15, #16, #17, #20, #21, #22, #23, #24, #25, #26, #27, #28, #30, #31, #32, #33, #34, #36, #37, #39, and #40).

[0104] The results of the BRET assay are shown in Figure 3. The vertical axis of the graph in Figure 3 represents the BRET ratio, and the # numbers above the graph indicate the HEK-KGA clone cell line number. The horizontal axis of the graph, "pre," represents cells before treatment with the test substance or control solvent, and "post" represents cells after treatment with the test substance or control solvent. The bars on the graph represent the average and individual values ​​for duplicate wells of the same treatment. Figures 3A-F show the results for all clone cell lines (HEK-KGA #3, #6, #18, #19, #29, and #35, respectively) in which luminescence was detected and the BRET ratio increased in the positive control substance-treated group after treatment (post) compared to before treatment (pre). This increase in the BRET ratio indicates that BRET occurred due to the binding reaction of β-arrestin 2 to the opioid κ receptor. Figures 3G-J show the results for all clonal cell lines (HEK-KGA #7, #12, #13, and #38, respectively) in which luminescence was detected and no change in the BRET ratio was observed in the positive control substance-treated group after treatment compared to both the pre-treatment group and the vehicle-treated group after treatment. Figure 3K shows the result for #40 as a representative example of a clonal cell line in which luminescence was not detected and BRET measurement was difficult.

[0105] The results in Figures 2 and 3 indicate that the results of the BRET assay for the positive control substance of each of the obtained clone cell lines could be classified into three types. In particular, it was shown that even among the clone cell lines in which fluorescence derived from NanoLuc luciferase was detected, there were clone cell lines in which an increase in the BRET ratio could be detected, i.e., in which the binding reaction of β-arrestin 2 to the opioid κ receptor induced by an opioid κ receptor agonist could be detected, and clone cell lines in which this reaction could not be detected.

[0106] (Example 3) Examination of the suitability of HEK-KGA cell clones for evaluating the binding response of β-arrestin 2 to the human opioid κ receptor All of the clonal cell lines (HEK-KGA #3, #6, #18, #19, #29, and #35) in which an increase in the BRET ratio was detected in Example 2 were used to evaluate whether they could properly detect the β-arrestin 2 binding response to the human opioid κ receptor induced by the concentration difference biasing compound and the intensity difference biasing compound.

[0107] The test substances used were a positive control substance ((-)-U-50488, final concentration 10 μM), nalfurafine hydrochloride, a concentration difference biasing compound, and intensity difference biasing compound A. The test substances were dissolved in DMSO to prepare 10 mM stock solutions, which were stored at −20°C or below until the day of use. As a control, the solvent (DMSO, final concentration 0.1%) was used instead of the test substance.

[0108] For BRET assays, use NanoBRET TM Nano-Glo (R) The BRET assay was performed using a BRET Detection System (Promega). The day before the BRET assay, cells stored frozen at -80°C were quickly thawed in a 37°C water bath and immediately mixed into the culture medium. The cells were centrifuged (150 × g, 5 minutes, room temperature), the supernatant was removed, and the cells were suspended in the seeding medium described in Example 2. 4 × 10 5 Dilute the cell suspension to 1 / 1000 of the original volume of 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.

[0109] 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%.

[0110] 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. Three minutes after treatment with the test substance or solvent, 25 μL / well of 5x Nano-Glo substrate solution was added. BRET was then measured 5 minutes after treatment with the test substance or solvent. BRET measurements were performed using EnVision 2105 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.

[0111] Furthermore, based on the BRET ratio, the BRET induction rate was calculated using the following formula.

[0112]

number

[0113] This BRET induction rate is expressed as the percentage of the BRET ratio of each test substance treatment group relative to the mean BRET ratio in the solvent treatment group, which is defined as a BRET induction rate of 0%, and the mean BRET ratio in the positive control substance treatment group using (-)-U-50488, a concentration difference biasing compound, which is defined as a BRET induction rate of 100%.

[0114] 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).

[0115] The results of the BRET assay for each clone cell line are shown in Figure 4. The horizontal axis of the graph represents the concentration of the test substance (log (mol / L)), and the vertical axis represents the BRET induction rate (%). The # numbers at the top of the graph indicate the HEK-KGA clone cell line number. Each plot represents the mean ± standard deviation of duplicate wells for the same treatment. As shown in Figure 4, for all clone cell lines evaluated, the BRET induction rate increased with increasing concentrations of the test substances (concentration difference biasing compounds and intensity difference biasing compounds), confirming that the binding reaction of β-arrestin 2 to the human opioid κ receptor occurs in a test substance concentration-dependent manner. Of these, HEK-KGA #3, #6, #29, and #35 exhibited characteristics suitable for evaluation in terms of uniformity within the same treatment and regression to a sigmoid curve. On the other hand, HEK-KGA #18 exhibited poor uniformity within the same treatment, and HEK-KGA #19 exhibited poor regression to a sigmoid curve.

[0116] To select the most suitable clone from HEK-KGA #3, #6, #29, and #35, 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:

[0117]

number

[0118] The results are shown in Table 3. The BRET change rate of HEK-KGA #35 was 1.33, the highest.

[0119] [Table 3]

[0120] EC of cell line HEK-KGA #35 50and E max The calculation results are shown in Table 4.

[0121] [Table 4]

[0122] (Example 4) Comparison of expression levels of the human opioid κ receptor gene in HEK-KGA cell clones In Example 2, the clone cell lines confirmed to express NanoLuc luciferase are thought to also express the human opioid κ receptor fused with NanoLuc luciferase, but an increase in the BRET ratio was not detected in some clone cell lines (HEK-KGA #7, #12, #13, and #38). Therefore, to explore the factors behind the difference in whether BRET was detectable or not, the expression level of the human opioid κ receptor gene was quantified by real-time PCR for all clones confirmed to express NanoLuc luciferase.

[0123] Measurement of the expression level of the human opioid κ receptor gene using real-time PCR was carried out as follows. The cells were washed with D-PBS(-) and 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 D-PBS(-). The number of cells was counted, and 4 × 10 6The cell suspension containing 100 cells was collected and centrifuged again (150 × g, 5 minutes, room temperature). The supernatant was removed, and the resulting cell pellet was stored at -80°C. QIAzol Lysis Reagent (QIAGEN) was added to the cell pellet, and total RNA was extracted using QIAshredder and miRNeasy Mini Kit (both QIAGEN) according to the manufacturer's protocol. The extracted total RNA, adjusted to 0.8–1.0 μg, was used as a template for reverse transcription PCR using the reverse transcriptase ReverTra Ace® (TOYOBO). The synthesized cDNA was used as a template and mixed with PowerUP SYBR Green Master Mix (Thermo Fisher Scientific) along with primers. Real-time PCR was performed for up to 40 cycles using a 7500 Fast Real-Time PCR system (Thermo Fisher Scientific). The temperature cycle conditions for real-time PCR were as follows: an initial denaturation step at 95°C for 10 minutes, followed by a 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. The sequences of the primers used were as follows: Human opioid kappa receptor gene (hOPRK1): Forward primer 5'-GCTGCTCTCTCCAGCTATTACTTCT-3' (SEQ ID NO: 14) Reverse primer 5'-GAGTCCGAAATACAGTTCAGGATCCTGC-3' (SEQ ID NO: 15) Human endogenous housekeeping gene GAPDH: Forward primer 5'-AATCCCATCACCATCTTCCA-3' (SEQ ID NO: 16) Reverse primer 5'-GGCGGAGATGACCCTT-3' (SEQ ID NO: 17)

[0124] The ΔCt value was calculated by subtracting the Ct value of the human opioid κ receptor gene from the GAPDH cycle (Ct) value of each well, and the expression level of the human opioid κ receptor gene in each clone cell line was calculated as 2 ΔCt The values ​​were calculated. ΔCt The values ​​were calculated similarly for the host cells, HEK293 cells. ΔCt The value obtained by dividing the expression level by the value was used as a relative value of the expression level of the human opioid κ receptor gene for comparison of the expression levels.

[0125] The results are shown in Figure 5. The horizontal axis of the graph represents untransformed HEK293 cells (HEK) or transformed HEK-KGA clone cell lines, and the vertical axis and the numbers in the figure represent the relative expression levels of the human opioid κ receptor gene in each clone cell line, when the expression level of the human opioid κ receptor gene in untransformed HEK293 cells is defined as 1. The relative values ​​of the human opioid κ receptor gene expression levels of cell lines HEK-KGA #3, #6, #29, and #35, which were determined to have characteristics suitable for evaluation in Example 3, were relatively high, at 1500 or higher, whereas the relative values ​​of the human opioid κ receptor gene expression levels of cell lines #18 and #19, which were determined not to have characteristics suitable for evaluation, and cell lines HEK-KGA #7, #12, #13, and #38, in which BRET was not detected, were lower, at less than 1500. This indicates that for the above-mentioned transformed cells to be suitable for evaluating the β-arrestin 2 binding response to the human opioid κ receptor, the opioid κ receptor gene expression level should be 1,500-fold or more higher than that of untransformed HEK293 cells.

[0126] Based on these results, HEK-KGA #35 was selected as the most suitable cell line for evaluating the β-arrestin2 binding response to the opioid κ receptor agonist. HEK-KGA #35 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-04098).

[0127] (Example 5) Examination of test conditions for evaluating β-arrestin 2 binding response to opioid κ receptor To better assess the response of biasing compounds, test conditions were explored.

[0128] NanoBRET TM Nano-Glo (R) The product protocol for the Detection System (Promega) states that the medium used for medium changes and compound solution preparation is Opti-MEM I Reduced Serum Medium, no phenol red (Thermo Fisher Scientific), which contains 4% FBS, the same as the seeding medium (hereinafter referred to as "conditions described in the protocol"). However, to reduce the effects of background increase due to components contained in FBS under the conditions described in the protocol and the effects of pH changes caused by the need to take the cell plate out of the carbon dioxide incubator from the time of measurement preparation to the time of measurement, we used 1% GlutaMAX, which does not contain FBS. TM We investigated conditions using CO2-independent medium (Thermo Fisher Scientific) supplemented with Calcium Supplement (Thermo Fisher Scientific) (hereinafter referred to as "modified medium conditions").

[0129] First, the response to 10 μM (-)-U-50488, a positive control substance belonging to the concentration-difference biasing compound, was measured by BRET assay using HEK-KGA #35. BRET measurements were performed using Opti-MEM I Reduced Serum Medium, no phenol red (Thermo Fisher Scientific) containing 4% FBS as the measurement medium (conditions described in the protocol) or 1% GlutaMAX TMCO2-independent medium (FBS-free; modified medium conditions) supplemented with Supplement (Thermo Fisher Scientific) was used. Otherwise, the BRET assay and measurements were performed as in Example 2. The BRET rate change was calculated as in Example 3. The difference in BRET ratio was calculated by subtracting the mean BRET ratio in the solvent-treated group from the mean BRET ratio in the positive control substance-treated group. The results are shown in Table 5.

[0130] [Table 5]

[0131] Compared to when the measurement medium conditions described in the protocol were used, when the modified medium conditions were used, both the BRET change rate and the difference in the BRET ratio increased. This indicates that the modified medium conditions (using CO2-independent medium) are superior for quantitative evaluation and are easier to detect the β-arrestin2 binding reaction to the opioid κ receptor.

[0132] Furthermore, a BRET assay was performed using the same concentration difference biasing compound nalfurafine hydrochloride used in Example 3 under the conditions described in the protocol and under modified medium conditions, and the results were compared. Except for using the conditions described in the protocol or the modified medium conditions, the BRET assay, BRET measurement, calculation of the BRET ratio and BRET induction rate, regression to a four-parameter sigmoid curve, and 50% effective concentration (EC 50 ) and maximum response (E max The calculation of the BRET induction rate and the creation of graphs were carried out in the same manner as in Example 3. 50 ) and maximum response (E max ) are shown in Table 6, and this was taken as the level of β-arrestin 2 activation via the opioid κ receptor (β-arrestin 2 activity).

[0133] [Table 6]

[0134] On the other hand, Nakao et al., European Journal of Pharmacology, 2012, Vol. 695, p. 57-61, reported that the EC20 values ​​of nalfurafine hydrochloride, a compound with a concentration difference bias, showed the G protein activation level (G protein activity) via the opioid kappa receptor. 50 was 0.00940 nM (Table 2).

[0135] Under the conditions described in the protocol of this example and the modified medium conditions, the EC 50 The EC values ​​were 0.194 nM and 0.404 nM, respectively, a 2.08-fold increase in the modified medium conditions compared to the conditions described in the protocol. These results indicate that the EC values, which represent the level of β-arrestin activation measured in the modified medium conditions, are 50 The values ​​are the known EC values ​​that indicate the level of G protein activation via the opioid κ receptor described above. 50 This suggests that the difference between the two values ​​shifted in the increasing direction, indicating that the modified culture conditions are a method that is easy to detect concentration difference bias.

[0136] In addition, the BRET induction rate E max The efficiencies were 89.7% and 104.0% under the conditions described in the protocol and the modified medium conditions, respectively (Table 6). These results indicate that the concentration-difference-biasing compound nalfurafine hydrochloride used in this example is known to be a full agonist, but not a partial agonist, for the above-mentioned β-arrestin binding reaction (β-arrestin 2 activation via the opioid κ receptor). However, under the conditions described in the protocol, the data showed a partial agonism of 89.7%. This revealed that the conditions described in the protocol may make it difficult to distinguish between concentration-difference-biasing compounds and intensity-difference-biasing compounds. Therefore, modified medium conditions were shown to be more suitable for evaluating biasing compounds when it comes to distinguishing between concentration-difference-biasing compounds and intensity-difference-biasing compounds. [Industrial Applicability]

[0137] The cells and methods of the present invention can detect the β-arrestin 2 binding reaction to the opioid κ receptor induced by a test substance with high accuracy and little variability, and are therefore useful for screening opioid κ receptor agonists that have weak β-arrestin 2 pathway activation ability and reduced side effects.

[0138] <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 kappa receptor protein SEQ ID NO: 6: Human opioid kappa 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 VB210518-1359sty SEQ ID NO: 12: Nucleotide sequence of expression vector VB210518-1378<em>f SEQ ID NO: 13: Nucleotide sequence of expression vector VB210518-1365eqk SEQ ID NO: 14: Forward primer for amplifying the human opioid κ receptor gene SEQ ID NO: 15: Reverse primer for amplifying the human opioid κ receptor gene SEQ ID NO: 16: Forward primer for amplifying the human endogenous housekeeping gene GAPDH SEQ ID NO: 17: Reverse primer for amplifying the human endogenous housekeeping gene GAPDH

Claims

1. a gene encoding a fusion protein containing an opioid κ 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 opioid κ receptor-containing fusion protein or the β-arrestin 2-containing fusion protein, 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 κ receptor encodes a fusion protein in which the luciferase is fused to the C-terminus of the opioid κ receptor; The cultured transformed animal 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. 2. The transformed animal cultured cell according to claim 1, wherein the gene expression level of the opioid κ receptor is 1500 times or more as compared with the gene expression level of the opioid κ receptor in a corresponding non-transformed animal cultured cell.

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-KGA #35 having accession number NITE AP-04098.

6. 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 κ 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 κ receptor as a level of β-arrestin 2 activation via the opioid κ receptor, and comparing it with a complete level of β-arrestin 2 activation via the opioid κ receptor; A method for evaluating the level of β-arrestin 2 activation mediated by an opioid κ receptor by a test substance, comprising:

7. Serum-free CO ２ The method according to claim 6, wherein the test substance is contacted with the cultured transformed animal cells in an independent medium.

8. Measuring the level of β-arrestin 2 activation mediated by the opioid κ receptor by a test substance using the method of claim 6; comparing the measured β-arrestin 2 activation level with the G protein activation level via the opioid κ receptor and / or the full activation level of β-arrestin 2 via the opioid κ receptor to select a test substance that exhibits bias in intracellular signal transduction via the opioid κ receptor; A method for screening for an opioid κ receptor agonist having bias, comprising:

9. 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 κ receptor, comprising: i) introducing into cultured animal cells a gene encoding a fusion protein comprising an opioid κ 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 κ receptor-containing fusion protein or the β-arrestin 2-containing fusion protein, 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) selecting from the obtained transformed cells, transformed cells in which the expression level of the opioid κ receptor gene is 1500 times or more compared to the expression level of the opioid κ receptor gene in the corresponding non-transformed animal cultured cells; A method comprising:

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