Mutant G protein-coupled receptors

Abstract

To provide a chemogenetic technique that can be applied to a wider range of G-protein-coupled receptors and has higher biological safety.SOLUTION: A variant G-protein-coupled receptor includes a substituent to at least one amino acid residue selected from the group consisting of a bulky amino acid residue, a small amino acid residue, and an amino acid residue to change the presence / absence or the positivity / negativity of a charge in a region around a storage cysteine residue of extracellular loop 2.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a mutant G protein-coupled receptor and a chemical genetics technique using the same. [Background technology]

[0002] The human brain contains over 100 billion neurons and 10 times as many glial cells (cells other than neurons) that transmit information. Advances in neuroscience have revealed the functions of each part and each type of cell. However, the role of each protein in each neural circuit remains unclear. Elucidating this will not only lead to an understanding of higher brain functions, but will also lead to the development of molecular targeted drugs for psychiatric disorders, which is an unexplored field. To achieve this, there is an urgent need to develop methods to control the activity of nerve cells in a site-specific and cell-type-selective manner. To date, methods known as optogenetics and chemogenetics have been developed as a means of controlling specific cells.

[0003] Optogenetics is a method to control only cells expressing rhodopsin by light, using a receptor called rhodopsin that undergoes structural changes in response to light. This method uses light, making it possible to control neural activity in a matter of microseconds to milliseconds, and it can also be used in free-ranging animals. However, optogenetics is an invasive method that requires inserting optical fibers into the brain, and it cannot be applied to deep tissues where light cannot reach.

[0004] The DREADD method, a type of chemogenetics, is a method that can overcome these shortcomings. The DREADD method is a non-invasive method that uses artificial G protein-coupled receptors that are selectively activated by specific compounds (artificial ligands). However, the DREADD method only artificially induces downstream signals from artificial G protein-coupled receptors, and is not suitable for functional analysis of endogenous G protein-coupled receptors, nor is it expected to be applied to drug discovery. As a next-generation technology that will lead to the development of molecular targeted drugs, new chemical genetics techniques are needed to elucidate the functions of target receptors that are endogenously expressed in cells of interest. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nature Chemistry. 2016 Oct; 8(10): 958-967. Summary of the Invention [Problem to be solved by the invention]

[0006] Under these circumstances, the present inventor developed a receptor-selective chemogenetics method called coordination chemogenetics (also known as OcCC) in 2016 (Non-Patent Document 1). This method involves adding a mutation to a receptor to enable it to coordinate with a metal complex, and controlling activity by inducing a structural change. This method allows artificial control using a metal complex (palladium bipyridyl complex) while maintaining the original receptor function (ligand binding ability and response). However, the pharmacokinetics of the metal complex used is unknown, and considering the physical properties of the metal complex, there are concerns about its inability to pass through the blood-brain barrier and its toxicity, making it difficult to apply this methodology to individual animals. In addition, although there are 200 to 300 types of G protein-coupled receptors, this method can only be applied to a very small number of G protein-coupled receptors that have large ligand-binding sites outside the cell.

[0007] Therefore, an objective of the present invention is to provide a chemogenetics technique that can be applied to a wider variety of G protein-coupled receptors and is highly safe for the living body. [Means for solving the problem]

[0008] It is known that the region surrounding the conserved cysteine ​​residue in the extracellular loop 2 of the G protein-coupled receptor (the cysteine ​​residue that forms an SS bond with the conserved cysteine ​​residue in the transmembrane domain 3) is conserved. In addition, the endogenous ligand of the G protein-coupled receptor recognizes the ligand-binding domain exposed outside the cell (in the case of class C) or a relatively deep part of the transmembrane domain, while the artificial ligand recognizes a position relatively close to the extracellular loop 2 (a relatively shallow position in the transmembrane domain). From this, it was thought that the affinity of the artificial ligand could be changed (as a result, the G protein signal strength could be changed) by replacing the amino acid residue in the region surrounding the conserved cysteine ​​residue in the extracellular loop 2 with a bulky amino acid residue, a small amino acid residue, or an amino acid residue that changes the presence or absence or positive / negative charge. If the affinity of the artificial ligand differs between the mutant type and the non-mutated type (for example, the wild type), for example, by administering the artificial ligand to an animal individual containing cells expressing the mutant type and cells expressing the non-mutated type, a G protein signal can be induced specifically in one of the cells.

[0009] As a result of intensive research, the present inventors have found that the above hypothesis is universally applicable to G protein-coupled receptors. Based on this finding, the present inventors have further researched and completed the present invention. That is, the present invention includes the following aspects.

[0010] Item 1. A mutant G protein-coupled receptor comprising a substitution with at least one amino acid residue selected from the group consisting of bulky amino acid residues, small amino acid residues, and amino acid residues that change the presence or absence or positive / negative charge in the region surrounding the conserved cysteine ​​residue in extracellular loop 2.

[0011] Item 2. The mutant G protein-coupled receptor according to Item 1, wherein the region surrounding the conserved cysteine ​​residue is a region consisting of 20 amino acid residues consisting of 10 amino acid residues on the N-terminal side and 10 amino acid residues on the C-terminal side of the conserved cysteine ​​residue.

[0012] Item 3. The mutant G protein-coupled receptor according to Item 1 or 2, wherein the bulky amino acid residue is an aromatic amino acid residue, and the small amino acid residue and the amino acid residue that changes the presence or absence or polarity of the charge are alanine residues and / or glycine residues.

[0013] Item 4. The mutant G protein-coupled receptor according to any one of Items 1 to 3, wherein the G protein-coupled receptor is a class C receptor or a class A receptor.

[0014] Item 5. The mutant G protein-coupled receptor according to any one of Items 1 to 4, wherein the G protein-coupled receptor is a metabotropic glutamate receptor or a histamine receptor.

[0015] Item 6. A polynucleotide comprising a coding sequence for the mutant G protein-coupled receptor according to any one of Items 1 to 5.

[0016] Item 7. A cell comprising the polynucleotide according to Item 6.

[0017] Item 8. A non-human animal comprising the cell according to Item 7.

[0018] Section 9. General formula (1):

[0019] [ka]

[0020] [In the formula: R 1 are the same or different, -CH(-R 11 )2(R 11 are the same or different and each represents an alkyl group having 2 or more carbon atoms.) -(CH2) 0-4 -R 12 (R12 represents an optionally substituted carbocycle, or represents a hydrogen atom (provided that all R 1 (Except when R is a hydrogen atom.) 2 represents a halogen atom, an alkyl group, or an alkoxy group. A compound represented by the formula:

[0021] Section 10. General formula (2): [ka]

[0022] [In the formula: R 3 are the same or different and each represents an alkyl group, -(CH2) 0-4 -R 31 (R 31 represents an optionally substituted carbocycle, or represents a hydrogen atom (provided that all R 3 (Except when R is a hydrogen atom.) 4 represents a halogen atom, an alkyl group, or an alkoxy group. A compound represented by the formula:

[0023] Item 11. (i) A step of administering an artificial ligand of the non-mutated G protein-coupled receptor to a non-human animal comprising a cell 1 expressing the mutant G protein-coupled receptor according to any one of items 1 to 5 and a cell 2 expressing a non-mutated G protein-coupled receptor. A method for inducing a G protein signal specifically in the cell 1 or in the cell 2, comprising the steps of:

[0024] Item 12. The method according to item 11, wherein the G protein-coupled receptor is a metabotropic glutamate receptor or a histamine receptor, and the artificial ligand is the compound according to item 9, a salt thereof, or a solvate thereof, or the artificial ligand is the compound according to item 10, a salt thereof, or a solvate thereof.

[0025] Item 13. (a) A step of allowing a test substance to act on a cell x expressing a mutant G protein-coupled receptor containing a substitution of an amino acid residue in the region surrounding the conserved cysteine ​​residue of extracellular loop 2, and a cell y expressing a non-mutated G protein-coupled receptor. (b) detecting a G protein signal after step (a); (c) selecting a test substance that causes a difference between the intensity of the G protein signal in the cell x and the intensity of the G protein signal in the cell y; A method for screening an artificial ligand, comprising:

[0026] Item 14. The screening method according to Item 13, wherein the artificial ligand is an artificial ligand for use in the method according to Item 11. Effect of the Invention

[0027] According to the present invention, it is possible to provide a chemogenetics technology that can be applied to a wider variety of G protein-coupled receptors and has higher biological safety. Specifically, it is possible to provide a mutant G protein-coupled receptor for use in the technology. Furthermore, it is possible to provide an artificial ligand that can be suitably used in one embodiment of the technology. [Brief description of the drawings]

[0028] [Figure 1] 1 shows the results of Ca2+ fluorescence imaging in Test Example 2. The vertical axis shows the fluorescence intensity ratio at 340 nm and 380 nm, and the horizontal axis shows the final FITM concentration. [Diagram 2] 1 shows the results of Ca2+ fluorescence imaging in Test Example 4. The vertical axis shows the fluorescence intensity ratio at 340 nm and 380 nm, and the horizontal axis shows the final concentration of compound 2. [Diagram 3]1 shows the results of Ca2+ fluorescence imaging in Test Example 4. The vertical axis shows the relative value of the fluorescence intensity ratio at 340 nm to 380 nm, with the ratio at 0 μM of FITM derivative (compound 2, compound 3, or compound 4) set to 1, and the horizontal axis shows the final concentration of FITM derivative (compound 2, compound 3, or compound 4). The graph on the left shows the results when wild-type mGluR1 was used, and the graph on the right shows the results when mGluR1 mutants were used. [Figure 4] 1 shows the results of Ca2+ fluorescence imaging in Test Example 5. The vertical axis shows the ratio of fluorescence intensities at 340 nm and 380 nm, and the horizontal axis shows the final concentration of glutamate. The upper graph shows the results when wild-type mGluR1 was used, and the lower graph shows the results when mGluR1 mutants were used. [Diagram 5] FIG. 1 shows an outline of a method for constructing an H1R mutant expression vector by overlap extension PCR (Test Example 6). [Figure 6] 1 shows the results of Ca2+ fluorescence imaging in Test Example 8. The vertical axis shows the fluorescence intensity ratio at 340 nm and 380 nm, and the horizontal axis shows the final concentration of Lvc-Diisopro. [Figure 7] 1 shows the results of Ca2+ fluorescence imaging in Test Example 8. The vertical axis shows the fluorescence intensity ratio at 340 nm and 380 nm, and the horizontal axis shows the final concentration of Lvc-Adam. [Figure 8] 1 shows the results of Ca2+ fluorescence imaging in Test Example 8. The vertical axis shows the fluorescence intensity ratio at 340 nm and 380 nm, and the horizontal axis shows the final concentration of Lvc-1-MeNaph. [Figure 9] 1 shows the results of Ca2+ fluorescence imaging in Test Example 8. The vertical axis shows the fluorescence intensity ratio at 340 nm and 380 nm, and the horizontal axis shows the final concentration of Lvc-Dipro. [Figure 10] 1 shows the results of Ca2+ fluorescence imaging in Test Example 10. The vertical axis shows the fluorescence intensity ratio at 340 nm and 380 nm, and the horizontal axis shows the final concentration of Lvc-Diisopro. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0030] The "identity" of an amino acid sequence refers to the degree of amino acid sequence agreement between two or more comparable amino acid sequences. Thus, the higher the agreement between two amino acid sequences, the higher the identity or similarity between those sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (S. Karlin, SF Altschul. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes." Proc. Natl. Acad. Sci. USA 87, 2264-2268 (1990); S. Karlin, SF Altschul. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc. Natl. Acad. Sci. USA 90, 5873-5877 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of base sequences is also defined in the same manner as above.

[0031] In this specification, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is considered to be conservative substitution. In addition, substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also considered to be conservative substitutions.

[0032] In the present specification, nucleotides such as DNA and RNA may be chemically modified as shown below. To prevent degradation by hydrolases such as nucleases, the phosphoric acid residue (phosphate) of each nucleotide may be replaced with a chemically modified phosphoric acid residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. In addition, BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety, can also be preferably used.

[0033] 2. Mutant G protein-coupled receptors In one aspect, the present invention relates to a mutant G protein-coupled receptor (sometimes referred to herein as the "mutant G protein-coupled receptor of the present invention") comprising a substitution with at least one amino acid residue selected from the group consisting of bulky amino acid residues, small amino acid residues, and amino acid residues that change the presence or absence or positive / negative charge in the region surrounding the conserved cysteine ​​residue in extracellular loop 2. This will be described below.

[0034] First, a G protein-coupled receptor (sometimes simply referred to as a "G protein-coupled receptor" or a "non-mutated G protein-coupled receptor") before "substitution with at least one amino acid residue selected from the group consisting of bulky amino acid residues, small amino acid residues, and amino acid residues that change the presence or absence or polarity of a charge in the region surrounding the conserved cysteine ​​residue in extracellular loop 2" will be described.

[0035] G protein-coupled receptors are receptors that couple with G proteins to transmit signals from the outside world into cells. G protein-coupled receptors have a common structure that penetrates the cell membrane seven times and constitute the G protein-coupled receptor superfamily. G protein-coupled receptors are classified into six classes, class A, class B, class C, class D, class E, and class F, based on similarities in amino acid sequence and function.

[0036] The origin of the G protein-coupled receptor is not particularly limited, and examples of the origin of the G protein-coupled receptor include animals, such as various mammals, including humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.

[0037] Examples of G protein-coupled receptors include metabotropic glutamate receptors (e.g., mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7, mGluR8, etc.), adenosine receptors, β-adrenergic receptors, neurotensin receptors, muscarinic acid receptors, 5-hydroxytryptamine receptors, adrenergic receptors, anaphylatoxin receptors, angiotensin receptors, apelin receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemokine receptors, cholecystokinin receptors, dopamine receptors, endothelin receptors, free fatty acid receptors, bile acid receptors, galanin receptors, motilin receptors, ghrelin receptors, glycoprotein hormone receptors, GnRH receptors, histamine receptors, KiSS1-derived peptide receptors, leukotriene and lipoxin receptors, lysophospholipid receptors, melanin-concentrating hormone receptors, and the like. These include acetylcholine receptors, melanocortin receptors, melatonin receptors, neuromedin U receptors, neuropeptide receptors, N-formyl peptide family receptors, nicotinic acid receptors, opioid receptors, G protein-coupled receptor-like receptors, orexin receptors, P2Y receptors, peptide P518 receptors, platelet-activating factor receptors, prokineticin receptors, prolactin-releasing peptide receptors, prostanoid receptors, protease-activated receptors, relaxin receptors, somatostatin receptors, SPC / LPC receptors, tachykinin receptors, trace amino acid receptors, thyrotropin-releasing hormone receptors, eutrophin receptors, vasopressin / oxytocin receptors, orphan GPCRs, calcitonin receptors, corticotropin-releasing factor receptors, glucagon receptors, parathyroid receptors, VIP / PACAP receptors, LNB7TM receptors, GABAB receptors, and calcium sensor receptors. Among these, preferred examples include metabotropic glutamate receptors, histamine receptors, etc., and more preferred examples of these include mGluR1, hH1R, etc.

[0038] In one embodiment of the present invention, it is preferable to adopt a G protein-coupled receptor whose crystal structure is known. This makes it possible to determine the amino acid residues to be substituted with higher accuracy, as described below. The crystal structure is published in the Protein Data Bank (PDB) (https: / / www.rcsb.org / ). Examples of G protein-coupled receptors whose crystal structure is known include β1-Adrenergic receptor, β2-Adrenergic receptor, Adenosine A, and β1-Adrenergic receptor. 2A Adenosine A 2A receptor), Dopamine D2 receptor, Dopamine D3 receptor, Histamine H1 receptor, Muscarinic M2 receptor, Muscarinic M3 receptor, Sphingosine 1-phosphate receptor, Serotonin 5-HT 1B receptor (serotonin 5-HT 1B receptor), Serotonin 5-HT 2B receptor (serotonin 5-HT 2B receptor), Serotonin 5-HT3 receptor, Neurotensin receptor 1, Chemokine receptor 4, Chemokine receptor 5, μ-Opioid receptor, P2Y 12 Purinergic receptor (P2Y 12Class A receptors include purinergic receptors, as well as metabotropic glutamate receptor 1, metabotropic glutamate receptor 5, and GABA receptors. B receptor (GABA B Class C receptors such as GPCRs with β-glucosamine hydrochloride (GPCRs with β-glucosamine hydrochloride) and class C receptors such as class C receptors and orexin receptors. In addition to the above, structural biological information on many other GPCRs (structural information obtained from X-ray crystallography and single particle analysis using cryo-electron microscopy) has been reported. By utilizing this information, it is possible to determine with higher accuracy the amino acid residues to be substituted, as described below.

[0039] The amino acid sequences of various G protein-coupled receptors are known. For example, they can be obtained from known databases (for example, NCBI: https: / / www.ncbi.nlm.nih.gov / ). Specifically, for example, rat-derived mGluR1 includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Accession Number: NM_017011.1).

[0040] The G protein-coupled receptor may have a mutation such as an amino acid substitution, deletion, addition, or insertion, relative to the wild-type G protein-coupled receptor, so long as the property of inducing a G protein signal by binding to a ligand is not significantly reduced. The mutation is preferably a substitution, more preferably a conservative substitution. When the G protein-coupled receptor has an amino acid mutation, the amino acid sequence has, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more identity to the amino acid sequence of the wild-type G protein-coupled receptor.

[0041] Preferred specific examples of the G protein-coupled receptor include the proteins described in the following (a) and (b): (a) a protein consisting of a wild-type mGluR1 amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO:1); and (b) a protein consisting of an amino acid sequence having 85% or more identity with the wild-type mGluR1 amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO:1); At least one selected from the group consisting of:

[0042] In the above (b), the identity is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more. The number of mutated amino acids in the above (b) and (d) is, for example, 1 to 30, preferably 1 to 15, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 2.

[0043] The G protein-coupled receptor may be chemically modified so long as its ability to induce a G protein signal upon ligand binding is not significantly reduced.

[0044] G protein-coupled receptors have a C-terminus that is either a carboxyl group (-COOH) or a carboxylate (-COO - ), amide (-CONH2) or ester (-COOR).

[0045] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0046] In the G protein-coupled receptor, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester may be, for example, the C-terminus ester described above.

[0047] Furthermore, in G protein-coupled receptors, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., C group such as formyl group or acetyl group). 1-6 Alkanoyl C 1-6 those protected by suitable protecting groups (e.g., C groups such as formyl and acetyl groups); those in which the N-terminal glutamine residue that may be cleaved in vivo is pyroglutamic-substituted; and those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) are protected by suitable protecting groups (e.g., C groups such as formyl and acetyl groups). 1-6 Alkanoyl group etc. 1-6 Also included are those protected by an acyl group, or conjugated proteins such as so-called glycoproteins to which sugar chains are bound.

[0048] The G protein-coupled receptor may be one (e.g., a fusion protein) to which a linker, an epitope tag, a fluorescent protein, a peptide that facilitates purification, a cleavable linker peptide, etc., is added. Examples of tags include a histidine tag, a FLAG tag, and an HA tag.

[0049] The mutant G protein-coupled receptor of the present invention is a mutant of the above-mentioned "G protein-coupled receptor" which comprises a substitution with at least one amino acid residue selected from the group consisting of bulky amino acid residues, small amino acid residues, and amino acid residues that change the presence or absence or positive / negative charge in the region surrounding the conserved cysteine ​​residue in extracellular loop 2.

[0050] Extracellular loop 2 is the second loop from the N-terminus of the three extracellular loops among the six loops formed in the seven-transmembrane structure of G protein-coupled receptors (commonly referred to as "ECL2").

[0051] The conserved cysteine ​​residue in extracellular loop 2 is a cysteine ​​residue that forms an S—S bond with a conserved cysteine ​​residue in transmembrane domain 3 (the third domain from the N-terminus of the seven transmembrane domains in the cell membrane).

[0052] The extracellular loop 2 and the conserved cysteine ​​residues can be easily determined based on publicly known amino acid sequence information, structural analysis information, and the like. For example, the conserved cysteine ​​residue in rat-derived mGluR1 (amino acid sequence: SEQ ID NO: 1) is the 746th amino acid residue from the N-terminus, and therefore, the conserved cysteine ​​residues in mGluR1 and other metabotropic glutamate receptors in other species can also be easily determined based on this information (e.g., by alignment analysis). Furthermore, since the region surrounding the conserved cysteine ​​residue in extracellular loop 2 is conserved, the conserved cysteine ​​residues in various G protein-coupled receptors can also be easily determined based on the above information (and / or similar information in other G protein-coupled receptors).

[0053] The region around the conserved cysteine ​​residue is not particularly limited as long as it is a region that is highly conserved in G protein-coupled receptors. The region around the conserved cysteine ​​residue is preferably a region consisting of 20 amino acid residues, consisting of 10 amino acid residues on the N-terminus side and 10 amino acid residues on the C-terminus side of the conserved cysteine ​​residue. More preferably, the region is a region consisting of 10 amino acid residues, consisting of 5 amino acid residues on the N-terminus side and 5 amino acid residues on the C-terminus side of the conserved cysteine ​​residue.

[0054] In one embodiment of the present invention, the region is more preferably a region consisting of 6 amino acid residues, consisting of 1 amino acid residue on the N-terminus and 5 amino acid residues on the C-terminus of the conserved cysteine ​​residue. In one embodiment of the present invention, the region is even more preferably a region consisting of 4 amino acid residues on the C-terminus of the conserved cysteine ​​residue. In one embodiment of the present invention, the region is even more preferably a region consisting of 3 amino acid residues on the C-terminus of the conserved cysteine ​​residue.

[0055] In one embodiment of the present invention, the region is more preferably a region consisting of 6 amino acid residues consisting of 5 amino acid residues on the N-terminal side and 1 amino acid residue on the C-terminal side of the conserved cysteine ​​residue. In one embodiment of the present invention, the region is even more preferably a region consisting of 4 amino acid residues on the N-terminal side of the conserved cysteine ​​residue. In one embodiment of the present invention, the region is even more preferably a region consisting of 3 amino acid residues on the N-terminal side of the conserved cysteine ​​residue.

[0056] The type of amino acid residue to be substituted in the region surrounding the conserved cysteine ​​residue is not particularly limited.

[0057] When replacing with a bulky amino acid residue, the amino acid residue to be replaced is preferably an amino acid residue with a relatively small molecular weight, such as glycine, alanine, cysteine, serine, threonine, valine, isoleucine, leucine, etc., and preferably glycine, alanine, cysteine, serine, threonine, valine, etc.

[0058] When replacing with a small amino acid residue, the amino acid residue to be replaced is preferably an amino acid residue with a relatively large molecular weight, such as phenylalanine, tyrosine, histidine, tryptophan, proline, aspartic acid, glutamic acid, cysteine, methionine, lysine, arginine, asparagine, glutamine, serine, threonine, isoleucine, leucine, valine, etc.

[0059] The number of amino acid residues to be substituted in the region surrounding the conserved cysteine ​​residue is not particularly limited, and may be, for example, 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 1.

[0060] The position of the amino acid residue to be substituted in the region surrounding the conserved cysteine ​​residue is not particularly limited. In a preferred embodiment, an amino acid residue whose side chain faces the side of the artificial ligand to be bound can be identified based on structural information or the like. By substituting such an amino acid residue with a bulkier amino acid residue, the affinity of the artificial ligand can be further reduced due to steric repulsion.

[0061] There are various known artificial ligands that act on various G protein-coupled receptors. For example, 4-fluoro-N-(4-(6-(isopropylamino)pyrimidin-4-yl)thiazol-2-yl)-N-methylbenzamide (FITM) for metabotropic glutamate receptors, 2-(4-(carbokyethyl)-phenylethylamino)adenosine-5'-N-ethyluronamide (CGS21680) for adenosine A2A receptors, and 8,8'-(Carbonylbis(imino-3,1-phenylenecarbonylimino(4-methyl-3,1-phenylene)carbonylimino))di(1,3,5-naphthalenetrisulfonic acid) for P2Y receptors. acid) (suramin), N,N-Dimethyl-3-(6,11-dihydrodibenz(b,e)oxepin-11-ylidene)propylamine (doxepin) for histamine receptors, 11-((4-methylpiperazin-1-yl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one (pirenzepine) for muscarinic receptors, and N-((1R,5S)-8-methyl-8-azabicyclo(3.2.1)oct-3-yl)-2-oxo-3-(propan-2-yl)-2,3-dihydro-1H-benzimidazole-1-carboxamide hydrochloride (BIMU8) for serotonin receptors. In addition, artificial ligands have been reported in various publications, such as publication 1 regarding artificial ligands for adenosine A2A receptors (M. de Lera Ruiz, et al. J. Med. Chem. 57, 3623-3650 (2014)) and publication 2 regarding artificial ligands for metabotropic glutamate receptors (CM Niswender, et al. Annu. Rev. Pharmacol. Toxicol. 50, 295-322 (2010)).

[0062] The molecular weight of the artificial ligand is, for example, 100 to 3,000, 100 to 2,000, 150 to 1,000, 200 to 700, or 200 to 500.

[0063] The amino acid residue after substitution is at least one type of amino acid residue selected from the group consisting of bulkier amino acid residues, small amino acid residues, and amino acid residues whose electric charge is changed to either positive or negative.

[0064] The bulky amino acid residue is not particularly limited and includes both natural amino acid residues and artificial amino acid residues. Preferred examples of the bulky amino acid residue include aromatic amino acid residues.

[0065] The aromatic amino acid residue is not particularly limited as long as it is an amino acid residue having an aromatic group on the side chain (preferably an aromatic group at the end of the side chain). The aromatic group is not particularly limited, and examples thereof include aryl groups having 6 to 20, preferably 6 to 12, and more preferably 6 to 8 ring carbon atoms, specifically monocyclic or polycyclic aryl groups such as phenyl, hydroxyphenyl, naphthyl, biphenyl, pentalenyl, indenyl, anthranyl, tetracenyl, pentacenyl, pyrenyl, perylenyl, fluorenyl, and phenanthryl groups, and heteroaryl groups having 3 to 20, preferably 3 to 12, and more preferably 7 to 11 ring atoms (preferably a heteroaryl group in which the heteroatom is a nitrogen atom), Specific examples of such heteroaryl groups include monocyclic or polycyclic heteroaryl groups such as a pyrrolyl group, a pyridyl group, a pyrrolidyl group, a piperidyl group, an imidazolyl group, a pyrazolyl group, a pyrazyl group, a pyrimidyl group, a pyridazyl group, a piperazyl group, a triazinyl group, an oxazolyl group, an isoxazolyl group, a morpholyl group, a thiazolyl group, an isothiazolyl group, a furanyl group, a thiophenyl group, an indolyl group, a quinolyl group, an isoquinolyl group, a benzimidazolyl group, a quinazolyl group, a phthalazyl group, a purinyl group, a pteridyl group, a benzofuranyl group, a coumaryl group, a chromonyl group, and a benzothiophenyl group.

[0066] The aromatic group is preferably a heteroaryl group, more preferably a heteroaryl group having a nitrogen atom as a heteroatom, even more preferably a heteroaryl group having 1 to 3 (preferably 1) nitrogen atoms as a heteroatom, still more preferably a heteroaryl group having 7 to 11 ring atoms and having one nitrogen atom as a heteroatom, and particularly preferably an indolyl group.

[0067] In a preferred embodiment of the aromatic amino acid residue, the side chain is represented by the formula (A): -L A -R A (In the formula: L A R represents an optionally substituted alkylene group. A represents an aromatic group. - represents a single bond.

[0068] L A The alkylene group represented by the formula (I) is not particularly limited, and examples thereof include linear or branched (preferably linear) alkylene groups having 1 to 6 carbon atoms, preferably 1 to 4, more preferably 1 to 2, and even more preferably 1. Examples of the alkylene group include a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, and a hexylene group.

[0069] L A The alkylene group represented by may be substituted. The substituent is not particularly limited, and examples thereof include an amino group, a carboxyl group, a carbamoyl group, and a hydroxyl group. The number of the substituent is not particularly limited, and is, for example, 0 to 3, preferably 0 to 1, and more preferably 0.

[0070] R A The aromatic group represented by the formula (I) is the same as the aromatic group described above.

[0071] Specific examples of aromatic amino acid residues include tryptophan residues, phenylalanine residues, tyrosine residues, histidine residues, thyroxine residues, etc., preferably tryptophan residues, phenylalanine residues, tyrosine residues, etc., more preferably tryptophan residues.

[0072] The small amino acid residue is not particularly limited and includes both natural and artificial amino acid residues. Suitable small amino acid residues include glycine residues, alanine residues, serine residues, and the like.

[0073] The amino acid residue for changing the presence or absence of charge or the positive / negative charge is not particularly limited, so long as it is an amino acid residue for which the presence or absence of charge is changed (e.g., from charged to uncharged, or from uncharged to charged) or the positive / negative charge is changed (e.g., from positive to negative, or from negative to positive) from the amino acid residue to be substituted. For example, when the amino acid to be substituted is a charged amino acid such as lysine or glutamic acid, it may be substituted with an uncharged amino acid such as alanine or glycine.

[0074] The mutant G protein-coupled receptor of the present invention may be in the form of a pharma- ceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is a pharma- ceutically acceptable salt, and both acid salts and basic salts can be used. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0075] The mutant G protein-coupled receptor of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharma- ceutically acceptable, and examples of the solvent include water, ethanol, glycerol, and acetic acid.

[0076] The mutant G protein-coupled receptor of the present invention can be easily produced by known genetic engineering techniques, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques.

[0077] 3. Polynucleotides, cells, and non-human animals In one aspect, the present invention relates to a polynucleotide (sometimes referred to as the "polynucleotide of the present invention" in this specification) comprising a coding sequence of the mutant G protein-coupled receptor of the present invention. This will be described below.

[0078] The coding sequence of the mutant G protein-coupled receptor of the present invention is not particularly limited, so long as it is a polynucleotide consisting of a base sequence encoding the mutant G protein-coupled receptor of the present invention.

[0079] In one embodiment, the polynucleotide of the present invention comprises an expression cassette for a mutant G protein-coupled receptor of the present invention.

[0080] The expression cassette for the mutant G protein-coupled receptor of the present invention is not particularly limited as long as it is a polynucleotide capable of expressing the mutant G protein-coupled receptor of the present invention in cells. A typical example of the expression cassette for the mutant G protein-coupled receptor of the present invention is a polynucleotide comprising a promoter and a coding sequence for the mutant G protein-coupled receptor of the present invention placed under the control of the promoter.

[0081] The species of origin of the target cells is not particularly limited, and examples thereof include bacteria such as Enterobacteriaceae bacteria, fungi such as yeast, animals, and plants. Examples of animals include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer, as well as non-mammalian vertebrates and invertebrates. In a preferred embodiment of the present invention, examples of the species of origin of the target cells include mammals, Escherichia coli, yeast, Bacillus subtilis, zebrafish, medaka, and insects. The types of cells are also not particularly limited, and examples of the cells are cells derived from various tissues or with various properties, such as blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0082] The promoter contained in the expression cassette of the mutant G protein-coupled receptor of the present invention is not particularly limited and can be appropriately selected depending on the target cell. As the promoter, for example, various pol II promoters can be used. As the pol II promoter, there is no particular limitation, but examples thereof include CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, etc. Other examples of the promoter include tryptophan promoters such as trc and tac, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, tetracycline-inducible promoter, etc.

[0083] The expression cassette of the mutant G protein-coupled receptor of the present invention may contain other elements (e.g., a multiple cloning site (MCS), a drug resistance gene, a replication origin, an enhancer sequence, a repressor sequence, an insulator sequence, a reporter protein (e.g., a fluorescent protein, etc.) coding sequence, a drug resistance gene coding sequence, etc.) as necessary. The MCS is not particularly limited as long as it contains a plurality of restriction enzyme sites (e.g., 2 to 50, preferably 2 to 20, more preferably 2 to 10). When the mutant G protein-coupled receptor of the present invention does not contain a functional domain, the MCS can be used for inserting a coding sequence for any functional domain.

[0084] Examples of drug resistance genes include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, and a puromycin resistance gene.

[0085] The reporter protein is not particularly limited as long as it is a luminescent (color-producing) protein that emits light (color) upon reaction with a specific substrate, or a fluorescent protein that emits fluorescence upon excitation light. Examples of luminescent (color-producing) proteins include luciferase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, etc., and examples of fluorescent proteins include GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, JRed, KillerRed, Cherry, HcRed, mPlum, etc.

[0086] The expression cassette of the mutant G protein-coupled receptor of the present invention may constitute an expression vector by itself or together with other sequences. The type of vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids; virus vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus. In addition, examples of the vector include ColE1-based plasmids, such as pBR322 derivatives in Escherichia coli, pACYC-based plasmids having p15A origin, pSC-based plasmids, and mini-F plasmids derived from F factors such as Bac-based plasmids.

[0087] The polynucleotide of the present invention can be easily prepared according to known genetic engineering techniques, for example, by using PCR, restriction enzyme digestion, DNA ligation techniques, and the like.

[0088] In one aspect, the present invention relates to a cell (sometimes referred to as "cell of the present invention" in the present specification) containing the polynucleotide of the present invention. The cell is similar to the subject cell described above. The polynucleotide of the present invention is present within the genome of the cell of the present invention or outside the genome.

[0089] Furthermore, in one aspect, the present invention relates to a non-human animal (sometimes referred to as "animal of the present invention" in the present specification) comprising the cell of the present invention. The animal is the same as the animal described above. As the animal of the present invention, preferably, a non-human animal used in the induction method of the present invention described below can be adopted.

[0090] 4. Artificial Ligands In one aspect, the present invention comprises:

[0091] [ka]

[0092] The present invention relates to a compound represented by the following formula (1): In one aspect, the present invention comprises:

[0093] [ka]

[0094] The present invention relates to a compound represented by the following formula (1):

[0095] These will be explained below.

[0096] R 1 are the same or different, -CH(-R 11 )2, -(CH2) 0-4 -R 12 or a hydrogen atom (however, all R 3 (Except when R is a hydrogen atom.) 1 Preferably, one of R is a hydrogen atom. 1 is preferably -CH(-R 11 )2.

[0097] R 11 are the same or different and each represents an alkyl group having 2 or more carbon atoms. The alkyl group may be either linear or branched (preferably linear). The number of carbon atoms in the alkyl group is, for example, 2 to 8. The number of carbon atoms is preferably 2 to 5, more preferably 2 to 3, and particularly preferably 2. Specific examples of the alkyl group include an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group.

[0098] R 12 R represents a carbocyclic ring which may be substituted. The number of carbon atoms constituting the carbocyclic ring is not particularly limited, but is, for example, 3 to 12, and preferably 5 to 8.12 Examples of the substituent include aromatic groups such as a phenyl group, a naphthyl group, a biphenyl group, a pentalenyl group, an indenyl group, an anthranyl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perylenyl group, a fluorenyl group, and a phenanthryl group, cycloalkyl groups such as cyclohexyl, and aliphatic groups such as an adamantyl group. The substituent of the carbon ring is not particularly limited, and examples thereof include an alkyl group.

[0099] R 1 Inside "-(CH2) 0-4 The number of methylene groups in "-" is preferably 1 to 3, and more preferably 1 to 2.

[0100] R 2 R is a halogen atom, an alkyl group, or an alkoxy group. 2 is preferably a halogen atom.

[0101] R 2 Examples of the halogen atom represented by the formula (I) include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. Among these, a fluorine atom is preferred.

[0102] R 2 The alkyl group represented by the formula (I) includes both linear and branched alkyl groups (preferably linear). The number of carbon atoms in the alkyl group is, for example, 1 to 6. The number of carbon atoms is preferably 1 to 4, more preferably 1 to 2, and particularly preferably 1. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group.

[0103] R 2The alkoxy group represented by the formula (I) includes both linear and branched ones (preferably linear). The number of carbon atoms in the alkoxy group is, for example, 1 to 6. The number of carbon atoms is preferably 1 to 4, more preferably 1 to 2, and particularly preferably 1. Specific examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, and the like.

[0104] R 3 are the same or different and each represents an alkyl group, -(CH2) 0-4 -R 31 or a hydrogen atom (however, all R 3 (Except when R is a hydrogen atom.) 1 Preferably, both are alkyl groups.

[0105] R 3 The alkyl group represented by the formula (I) includes both linear and branched alkyl groups (preferably branched). The number of carbon atoms in the alkyl group is, for example, 1 to 8. The number of carbon atoms is preferably 2 to 5, more preferably 2 to 3, and particularly preferably 3. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group.

[0106] R 31 represents an optionally substituted carbocycle. For the carbocycle, R 12 This is similar to the carbocycle represented by the formula:

[0107] R 3 Inside "-(CH2) 0-4 The number of methylene groups in "-" is preferably 0 to 3, and more preferably 0 to 2.

[0108] R 4 is a halogen atom, an alkyl group, or an alkoxy group.

[0109] R 4 Examples of the halogen atom represented by the formula (I) include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. Among these, a chlorine atom is preferred.

[0110] R 4 For the alkyl group represented by R 2 This is the same as the alkyl group represented by the following formula:

[0111] R 4 For the alkoxy represented by R 2 This is the same as the alkoxy represented by the formula:

[0112] Salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and both acid salt and basic salt can be adopted.For example, examples of acid salt include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, paratoluenesulfonate, etc.; amino acid salts such as aspartate, glutamate, etc. In addition, examples of basic salt include alkali metal salts such as sodium salt, potassium salt, etc.; alkaline earth metal salts such as calcium salt, magnesium salt, etc.

[0113] The solvent is not particularly limited as long as it is pharma- ceutical acceptable, and examples thereof include water, ethanol, glycerol, acetic acid, and the like.

[0114] The above compound can be synthesized by various methods. As an example, the artificial ligand 1 of the present invention can be synthesized by combining the compound 1 used in the test example 3 described later and the compound represented by the general formula (3): (R 1 -)2NH[in the formula:R 1 is the same as above.]. In addition, as another example, the artificial ligand 2 of the present invention can be synthesized by reacting levocetirizine with a compound represented by the general formula (4): (R 3 -)2NH[in the formula:R 1is the same as above. The compound represented by the formula (3) can be synthesized by reacting a compound represented by the formula (4). The amount of the compound represented by the formula (3) and the compound represented by the formula (4) to be used is preferably 1 to 7 moles, more preferably 1 to 5 moles, per mole of compound 1 or levocetirizine, from the viewpoint of yield, ease of synthesis, and the like. This reaction is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, and examples thereof include N,N-dimethylformamide, dichloromethane, acetonitrile, tetrahydrofuran, acetone, toluene, and the like. The solvent may be used alone or in combination. This reaction is preferably carried out in the presence of a base. The base is not particularly limited, and for example, a wide variety of known bases can be used. Specific examples of the base include potassium carbonate, diisopropylethylamine (DIPEA), and the like. The base may be used alone or in combination of two or more. The amount of the base to be used is preferably 1 to 2 moles per mole of compound 1 or levocetirizine, for example. The reaction can be carried out under heating, at room temperature, or under cooling, and is usually preferably carried out at 70 to 100°C. The reaction time is not particularly limited, and can usually be 1 to 30 hours. The progress of the reaction can be monitored by a conventional method such as chromatography. After completion of the reaction, the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0115] The above compound can be suitably used in one embodiment of the induction method of the present invention described below.

[0116] 5. Chemogenetics In one aspect, the present invention relates to (i) a method for inducing a G protein signal specifically in cell 1 or cell 2, comprising a step of administering an artificial ligand of a non-mutated G protein-coupled receptor to a non-human animal comprising cell 1 expressing a mutant G protein-coupled receptor of the present invention and cell 2 expressing a non-mutated G protein-coupled receptor (also referred to as the "inducing method of the present invention" in this specification). This will be described below.

[0117] The cell 1 can be obtained by introducing the polynucleotide of the present invention. In the cell 1, the expression of a non-mutated G protein-coupled receptor is preferably suppressed. In one embodiment of the present invention, a mutation is introduced into the wild-type G protein-coupled receptor gene in the cell 1 so that the expression of the wild-type G protein-coupled receptor is deleted or reduced. "Deletion" means that the expression level of the G protein-coupled receptor is below the detection limit for a sample obtained from the cell 1. In addition, "reduction" means that the expression level of the G protein-coupled receptor is lower than the activity and / or expression level of the wild-type G protein-coupled receptor gene before the mutation is introduced (e.g., 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, 1 / 1000, 1 / 2000, 1 / 5000, 1 / 10000 or less).

[0118] The suppression of gene expression can be evaluated by detecting at least one of the mRNA transcribed from the target gene and the protein encoded by the target gene. Specifically, when evaluating based on mRNA, the amount of mRNA transcribed from the target gene can be measured by Northern hybridization, RT-PCR, quantitative PCR, microarray, etc. using primers or probes specific to the target gene. When evaluating based on protein, the amount of expression of the protein can be measured by immunological methods such as Western blotting and ELISA using an antibody specific to the protein encoded by the target gene.

[0119] When a mutation is introduced into a G protein-coupled receptor gene, the mutation is not particularly limited as long as it is a mutation that causes a loss or decrease in expression of the G protein-coupled receptor gene. Examples of the mutation include gene loss, a mutation in a protein coding region, a mutation in a splicing regulatory region, and a mutation in an expression control region (e.g., a promoter, an activator, an enhancer, etc.). Among these, gene loss is preferable. In addition, it is preferable that the cell 1 has the mutation in both of the paired chromosomes.

[0120] Introduction of a mutation into a G protein-coupled receptor gene can be achieved by, for example, gene knockout using artificial nucleases (TALEN, Cas protein), gene knockout using ES cells into which a targeting vector containing at least a part of a G protein-coupled receptor gene has been introduced by homologous recombination, gene knockdown using shRNA, antisense, ribozyme, etc. against the G protein-coupled receptor gene, radiation exposure, administration of chemical mutagens, transposon introduction, etc. Those skilled in the art can apply conventionally known methods to construct targeting vectors used for gene knockout, gene knockdown, and gene mutation introduction, design and synthesis of shRNA, antisense, ribozyme, etc., and introduction of DNA into ES cells, unfertilized eggs, fertilized eggs, embryos, primordial germ cells, etc.

[0121] Cell 2 may be, for example, a normal cell that has not been subjected to treatment such as gene mutation introduction, or may be a cell obtained by introducing an expression cassette for another non-mutated G protein-coupled receptor into a cell in which the expression of the above-mentioned non-mutated G protein-coupled receptor is suppressed.

[0122] In one embodiment of the present invention, a non-human animal comprising cell 1 and cell 2 can be obtained, for example, by producing an animal in which the wild-type G protein-coupled receptor gene in all cells has been replaced with the mutant G protein-coupled receptor gene of the present invention by recombinant techniques using an artificial nuclease system, a targeting vector, or the like, and then expressing a non-mutated G protein-coupled receptor only in specific cells in the animal (for example, by using a cell type-specific promoter, or the like).

[0123] In one embodiment of the present invention, a non-human animal comprising cell 1 and cell 2 can be obtained, for example, by expressing a mutant G protein-coupled receptor only in specific cells (e.g., using a cell type-specific promoter, etc.).

[0124] The method of administering the artificial ligand is not particularly limited, and various methods can be adopted. The route of administration is not particularly limited as long as the desired effect can be obtained, and includes enteral administration such as oral administration, tube feeding, and enema administration; parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration; and the like.

[0125] In one embodiment of the present invention, the artificial ligand binds to the non-mutated G protein-coupled receptor in cell 2, whereas the affinity between the artificial ligand and the mutant G protein-coupled receptor of the present invention is low in cell 1, so that the binding amount between the two is reduced. In another embodiment of the present invention, the artificial ligand binds to the mutant G protein-coupled receptor of the present invention in cell 1, whereas the affinity between the artificial ligand and the non-mutated G protein-coupled receptor is low in cell 2, so that the binding amount between the two is reduced. Therefore, by the step (i), it is possible to induce a G protein signal specifically in cell 1 (preferentially in cell 1) or specifically in cell 2 (preferentially in cell 2). That is, in one embodiment of the present invention, when the artificial ligand is an agonist, it is possible to induce a G protein signal specifically in cell 2, and when the artificial ligand is an antagonist, it is possible to induce a G protein signal specifically in cell 1. In addition, in another embodiment of the present invention, when the artificial ligand is an agonist, it is possible to induce a G protein signal specifically in cell 1, and when the artificial ligand is an antagonist, it is possible to induce a G protein signal specifically in cell 2.

[0126] Specifically means, for example, that the G protein signal intensity in cell 1 (or cell 2) is 2 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, 200 times or more, 500 times or more, or 1000 times or more than the G protein signal intensity in cell 2 (or cell 1).

[0127] The G protein signal strength can be measured, for example, by directly or indirectly measuring the amount of a second messenger (e.g., calcium ion, cyclic AMP, inositol triphosphate, etc.). The measurement method is not particularly limited, and a method according to or similar to a known method can be adopted.

[0128] In a preferred embodiment of the present invention, a metabotropic glutamate receptor is used as the G protein-coupled receptor, and the artificial ligand of the present invention is used as the artificial ligand, thereby making it possible to further increase the specificity of G protein signal induction.

[0129] Matters other than those described above in this section are as described in the preceding sections.

[0130] 6. Reagents and kits The mutant G protein-coupled receptor of the present invention, the polynucleotide of the present invention, the cell of the present invention, the non-human animal of the present invention, and the artificial ligand of the present invention can be suitably used in the induction method of the present invention. From this viewpoint, in one aspect, the present invention relates to a reagent or kit for use in the induction method of the present invention, which contains at least one selected from the group consisting of the mutant G protein-coupled receptor of the present invention, the polynucleotide of the present invention, the cell of the present invention, the non-human animal of the present invention, and the artificial ligand of the present invention.

[0131] The reagent may further contain other components as necessary, including, but not limited to, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, and the like.

[0132] The kit may contain, as necessary, other materials, reagents, instruments, etc. necessary for producing the cells or non-human animals of the present invention, carrying out the induction method of the present invention, such as virus preparation reagents, gene introduction reagents, cells, buffer solutions, etc.

[0133] 7. Screening Method In one aspect, the present invention relates to a method for screening an artificial ligand (sometimes referred to as the "screening method of the present invention" in this specification), which comprises the steps of: (a) allowing a test substance to act on a cell x expressing a non-mutated G protein-coupled receptor and a cell y expressing a mutant G protein-coupled receptor including a substitution of an amino acid residue in a region around a conserved cysteine ​​residue in extracellular loop 2; (b) detecting a G protein signal after the step (a); and (c) selecting a test substance for which the intensity of the G protein signal in the cell x is different from that in the cell y. This will be described below.

[0134] Cell x is similar to cell 1, except that the amino acid residue after substitution in the mutant G protein-coupled receptor is not limited. Cell y is similar to cell 2.

[0135] The type of test substance is not particularly limited as long as it can be a candidate for a therapeutic drug, and examples thereof include proteins, peptides, non-peptide compounds (nucleotides, amines, carbohydrates, lipids, etc.), organic low molecular weight compounds, inorganic low molecular weight compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc.

[0136] Examples of G protein signals include second messengers (e.g., calcium ion, cyclic AMP, inositol triphosphate, etc.) The measurement method is not particularly limited, and a method according to or similar to a known method can be used.

[0137] The difference in intensity serving as the selection criterion in step (c) is, for example, 2-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 500-fold or more, or 1000-fold or more.

[0138] The selected test substance can be suitably used as an artificial ligand, particularly as an artificial ligand for use in the inducing method of the present invention.

[0139] Matters other than those described above in this section are as described in the preceding sections. EXAMPLES

[0140] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0141] Test Example 1. Creation of rat-derived mGluR1 mutants It is known that the region surrounding the conserved cysteine ​​residue in extracellular loop 2 of a G protein-coupled receptor (the cysteine ​​residue that forms an SS bond with the conserved cysteine ​​residue in transmembrane domain 3) is conserved. In addition, endogenous ligands of G protein-coupled receptors recognize the ligand-binding domain exposed to the outside of the cell (in the case of class C) or a relatively deep part of the transmembrane domain, whereas artificial ligands recognize a position relatively close to extracellular loop 2 (a relatively shallow position in the transmembrane domain). From this, it was thought that the affinity of the artificial ligand could be reduced (and as a result, the G protein signal strength could be changed) by replacing the amino acid residues in the region surrounding the conserved cysteine ​​residue in extracellular loop 2 with bulky amino acid residues. To put this hypothesis into practice, we constructed an expression plasmid for an mGluR1 mutant in which the conserved cysteine ​​residue in extracellular loop 2 (amino acid residue at position 746 from the N-terminus of SEQ ID NO:1) and the surrounding region (amino acid residue at position 748 from the N-terminus of SEQ ID NO:1: threonine residue) were replaced with tryptophan residues in the amino acid sequence of rat-derived mGluR1 (amino acid sequence: SEQ ID NO:1, coding sequence: SEQ ID NO:2).

[0142] The drug resistance gene was removed from pcDNA3.1 (ThermoFisher) using the restriction enzyme PvuII, and a 3.3 kbp expression vector (pCDM) was prepared using the DNA Ligation Kit Ver.2.1 (Takara Bio). A rat-derived mGluR1 cDNA (Reference 3: S. Kiyonaka, et al. Nat. Chem. 8, 958-967 (2016)) was subcloned into the resulting pCDM expression vector to create a plasmid (pCDM-mGluR1). This was used as a PCR template to introduce mutations using the Q5 site-directed mutagenesis kit (New England Biolabs). Mutations were introduced according to the attached manual, and an expression plasmid (pCDM-mGluR1-T748W) of mGluR1 with the desired mutation was obtained.

[0143] Test Example 2. Detection of G protein signal 1 The mGluR1 mutant and wild-type mGluR1 obtained in Test Example 1 were treated with an artificial ligand (FITM) and an endogenous ligand (glutamic acid), and the G protein signal intensity was measured. Specifically, the measurement was performed as follows.

[0144] [ka]

[0145] Test Example 2-1. Cultivation of HEK293 cells HEK293 cells (ATCC) were maintained in a 37°C incubator under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0146] Test Example 2-2. Transfection Transient transfection of mGluR1 expression plasmid (pCDM-mGluR1 or pCDM-mGluR1-T748W) into HEK293 cells was performed using DMEM-GlutaMAX medium supplemented with 10% dialyzed-FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin, Viafect Transfecton Reagent (Promega) as a transfection reagent according to the Promega manual. pEGFP-F (Clontech) and pDsRed monomer-N1 (Clontech) were used as expression markers, and transfected simultaneously with the mGluR1 expression plasmid. Ca 2+ Before imaging, HEK293 cells after 24–48 h of incubation were seeded onto coverslips (Matsunami) coated with poly-L-lysine (Sigma-Aldrich).

[0147] Test Example 2-3.Ca 2+ Fluorescence imaging HEK293 cells seeded on glass were stained with 5 μM Fura 2-AM (Dojindo) in the medium for 20-30 min. Fura 2 fluorescence in HEK293 cells was measured in HBS (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 11.5 mM glucose, 20 mM HEPES at pH 7.4). FITM was dissolved in HBS from a 1000× DMSO stock and added to HEK293 cells 3 min before the addition of glutamate. Fluorescence images were captured every 5 seconds using a fluorescence microscope (IX71, Olympus) equipped with a CMOS camera (ORCA-flash4.0, Hamamatsu Photonics) attached to a xenon lamp, and the fluorescence intensity ratio (340 nm / 380 nm) of each ROI on the image was analyzed using the imaging system cellSens (Olympus). To streamline the experiment, two types of HEK293 cells expressing two different types of mGluR1 (mutant or wild type) were seeded simultaneously on a cover glass. The two types of mGluR1 expressed in the cells can be distinguished from the fluorescence of the co-expressed fluorescent protein.

[0148] result The results are shown in Figure 1. The hypothesis that the affinity of the artificial ligand can be reduced by substituting an amino acid residue in the region surrounding the conserved cysteine ​​residue in extracellular loop 2 with a bulky amino acid residue was verified.

[0149] In addition, similar results were obtained when similar tests were performed using G protein-coupled receptors other than mGluR1 and artificial ligands other than FITM, indicating that the above hypothesis can also be applied to combinations of other G protein-coupled receptors and artificial ligands.

[0150] Test Example 3. Synthesis of FITM derivatives We thought that by modifying a part of the structure of the artificial ligand FITM to make it bulky, we could further increase the difference in affinity between the wild-type G protein-coupled receptor and the mutant G protein-coupled receptor in which the amino acid residues in the region surrounding the conserved cysteine ​​residue in extracellular loop 2 were replaced with bulky amino acid residues. Therefore, we first prepared various FITM derivatives.

[0151] Test Example 3-1. Reagents and measuring equipment used to identify compounds The reagents used were purchased from Tokyo Chemical Industry Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Sigma-Aldrich Co., Ltd., and Kanto Chemical Co., Ltd. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Biospin Avance III HD (300 MHz for 1 H NMR and 500 MHz for 1 H NMR (H NMR) and mass spectra (MS) were obtained by electrospray ionization mass spectrometry (ESI-MS) using a Bruker Daltonics compact at the Nagoya University Chemical Measurement Equipment Laboratory.

[0152] Test Example 3-2. Synthesis of Compound 2

[0153] [ka]

[0154] Compound 1 was synthesized with reference to the literature 4 (A. Satoh, et al. Bioorg. Med. Chem. Lett. 19, 5464-5468 (2009).). Compound 1 (50 mg, 0.143 mml, 1 eq.) and potassium carbonate (27 mg, 0.195 mmol, 1.36 eq.) were dissolved in dry N,N-dimethylformamide (DMF) (5 ml), 3-aminopentane (40 μl, 30 mg, 0.344 mmol, 2.41 eq.) was added, and the mixture was heated and stirred at 90°C for 22.5 hours under a nitrogen atmosphere. The reaction solution was evaporated to remove the DMF, and ethyl acetate and saturated aqueous ammonium chloride were added to the residue and the mixture was separated twice. A saturated aqueous sodium chloride solution was added to the obtained organic phase and the mixture was further separated. The organic phase was dried over magnesium sulfate, filtered to remove salts, and the filtrate was concentrated by evaporation. The residue was purified by silica gel column chromatography (chloroform, chloroform / ethyl acetate = 50 / 1, 25 / 1) and preparative thin layer chromatography (chloroform / ethyl acetate = 1 / 1) to give the desired product as a white solid in 16% yield (9 mg, 0.023 mmol).

[0155] 1 H NMR (300 MHz, CD3OD): δ 8.35 (d, J = 1.1 Hz, 1H), 7.88 (s, 1H), 7.71 (dd, J = 5.3, 8.9 Hz, 2H), 7.28 (t, J = 8.9 Hz, 2H), 7.23 (brs, 1H), 3.74 (s, 3H), 1.73-1.44 (m, 4H), 0.95 (t, J= 7.4 Hz, 6H); HRMS (ESI + ): calcd for [M + H] + (C 20 H 23 FN5OS) 400.1602, found 400.1607.

[0156] Test Example 3-3. Synthesis of Compound 3

[0157] [ka]

[0158] The synthesis was carried out in the same manner as in Test Example 3-2, except that an appropriate compound was used instead of 3-aminopentane. The target product was obtained as a white solid in a yield of 32% (14 mg, 33.4 mmol).

[0159] 1 H NMR (500 MHz, CDCl3): δ 8.63 (s, 1H), 7.93 (s, 1H), 7.61 (dd, J = 5.2, 8.8 Hz, 2H), 7.38-7.34 (m, 4H), 7.32-7.28 (m,1H), 7.19 (t, J = 8.8 Hz, 2H), 7.13 (s, 1H), 5.35 (brs, 1H, NH), 4.64 (s, 2H), 3.72 (s, 3H); HRMS (ESI + ): calcd for [M + H] + (C 22 H 19 FN5OS) 420.1289, found 420.1286.

[0160] Test Example 3-4. Synthesis of Compound 4

[0161] [ka]

[0162] The synthesis was carried out in the same manner as in Test Example 3-2, except that an appropriate compound was used instead of 3-aminopentane. The target product was obtained as a white solid in 88% yield (52 mg, 0.122 mmol).

[0163] 1H NMR (300 MHz, CDCl3): δ 8.56 (brs, 1H), 7.92 (s, 1H), 7.61 (dd, J = 5.2, 8.9 Hz, 2H), 7.20 (t, J = 8.9 Hz, 2H), 7.07 (d, J = 1.1 Hz, 1H), HRMS (ESI + ): calcd for [M + H] + (C 22 H 25 FN5OS) 426.1758, found 426.1750.

[0164] Test Example 3-5. Synthesis of Compound 5

[0165] [ka]

[0166] The synthesis was carried out in the same manner as in Test Example 3-2, except that an appropriate compound was used instead of 3-aminopentane. The target product was obtained as a white solid in a yield of 94% (56 mg, 0.127 mmol).

[0167] 1H NMR (300 MHz, CDCl3):δ 8.57 (brs, 1H), 7.93 (s, 1H), 7.61 (dd, J = 5.2, 8.8 Hz, 2H), 7.20 (t, J = 8.8 Hz, 2H), 7.07 (d, J = 1.0 Hz, 1H), 5.02 (brs, 1H, NH), 3.75 (s, 3H), 3.41 (brs, 2H), 1.78-1.64 (m, 6H), 1.55 (dt, J = 7.0, 7.5 Hz, 2H), 1.44-1.36 (m, 1H), 1.29-1.12 (m, 2H), 1.02-0.91 (m, 2H); HRMS (ESI + ): calcd for [M + H] + (C 23 H 27 FN5OS) 440.1915, found 440.1915.

[0168] Test Example 4. Detection of G protein signal 2 The mGluR1 mutant and wild-type mGluR1 obtained in Test Example 1 were treated with an artificial ligand (FITM derivative: Test Example 3) and an endogenous ligand (glutamic acid), and the G protein signal intensity was measured.

[0169] The results are shown in Figures 2 and 3. By modifying a part of the structure of the artificial ligand FITM to make it bulky, it was possible to further increase the difference in affinity between the wild-type G protein-coupled receptor and the mutant G protein-coupled receptor in which amino acid residues in the region surrounding the conserved cysteine ​​residue in extracellular loop 2 were replaced with bulky amino acid residues.

[0170] Test Example 5. Detection of G Protein Signals 3 The endogenous ligand (glutamic acid) alone was allowed to act on the mGluR1 mutant and wild-type mGluR1 obtained in Test Example 1, and the G protein signal intensity was measured.

[0171] The results are shown in Figure 4. Figure 4 shows that the introduction of mutations into the extracellular loop 2 site does not impair the responsiveness to the endogenous ligand (glutamic acid).

[0172] Test Example 6. Preparation of human-derived H1R mutant 1 An expression plasmid for an hH1R mutant was prepared in which the conserved cysteine ​​residue in extracellular loop 2 (amino acid residue 180 from the N-terminus of SEQ ID NO: 3) in the surrounding region (amino acid residue 178 from the N-terminus of SEQ ID NO: 3: aspartic acid residue, and amino acid residue 179 from the N-terminus of SEQ ID NO: 3: lysine residue) was replaced with alanine residues in human H1R (amino acid sequence: SEQ ID NO: 3, coding sequence: SEQ ID NO: 4). Specifically, it was prepared as follows. The drug resistance gene was removed from pcDNA3.1 (ThermoFisher) using the restriction enzyme PvuII, and a 3.3 kbp expression vector was prepared using the DNA Ligation Kit Ver.2.1 (Takara Bio) (pCDM vector). The promoter of this gene expression vector is the CMV promoter derived from cytomegalovirus, which strongly expresses genes subcloned downstream of the promoter. However, in this experiment, to suppress excessive expression of H1R, the CMV promoter of the pCDM vector was replaced with the CMVd3 promoter of the pFN24A HaloTag CMVd3 Flexi Vector (Promega), and the H1R cDNA was subcloned to prepare an H1R expression vector, pCDM-d3-H1R. The H1R mutant expression vector was created using overlap extension PCR (Figure). First, PCR was performed using the mutated primers to obtain two PCR products with complementary mutation sites. PCR was then performed using these two PCR products as templates to obtain a PCR product with a full-length cDNA, which was then subjected to restriction enzyme treatment with NheI and NotI to obtain the mutated cDNA. This was then subcloned into the pCDM-d3 vector to create an expression vector for the H1R mutant.

[0173] Test Example 7. Synthesis of levocetirizine derivatives Part of the structure of levocetirizine, a third-generation antihistamine that acts as an antagonist of the histamine H1 receptor (H1R), has been modified to make it bulkier.

[0174] Test Example 7-1. Reagents and measuring equipment used to identify compounds The reagents used were purchased from Tokyo Chemical Industry Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Sigma-Aldrich Co., Ltd., and Kanto Chemical Co., Ltd. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Biospin Advance III HD (300 MHz for 1 H NMR) and Bruker Biospin Advance III HD (500 MHz for 1 1 H NMR. Abbreviations: DMF; N,N-dimethylformamide, HBTU; 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hemifluorophosphate, DIPEA; N,N-diisopropylethylamine.

[0175] Test Example 7-2. Synthesis of Lvc-Diisopro

[0176] [ka]

[0177] Levocetirizine·2HCl 45 mg (0.10 mmol, 1.0 eq) and HBTU 50 mg (0.13 mmol, 1.4 eq) were placed in a 25 mL two-necked flask, and degassed and purged with nitrogen. After purging, dry DMF 3 mL and DIPEA 60 μL (0.35 mmol, 3.6 eq) were added by syringe and stirred at room temperature for 56 minutes. After stirring, diisopropylamine 60 μL (0.43 mmol, 4.4 eq) was added by syringe and stirred at room temperature for 3 hours. After stopping the reaction and distilling off the solvent, the product was dissolved in chloroform and separated four times with 5% aqueous sodium carbonate solution. Only the chloroform phase was collected and dehydrated with anhydrous sodium sulfate, and the filtrate was collected by suction filtration. The solvent was distilled off from the collected filtrate. The crude product was purified by silica gel column chromatography (chloroform → chloroform:methanol = 99:1 → chloroform:methanol = 8:1), and the purified product was dissolved in CDCl3, 1 H NMR was measured (yield 46 mg, 100%, colorless transparent oil).

[0178] 1 H NMR (500 MHz CDCl3) δ: 7.36-7.34 (m, 4H), 7.27-7.16 (m, 5H), 4.20 (s, 1H), 4.07 (s, 2H), 3.99 (m, 1H), 3.63 (t, J = 5.8 Hz, 2H), 3.39 (m, 1H), 2.62 (t, J = 5.8 Hz, 2H), 2.52 (br, 4H), 2.41 (br, 4H), 1.90 (d, J = 6.5 Hz, 6H), 1.16 (d, J = 6.5 Hz, 6H).

[0179] Test Example 7-3. Synthesis of Lvc-Adam

[0180] [ka]

[0181] In a 25 mL two-necked flask, 45 mg (0.10 mmol, 1.0 eq) of Levocetirizine·2HCl and 50 mg (0.13 mmol, 1.4 eq) of HBTU were placed, and the mixture was degassed and replaced with nitrogen. After replacement, 2 mL of DMF and 60 μL (0.35 mmol, 3.6 eq) of DIPEA were added by syringe, and the mixture was stirred at room temperature for 40 minutes. During this time, the reaction solution changed from transparent to light brown. After stirring, a solution of 26 mg (0.17 mmol, 1.8 eq) of 1-adamantanamine dissolved in 1 mL of DMF was added by syringe, and the mixture was stirred at room temperature for 3 hours and 45 minutes. After the reaction was stopped and the solvent was distilled off, the mixture was dissolved in chloroform, and the mixture was separated three times with 5% aqueous sodium carbonate solution. Only the chloroform phase was collected and dehydrated with anhydrous sodium sulfate, and the filtrate was collected by suction filtration, and the solvent was distilled off from the filtrate. The crude product was purified by silica gel column chromatography (chloroform → chloroform:methanol = 100:1 → chloroform:methanol = 50:1 → chloroform:methanol = 25:1), and the purified product was dissolved in CDCl3, 1 H NMR was measured (yield 51 mg, 100%, brown oil).

[0182] 1 H NMR (300 MHz CDCl3) δ: 7.38-7.34 (m, 4H), 7.29-7.19 (m, 5H), 6.29 (br, 1H), 4.20 (s, 1H), 3.82 (s, 1H), 3.59 (t, J = 5.7 Hz, 2H), 2.60 (t, J = 5.7 Hz, 2H), 2.53 (br, 4H), 2.42 (br, 4H), 2.07 (br, 3H), 2.00 (m, 6H), 1.68 (t, J = 2.7 Hz, 6H).

[0183] Test Example 7-4. Synthesis of Lvc-1-MeNaph

[0184] [ka]

[0185] 50 mg (0.11 mmol, 1.0 eq) of Levocetirizine·2HCl and 82 mg (0.21 mmol, 1.9 eq) of HBTU were placed in a 25 mL two-necked flask, and the flask was degassed and purged with nitrogen. After the purging, 4 mL of DMF and 60 μL (0.35 mmol, 3.3 eq) of DIPEA were added by syringe and stirred at room temperature for 50 minutes. During this time, the reaction solution changed from transparent to light yellow. After stirring, 20 μL (0.14 mmol, 1.3 eq) of 1-Naphthylmethylamine was added by syringe and stirred at room temperature for 22 hours. After stopping the reaction and evaporating the solvent, the product was dissolved in chloroform and separated once with an aqueous solution of sodium hydroxide (1 mM). Only the chloroform phase was collected and dehydrated with anhydrous sodium sulfate, and the filtrate was collected by suction filtration. The solvent was evaporated from the collected filtrate. The crude product was purified by silica gel column chromatography (chloroform → chloroform:methanol = 19:1), and the purified product was dissolved in CDCl3, 1 H NMR was measured (yield 11 mg, 19%, yellow-brown oil).

[0186] 1 H NMR (500 MHz CDCl3) δ: 8.01 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.79 (br, 1H, l), 8.01 (d, J = 8.5 Hz, 1H), 7.59-7.35 (m, 4H), 7.23-7.13 (m, 9H), 4.97 (d, J = 5.5 Hz, 2H), 4.07 (s, 2H), 3.65 (s, 1H), 3.56 (t, J = 5.0 Hz, 2H), 2.46 (t, J = 5.0 Hz, 2H), 2.31 (br, 4H), 1.99 (br, 4H).

[0187] Test Example 7-5. Synthesis of Lvc-Dipro

[0188] [ka]

[0189] In a 25 mL two-necked flask, 45 mg (0.10 mmol, 1.0 eq) of Levocetirizine·2HCl and 50 mg (0.13 mmol, 1.4 eq) of HBTU were placed, and the mixture was degassed and replaced with nitrogen. After replacement, 4 mL of dry DMF and 60 μL (0.35 mmol, 3.6 eq) of DIPEA were added by syringe, and the mixture was stirred at room temperature for 40 minutes. During this time, the mixture changed from transparent to light brown. After stirring, 50 μL (0.37 mmol, 3.8 eq) of dipropylamine was added by syringe, and the mixture was stirred at room temperature for 4 hours and 35 minutes. After stopping the reaction and evaporating the solvent, the mixture was dissolved in chloroform, and the mixture was separated twice with an aqueous solution of sodium hydroxide (pH 11). Only the chloroform phase was collected and dehydrated with anhydrous sodium sulfate, and the filtrate was collected by suction filtration. The solvent was removed from the collected filtrate. The crude product was purified by silica gel column chromatography (chloroform → chloroform:methanol = 49:1), and the purified product was dissolved in CDCl3, 1 H NMR was measured (yield 40 mg, 87% yield, yellow oil).

[0190] 1 H NMR (500 MHz CDCl3) δ: 7.37-7.34 (m, 4H), 7.29-7.17 (m, 5H), 4.26 (s, 1H), 4.20 (s, 2H), 3.72 (t, J = 5.5 Hz, 2H), 3.19 (dt, J = 7.5, 8.8 Hz, 4H), 2.85 (t, J = 5.5 Hz, 2H), 2.79 (br, 4H), 2.52 (br, 4H), 1.59-1.52 (m, 4H), 0.89 (dd, J = 7.5, 8.5 Hz, 6H).

[0191] Test Example 8. Detection of G protein signal 4 The artificial ligand (levocetirizine derivative: Test Example 7) was allowed to act on the hH1R mutant and wild-type hH1R obtained in Test Example 6, and the G protein signal intensity was measured. Specifically, it was carried out as follows.

[0192] <Culture of HEK293 cells> HEK293 cells (ATCC) were maintained in an incubator at 37 °C under 5% CO2 conditions using Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 100 unit / mL penicillin, and 100 μg / ml streptomycin as the culture medium.

[0193] <Transfection> Transient transfection of plasmids (pCDM-d3-H1R, pCDM-d3-H1R-D178A / K179A, pCDM-d3-H1R-D178G / K179G) into HEK293 cells was performed according to the Promega manual using Viafect Transfecton Reagent (Promega) as the transfection reagent with DMEM-GlutaMAX medium supplemented with 10% Dialyzed-FBS, 100 unit / mL penicillin, and 100 μg / ml streptomycin. pCDM-d1-EGFP-F, pDsRed monomer-N1 (Clontech), piRFP670-N1 (Addgene) were transfected simultaneously with pCDM-mGlu1, and these fluorescent proteins were used as expression markers. Also, Ca 2+ Before performing imaging, HEK293 cells after 24 - 48 hours of incubation were seeded on cover glasses (Matsunami) coated with poly-L-lysine (Sigma-Aldrich).

[0194] <Ca 2+ <Fluorescence imaging> HEK293 cells seeded on glass were stained with 5 μM Fura 2-AM (Dojindo) in the medium for 20-30 min. Fura 2 fluorescence in HEK293 cells was measured in HBS (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 11.5 mM glucose, 20 mM HEPES at pH 7.4). Levocetirizine or levocetirizine derivatives were dissolved in HBS from 1000× DMSO stock and added to HEK293 cells 4 min before the addition of histamine. Fluorescence images were captured every 5 seconds using a fluorescence microscope (IX71, Olympus) equipped with a CMOS camera (ORCA-flash4.0, Hamamatsu Photonics) attached to a xenon lamp, and the fluorescence intensity ratio (340 nm / 380 nm) of each ROI on the image was analyzed using the imaging system cellSens (Olympus). To streamline the experiment, three types of HEK293 cells expressing three different types of H1R (mutant or wild type) were seeded simultaneously on a cover glass. The three types of H1R expressed in the cells were identified from the fluorescence of the co-expressed fluorescent protein.

[0195] <Result> The results are shown in Figures 6 to 9. By substituting amino acid residues in the region surrounding the conserved cysteine ​​residue in extracellular loop 2 with smaller amino acid residues, the difference in affinity between the obtained mutant G protein-coupled receptor and the artificial ligand and the affinity between the wild-type G protein-coupled receptor and the artificial ligand could be increased.

[0196] Test Example 9. Preparation of human-derived H1R mutant 2 An expression plasmid for an hH1R mutant was prepared in which the conserved cysteine ​​residue in extracellular loop 2 (amino acid residue 180 from the N-terminus of SEQ ID NO: 3) in the surrounding region (amino acid residue 178 from the N-terminus of SEQ ID NO: 3: aspartic acid residue, and amino acid residue 179 from the N-terminus of SEQ ID NO: 3: lysine residue) in human-derived H1R (amino acid sequence: SEQ ID NO: 3; coding sequence: SEQ ID NO: 4) was replaced with glycine residue and glycine residue, respectively.

[0197] Test Example 10. Detection of G Protein Signals 5 The hH1R mutant obtained in Test Example 9 and the wild-type hH1R were treated with an artificial ligand (levocetirizine derivative: Test Example 7) and an endogenous ligand (histamine), and the G protein signal intensity was measured. Specifically, the same procedure as in Test Example 8 was used. The results are shown in Figure 10.

Claims

1. A mutant G protein-coupled receptor comprising a substitution with at least one amino acid residue selected from the group consisting of bulky amino acid residues, small amino acid residues, and amino acid residues that change the presence or absence or positive / negative charge in a region surrounding a conserved cysteine ​​residue in extracellular loop 2, the conserved cysteine ​​residue in the extracellular loop 2 is a cysteine ​​residue that forms an S-S bond with a conserved cysteine ​​residue in transmembrane domain 3 (the third domain from the N-terminus of the seven transmembrane domains in the cell membrane); the region surrounding the conserved cysteine ​​residue is a region consisting of 20 amino acid residues consisting of 10 amino acid residues on the N-terminal side and 10 amino acid residues on the C-terminal side of the conserved cysteine ​​residue, the bulky amino acid residue is an aromatic amino acid residue, and the small amino acid residue and the amino acid residue that changes the presence or absence or positive / negative charge are alanine residues and / or glycine residues; The G protein-coupled receptor is a metabotropic glutamate receptor or a histamine receptor. Mutant G protein-coupled receptors.

2. A polynucleotide comprising a coding sequence for the mutant G protein-coupled receptor of claim 1.

3. A cell comprising the polynucleotide of claim 2.

4. A non-human animal comprising the cell of claim 3.

5. General formula (1): 【Chemistry 1】 [In the formula: R 1 are the same or different and are -CH(-R 11 ) 2 (R 11 are the same or different and each represents an alkyl group having 2 to 8 carbon atoms.), -(CH 2 ) 0-4 -R 12 (R 12 represents a carbon ring having 5 to 8 carbon atoms which may be substituted.) or represents a hydrogen atom (provided that all R 1 (Except when R is a hydrogen atom.) 2 represents a halogen atom. A compound represented by the formula:

6. General formula (2): 【Chemistry 2】 [In the formula: R 3 are the same or different and are -(CH 2 ) 0-4 -R 31 (R 31 represents an optionally substituted carbocycle, the carbocycle being a naphthyl group or an adamantyl group; or represents a hydrogen atom (provided that all R 3 (Except when R is a hydrogen atom.) 4 represents a halogen atom. A compound represented by the formula:

7. (i) administering an artificial ligand of the non-mutated G protein-coupled receptor to a non-human animal comprising a cell 1 expressing the mutant G protein-coupled receptor of claim 1 and a cell 2 expressing a non-mutated G protein-coupled receptor. A method for inducing a G protein signal specifically in the cell 1 or the cell 2, comprising: The artificial ligand is General formula (1): 【Chemistry 3】 [In the formula, R 1 is the same or different and represents -CH(-R 11 ) 2 (R 11 is the same or different and represents an alkyl group having 2 to 8 carbon atoms), -(CH 2 ) 0-4 -R 12 (R 12 represents a carbocyclic ring having 5 to 8 carbon atoms which may be substituted), or a hydrogen atom (excluding the case where all R 1 s are hydrogen atoms). R 2 represents a halogen atom.] or a salt thereof, or a solvate thereof, General formula (2): 【Chemistry 4】 [In the formula, R 3 is the same or different and represents -(CH 2 ) 0-4 -R 31 (R 31 represents an optionally substituted carbocyclic ring, which is a naphthyl group or an adamantyl group), or a hydrogen atom (excluding the case where all R 3 are hydrogen atoms); and R 4 represents a halogen atom.] A compound represented by the formula: method.

8. A composition for use in the method of claim 7, comprising an artificial ligand, The artificial ligand is General formula (1): 【Chemistry 5】 [In the formula, R 1 is the same or different and represents -CH(-R 11 ) 2 (R 11 is the same or different and represents an alkyl group having 2 or more carbon atoms), -(CH 2 ) 0-4 -R 12 (R 12 represents a carbocycle which may be substituted), or a hydrogen atom (excluding the case where all of R 1 are hydrogen atoms). R 2 represents a halogen atom, an alkyl group, or an alkoxy group. or a salt thereof, or a solvate thereof, General formula (2): 【Chemistry 6】 [In the formula, R 3 is the same or different and represents an alkyl group, —(CH 2 ) 0-4 —R 31 (R 31 represents an optionally substituted carbocycle), or a hydrogen atom (excluding the case where all of R 3 are hydrogen atoms). R 4 represents a halogen atom, an alkyl group, or an alkoxy group. A compound represented by the formula: composition.

9. (a) applying a test substance to a cell x expressing a mutant G protein-coupled receptor containing a substitution of an amino acid residue in the region surrounding the conserved cysteine ​​residue in extracellular loop 2, and a cell y expressing a non-mutated G protein-coupled receptor; (b) detecting a G protein signal after step (a); (c) selecting a test substance for which the intensity of the G protein signal in the cell x is different from the intensity of the G protein signal in the cell y; A method for screening an artificial ligand, comprising: the conserved cysteine ​​residue in the extracellular loop 2 is a cysteine ​​residue that forms an S-S bond with a conserved cysteine ​​residue in transmembrane domain 3 (the third domain from the N-terminus of the seven transmembrane domains in the cell membrane); the region surrounding the conserved cysteine ​​residue is a region consisting of 20 amino acid residues consisting of 10 amino acid residues on the N-terminal side and 10 amino acid residues on the C-terminal side of the conserved cysteine ​​residue, The substitution of the amino acid residue is a substitution with at least one type of amino acid residue selected from the group consisting of bulky amino acid residues, small amino acid residues, and amino acid residues that change the presence or absence or positive / negative charge, the bulky amino acid residue is an aromatic amino acid residue, and the small amino acid residue and the amino acid residue that changes the presence or absence or positive / negative charge are alanine residues and / or glycine residues; the G protein-coupled receptor is a metabotropic glutamate receptor or a histamine receptor; The artificial ligand is General formula (1): 【Chemistry 7】 [In the formula, R 1 is the same or different and represents -CH(-R 11 ) 2 (R 11 is the same or different and represents an alkyl group having 2 to 8 carbon atoms), -(CH 2 ) 0-4 -R 12 (R 12 represents a carbocyclic ring having 5 to 8 carbon atoms which may be substituted), or a hydrogen atom (excluding the case where all R 1 s are hydrogen atoms). R 2 represents a halogen atom.] or a salt thereof, or a solvate thereof, General formula (2): 【Chemistry 8】 [In the formula, R 3 is the same or different and represents -(CH 2 ) 0-4 -R 31 (R 31 represents an optionally substituted carbocyclic ring, which is a naphthyl group or an adamantyl group), or a hydrogen atom (excluding the case where all R 3 are hydrogen atoms); and R 4 represents a halogen atom.] A compound represented by the formula: Screening methods.

10. The screening method according to claim 9, wherein the artificial ligand is an artificial ligand for use in the method according to claim 7.

Images