WO2002088182A1 - Nouvelle proteine des recepteurs couples aux proteines-g et adn associe - Google Patents

Nouvelle proteine des recepteurs couples aux proteines-g et adn associe Download PDF

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Publication number
WO2002088182A1
WO2002088182A1 PCT/JP2002/004215 JP0204215W WO02088182A1 WO 2002088182 A1 WO2002088182 A1 WO 2002088182A1 JP 0204215 W JP0204215 W JP 0204215W WO 02088182 A1 WO02088182 A1 WO 02088182A1
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Prior art keywords
protein
receptor protein
salt
present
coupled receptor
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PCT/JP2002/004215
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English (en)
Japanese (ja)
Inventor
Takeo Moriya
Takashi Ito
Yasushi Shintani
Nobuyuki Miyajima
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Takeda Chemical Industries, Ltd.
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Publication of WO2002088182A1 publication Critical patent/WO2002088182A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4719—G-proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein derived from mouse brain or a salt thereof, and DNA encoding the same.
  • G protein conjugated guanine nucleic acid-binding protein
  • G protein-coupled receptor proteins are present on the surface of each functional cell in living cells and organs, and are physiologically targeted as molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. It plays an important role.
  • the receptor transmits a signal into the cell via binding to a physiologically active substance, and this signal causes various reactions such as suppression of activation and activation of the cell.
  • physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or bioactive substances.
  • physiologically active substances are present at various sites in the body, and regulate their physiological functions through the corresponding receptor proteins.
  • receptor proteins There are many unknown hormones, neurotransmitters and other physiologically active substances in the living body, and the structure of these receptor proteins has not yet been reported. Furthermore, it is often unknown whether subtypes exist in known receptor proteins.
  • Clarifying the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins is a very important tool for the development of pharmaceuticals including agonists and antagonists against receptor proteins.
  • the functions of receptor protein genes expressed in vivo were elucidated, and these were clarified. It was necessary to express it in an appropriate expression system.
  • the G protein-coupled receptor is useful for searching for a new ligand (a physiologically active substance) using its signal transduction action as an index, and for searching for an agonist or an agonist for the receptor.
  • a physiological ligand a physiologically active substance
  • an agonist or an antagonist to the receptor is analyzed. It is also possible to make evening gonists.
  • These ligands, agonists, and antagonists for the receptor can be expected to be used as preventive / therapeutic or diagnostic agents for diseases associated with dysfunction of G protein-coupled receptors.
  • a decrease or enhancement of the function of the receptor in the living body based on a gene mutation of a G protein-coupled receptor often causes some disease.
  • the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation in the gene
  • the gene of the receptor is a prophylactic / therapeutic agent for a disease associated with dysfunction of the receptor. And can be applied to diagnostics.
  • the present invention provides a novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide encoding the G protein-coupled receptor protein or a partial peptide thereof (DNA, RNA, and the like) (D, NA, RNA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, the G protein A method for producing a conjugated receptor protein or a salt thereof, an antibody against the G protein-coupled receptor protein or a partial peptide or a salt thereof, a compound that changes the expression level of the G protein-coupled receptor protein, A method for determining a ligand for the G protein-coupled receptor, comprising: changing the binding between the ligand and the G protein-coupled receptor protein Method for screening a compound (antagonist, agonist) or a salt thereof, the binding between a lig
  • the present inventors have conducted extensive research and have succeeded in isolating cDNA encoding a novel G protein-combined receptor Yuichi protein derived from mouse brain and analyzing its entire nucleotide sequence. . When this nucleotide sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were identified on the hydrophobicity plot shown in FIG. 1, and the protein encoded by these cDNAs was transmembrane-seven times. Type G protein-coupled receptor. The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention. That is, the present invention
  • a G protein-coupled receptor protein or a salt thereof comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1;
  • G protein-coupled receptor protein or a salt thereof, which comprises the amino acid sequence represented by SEQ ID NO: 1;
  • polynucleotide according to (4) which is DNA
  • polynucleotide according to the above (5) having the nucleotide sequence represented by SEQ ID NO: 2;
  • the antibody according to (12) which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to (1);
  • the G protein-coupled receptor protein described in (1) above which can be obtained by using the G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof.
  • a ligand for the salt is a ligand for the salt.
  • the G protein-coupled receptor protein according to (1) wherein the G protein-coupled receptor protein according to (1) or the partial peptide according to (3) or a salt thereof is used.
  • a method for determining a ligand for a salt thereof wherein (18) the G protein-coupled receptor protein described in the above (1) or the partial peptide described in the above (3) or a salt thereof is used.
  • the G protein-coupled receptor protein described in (1) above which comprises the G protein-coupled receptor protein described in (1) or the partial peptide or salt thereof described in (3).
  • the binding between the salt and the ligand A kit for screening a compound to be changed or a salt thereof,
  • a pharmaceutical comprising a compound or a salt thereof that alters the expression level of the G protein-coupled receptor protein according to (1), which can be obtained by using the screening method according to (27);
  • a medicine comprising a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein described in (1) above in a cell membrane obtainable by using the screening method described in (28).
  • amino acid sequence represented by SEQ ID NO: 1 (2) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably 1 to 10) An amino acid sequence in which several (1 to 5) amino acids have been deleted, and 3 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30) More preferably about 1 to 10, more preferably several (1 to 5) amino acids; 4 one or two or more (preferably An amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids have been substituted with other amino acids; or
  • the ligand is, for example, angiotensin, bombesin, cannapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasoprescin, oxitocin, PAC ⁇ (eg, PACAP 27, PACAP 38) , Secretin, glucagon, calcitonin, adrenomedullin, somatostin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal Polypeptide), somatostin, dopamine, motilin, amylin, bradykinin, CGRP (Calcitonin gene relayed peptide), leukotriene, pancreatin, prostaglandin, tropoxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GR ⁇ a, GROi3, GROa,
  • a method for screening a compound or a salt thereof that alters the binding property between a ligand that binds to a G protein-coupled receptor protein or a salt thereof according to (1) above (43) (i) contacting the labeled ligand with a G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (8) above; (Ii) Labeling in the case where the labeled ligand and the test compound were brought into contact with the G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (8) above; The amount of binding of the ligand to the G protein-coupled receptor protein is measured and compared, and the binding between the ligand and the G protein-coupled receptor protein or salt thereof described in (1) above is determined.
  • a method of screening the compound to be changed or a salt thereof (43) (i) contacting the labeled ligand with a G protein-coupled receptor protein expressed on the cell membrane of the transformant by cul
  • a compound that activates the G protein-coupled receptor protein or its salt according to (1) was expressed in the cell membrane of the transformant by culturing the transformant according to (8).
  • the compound that activates the G protein-coupled receptor protein or a salt thereof and a test compound are transformed with the transformant described in (7) above.
  • a method for screening a compound that changes the binding property between a ligand that binds to a G protein-coupled receptor protein or a salt thereof according to the above (1) or a salt thereof,
  • chemokine Subfamily 1 C chemokine subfamily such as lymphotactin; CX3C chemokine subfamily such as iractalkine, etc.
  • endothelin enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) ), Sphingosine 1-phosphate, lysophosphatidylserine, sphingosylphosphorylcholine, lysophosphatidylcholine, steroids, bile acids, isoprenoids, arachidonic acid metabolites, amines, amino acids Nucleotide, nucleoside, a saturated fatty acid or unsaturated fatty acid above (44) or (45) the screening method described in
  • a medicament comprising the salt,
  • FIG. 1 is a hydrophobicity plot of TGR38. BEST MODE FOR CARRYING OUT THE INVENTION
  • the G protein-coupled receptor protein of the present invention may have an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Contains Recept Yuichi protein.
  • the receptor protein of the present invention includes, for example, all cells (eg, spleen cells, neurons) of human mammals (eg, guinea pigs, rats, mice, rabbits, bushes, sheep, horsetails, monkeys, etc.) , Glial cells, kidney
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 50% or more, preferably about 60% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1. Is about 70% or more, more preferably about 80% Above all, preferably, an amino acid sequence having about 90% or more, most preferably about 95% or more homology is mentioned.
  • Examples of the protein of the present invention containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 And a protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1.
  • Such activities include, for example, ligand binding activity, signal transduction action and the like. Substantially the same means that their activities are the same in nature. Therefore, activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the measurement of the activity such as the ligand binding activity and the signal information transmission activity can be performed according to a known method.
  • the measurement can be performed according to a ligand determination method or a screening method described later. it can.
  • the receptor protein of the present invention includes: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably about 1 to 10, more preferably An amino acid sequence in which several (1 to 5) amino acids have been deleted; (2) one or more (preferably about 1 to 30, more preferably 1 to 10) amino acid sequences represented by SEQ ID NO: 1 An amino acid sequence to which about, more preferably several (1 to 5) amino acids have been added; 3 SEQ ID NO: 1 or 2 or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1 , More preferably about 1 to 10, more preferably several (i to 5) amino acids in which the amino acid sequence is substituted with another amino acid, or a protein containing an amino acid sequence in which they are combined. Also used That.
  • the amino acid sequence of the receptor protein is N-terminal (amino terminal) at the left end and C-terminal (terminal end of lipoxyl) at the right end according to the convention of peptide labeling.
  • the receptor protein of the present invention including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal carboxy group. Or a carboxylate (one C00—), an amide (_C0NH 2 ) or an ester (one C00R).
  • R in the ester e.g., methyl, Echiru, n- propyl, C, such as isopropyl or n- butyl
  • _ 6 alkyl groups for example, shea Kuropenchiru
  • C 3 _ 8 cycloalkyl group such as cyclohexyl , for example, Hue sulfonyl
  • C 6 _ 12 Ariru groups such as single-naphthyl, for example, benzyl, phenylene Lou C such as phenethyl, _ 2 alkyl or ⁇ - naphthylmethyl ⁇ - Na Fuchiru C
  • Bok 2 alkyl groups such as such as C 7 _ 14 other Ararukiru groups, such as pivaloyl Ruo carboxymethyl group which is generally used as an oral ester.
  • the receptor protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus
  • a protein in which the carboxyl group is amidated or esterified is also included in the receptor protein of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
  • the receptions evening one protein of the present invention is the protein mentioned above, Amino groups Mechionin residues of ⁇ -terminal protecting group (for example, C, such as formyl group, which C 2 _ 6 Arukanoiru group of Asechiru, _ 6- glycyl group, etc.), the glutamyl group generated by cleavage of the terminal end in vivo and the glutamine oxidation of the mouth, the substituent on the side chain of the amino acid in the molecule (eg, SH, amino group, imidazo Ichiru group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C 2 such Asechiru - such as C, such as 6 Arukanoiru group, _ 6 ⁇ Shi Le group) And complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • ⁇ -terminal protecting group for example, C, such as formyl group, which C 2 _ 6 Arukan
  • Examples of the receptor protein of the present invention include, for example, a receptor protein containing an amino acid sequence represented by SEQ ID NO: 1 and the like.
  • the partial peptide of the receptor protein of the present invention may be any partial peptide of the receptor protein of the present invention described above.
  • the receptor protein molecules of the present invention those which are exposed outside the cell membrane and have substantially the same activity can be used.
  • substantially the same activity indicates, for example, ligand binding activity.
  • the measurement of the ligand binding activity can be performed in the same manner as described above.
  • the partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 is an extracellular region (hydrophilic region) in the hydrophobic plot shown in FIG. It is a peptide that contains the part that was analyzed. Further, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used. A peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used.
  • the number of amino acids in the amino acid sequence of the partial peptide of the present invention is at least 20 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences constituting the receptor protein of the present invention. .
  • a substantially identical amino acid sequence refers to an amino acid sequence having about 50% or more, preferably about 60% or more, more preferably about 70% or more, still more preferably about 80% or more, and still more preferably about 90% or more.
  • the amino acid sequence has the above, most preferably about 95% or more homology.
  • amino acid sequence of the partial peptide of the present invention 1) one or more (preferably about 1 to 10, and more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted; (2) One or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are included in the above amino acid sequence. (3) One or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the above amino acid sequence are replaced with other amino acids May be substituted. Further, any two or more selected from the above (1) to (3) may be appropriately combined.
  • the partial peptide of the present invention C-terminal, the partial peptide of the present invention, the force Rupokishiru group rather - C00H), the force Rupokishireto (one C00-), amide (- C0Nh 2) or an ester (which may be either single C00R) (R is The meaning is as defined above).
  • R is The meaning is as defined above.
  • the partial peptide of the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminus, those having a lipoxyl group amidated or esterified may also be used in the present invention. Included in partial peptides.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the partial peptide of the present invention has the same structure as the above-mentioned receptor protein of the present invention, in which the amino group of the N-terminal methionine residue is protected with a protecting group, but the N-terminal is cleaved in vivo.
  • Gin generated by pyroglutamine oxidation amino acids in which the substituent on the side chain of the amino acid is protected by an appropriate protecting group, or complex peptides such as so-called bran peptides to which sugar chains are bound are also included. It is.
  • Examples of the salt of the receptor protein or its partial peptide of the present invention include physiologically acceptable salts with acids or bases, and especially physiologically acceptable salts.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid) are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid are used.
  • the receptor protein of the present invention or a salt thereof can be produced from the above-described human or mammalian cells or tissues by a known method for purifying a receptor protein.
  • the receptor protein of the present invention described later can be prepared. It can also be produced by culturing a transformant containing the encoding DNA.
  • the protein can be produced by the protein synthesis method described later or according to the method.
  • the receptor protein of the present invention or a salt thereof is produced from human or mammalian tissues or cells
  • the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the extract is subjected to chromatography.
  • chromatography Eg, reversed phase chromatography, ion exchange chromatography, or a combination thereof
  • the receptor protein of the present invention or a salt thereof can be purified and isolated.
  • a commercially available resin for protein synthesis can be used.
  • resins include, for example, chloromethyl resin, hydroxymethyl Resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamide methyl resin, polyacrylamide Resin, 4- (2 ′, 4′-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-1-Fmocaminoethyl) phenoxy resin and the like.
  • amino acids having a suitably protected amino group and side chain functional group are condensed on the resin in accordance with the amino acid sequence of the target protein or peptide according to various known condensation methods.
  • the protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or its amide You.
  • the protected amino acid may be added directly to the resin along with a racemization inhibitor (eg, HOBtHOOBt), or may be added to the symmetric acid anhydride or HOBt ester or HOOBt.
  • the amino acid is condensed by adding the activated amino acid to the resin after activating the protected amino acid in advance as an ester.
  • the solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone
  • halogenated hydrocarbons such as methylene chloride and chloroform
  • alcohols such as trifluorophenol Sulfoxides such as dimethylsulfoxide
  • amines such as pyridine
  • ethers such as dioxane and tetrahydrofuran
  • nitriles such as acetonitrile and propionitrile
  • esters such as methyl acetate and ethyl acetate
  • An appropriate mixture or the like is used.
  • the reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 20 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5- to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, Yuichi Sharipentyloxycarbonyl, Isopolnylooxycarponyl, 4-methoxybenzyloxylponyl, C11Z, Br—Z, Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the carboxyl group can be, for example, alkyl esterified (for example, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.).
  • alkyl esterified for example, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
  • branched or cyclic alkyl esterification branched or cyclic alkyl esterification
  • aralkyl esterification eg, benzyl ester, 412 trobenzyl ester, 4-methoxybenzyl ester, chlorobenzene ester, benzhydryl esterification
  • fenasi It can be protected by esterification, benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide, or the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for this esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarponyl group, an ethoxycarbonyl group and the like are used.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B z 1, G 1 2 -Bz 2-nitrobenzyl, Br- ⁇ , tert-butyl and the like are used.
  • protecting group for histidine imidazole for example, Tos, 4-methoxy2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Examples of the activated form of the raw oxypoxyl group include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 —Esters with dinitrophenol, cyanomethyl alcohol, paranitrophenol, H—NB, N-hydroxysuccinimide, N-hydroxyphenolimide, HOB t)].
  • alcohol eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 —Esters with dinitrophenol, cyanomethyl alcohol, paranitrophenol, H—NB, N-hydroxysuccinimide, N-hydroxyphenolimide, HOB t
  • the activated amino group of the raw material for example, a corresponding phosphoric amide is used.
  • the protection of functional groups that should not be involved in the reaction of the raw materials and the protecting groups, and the removal of the protecting groups, the activation of the functional groups involved in the reaction, etc. are known groups or known groups. It can be appropriately selected from the means.
  • an ⁇ -functional propyloxyl group of the C-terminal amino acid is protected by amidation, and a peptide (protein) chain is extended to a desired chain length on the amino group side.
  • a protein was prepared by removing only the protecting group of the ⁇ -amino group at the ⁇ -terminal of the peptide chain and a protein from which only the protecting group of the C-terminal lipoxyl group was removed.
  • a method of condensing in water can also be used. The condensation is performed in the same manner as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein.
  • the crude protein is purified using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • an ester of a protein for example, after condensing the ⁇ -hydroxyl group of the C-terminal amino acid with a desired alcohol to form an amino acid ester, the desired ester is obtained in the same manner as in the case of the amide of the protein.
  • An ester of protein can be obtained.
  • the partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method, or by cleaving the receptor protein of the present invention with an appropriate peptide.
  • the peptide synthesis method include a solid phase synthesis method and a liquid phase synthesis method.
  • the partial peptide or amino acid that can constitute the receptor protein of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is removed to produce the desired partial peptide. can do.
  • the condensation and the elimination of the protecting group are performed, for example, according to the methods described in the following 1 to 5.
  • the partial peptides obtained in this way can be purified by conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, It can be purified and isolated by recrystallization or a combination thereof.
  • the partial peptide obtained by the above method is free, it can be converted to an appropriate salt by a known method.On the contrary, when the partial peptide is obtained as a salt, it can be converted to a free form by a known method. Can be converted.
  • the polynucleotide encoding the receptor protein of the present invention may be any polynucleotide containing a nucleotide sequence (DNA or RNA, preferably DNA) encoding the above-described receptor protein of the present invention. Good.
  • the polynucleotide include RNA such as DNA and mRNA encoding the receptor protein of the present invention. These may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
  • a known method for example, the method described in Experimental Medicine Special Edition “New PCR and Its Application” 15 (7), 1997 or a method analogous thereto, specifically Can quantify the mRNA of the receptor protein of the present invention by a method such as TaqManPCR.
  • Examples of the DNA encoding the receptor protein of the present invention include genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • the DNA can also be directly amplified by the reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using the total RNA or mRNA fraction prepared from the above-mentioned cell'tissue. .
  • the DNA encoding the receptor protein of the present invention for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a DNA having the nucleotide sequence represented by SEQ ID NO: 2 And a DNA that hybridizes under high stringent conditions and has substantially the same activity (eg, ligand binding activity, signal transduction action, etc.) as the receptor protein of the present invention. And DNA encoding the same.
  • Examples of the DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, preferably about 80% of the nucleotide sequence represented by SEQ ID NO: 2.
  • DNA containing a nucleotide sequence having a homology of at least about 90% or more, more preferably at least about 95% or more is used.
  • Hybridization can be performed by a known method or a method analogous thereto, such as the method described in Molecular 'Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be done according to. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Hybridization is preferably performed according to high stringency conditions.
  • the high stringency conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Is shown. In particular, a condition in which the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
  • DNA encoding the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1 DNA containing the base sequence represented by SEQ ID NO: 2, etc. is used.
  • a part of the nucleotide sequence of DNA encoding the receptor protein of the present invention or a polynucleotide containing a part of a nucleotide sequence complementary to the DNA is a partial peptide of the present invention described below. It is used not only to include the encoding DNA, but also to include RNA.
  • RNA-coupled receptor protein gene replication or expression of a G protein-coupled receptor protein gene.
  • Antisense 'polynucleotides that can inhibit expression can be designed and synthesized based on cloned or determined DNA sequence information encoding G protein-coupled receptor proteins. .
  • Such a polynucleotide can hybridize with the RNA of the G protein-coupled receptor protein gene and inhibit the synthesis or function of the RNA, or it can inhibit the synthesis or function of the G protein-coupled receptor protein.
  • the expression of G protein-coupled receptor protein gene can be regulated and controlled through the interaction with NA.
  • Polynucleotides that are complementary to the selected sequence of the G protein-coupled receptor protein-related RNA and that can specifically hybridize to G-protein-coupled receptor protein-related RNA are those that are used in vivo and in vitro. It is useful for regulating and controlling the expression of protein-coupled receptor protein gene, and is also useful for treating or diagnosing diseases and the like.
  • the 5'-end hairpin loop, 5'-end 6-base spare repeat, 5'-end untranslated region, 5'-end untranslated region, polypeptide translation termination codon, protein coding region, 0RF translation start of the G protein-coupled receptor protein protein gene Codons, 3 'untranslated regions, 3' palindromes, and 3 'hairpin loops may be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene may be selected. sell.
  • the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region that is, the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target can be said to be “antisense”.
  • Other polymers eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers containing special bonds provided that the polymers are paired with bases such as those found in DNA or RNA And nucleotides having a configuration permitting base attachment).
  • RNA hybrid double-stranded D NA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or DNA: RNA hybrid, and may be an unmodified polynucleotide (or an unmodified oligonucleotide) or a known polynucleotide.
  • Modified polynucleotides [e.g., labeled, capped, methylated, substituted for one or more natural nucleotides with analogs, known in the art, intramolecular Nucleotide-modified (eg, having an uncharged bond (eg, methyl phosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphophosphate, etc.) (Eg, protein (nuclease, nuclease / inhibitor, toxin, antibody) Those having side groups such as signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), those having inter-current compounds (eg, acridine, psoralen), Compounds containing chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.
  • nucleoside include, in addition to purine and pyrimidine bases, modified heterocyclic bases (eg, methylated purine and pyrimidine, acylated purine and pyrimidine bases). Pyrimidine).
  • the "nucleoside”, “nucleotide” and “nucleic acid” may have a modified sugar moiety, one or more hydroxyl groups are substituted with a halogen or an aliphatic group, or an ether or an amine. It may be converted to a functional group.
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, for example, a nucleic acid resistant to decomposition of a sulfur derivative ⁇ thiophosphate derivative, polynucleoside amide ⁇ oligonucleoside amide, and the like.
  • the antisense nucleic acid of the present invention makes the antisense nucleic acid more stable in cells, enhances the cell permeability of the antisense nucleic acid, and increases the affinity for the target sense strand. That the toxicity of the antisense nucleic acid is smaller 421S
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in special forms such as ribosomes and microspheres, or applied by gene therapy. Or can be given in additional form.
  • the adduct used in the addition form may be a polycation such as polylysine, which acts to neutralize the charge of the phosphate skeleton; enhances interaction with the cell membrane, and increases nucleic acid uptake.
  • Hydrophobic substances such as lipids (for example, phospholipids, cholesterol, etc.), may be mentioned.
  • the protein is preferably cholesterol or a derivative thereof (for example, cholesteryl chromate formate, cholic acid, etc.), and these can be attached to the 3 ′ end or 5 ′ end of nucleic acid. It can be attached via a base, sugar, or intramolecular nucleoside bond.
  • a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid to prevent degradation by nucleases such as exonuclease and RNase.
  • capping groups include hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of a G protein-coupled receptor protein. it can.
  • the nucleic acid can be applied to cells by various known methods. '
  • the antisense polynucleotide having 27 columns has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide (eg, DNA) of the present invention, and the polynucleotide (eg, DNA) Any polynucleotide may be used as long as it has an action capable of suppressing the expression of.
  • antisense polynucleotide antisense DNA is preferable.
  • the nucleotide sequence substantially complementary to the polynucleotide of the present invention is, for example, a nucleotide sequence complementary to the polynucleotide of the present invention (eg, DNA) (ie, the polynucleotide of the present invention).
  • the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention eg, the nucleotide sequence near the initiation codon
  • Antisense polynucleotides having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the complementary strand are suitable.
  • Antisense polynucleotides having a portion are also included.
  • the antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
  • phosphate residues (phosphate) of each nucleotide constituting the antisense polynucleotide May be substituted with a chemically modified phosphate residue such as, for example, phosphorothioate, methylphosphonate, or phosphorodithioate.
  • phosphate residues such as, for example, phosphorothioate, methylphosphonate, or phosphorodithioate.
  • the DNA encoding the partial peptide of the present invention may be any as long as it contains the above-described nucleotide sequence encoding the partial peptide of the present invention.
  • a genomic DNA a genomic DNA library, Any of the above-described cDNA derived from cells and tissues, the above-described cDNA library derived from cells and tissues, and synthetic DNA may be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • the DNA can also be directly amplified by the RT-PCR method using the mRNA fraction prepared from the cells and tissues described above.
  • the DNA encoding the partial peptide of the present invention includes, for example, (1) a DNA having a partial nucleotide sequence of DNA having a base sequence represented by SEQ ID NO: 2, or (2) A protein having DNA that hybridizes under high stringent conditions with the DNA represented by SEQ ID NO: 2 and having substantially the same activity as the partial peptide of the present invention (eg, ligand binding activity, signal transduction activity, etc.)
  • a DNA having a partial nucleotide sequence of the DNA encoding DNA is used.
  • Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more, and more preferably the base sequence represented by SEQ ID NO: 2
  • DNA containing a nucleotide sequence having a homology of about 90% or more, more preferably about 95% or more may be used.
  • Cloning of a DNA encoding the receptor protein of the present invention or a partial peptide thereof may be performed by encoding the peptide of the present invention.
  • Amplification by PCR using synthetic DNA primers having a partial nucleotide sequence of the DNA nucleotide sequence, and DNA incorporated into an appropriate vector Examples include a method of hybridizing with a DNA fragment encoding a part or all of the region or a DNA labeled with a synthetic DNA. Hybridization can be carried out, for example, according to the method described in ui in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual.
  • Substitution of the nucleotide sequence of DNA is performed using PCR or a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), and the like.
  • the method can be performed according to a known method such as the gapped duplex method, the Kunkel method, or the like, or a method analogous thereto.
  • the DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be used after digestion with a restriction enzyme or addition of a linker if desired.
  • the DNA may have ATG as a translation initiation codon at its 5 'end and may have TAA, TGA or TAG as a translation stop codon at its 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • the expression vector of the receptor protein of the present invention is, for example, (i) excising a DNA fragment of interest from, for example, cDNA containing DNA encoding the receptor protein of the present invention; It can be produced by ligating downstream of a promoter in an expression vector.
  • examples of the expression vector include plasmids derived from Escherichia coli (eg, pCR4, pCR2.K PBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, UBHO, pTP5, pC194); Eg, pSH19, pSH15); bacteriophages such as human phage; animal viruses such as retrovirus, vaccinia virus, and baculovirus; PA11, pXTK pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. .
  • the promoter is not particularly limited, and may be appropriately selected depending on the host used for gene expression.
  • animal cells are used as hosts.
  • SR promoter SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • CMV promoter CMV promoter, SRo! Promo One Night, etc. are preferred.
  • trp promoter Isseki one, lac promoter one, re cA promoter one, AP L promoter, lpp promoter one coater is; when the host is Bacillus, S PO 1 promoter, SP02 promoter, penP promoter and the like; when the host is yeast, PH5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may contain, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like.
  • the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene (methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter sometimes abbreviated as Amp 1 ) , neomycin resistance gene (hereinafter sometimes abbreviated as Ne o 1 ", G418 resistance).
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host may be added to the N-terminal side of the receptor protein of the present invention.
  • the host is a genus Escherichia, a PhoA signal sequence, a ⁇ mpA signal sequence, etc .; if the host is a bacterium, a monoamylase signal sequence, a subtilisin-signal sequence, etc. If the host is yeast, MFa signal sequence, SUC2 signal sequence, etc .; if the host is animal cells, inulin signal sequence, ⁇ -interferon signal sequence, antibody molecule A signal array and the like can be used. 4215
  • a transformant can be produced by introducing the thus-produced vector containing DNA encoding the receptor protein of the present invention into a host.
  • bacteria of the genus Escherichia bacteria of the genus Bacillus, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia examples include, for example, Escherichia coli Escherichia coli K12 DH1 [Processing's of the 'National Academy of Ob-Sciences of the The Prosp. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research], (Nucleic Acids Research), 9, 309 (1981) 3, JA221 [Janal of Ob.
  • Bacillus spp. include Bacillus subtilis MI114 [Gene, 24, 255 (1983) 3, 207-21 [Journal of Biochemistry, 95, 87] (1984)].
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R-, Sono-11A, DKD-5D, 20B-12; Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris and the like.
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R-, Sono-11A, DKD-5D, 20B-12; Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris and the like.
  • Insect cells include, for example, when the virus is Ac NPV, a cell line derived from the larvae of night moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, and egg derived from Trichoplusia ni egg High Five TM cells, cells derived from Mamestra brassicae, cells derived from Estigmena acrea, etc. are used.
  • a silkworm-derived cell line (Bombyx mori N; BmN cell) or the like is used.
  • S: f cell examples include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)) and the like. Used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), and dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr-) cell). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells.
  • Transformation of a genus Escherichia is described, for example, in Proc. Natl. Acad. Sci. USA, 69, Proc. Natl. Acad. Sci. USA. Vol., 2110 (1972) and Gene (17), 107 (1982).
  • Transformation of a bacterium of the genus Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
  • Yeast transformation is described, for example, in Methods in Enzymology, Vol. 194, 182—187 (1991), Processings of the National Academy of Sciences. Natl. Acad. Sci. USA, 75, 1929 (1978).
  • Transformation of insect cells or insects can be carried out, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • Transformation of animal cells can be performed, for example, according to the method described in Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do it. Thus, a transformant transformed with the expression vector containing the DNA encoding the G protein-coupled receptor protein is obtained.
  • the G protein-coupled receptor protein of the present invention can be produced by culturing the transformant in a medium suitable for the host.
  • a liquid medium is preferable as a medium used when culturing a transformant whose host is a genus Escherichia or Bacillus.
  • a medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant.
  • the carbon source for example, glucose, dextrin, soluble starch, sucrose, etc .
  • the nitrogen source for example, ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal
  • inorganic or organic substances such as a nutrient extract
  • examples of the inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • the medium may contain yeast extract, vitamins, growth promoting factors and the like.
  • the pH of the medium is preferably about 5-8.
  • M9 medium containing glucose and casamino acid As a medium used for culturing a transformant whose host is a bacterium belonging to the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Mi Her, Journal of Experimen 'in' Molecular • Genetics (Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • an agent such as 3 / 8-indolylacrylic acid may be added to the medium.
  • the culturing is usually performed at about 30 to 40 ° C for about 6 to 24 hours. If necessary, ventilation or stirring may be performed.
  • a medium used for culturing a transformant in which the host is yeast for example, Burkholder's minimum medium [Bostian, KL et al., "Procedures of the National Co., Ltd.” Academy of Sciences Natl. Acad. Sci. USA, 77, 4505 (1980) J; SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc.
  • the pH of the medium is about 5-8.
  • the cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours. If necessary, ventilation or stirring may be performed.
  • the culture medium used for culturing insect cells or transformants whose insect host is, for example, Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) Examples thereof include those to which additives such as 10% serum are added as appropriate.
  • the pH of the medium is preferably about 6.2 to 6.4.
  • the culture is usually performed at about 27 ° C for about 3 to 5 days. If necessary, ventilation or stirring may be performed.
  • the G protein-coupled receptor protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the receptor protein of the present invention thus obtained can be separated and purified, for example, by the following method.
  • the buffer may contain a protein denaturant such as urine or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • the receptor protein is secreted into the culture solution during the culturing of the transformant, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the culture supernatant is collected.
  • the receptor protein of the present invention can be separated.
  • the receptor extract protein can be purified by subjecting the thus obtained crude extract or culture supernatant to known separation and purification methods.
  • known separation and purification methods include, for example, methods using solubility such as salting out and solvent precipitation; dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method using a difference in hydrophobicity such as an isoelectric point
  • a method using a difference in isoelectric points such as an isoelectric point method and a perturbation method are used.
  • the receptor protein obtained in this way is a free form, it can be converted to a salt by a known method or a method analogous thereto.
  • the receptor protein is a salt, it can be converted into a free form or another salt by a known method or a method analogous thereto.
  • the receptor protein produced by the transformant can be arbitrarily modified or the polypeptide can be partially removed by allowing a suitable protein modifying enzyme to act on the receptor protein before or after purification.
  • a suitable protein modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the activity of the receptor protein of the present invention or a salt thereof thus obtained can be determined by a binding experiment with a labeled ligand and a specific antibody.
  • An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof may be obtained by using the receptor protein of the present invention as an antigen. It can be produced according to a known antibody or antiserum production method. Hereinafter, the method for producing an antibody against the receptor protein of the present invention will be described in detail.
  • the receptor protein or the like of the present invention is administered to a mammal at a site capable of producing an antibody upon administration, itself or together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be used to enhance antibody production upon administration.
  • Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
  • a mammal immunized with the antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
  • an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the measurement of the antibody titer in the antiserum can be performed, for example, by reacting the below-described labeled receptor protein or the like with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Kayla and Milstein [Nature, 256, 495 (1975)].
  • a fusion accelerator may be, for example, polyethylene daricol (PEG) or Sendai virus. Among them, PEG is preferred.
  • myeloma cells examples include NS-1, P3U1, SP2 / 0 and the like, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells is about 1: 1 to 20: 1.
  • the fusion operation is carried out in the presence of PEG at a concentration of about 10 to 80% (preferably, PEG 1000 to> EG6000) at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about ⁇ ⁇ to 10 minutes. Can be implemented efficiently.
  • Screening of the monoclonal antibody-producing hybridoma is performed according to a known method.
  • Such methods include, for example, adding a hybridoma culture supernatant to a solid phase (eg, microplate) on which an antigen such as a receptor protein is adsorbed directly or together with a carrier, and then adding a radioactive substance or an enzyme.
  • a method for detecting the monoclonal antibody bound to the solid phase by adding the anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice)
  • a method in which a hybridoma culture supernatant is added to a solid phase to which an antibody or protein A is adsorbed, and a receptor protein or the like labeled with a radioactive substance, an enzyme, or the like is added to detect a monoclonal antibody bound to the solid phase. Is mentioned.
  • Monoclonal antibodies can be selected by using a medium for selection and breeding according to a known method or a method analogous thereto.
  • the selection of monoclonal antibodies is usually performed in animal cell culture media supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as a hybridoma can grow, and specifically, RPMI containing 1 to 20%, preferably 10 to 20% fetal bovine serum. 1640 medium, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) .
  • the culture temperature is usually 20 to 40 ° (:, preferably about 37 ° C.)
  • the culture time is usually 5 days to 3 weeks, preferably 1 week Between ⁇ 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known immunoglobulin separation and purification methods (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, adsorption / desorption using an ion exchanger (eg, DEAE), Centrifugation, gel filtration, antigen-binding solid phase or specific purification method in which only antibodies are collected using an active adsorbent such as protein A or protein G, and the bonds are dissociated to obtain antibodies.
  • immunoglobulin separation and purification methods eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, adsorption / desorption using an ion exchanger (eg, DEAE), Centrifugation, gel filtration, antigen-binding solid phase or specific purification method in which only antibodies are collected using an active adsorbent such as protein A or protein G, and the bonds are dissociated to obtain antibodies.
  • the polyclonal antibody of the present invention can be produced by a known method or a method analogous thereto.
  • the polyclonal antibody of the present invention can be used to immunize a mammal, for example, using an immunizing antigen (an antigen such as the receptor protein of the present invention) in the same manner as in the method for producing a monoclonal antibody described above.
  • the antibody can be produced by collecting the antibody-containing substance against the receptor protein of the present invention and separating and purifying the antibody.
  • the immunizing antigen (antigen such as receptor protein of the present invention) may be a complex with carrier protein.
  • the type of the carrier protein and the mixing ratio of the carrier protein and the immunizing antigen are not particularly limited as long as the antibody can be efficiently produced against the immunizing antigen immunized by cross-linking the carrier.
  • a carrier protein such as serum serum albumin, serum cycloglobulin, keyhole, lysine hemocyanin, etc. may be mixed in a weight ratio of about 0.1 to 20, preferably about 1 to 5, with respect to 1 immunizing antigen. It is preferable to ring.
  • the coupling between the immunizing antigen and the carrier protein can be performed using various condensing agents.
  • the condensing agent include datalaldehyde, carposimid, maleimide active ester, and an active ester reagent containing a thiol group and a dithioviridyl group.
  • the immunizing antigen (including the complex of the immunizing antigen and the carrier protein) is supplied to the mammal at a site where antibody production is possible, together with the carrier itself or a diluent. Is administered. To increase antibody production during administration, complete:
  • Subjuvant or incomplete Freund's adjuvant may be used. Administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected preferably from blood, such as blood, ascites, and the like of a mammal immunized by the above method.
  • the polyclonal antibody titer in the antiserum can be measured by the same method as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be performed according to the above-mentioned known method of separating and purifying immunoglobulin.
  • the receptor encoding the receptor protein of the present invention or its salt, its partial peptide or its salt, and the receptor protein or its partial peptide are encoded by (1) a ligand for the G protein-coupled receptor protein of the present invention. (2) a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the G protein-coupled receptor protein of the present invention; (3) a gene diagnostic agent; and (4) a drug of the present invention. (5) A method for screening a compound that changes the expression level of a receptor protein or a partial peptide thereof, (5) prevention and / or prevention of various diseases containing a compound that changes the expression level of the receptor protein or a partial peptide thereof of the present invention.
  • Therapeutic agent (6) Quantitative method of ligand for G protein-coupled receptor protein of the present invention, (7) G protein poorly coupled receptor protein of the present invention and Riga (8) Compounds that alter the binding between the G protein-coupled receptor protein of the present invention and the ligand (agonists, antagonists) A prophylactic and / or therapeutic agent for various diseases, comprising: (9) quantification of the receptor protein of the present invention or its partial peptide or a salt thereof; (10) the receptor protein of the present invention in a cell membrane or its partial peptide (Ii) a method for screening and / or treating various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane; ) Neutralization of the receptor protein of the present invention or its partial peptide or a salt thereof with an antibody, (13) G of the present invention Used like for the production of non-human transformer diethyl nick animals with DN Alpha encoding white matter coupled receptions evening one protein Can be
  • the binding of a ligand to a G-protein-coupled receptor specific to a human mammal can be improved.
  • the compound to be changed eg, agonist, angyo gonist, etc.
  • the agonist or angyo gonist can be used as an agent for preventing or treating various diseases.
  • the receptor protein or the partial peptide of the present invention or a salt thereof, the DNA encoding the receptor protein of the present invention or the partial peptide thereof (hereinafter, may be abbreviated as the DNA of the present invention), the receptor protein of the present invention, and the like.
  • the use of the antibody (hereinafter, may be abbreviated as the antibody of the present invention) for the antibody is specifically described below.
  • the receptor protein of the present invention or its salt or the partial peptide or its salt of the present invention can be used as a reagent for searching or determining a ligand (agonist) for the receptor protein of the present invention or its salt.
  • the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof with a test compound. I will provide a.
  • Test compounds include known ligands (for example, angiotensin, bombesin, cannapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasoprescin, oxytocin, PACAP (eg, PACAP 27, PACAP 38), secretin, glucagon, calcitonin, 7-drenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (basoactive intestinal and related polypeptide), somatos, dopamine, motilin, Amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatin, pro Staglandin, tropoxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROa, GROj3, GRA, NAP-2, ENA-78, GCP-2
  • the ligand determination method of the present invention uses the receptor protein of the present invention or its partial peptide or a salt thereof, or constructs a recombinant receptor-protein expression system, and Receptor binding using the ATS system is used to bind to the receptor protein of the present invention and to stimulate cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca "release, intracellular cAMP production, intracellular c A compound having an activity of promoting or inhibiting GMP production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, activation of c-fos, decrease of pH, etc.
  • cell stimulating activity eg, arachidonic acid release, acetylcholine release, intracellular Ca "release, intracellular cAMP production, intracellular c
  • a test compound for example, the amount of a test compound bound to the receptor protein or the partial peptide, It is characterized by measuring cell stimulating activity and the like.
  • the present invention provides
  • the receptor of the labeled test compound A method for determining a ligand for a receptor protein of the present invention, which comprises measuring the amount of binding to an evening protein or a salt thereof;
  • ⁇ Cellular stimulating activity via the receptor protein when the test compound is brought into contact with cells containing the receptor protein of the present invention eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular c AMP generation, intracellular c GMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-1: activation of fos, reduction or activation of pH Activity, etc.
  • a method for determining a ligand for the receptor protein or a salt thereof of the present invention eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular c AMP generation, intracellular c GMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-1: activation of fos, reduction or activation of pH Activity, etc.
  • Activity for example, arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, and a method for determining a ligand for the receptor protein or a salt thereof of the present invention, which comprises measuring the activity of promoting or suppressing fos activation or pH reduction.
  • any receptor protein used in the ligand determination method may be used as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention.
  • Receptacle protein is suitable.
  • the above-described expression method is used. It is preferable to express DNA encoding the receptor protein in mammalian cells or insect cells.
  • cDNA is usually used, but is not necessarily limited thereto.For example, even if a gene fragment or synthetic DNA is used, Good.
  • the DNA fragment should be transferred to a nucleus belonging to baculovirus using an insect as a host.
  • Polyhedrin promoter of somatic disease virus somatic disease virus (nuclear polyhedros is virus; NPV), promoter derived from SV40, promoter of retrovirus, metamouth thionine promoter, human heat shock promoter, cytomegalovirus promoter It is preferable to incorporate it downstream, such as the SR promoter.
  • the quantity and quality of the expressed receptor can be determined by known methods, for example, as described in the literature [Nambi, P. et al., The 'Journal' of 'Biological' Chemistry (J. Biol. Cem.), 267, 19555- 19559, 1992].
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof used in the ligand determination method of the present invention can be prepared according to a known method.
  • the protein may be a purified receptor protein or its partial peptide or a salt thereof, or may be a cell containing the receptor protein or a cell membrane fraction thereof.
  • the cells When cells containing the receptor protein of the present invention are used in the ligand determination method of the present invention, the cells may be immobilized with daltaraldehyde, formalin, or the like. Immobilization can be performed according to a known method.
  • the cell containing the receptor protein of the present invention means a host cell expressing the receptor protein of the present invention.
  • the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, and animal cells. Are used.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by crushing cells and then obtained by a known method.
  • Methods for crushing cells include crushing cells with a Potter-Elvehj em homogenizer, crushing method using a single ring blender ⁇ polytron (Kinematica), crushing method using ultrasonic waves, and French press.
  • a crushing method in which cells are ejected from a fine nozzle while applying pressure, and the like, may be mentioned.
  • the cell membrane thus obtained can be fractionated by a centrifugal fractionation method such as a differential centrifugation method or a density gradient centrifugation method.
  • the cell lysate is centrifuged at low speed (500-3000 rpm) for a short time (typically, about 1 minute to 10 minutes), and the supernatant is further centrifuged at high speed (15,000 to 30,000 rpm) for 30 minutes to 2 hours.
  • a cell membrane fraction can be obtained.
  • the cell membrane fraction contains a large amount of expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of receptions evening one protein of a cell and in their membrane fraction containing the receptor protein, per cell preferably to 10 8 molecules, more preferably to 107 molecules.
  • the higher the expression level the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to screen a large number of test compounds in the same lot.
  • an appropriate receptor protein fraction and a labeled test compound are required.
  • the receptor protein fraction is preferably a natural receptor protein fraction, or a recombinant receptor fraction having the same activity as the fraction.
  • “equivalent activity” means equivalent ligand binding activity, signal transduction action, and the like.
  • the labeled test compound, [3 H], [125 I], [14 C] or [35 S] angiotensin were identified respectively labels such as bombesin, Kan'nabi maytansinoid, cholecystokinin, glutamine, serotonin , Melatonin, new oral peptide Y, opioid, purine, vasopressin, oxitocin, ⁇ CAP (eg, PACAP 27, PACAP 38), secretin, glucagon, calcitonin, 7-drenomediulin, somatostatin, GHRH, CRF, ACTH, GRP , PTH, VIP (Vasoactive Intestinal and Retained Polypeptides), Somatos-tin, Dopamine, Motilin, Amylin, Bradykinin, CGRP (Calcitonin Gene-Related Peptide), Leukotriene, Pancreatin, Prostaglandin, Trompoxane, Adenosine, 7-d
  • CX3 chemokine subfamily of fractalkine, etc. CX3 chemokine subfamily of fractalkine, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-lin Acid, lysophosphatidylserine, sphingosyl phosphoryl Choline, lysophosphatidylcholine, steroids, bile acids, isoprenoids, ⁇ Rakidon acid metabolites, amines, amino acids, nucleotides, nucleosides, etc. saturated or unsaturated fatty acids are preferred.
  • LPA lysophosphatidic acid
  • sphingosine 1-lin Acid lysophosphatidylserine
  • sphingosyl phosphoryl Choline lysophosphatidylcholine
  • steroids bile acids
  • a ligand for the receptor protein of the present invention or a salt thereof
  • cells or a membrane fraction of the cells containing the receptor protein of the present invention are suspended in an appropriate buffer.
  • the buffer is not particularly limited as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer of ⁇ 4 to 10 (preferably pH 6 to 8) or a tris-monohydrochloride buffer.
  • proteins such as surfactants such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, dexcholate, and serum albumin and gelatin can be added to the buffer. Good.
  • protein inhibitors such as SF, leptin, E-64 (manufactured by Peptide Research Institute), and pepstatin were added to the buffer to suppress the degradation of the receptor and ligand by the protease. You may.
  • the reaction solution is filtered through a glass fiber filter paper, etc., and the glass fiber filter paper is washed with an appropriate amount of buffer (a buffer similar to a single receptor), and the radioactivity remaining on the glass fiber filter paper is measured by a liquid scintillation counter. Or, it is measured in the evening of the county.
  • the test compound having a count (B-NSB) of less than 0 cpm obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) exceeds the receptor protein of the present invention or its protein. It can be selected as a ligand for salt.
  • the cell stimulating activity for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP Production, phosphoric acid production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-
  • the activity of promoting or suppressing the activation of fos, the decrease of pH, etc. can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like.
  • the assay Prior to ligand determination, replace with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Then, the generated product is quantified according to each method. If the production of a substance that serves as an indicator of cell stimulating activity (for example, arachidonic acid) cannot be confirmed by the degrading enzyme contained in the cells, the assay may be performed by adding an inhibitor for the degrading enzyme. Good. In addition, activities such as inhibition of cAMP production can be detected as an activity of inhibiting production of cells whose basic production has been increased by forskolin or the like.
  • a substance that serves as an indicator of cell stimulating activity for example, arachidonic acid
  • the kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof includes a receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, a cell containing the receptor protein of the present invention, or It contains the membrane fraction of cells containing the receptor protein of the present invention.
  • kits for determining a ligand of the present invention include the following.
  • CHO cells expressing the receptor protein of the present invention 12-well plates and passaged 5 10 5 holes, 37, 5% C0 2, with 95% ai r 2 days followed by culturing.
  • Examples of the ligand capable of binding to the receptor protein of the present invention or a salt thereof include substances specifically present in the hypothalamus, cerebral cortex, colon cancer, lung cancer, cardiac placenta, lung, and the like. Specifically, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, pudding, vasoprescin, oxitocin, PACAP (e.g., PACAP 27, PACAP 38), secretin, Glucagon, calcitonin, adrenomedullin, somatos quintin, GHRH, CRF, ACTH, GRP, PTH, VIP (basoactive intestinal and lit polypeptide), somatos quintin, dopamine, motilin, amylin, bradykinin, CGRP (Calcitonin Gene Relay Peptide, leukotriene, pancreatin, prostaglan
  • LPA lysophosphatidic acid
  • sphingosin monophosphate lysophosphatidylserine
  • sphingosylphosphorylcholine lysophosphatidylcholine
  • steroids bile acids
  • isoprenoids araki Metabolites of donic acid, amines, amino acids, nucleotides, nucleosides, saturated or unsaturated fatty acids, and the like.
  • the receptor protein of the present invention or the DNA encoding the receptor protein of the present invention can be used in accordance with the action of the ligand. It can be used as a medicament such as a prophylactic and / or therapeutic agent for diseases associated with receptor protein dysfunction.
  • the receptor protein of the present invention may be administered to a patient to supplement the amount of the receptor protein, or (i) administering the receptor protein of the present invention to the patient.
  • DNA encoding the receptor protein of the present invention is useful as an agent for preventing and / or treating a disease associated with dysfunction of the safe and low-toxic receptor protein of the present invention.
  • the receptor protein of the present invention is a novel seven-transmembrane receptor protein having approximately 27% homology at the amino acid sequence level with 5HT1D (serotonin receptor).
  • the receptor protein of the present invention or the DNA encoding the receptor protein may be a central disease (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.), an internal sulcus disease (eg, hypertension, gonad dysfunction) , Thyroid dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, lipid metabolism disorders, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder) It is used as a medicament useful for prevention, Z or treatment of cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., heart disease (eg, angina, myocardial infarction, etc.).
  • a central disease eg, depression, Alzheimer's disease, dementia, eating disorder, etc.
  • an internal sulcus disease eg, hypertension, gonad dysfunction
  • Thyroid dysfunction e.g., pituitary dysfunction
  • metabolic diseases eg, diabetes, lipid metabolism disorders, hyper
  • the receptor protein of the present invention When used as a medicine, it can be formulated according to a method commonly used in the technical field of formulation.
  • the DNA of the present invention when used as a medicine, the DNA of the present invention may be used as it is, or a retrovirus vector, an adenovirus vector, an adenovirus-associated virus vector, or the like. After insertion into an appropriate vector, a pharmaceutical preparation can be prepared in the same manner as the receptor protein of the present invention.
  • the DNA of the present invention can be administered as it is, or together with an auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
  • Examples of the dosage form of the drug include oral preparations such as sugar-coated tablets, capsules, elixirs, and micropulcers, and parenteral preparations such as injections, if necessary.
  • oral preparations such as sugar-coated tablets, capsules, elixirs, and micropulcers
  • parenteral preparations such as injections, if necessary.
  • These preparations include, for example, (1) a receptor protein of the present invention or (2) DNA encoding the receptor protein, a preparation additive, for example, a physiologically acceptable carrier, a flavoring agent, an excipient, a vehicle. It can be manufactured by mixing with preservatives, stabilizers, binders and the like.
  • the content of the receptor protein or DNA of the receptor of the present invention in these preparations is determined by the following formula of the present invention. 2 04215
  • additives used in the manufacture of disintegrants and capsules include, for example, binders such as gelatin, cornstarch, tragacanth, and arabic gum; shaping agents such as crystalline cellulose; cornstarch, gelatin, alginic acid, and the like. Swelling agents; lubricating agents such as magnesium stearate; sweeteners such as sucrose, lactose or sachets; and phosphorus and other sweeteners (j; flavoring agents such as peppermint, cocoa oil or cherry), and the like.
  • liquid carriers such as oils and fats may be further contained.
  • aqueous vehicles such as water for injection and saline
  • oily vehicles such as vegetable oils such as sesame oil, coconut oil, and soybean oil.
  • isotonic agents such as butdu sugar, D-sorbitol, D-mannitol, and sodium chloride; alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), A solubilizing agent such as a zwitterionic surfactant (eg, polysorbate 80 TM, HCO-50) may be used.
  • a solubilizing agent such as benzyl benzoate or benzyl alcohol may be used.
  • the injection solution is usually filled in a suitable ampoule.
  • formulation additives examples include a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, prochloride hydrochloride, etc.), a stabilizer (eg, human serum albumin) , Polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, benzalkonium chloride, prochloride hydrochloride, etc.
  • a stabilizer eg, human serum albumin
  • preservatives eg, benzyl alcohol, phenol, etc.
  • the preparations thus obtained have low toxicity, they are safe for humans and mammals (for example, rats, mice, rabbits, sheep, sheep, bush, horses, cats, dogs, monkeys, etc.). Can be administered.
  • the dosage of the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method and the like.
  • oral administration for example, about 0.1 per day for a cancer patient (body weight 60 kg). 100100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • the receptor of the present invention The dose of the protein is, for example, usually about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day for a cancer patient (body weight 60 kg) in the case of intravenous injection. Preferably about 0.1 to 10 mg.
  • the dosage of the DNA of the present invention varies depending on the administration subject, the target organ, the condition, the administration method, and the like.
  • oral administration for example, about 0.1 to 10 mg / day for a cancer patient (body weight 60 kg). 100 mg, preferably about 1.0 to 50 mg, more preferably thread 1. (! To 20 mg.
  • the dose of the DNA of the present invention is, for example, in the case of intravenous injection of an injection. For example, it is usually about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day for a cancer patient (assuming a body weight of 60 kg).
  • the DNA of the present invention can be used as a probe to produce the receptor protein of the present invention in humans or mammals (eg, rat, mouse, rabbit, rabbit, sheep, bush, rabbit, cat, dog, monkey, etc.).
  • Abnormality (genetic abnormality) of DNA or mRNA encoding the partial peptide can be detected, for example, damage, mutation or reduced expression of the DNA or mRNA, or DNA or mRNA of the DNA or mRNA. It is useful as a diagnostic agent for genes such as increased or overexpressed genes. '
  • the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processing's Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)) Can be implemented.
  • the receptor of the present invention can be used. It can be used for screening a compound that changes the expression level of one protein or its partial peptide.
  • the present invention relates to, for example, (i) non-human mammal blood, 2 specific organs, 3 tissues or cells isolated from organs, or (ii) transformants of the present invention.
  • the measurement of the mRNA amount of the receptor protein of the present invention or its partial peptide is specifically performed as follows.
  • non-human mammals eg, mouse, rat, rabbit, sheep, pigeon, pig, cat, cat, dog, monkey, etc., more specifically, dementia rat, obese mouse, atherosclerotic rabbit, Drugs (eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.) After a certain period of time, blood or a specific organ (eg, brain, lung, colon, etc.), or tissue or cells isolated from the organ is obtained.
  • a specific organ eg, brain, lung, colon, etc.
  • the mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells can be obtained, for example, by extracting mRNA from cells or the like by a conventional method, and using a method such as TaqMan PCR. And can be analyzed by performing Northern blotting by known means.
  • a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the mRNA of the receptor protein of the present invention or the partial peptide thereof contained in the transformant is prepared. Can be quantified and analyzed in the same manner.
  • Screening for a compound that changes the expression level of the receptor protein or its partial peptide of the present invention comprises:
  • test compound is mixed with the medium, and after culturing for a certain period of time (1 to 7 days, preferably 1 to 3 days, more preferably 2 to 3 days) After that, the amount can be determined by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the transformant.
  • the compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of the receptor protein or a partial peptide thereof of the present invention.
  • the receptor of the present invention By increasing the expression level of Yuichi protein or its partial peptide, cell stimulating activity via G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca "release, intracellular CAMP Production, intracellular c-GMP production, inositol monophosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc.
  • G protein-coupled receptor eg, arachidonic acid release, acetylcholine release, intracellular Ca "release, intracellular CAMP Production, intracellular c-GMP production, inositol monophosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc.
  • B reducing
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein of the present invention.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein or the like of the present invention.
  • the compound or a salt thereof obtained by using the screening method of the present invention can be made into various preparations in the same manner as in the case of the above-mentioned receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in human mammals (for example, rats, mice, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered.
  • human mammals for example, rats, mice, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the dose of the compound or a salt thereof is usually, for example, about 0.01% per day for a cancer patient (60 kg body weight) when intravenously injected. 3030 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg.
  • the receptor protein of the present invention is considered to play some important role in vivo, such as central function. Therefore, the compound that changes the expression level of the receptor protein of the present invention or its partial peptide can be used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention.
  • the disease associated with dysfunction of the receptor protein of the present invention includes, for example, central diseases (eg, depression, Alzheimer's disease, dementia, eating disorders, etc.), endocrine diseases (eg, hypertension, gonad dysfunction, Thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, dyslipidemia, 'hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer) , Breast cancer, cervical cancer, colon cancer, rectal cancer, etc., and heart diseases (eg, angina pectoris, myocardial infarction, etc.).
  • central diseases eg, depression, Alzheimer's disease, dementia, eating disorders, etc.
  • endocrine diseases eg, hypertension, gonad dysfunction, Thyroid dysfunction, pituitary dysfunction, etc.
  • metabolic disorders eg, diabetes, dyslipidemia, 'hyperlipidemia, etc.
  • cancer eg, non-small cell lung cancer, ova
  • the compound is useful for preventing diseases associated with dysfunction of the receptor protein of the present invention. 02 04215
  • the compound When used as 56 and Z or a therapeutic agent, the compound can be made into various preparations in the same manner as in the case of the receptor protein of the present invention described above.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and mammals (for example, rats, mice, puppies, higgs, bushes, cats, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the dose of the compound or a salt thereof may be, for example, in the case of intravenous injection, usually about 0.01% per day for a cancer patient (body weight 60 kg). 3030 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg.
  • the quantification method of the present invention can be carried out, for example, by combining with the 'competition method'. That is, the ligand concentration in the sample can be measured by bringing the sample into contact with the receptor protein of the present invention or the like. Specifically, for example, the quantification method of the present invention can be carried out according to the method described in (1) or (2) below or a method analogous thereto.
  • compounds eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.
  • Such compounds (a) G-protein coupled receptions evening through an cell-stimulating activity (e.g., Arakidon acid release, acetylcholine release, intracellular C a 24 Yu away, intracellular c AM P product, cells Compounds that have an activity of promoting or inhibiting intracellular cGMP production, inositol monophosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, activation of c-fos, pH reduction, etc.
  • B a compound not having the cell stimulating activity (so-called antagonist to the receptor protein of the present invention);
  • a compound that decreases the binding strength between the ligand and the G protein-coupled receptor protein of the present invention is preferably screened by the above-described ligand determination method).
  • the present invention relates to ( ⁇ ) the case where the receptor protein of the present invention or its partial peptide or a salt thereof is contacted with a ligand; and (ii) the receptor protein of the present invention or its partial peptide or a salt thereof. And a ligand and a test compound, and comparing the ligand with a receptor protein of the present invention or a partial peptide thereof or a salt thereof, or a salt thereof, A screening method is provided.
  • the screening method of the present invention is characterized in that, in the cases (i) and (ii), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, and the like are measured and compared.
  • the present invention provides
  • a compound that activates the receptor protein or the like of the present invention eg, a ligand for the receptor protein or the like of the present invention
  • a compound that activates a protein or the like and a test compound are brought into contact with cells containing the receptor protein or the like of the present invention, cell-stimulating activity via the receptor (for example, arachidonic acid release, acetylcholine release, Intracellular Ca 2+ release, intracellular CAMP generation, intracellular cGMP generation, inositol phosphate production, fluctuation of cell membrane potential, intracellular protein phosphorylation, activation of c-f0s, pH
  • the activity of promoting or suppressing the decrease in the activity of a ligand is measured and compared, and a compound that changes the binding property between the ligand and the receptor protein of the present invention or the like is characterized. Screening method for a substance or a salt thereof,
  • a compound that activates the receptor protein of the present invention (eg, a ligand for the receptor protein of the present invention) was expressed on the cell membrane by culturing a transformant containing the DNA of the present invention.
  • the receptor of the present invention expressed on the cell membrane by culturing a transformant containing the DNA of the present invention with a compound that activates the receptor protein or the like of the present invention and a test compound. Receptor-mediated cell stimulating activity upon contact with Yuichi protein etc.
  • a cell, tissue or cell membrane containing a G protein-coupled receptor receptor protein such as a rat is used prior to obtaining the receptor protein or the like of the present invention.
  • a candidate compound is obtained using the fraction (primary screening), and then a test is performed to confirm whether the candidate compound actually inhibits the binding of human G protein-coupled receptor protein to ligand (Secondary screening) was required. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins are also mixed, so it is difficult to actually directly screen an agonist or an antagonist for the target receptor protein.
  • secondary screening a test is performed to confirm whether the candidate compound actually inhibits the binding of human G protein-coupled receptor protein to ligand
  • mouse-derived receptor protein of the present invention eliminates the need for primary screening, and allows efficient screening of compounds that inhibit the binding between ligand and G-protein-coupling receptor protein. Can be. Furthermore, whether the screened compound is an agonist or an antagonist can be easily evaluated.
  • the receptor protein of the present invention used in the screening method of the present invention may be any as long as it contains the above-described receptor protein of the present invention.
  • Cell membrane fractions of mammalian organs containing proteins and the like are preferred. However, especially human organs are not available. Because it is extremely difficult, a mouse-derived receptor protein expressed in large amounts using a recombinant or the like is suitable for screening.
  • the above method is used to produce the receptor protein of the present invention and the like, but it is preferable to express the DNA of the present invention in mammalian cells and insect cells.
  • the DNA fragment encoding the protein portion of interest cDNA is used, but it is not necessarily limited thereto.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment In order to introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and to express them efficiently, the DNA fragment must be a nucleopolyhedron belonging to a baculovirus using an insect as a host.
  • NPV polyhedros is virus
  • NPV polyhedrin promoter promoter derived from SV40, retrovirus promoter overnight, metamouth thionine promoter, human heat shock promoter, cytomegalo It is preferable to incorporate it downstream such as a virus promoter and an SR promoter. Inspection of the amount and quality of the expressed receptor can be carried out by a known method, for example, as described in the literature [Namb i, P. et al., The Journal of Biological Biology Chemistry, vol. , Pp. 19555-19559, 1992].
  • the “receptor protein or the like of the present invention” used in the screening method of the present invention may be a receptor protein or the like purified according to a known method, a cell containing the receptor protein or the like, a cell membrane containing the receptor protein or the like. Any of fractions may be used.
  • the cells When cells containing the receptor protein or the like of the present invention are used in the screening method of the present invention, the cells may be immobilized with daltaraldehyde, formalin, or the like. Immobilization can be performed according to a known method. As the “cells containing the receptor protein or the like of the present invention” and the “cell membrane fraction”, those described in the method for determining a ligand of the present invention are used.
  • the amount of the receptor protein in the cell or membrane fraction containing the receptor protein or the like is preferably 10 3 to 10 molecules, more preferably 10 5 to 10 7 molecules per cell. It is.
  • an appropriate receptor protein fraction and a labeled ligand are required. is there.
  • the receptor protein fraction is preferably a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction activity and the like.
  • labeled ligand a labeled ligand, a labeled ligand analog compound, or the like is used.
  • ligands labeled with [ 3 H], [ 125 I], [ 14 C], [ 35 S] and the like are used.
  • a cell or a membrane fraction of the cell containing the receptor protein of the present invention is used.
  • the buffer is not particularly limited as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a tris-HCl buffer.
  • a surfactant such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, or dexcholate may be added to the buffer for the purpose of reducing non-specific binding.
  • a protease inhibitor such as PMSF, leptin, E-644 (manufactured by Peptide Research Institute), or peptide suptin may be added to the buffer for the purpose of suppressing the degradation of the receptor and ligand by the protease. .
  • a fixed amount (5000-500000 cpm) of the labeled ligand was added to 0.01 to 10 ml of the Recept Yuichi protein preparation thus obtained, and 4 to ⁇ -1 at the same time. M test compounds are allowed to coexist.
  • a large excess of unlabeled ligand was used instead of labeled ligand to determine the amount of non-specific binding (NSB). Coexist.
  • the reaction between the receptor protein preparation and the test compound or labeled ligand is performed at about 0 ° C to 50 ° C, preferably about 4 ° C to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 hours. It takes from a minute to three hours.
  • the reaction solution is filtered through a glass fiber filter paper, etc., and the glass fiber filter paper is washed with an appropriate amount of buffer (a buffer similar to the receptor protein sample). Then, the radioactivity remaining on the glass fiber filter paper is measured by a liquid scintillation counter. Measure with one or T-counter. Using the obtained measurement value, when the count (B. One NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B.) when there is no antagonist is 100%, the specific binding amount ( For example, a test compound having a B-NSB) of 50% or less can be selected as a candidate substance capable of competitive inhibition.
  • buffer a buffer similar to the receptor protein sample
  • the cell stimulating activity for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, cell Activity or suppression that promotes intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. Activity
  • the cell stimulating activity can be measured by known methods or using a commercially available measurement kit.
  • cells containing the 'receptor protein of the present invention and the like are cultured on a multi-well plate or the like. Before performing screening, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Then, the generated product is quantified according to each method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to the presence of a degrading enzyme contained in cells, the assay may be performed by adding an inhibitor against the degrading enzyme. Good. In addition, activities such as inhibition of CAMP production can be detected as production inhibitory effects on cells whose basic production has been increased by forskolin or the like.
  • a substance for example, arachidonic acid
  • cells expressing an appropriate receptor protein are required.
  • the cell line is preferably a cell line having the natural receptor protein of the present invention or the like, or a cell line expressing the above-mentioned recombinant receptor protein or the like.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
  • a screening kit for a compound or a salt thereof that alters the binding property of the ligand to the receptor protein of the present invention or the like comprises a cell containing the receptor protein of the present invention, the receptor protein of the present invention, or And those containing the membrane fraction of cells containing the receptor protein of the present invention.
  • Examples of the screening kit of the present invention include the following.
  • the solution may be sterilized by filtration through a 0.45 m filter and stored at 4 ° C, or may be prepared at use.
  • receptions evening CHO cells expressing an protein of the present invention passaged 5 10 5 cells on a 12-well plate, 37 ° (:, which was cultured for 2 days in 5% C0 2, 95% ai r .
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between a ligand and the receptor protein of the present invention.
  • a) Cell stimulating activity via G protein-coupled receptors eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation ,
  • G protein-coupled receptors eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation ,
  • a so-called agonist against the receptor protein of the present invention (a so-called agonist against the receptor protein of the present invention), ( b) the compound having no cell-stimulating activity (so-called antagonist to the receptor protein of the present invention); (c) the ligand and the compound of the present invention.
  • a compound that enhances the binding force to the G protein-coupled receptor protein or (d) a compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention.
  • Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, Fermentation products and the like may be mentioned, and these compounds may be novel compounds or known compounds.
  • the agonist against the receptor protein or the like of the present invention has the same activity as the physiological activity of the ligand for the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug according to the ligand activity.
  • the antagonist to the receptor protein or the like of the present invention can suppress the physiological activity of the ligand to the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug for suppressing the ligand activity.
  • the compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the receptor protein of the present invention or the like. It is.
  • a compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for reducing the physiological activity of the ligand for the receptor protein or the like of the present invention. is there.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention can be made into various preparations in the same manner as in the case of the receptor protein of the present invention described above.
  • the preparations obtained in this way are safe and low toxic, so they can be administered, for example, to humans and mammals (eg, rats, puppies, higgs, bush, puppies, cats, dogs, sals, etc.). can do.
  • mammals eg, rats, puppies, higgs, bush, puppies, cats, dogs, sals, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • oral administration for example, about 0 mg / day for a cancer patient (body weight 60 kg).
  • 1-100 mg preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the dose of the compound or a salt thereof is usually, for example, about 0.01 in a cancer patient (body weight 60 kg) per day when an injection is intravenously injected.
  • -30 mg preferably about 0.1-20111, more preferably about 0.1-10111.
  • the receptor protein of the present invention is considered to play some important roles in vivo, such as central function, circulatory function, digestive function, and cardiac function. Therefore, a compound that alters the binding property between the receptor protein of the present invention and the ligand (agonist, angonist) or a ligand for the receptor protein of the present invention is dysfunctional of the receptor protein of the present invention. It can be used as a prophylactic and / or therapeutic agent for diseases related to the disease.
  • diseases associated with dysfunction of the receptor protein of the present invention include, for example, central illness (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.) and endocrine diseases (eg, hypertension, gonad dysfunction, thyroid gland) Dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, dyslipidemia, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer) , Cervical cancer, colon cancer, rectal cancer, etc.), and heart diseases (eg, angina pectoris, myocardial infarction, etc.).
  • central illness eg, depression, Alzheimer's disease, dementia, eating disorder, etc.
  • endocrine diseases eg, hypertension, gonad dysfunction, thyroid gland
  • Dysfunction eg.
  • pituitary dysfunction e.
  • metabolic diseases eg, diabetes, dyslipidemia, hyperlipidemia, etc.
  • cancer eg, non
  • the compound or ligand when used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, the compound or ligand may be used as the above-described receptor protein of the present invention.
  • Various preparations can be prepared in the same manner as described above.
  • prophylactic / therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS preparation specifically targeting an organ or tissue in which the receptor protein of the present invention is highly expressed.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and mammals (for example, rats, mice, puppies, higgs, bushes, cats, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the compound or its salt
  • the dose is, for example, usually about 0.01 to 30 mg, preferably about 0.1 to 30 mg per day for a cancer patient (body weight 60 kg) when an injection is injected intravenously. 20 mg, more preferably about C-10 mg. (9) Determination of the receptor protein of the present invention or its partial peptide or its salt
  • the antibody of the present invention can specifically recognize the receptor protein and the like of the present invention, it is used for quantification of the receptor protein of the present invention in a test solution, particularly for quantification by a sandwich immunoassay. can do. That is, the present invention, for example,
  • the present invention provides a method for quantifying the receptor protein of the present invention in a test solution, characterized in that:
  • one antibody is an antibody that recognizes the N-terminal of the receptor protein or the like of the present invention
  • the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention.
  • the detection by tissue staining or the like can be performed. You can do it.
  • the antibody molecule itself may be used, or F (at) ′) 2 , Fab ′, or Fab fraction of the antibody molecule may be used.
  • the assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the antigen amount (for example, the amount of receptor protein) in the test solution Any method can be used as long as it is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • a method for measuring the displacement may be used.
  • the measuring method for example, nephelometry, a competitive method, an immunometric method and a sandwich method are preferably used, but the sandwich method described later is particularly preferable in terms of sensitivity and specificity.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ I4 C] and the like are used.
  • the enzyme a stable enzyme having a large specific activity is preferable.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • insolubilizing an antigen or an antibody a method using physical adsorption or a chemical bond usually used for insolubilizing or immobilizing a protein or an enzyme is used.
  • the carrier used for physical adsorption include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in reverse order, may be performed simultaneously, or may be performed with a time delay.
  • the same labeling agent as described above is used.
  • Insolubilization is performed in the same manner as described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one type, and a mixture of two or more types of antibodies is used for the purpose of improving the measurement sensitivity, etc. You may.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction an antibody having a different binding site such as a receptor protein is preferably used. That is, the antibodies used in the primary reaction and the secondary reaction are, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is other than the C-terminal For example, it is preferable to recognize the N end.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • a competition method the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen, (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation)
  • the amount of any of B, F and the label is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as the antibody
  • B / F separation is performed using a polyethylene glycol, a liquid phase method using a second antibody against the above antibody, etc .
  • an immobilized antibody is used as the first antibody, or
  • a soluble antibody is used, and an immobilization method using an immobilized antibody as the second antibody is used.
  • an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated, or Then, the unreacted labeled antibody is allowed to bind to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. .
  • the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured.
  • laser nephrometry using laser scattering is preferably used.
  • the measurement system for the receptor protein of the present invention or a salt thereof may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and procedures in each method. For such technical considerations, it is possible to refer to reviews and written documents. Specifically, Hiroshi Irie
  • the receptor protein of the present invention or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.
  • the antibody of the present invention can be used for specifically detecting the receptor protein of the present invention or the like present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used for purifying the receptor protein and the like of the present invention, detection of the receptor protein of the present invention in each fraction at the time of purification, and receptor of the present invention in test cells It can be used for analyzing the behavior of Yuichi proteins. (10) A method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention or its partial peptide or a salt thereof, a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane Skull of Can be used for reading.
  • the quantitative determination of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.
  • Non-human mammals eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc .; more specifically, demented rats, obese mice, arteriosclerotic rabbits
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, drugs, anti-obesity drugs, etc.
  • physical strikes After a certain period of time (eg, immersion stress, electric shock, light / dark, low temperature, etc.), blood, or a specific organ (eg, brain, lung, colon), or tissue isolated from the organ, Alternatively, obtain cells.
  • the obtained organ, tissue, or cell is suspended in, for example, an appropriate buffer (eg, Tris-HCl buffer, phosphate buffer, Hase buffer, etc.), and the organ, tissue, or cell is broken. Then, a cell membrane fraction is obtained by using a surfactant (eg, Triton X_100 TM, Tween-20 TM, etc.), and further using a ⁇ method such as centrifugation, filtration, or column fractionation.
  • an appropriate buffer eg, Tris-HCl buffer, phosphate buffer, Hase buffer, etc.
  • a cell membrane fraction is obtained by using a surfactant (eg, Triton X_100 TM, Tween-20 TM, etc.), and further using a ⁇ method such as centrifugation, filtration, or column fractionation.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by crushing cells and obtained by a known method.
  • Cell crushing methods include crushing cells with a Potter-Elvehj em-type homogenizer, crushing method using a Warlinda blender or a polytron (Kinematica); sonication crushing method; French press A crushing method in which cells are ejected from a thin nozzle while pressurizing with, for example, is mentioned.
  • the cell membrane thus obtained can be fractionated by a centrifugal fractionation method such as a differential centrifugation method or a density gradient centrifugation method.
  • the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30000 rpm) for 30 minutes to 2 hours.
  • a cell membrane fraction can be obtained.
  • the cell membrane fraction contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.
  • the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.
  • Such a sandwich immunoassay can be performed in the same manner as described above, and western blot analysis can be performed by known means.
  • a given time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour Before or after 6 hours) or after a certain time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour to 24 hours), or simultaneously with the drug or physical stress.
  • the receptor protein of the present invention or a portion thereof in the cell membrane (Ii)
  • the test compound is mixed in the medium, and after culturing for a certain period of time (after 1 day to 7 days, preferably Is one day later ⁇ 3 After more favorable Mashiku after 2 days to 3 days), or the receptor protein of the present invention on the cell membrane can be carried out by quantifying the amount of the part base peptide.
  • non-human mammals for example, mice, rats, rabbits, sheep, higgs, bush, birds, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteriosclerosis rabbits
  • Drugs eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • obtain blood or a specific organ eg, heart, placenta, lung, etc.
  • the obtained organs, tissues or cells are cut into tissue sections according to a conventional method, and immunostained using the antibody of the present invention.
  • the receptor protein of the present invention or its partial peptide on the cell membrane can be quantitatively or qualitatively determined. You can check the quantity.
  • the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or its partial peptide in a cell membrane, and specifically, (a ) By increasing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, the cell stimulating activity via G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release) Activates or suppresses intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc. (B) reducing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. Is a compound that decrease the cell
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein of the present invention.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein or the like of the present invention.
  • the compound or a salt thereof obtained by using the screening method of the present invention may be prepared into various preparations in the same manner as in the case of the receptor protein of the present invention described above. Can be.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered to humans and mammals (for example, rats, mice, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.). can do.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the dose of the compound or a salt thereof is usually, for example, in the case of intravenous injection, for example, a cancer patient (body 4215
  • the amount is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day based on 75 kg (60 kg).
  • a preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein or its partial peptide of the present invention in the cell membrane.
  • the receptor protein of the present invention is as described above, for example, heart or heart. It is considered to play some important role in vivo, such as function. Therefore, a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention.
  • diseases associated with dysfunction of the receptor protein of the present invention include, for example, central illness (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.) and endocrine diseases (eg, hypertension, gonad dysfunction, thyroid gland) Dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, dyslipidemia, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer) , Cervical cancer, colon cancer, rectal cancer, etc.), heart disease (eg, angina pectoris, myocardial infarction, etc.).
  • central illness eg, depression, Alzheimer's disease, dementia, eating disorder, etc.
  • endocrine diseases eg, hypertension, gonad dysfunction, thyroid gland
  • Dysfunction eg.
  • pituitary dysfunction e.
  • metabolic diseases eg, diabetes, dyslipidemia, hyperlipidemia, etc.
  • cancer eg, non-
  • the compound when used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, the compound may be a compound of the present invention described above. / Various preparations can be prepared in the same manner as in the case of the septa-protein.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered to humans and mammals (for example, rats, mice, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.). can do.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the dose of the compound or a salt thereof is usually, for example, about 0.01% per day for a cancer patient (60 kg body weight) when an injection is intravenously injected. ⁇ 30 mg, preferably about 0.1-20 mg, more preferably about 0.1 to 10 mg.
  • the neutralizing activity of the antibody against the receptor protein of the present invention or its partial peptide or a salt thereof against the receptor protein or the like means the activity of inactivating the signal transduction function involving the receptor protein. I do. Therefore, the antibody has a neutralizing activity due to signal transduction associated with the receptor protein, for example, cell stimulating activity via the receptor protein (for example, arachidonic acid release, acetylcholine release, intracellular C release). a Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, c-1: activation of fos, reduction of pH, etc. Activity or inhibitory activity). Therefore, the antibody of the present invention having a neutralizing activity can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein.
  • transgenic animals expressing the receptor protein and the like of the present invention can be produced.
  • animals include mammals (for example, rats, mice, rabbits, sheep, sheep, pigs, rabbits, cats, dogs, monkeys, etc.) (hereinafter sometimes abbreviated as animals). Mice, egrets and the like are preferred.
  • the DNA of the present invention When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells.
  • a gene construct linked to the downstream of various promoters capable of expressing the DNA of the present invention derived from an animal having high homology with the DNA in animal cells for example, The present invention is characterized by microinjection into fertilized eggs of egrets. 4215
  • 77 DNA-introduced animals that produce high levels of protein and other proteins can be produced.
  • the promoter for example, a virus-derived promoter, a ubiquitous expression promoter such as metamouth thionein and the like are used, and a promoter of a gene specifically expressed in the heart is preferable.
  • the DNA-introduced animal of the present invention can be bred in a normal breeding environment as a DNA-bearing animal after confirming that the gene is stably maintained by crossing. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the introduced gene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring can be obtained. It can be propagated to carry the DNA.
  • the animal into which the DNA of the present invention has been introduced expresses the receptor protein of the present invention at a high level, it is useful as an agonist or an animal for screening an antagonist against the receptor protein of the present invention.
  • the DNA-introduced animal of the present invention can also be used as a cell source for tissue culture.
  • the receptor protein of the present invention can be obtained. Etc. can be analyzed.
  • Cells of a tissue having the receptor protein or the like of the present invention are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture such as those derived from brain or peripheral tissues. be able to. Also, by using the cells, for example, it is possible to select a drug that enhances the function of various tissues.
  • the receptor protein of the present invention is isolated and purified therefrom. 4215
  • a medicine containing an antisense polynucleotide (nucleic acid) '' can complementarily bind to the polynucleotide (eg, DNA) of the present invention and suppress the expression of the polynucleotide (eg, DNA).
  • the antisense polynucleotide of the present invention has low toxicity and can suppress the function of the protein of the present invention or the polynucleotide of the present invention (eg, DNA) in a living body. It can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of receptor protein.
  • the disease associated with the dysfunction of the receptor protein of the present invention includes, for example, central illness (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.), endocrine disease (eg, hypertension, gonad dysfunction, thyroid gland) Dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, dyslipidemia, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer) , Cervical cancer, colon cancer, rectal cancer, etc.), and heart diseases (eg, angina pectoris, myocardial infarction, etc.).
  • central illness eg, depression, Alzheimer's disease, dementia, eating disorder, etc.
  • endocrine disease eg, hypertension, gonad dysfunction, thyroid gland
  • Dysfunction eg.
  • pituitary dysfunction e.
  • metabolic diseases eg, diabetes, dyslipidemia, hyperlipidemia, etc.
  • cancer eg,
  • the antisense polynucleotide when used as the therapeutic or prophylactic agent, the antisense polynucleotide may be formulated in the same manner as in the case of the DNA encoding the receptor protein of the present invention described above. it can.
  • the product obtained in this way has low toxicity and is orally or parenterally administered to humans or mammals (eg, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered in a controlled manner.
  • the antisense polynucleotide can be administered as it is or together with a physiologically acceptable carrier such as an auxiliary for promoting ingestion, using a gene gun or a catheter such as a hydrogel gel catheter.
  • a physiologically acceptable carrier such as an auxiliary for promoting ingestion, using a gene gun or a catheter such as a hydrogel gel catheter.
  • the dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.
  • the antisense polynucleotide of the present invention is administered to an organ (eg, liver, lung, heart, When administered topically to the kidney (eg kidney), the dose is about 0.1 to 100 mg per day for an adult (body weight 60 kg).
  • the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
  • HONB 1-Hydroxy-5-norpolene-2,3-dicarboximide.
  • FIG. 1 shows the amino acid sequence of a mouse-derived novel G protein-coupled receptor protein TGR38 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel mouse-derived G protein-coupled receptor protein TGR38 of the present invention.
  • the nucleotide sequence of Primer 1 used in the PCR reaction in Example 1 below was set to SEQ ID NO: 5
  • FIG. 1 shows the nucleotide sequence of the 5 ′ untranslated region of the DNA encoding the novel mouse-derived G protein-coupled receptor protein TGR38 of the present invention.
  • Example 1 shows the nucleotide sequence of the 3 ′ untranslated region of the DNA encoding the novel mouse-derived G protein-coupled receptor protein TGR38 of the present invention.
  • the transformant, Escherichia coli T0P10 / pCR2.1-TGR38, obtained in Example 1 below has been used since April 19, 2001 at 1-1-1 Higashi, Tsukuba City, Ibaraki Pref.
  • the 6th (zip code 305-8566) Independent National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary Depositary No.
  • Example 1 (Cloning of cDNA encoding G protein-coupled receptor Yuichi protein from mouse brain and determination of nucleotide sequence)
  • PCR was carried out using two primers, Primer 1 (SEQ ID NO: 3) and Primer 1 (SEQ ID NO: 4).
  • the above cDNA 1, Advantage-GC2 Polymerase Mix (CLONTECH) l xl, Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4) 0.5 M each, dNTPs 200 iM, and Advantage-GC2 Po
  • a reaction solution 50 JL61 consisting of 10 l of the buffer attached to the merase Mix and GC Melt 51 was used.
  • the PCR reaction product was subcloned into the plasmid vector pCR2.1 (Invitrogen) according to the method described in the T0P0-TA cloning kit (Invitrogen).
  • the resulting plasmid vector was introduced into E. coli T0P10, and added to LB agar medium containing ampicillin to select a clone having cDNA.
  • a cMA sequence SEQ ID NO: 2
  • the novel G protein-combined receptor protein containing an amino acid sequence obtained by translating the cDNA sequence was designated as TGR38. Further, the transformant was named Escherichia coli TOP10 / pCR2.1-TGR38. Industrial applicability
  • the G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, a polynucleotide encoding the receptor protein or a partial peptide thereof are: (2) Acquisition of antibodies and antisera, (3) Construction of a recombinant receptor protein expression system, (4) Development of a receptor-binding atsushi system using the same expression system and drug candidate compounds Screening, ⁇ ⁇ ⁇ ⁇ drug design based on comparison with structurally similar ligands and receptors, ⁇ ⁇ probes in genetic diagnosis, ⁇ ⁇ reagents for creating PCR primers, 7 transgenics. It can be used for animal production or as a drug such as a gene prevention and treatment agent.

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Abstract

L'invention concerne une protéine d'origine murine et un polynucléotide codant pour cette protéine, pouvant être utilisés : (1) pour sélectionner un ligand de cette protéine ; (2) dans des agents préventifs et/ou des médicaments destinés au traitement de maladies associées à une hypofonction de ladite protéine ; (3) pour sélectionner un composé (un agoniste, un antagoniste, etc.) capable de modifier les propriétés de fixation de ladite protéine sur un ligand ; etc.
PCT/JP2002/004215 2001-04-27 2002-04-26 Nouvelle proteine des recepteurs couples aux proteines-g et adn associe WO2002088182A1 (fr)

Applications Claiming Priority (2)

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JP2001-132201 2001-04-27
JP2001132201 2001-04-27

Publications (1)

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WO2002088182A1 true WO2002088182A1 (fr) 2002-11-07

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004065960A1 (fr) * 2003-01-17 2004-08-05 Takeda Pharmaceutical Company Limited Nouveau procede de criblage
CN111443209A (zh) * 2020-03-26 2020-07-24 中国中医科学院医学实验中心 一种筛选非激动剂型PPARγ配体的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0600278A2 (fr) * 1992-11-13 1994-06-08 Mitsubishi Chemical Corporation Protéine et gène du récepteur de glutamate
EP0711830A2 (fr) * 1994-10-13 1996-05-15 Takeda Chemical Industries, Ltd. Récepteur de la galanine murin et humain
EP0872550A2 (fr) * 1997-04-15 1998-10-21 Smithkline Beecham Corporation Clone HACCH94 de cADN qui code pour un récepteur humain 7-transmembranaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0600278A2 (fr) * 1992-11-13 1994-06-08 Mitsubishi Chemical Corporation Protéine et gène du récepteur de glutamate
EP0711830A2 (fr) * 1994-10-13 1996-05-15 Takeda Chemical Industries, Ltd. Récepteur de la galanine murin et humain
EP0872550A2 (fr) * 1997-04-15 1998-10-21 Smithkline Beecham Corporation Clone HACCH94 de cADN qui code pour un récepteur humain 7-transmembranaire

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004065960A1 (fr) * 2003-01-17 2004-08-05 Takeda Pharmaceutical Company Limited Nouveau procede de criblage
CN111443209A (zh) * 2020-03-26 2020-07-24 中国中医科学院医学实验中心 一种筛选非激动剂型PPARγ配体的方法
CN111443209B (zh) * 2020-03-26 2022-12-20 中国中医科学院医学实验中心 一种筛选非激动剂型PPARγ配体的方法

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