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WO2005049855A1 - Procede permettant de determiner un ligand - Google Patents

Procede permettant de determiner un ligand Download PDF

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Publication number
WO2005049855A1
WO2005049855A1 PCT/JP2004/017519 JP2004017519W WO2005049855A1 WO 2005049855 A1 WO2005049855 A1 WO 2005049855A1 JP 2004017519 W JP2004017519 W JP 2004017519W WO 2005049855 A1 WO2005049855 A1 WO 2005049855A1
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Prior art keywords
protein
receptor
cells
plasmid
ligand
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Japanese (ja)
Inventor
Ryo Fujii
Kazunori Nishi
Masataka Harada
Shuji Hinuma
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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Publication of WO2005049855A1 publication Critical patent/WO2005049855A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • the present invention relates to a method for determining a ligand of a receptor protein (orphan receptor protein) for which a ligand has not been determined.
  • G proteins guanine nucleotide-binding proteins
  • TMRs seven-transmembrane receptor proteins
  • G protein-coupled receptor proteins are present on the surfaces of various functional cells of living cells and organs, and are used as physiological targets as molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. Plays an important role.
  • the receptor transmits a signal into a cell through 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 exist in various parts of the body, and regulate their physiological functions through their corresponding receptor proteins.
  • receptor proteins There are many unknown hormones, neurotransmitters, and other physiologically active substances in the living body, and many of the structures of their receptor proteins have not yet been reported. Further, Many of the known receptor proteins do not have a subtype.
  • Clarifying the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins is a very important tool for drug development.
  • the functions of receptor protein genes expressed in vivo must be elucidated and expressed in an appropriate expression system. It was necessary to make it happen.
  • reporter gene assay an in vitro screening test using a ligand-dependent transcriptional activation mechanism of a target protein of a receptor protein is known (Chen W, Shields TS, Stork PJ, Cone RD). , Anal Biochem. 1995 Apr 10; 226 (2): 349-54.).
  • a reporter gene such as a luciferase gene, which is an indicator of receptor protein activation ability, is bound downstream of the enhancer element as a target gene. Create a plasmid (reporter plasmid).
  • the reporter plasmid is introduced into cells expressing the receptor protein, and the luciferase gene (reporter gene) expression level when treated with the chemical substance is used as an index to activate the receptor protein of the chemical substance. Measure performance.
  • reporter gene access can be achieved by introducing a plasmid that expresses the receptor protein into the cells simultaneously with the reporter plasmid. Examples of using the above-mentioned reporter gene Atsusei for determining the ligand of an orphan receptor protein using a cAMP response element as an enhancer element have been reported in WO2002 / 84286 and the like. And the serum response distribution as an enhancer element. It is not known that the sequence was used. Disclosure of the invention
  • the G protein-coupled receptor is useful for searching for a new physiologically active substance (that is, a ligand) and for searching for an agonist or antagonist for the receptor using the signal transduction action as an index.
  • a physiological ligand that is, a ligand
  • These ligands, agonists or antagonists to the receptor can be expected to be used as preventive and / or therapeutic or diagnostic agents for diseases associated with dysfunction of G protein-coupled receptors.
  • a decrease or enhancement of the function of a G protein-coupled receptor in a living body based on a gene mutation of the receptor often causes some disease.
  • administration of an antagonist or agonist to the receptor can be applied to gene therapy by introducing the receptor gene into a living body (or a specific organ) or introducing an antisense nucleic acid against the receptor gene.
  • the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation in the gene, and the receptor gene is used to prevent diseases associated with dysfunction of the receptor and / or It can also be applied to therapeutic and diagnostic agents.
  • a so-called stable cell line which stably expresses the receptor
  • stable cell line which stably expresses the receptor
  • Screening also requires a combination of multiple measurements, and if there are multiple test compounds, it would be time-consuming and difficult to perform.
  • conventional methods for determining the search for ligands and the like include (1) limited cell lines that can be used, (2) time-consuming establishment of the cell lines, and (3) multiple measurement methods. Due to the combination, there was a problem that it became difficult to perform it when the number of specimens increased. In order to solve these problems, it is desired to develop a method that can use various cell lines and that can measure ligand activity in a short time.
  • the present inventors have conducted intensive studies and have found that a special cell line is required by using the reporter gene Atssei using a serum response sequence as an enhancer element for the ligand determination method of the orphan receptor protein. It was found that the signal could be detected, and that the ligand could be determined with extremely high sensitivity and ease. The present investigators conducted further research based on this finding, and completed the present invention.
  • a method for determining a ligand for the receptor protein which comprises measuring the activity of the protein;
  • a test compound (1) a plasmid in which DNA encoding a reporter protein is linked downstream of a promoter containing a serum response element, and (2) a receptor protein whose ligand has not been determined The method according to the above (1), wherein the activity of the reporter protein of the cell containing the DNA-containing plasmid is measured.
  • the plasmid is a plasmid containing a gene encoding GaoA, a plasmid containing a DNA encoding G1 and a plasmid containing a gene encoding Gy2; 3) The method described,
  • the plasmid is a plasmid in which a gene encoding a TATA-like promoter and a reporter protein is linked downstream of a serum response element.
  • FIG. 1 shows changes in luciferase activity when various G protein-coupled receptors were transiently expressed in HeLa cells together with pSRE-Luc, and the ligand of each receptor was added.
  • FBS indicates calf serum.
  • FIG. 2 shows the effect of ligand-stimulated increase of luciferase activity when G protein is co-expressed with pSRE-Luc and neuropeptide Y receptor.
  • ⁇ indicates that no G protein was added.
  • GoA, ⁇ 1, and ⁇ 2 indicate Go, which is a species of G protein; oA, G1, and Go / 2, respectively.
  • the open par indicated by None indicates the case where the ligand neuropeptide Y was not added, and the black par indicated by 100 nM indicates the case where neuropeptide Y was added to the medium to a concentration of 100 nM.
  • the promoter of the present invention comprising a serum response element in the presence of a test compound. And a reporter protein characterized by measuring the activity of a reporter protein in a cell that expresses a receptor protein whose ligand has not been determined.
  • the Gand determination method is described in detail below.
  • the “promoter” of “plasmid in which a DNA encoding a reporter protein is linked downstream of a promoter containing a serum response element” includes, for example, a TATA-like region (eg, SV40 promoter, CMV promoter, thymidine kinase promoter of HSV).
  • a TATA-like region eg, SV40 promoter, CMV promoter, thymidine kinase promoter of HSV.
  • the TATA-like region or TATA box included in etc. is used.
  • a TATA-like region (TATA-like promoter) derived from the thymidine kinase promoter of HSV is used.
  • reporter protein for example, luciferase, jS-galactosidase, GFP, alkaline phosphatase and the like are used.
  • Luciferase or the like is used.
  • plasmid examples include a plasmid in which a TATA-like promoter is downstream of a serum response element and a DNA encoding luciferase is downstream thereof.
  • pSRE-Luc Invitrogen
  • the “plasmid in which a DNA encoding a reporter protein is linked downstream of a promoter containing a serum response element” can be produced according to a known method.
  • receptor protein for which a ligand has not been determined means a receptor protein which is thought to function as a receptor but has no known ligand binding thereto. It may be referred to as "fan receptor protein.”
  • the receptor protein used in the present invention includes, for example, all cells of humans and other mammals (eg, guinea pig, rat, mouse, rabbit, pig, sheep, sheep, monkey, monkey, etc.) (eg, spleen cells, nerves, etc.).
  • mammals eg, guinea pig, rat, mouse, rabbit, pig, sheep, sheep, monkey, monkey, etc.
  • spleen cells, nerves, etc. eg, spleen cells, nerves, etc.
  • Cells glial cells, knee i3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophage, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, $ osteoclasts, bone cells, osteoblasts, osteoclasts Cells, mammary gland Cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells), blood cells, or any tissue in which these cells are present, such as the brain, parts of the brain (eg, olfactory bulb) , Acrosomal nucleus, basal cerebral sphere, hippocampus, thalamus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum,
  • the polynucleotide encoding the receptor protein used in the present invention includes a nucleotide sequence encoding the receptor protein used in the present invention described above.
  • the polynucleotide is RNA such as DNA or mRNA encoding the receptor protein used in the present invention, and 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. If single stranded, it may be the sense strand (ie, coding strand) or the antisense strand (ie, non-coding strand).
  • the DNA encoding the receptor protein used in the present invention may be any of genomic DNA, genomic DNA library, cDNA derived from the above cells or tissues, cDNA library derived from the above cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of batteriophage, plasmid, cosmid and phagemid. Alternatively, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a preparation of the whole RA or mRNA fraction from the above cells or tissues.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • DNA completely encoding the receptor protein used in the present invention (1) Amplification by PCR using a synthetic DNA primer having a partial nucleotide sequence of the receptor protein used in the present invention.
  • DNA containing a target gene incorporated into an appropriate vector is labeled with DNA encoding a part or all of the receptor protein used in the present invention or synthetic DNA.
  • the method of high predication is, for example, molecular cloning (
  • DNA base sequence substitution can be performed using PCR or a known kit, such as Mutan TM -Super Express Km (Takara Shuzo) or Mutan TM -K (Takara Shuzo), using the ODA-LA PCR method, Gapped duplex method, or Kunkel method. It can be carried out according to a known method such as, 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 by digesting with a restriction enzyme or adding a linker, if desired.
  • the DNA may have ATG as a translation initiation codon on the 5'-terminal side, and may have TAA, TGA or TAG as a translation termination codon on the 3, terminal side. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the vector for expressing the receptor protein used in the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA (for example, cDNA) containing DNA encoding the receptor protein of the present invention; ) The DNA fragment can be produced by ligating the DNA fragment downstream of a promoter in an appropriate expression vector.
  • Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), and plasmids derived from yeast (eg, pSH19, pSH15)
  • Examples include pacteriophage such as phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, pAl-11, pXTl, pRc / CMV, pRc / RSV, and pcDNAI / Neo.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRct promoter SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • CMV Promo It is preferable to use an SRa promoter or the like.
  • the selection Ma one force one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp r) And a neomycin resistance gene (hereinafter sometimes abbreviated as Neo r , G418 resistance).
  • dhfr gene dihydrofolate reductase
  • MTX metalhotrexate
  • Amp r ampicillin resistant gene
  • Neo r neomycin resistance gene
  • Animal cells are particularly preferred as hosts.
  • animal cells include human HeLa cells, monkey cells COS-7, Vero, CHO cells, CHO (dhfr ”) cells, mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL.
  • Cells, human HEK293 cells, rat RH7777 cells, human HeLa-S cells, mouse C2C12 cells, rat A-10 cells, and the like are used, and more preferably, human HeLa cells are used.
  • Transformation of animal cells is 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). be able to.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122 vol., 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], etc. Is used.
  • the pH is about 6-8.
  • Cultivation is usually carried out at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the G protein-coupled receptor protein of the present invention can be produced in the transformant cells.
  • a G protein-coupled receptor protein or the like is used.
  • WO 96/05302 EP-A-711831, EP-A-789076, EP-A-1103563, EP-A-1103562, JP-A-8-154682, JP-A-8-283295, JP-A-8-196278, JP-A-8-245697, JP-A-8-266280, JP-A-9-51795, JP-A-9-121865, JP-A-9-2388686,
  • JP-A-9-2388686 JP-A-9-2388686
  • Examples of “cells” of “cells that contain a plasmid linked to a reporter protein-encoding DNA downstream of a promoter containing a serum response element and express a receptor protein for which a ligand has not been determined” include, for example, Animal cells are used.
  • animal cells include human HeLa cells, monkey cells COS-7, Vero, CHO cells, CHO (dhfr ”) cells, mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells
  • Examples include human HEL293 cells, rat RH7777 cells, human HeLa-S3 cells, mouse C2C12 cells, rat A-10 cells, etc.
  • human HeLa cells are used.
  • a plasmid containing DNA encoding the receptor protein for which a ligand has not been determined may be introduced into the cell.
  • plasmids include, for example, a promoter for expressing the receptor protein in eukaryotic cells, a drug resistance gene (for example, an ampicillin resistance gene) used as a selectable marker when proliferating in prokaryotes, and the like.
  • a contained plasmid can be used.
  • the above plasmid can be produced according to a known method, and is introduced into a cell according to a known method.
  • plasmids containing DNA encoding the reporter protein downstream of the promoter containing the enhancer region include commercially available plasmids that can be introduced into eukaryotic cells. Any of the plasmids can be used. A plasmid containing an enzyme gene whose enzyme activity can be detected by a known method is preferably used.
  • Enhancers include, for example, those derived from viruses such as SV40 and Nopiroma virus. Enhancer, LTR of retrovirus, serum response element (SRE), cAMP response element (cAMP response element (CRE), AP-1 (activating protein), NF- / cB (kappa B cell nuclear element response element), NFAT (activated T cell nuclear factor response element), TPA (12-0-tetradacanoylpholbol-13-acetate) response element (TRE) and the like are used, and preferably, serum response element is used.
  • SRE serum response element
  • CRE cAMP response element
  • AP-1 activating protein
  • NF- / cB kappa B cell nuclear element response element
  • NFAT activated T cell nuclear factor response element
  • TPA (12-0-tetradacanoylpholbol-13-acetate response element
  • the promoter for example, the TATA-like region or TATA pox contained in the SV40 promoter, the CMV promoter, the thymidine kinase promoter of HSV and the like are used, and the TATA-like region derived from the thymidine kinase promoter of HSV (TATA-like Promoter).
  • the cells may express two or more (preferably two to three) receptor proteins.
  • a G protein-coupled receptor protein is preferably used.
  • RFRP RF amide-related peptide
  • MelRl melatonin receptor 1
  • NPY5 neuropeptide Y receptor 5
  • AJ apelin receptor
  • NPGPR neuropeptide FF receptor
  • CBD1 Cannabinoid receptor 1
  • D2 dopamine receptor 2
  • GPR40 Gi / q-coupled long-chain fatty acid receptor
  • Gq-coupled norlepinephrine receptor ⁇ 1 ⁇ ( ⁇ ) and the like are used.
  • Similar characteristics include, for example, the expression level of a reporter protein when two or more receptor proteins are independently expressed.
  • the characteristics of each receptor protein can be distinguished by using the basal expression level of the reporter protein as an index when two or more receptor proteins are independently expressed.
  • the reporter proteins when expressing ligands by expressing two or more types of receptor proteins for which ligands have not been determined, the reporter proteins are classified into low, medium, and clearly high basal expression levels in advance. It is desirable to keep it. This is because, for example, there are two types of receptor proteins, one with high basal expression of reporter protein and the other with low basal expression of reporter protein. This is because, when a receptor protein of the same type is expressed, an increase in the reporter protein expression level becomes difficult to see when the latter is hit with a ligand. Therefore
  • a receptor protein with a high basal expression level of a reporter protein and a low receptor protein, and (2) a basal expression level of the reporter protein is comparable. It is preferable to express the receptor proteins in combination.
  • TGR-1 Japanese Patent Laid-Open No. 2002-078492
  • (a) Cell stimulating activity via receptor proteins eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, C-fos activation of any transcription factor, activity or inhibiting activity for promoting reduction, etc.
  • P H a compound having an expression induction activity, etc.
  • the cell-stimulating activity C
  • a compound that enhances the binding strength between the biologically active substance and the receptor protein used in the present invention or
  • a compound that enhances the binding strength between the biologically active substance and the receptor protein used in the present invention a compound or the like that reduces the amount.
  • the ligand may be a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, or the like.
  • the “test compound” includes known ligands (for example, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxotocin) , PACAP (eg, PACAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (passoactive 'intestinal' peptide), somatostatin, dopamine, motilin, Amylin, bradykinin, CGRP (force / lecitonin 'gene • Related peptide), leukotriene, pancreastatin, prostaglandin, tropoxane, adenosine, adrenaline, chemokine super Family (e.g., IL-
  • PACAP
  • ⁇ a cell containing a plasmid in which a DNA encoding a reporter protein is ligated downstream of a promoter containing a blood cell response element in the presence of a test compound and expressing a receptor protein whose ligand has not been determined
  • a promoter containing a blood cell response element in the presence of a test compound and expressing a receptor protein whose ligand has not been determined
  • a test compound In the presence of a test compound, (1) a plasmid in which a DNA encoding a reporter protein is linked downstream of a promoter containing a serum response element, and (2) a G protein ⁇ -subunit, ⁇ -subunit, and V-subunit Cells containing a plasmid containing DNA encoding one to three species and expressing a receptor protein (preferably a G protein-coupled receptor protein) whose ligand has not been determined are cultured, and the activity of the reporter protein is determined. Measure.
  • a test compound in the presence of a test compound, (1) a plasmid in which a DNA encoding a reporter protein is linked downstream of a promoter containing a serum response element, ( 2 ) a receptor protein whose ligand has not been determined (preferably a G protein ( 3 ) Plasmid containing DNA encoding 1-3 types selected from ⁇ -subunit, subunit and ⁇ -subunit of G protein The cells containing are cultured, and the activity of the reporter protein is measured. in this case,
  • the amount ratio of the plasmid containing DNA encoding the G protein ⁇ subunit is, for example, about 1 to 6: 4 to 10: 1: 1: 1, preferably 1: 9: 1: It is about 1: 1.
  • the activity of the reporter protein may be measured according to a known method, for example, a method described in an instruction manual attached to a commercial product.
  • the "G protein ⁇ subunit" for example, GaoA, Gatr, Gate, G o; olf, Gas, GaoB, Lr oC ⁇ ail, a ⁇ 2 ⁇ i3 ⁇ J ⁇ 3 ⁇ 4 Z, ⁇ aq s Gall, G a 12 , Gal3, Gal6 (Gal5) and the like.
  • G aoA GaoA, Gatr, Gate, G o; olf, Gas, GaoB, Lr oC ⁇ ail, a ⁇ 2 ⁇ i3 ⁇ J ⁇ 3 ⁇ 4 Z, ⁇ aq s Gall, G a 12 , Gal3, Gal6 (Gal5) and the like.
  • G aoA Preferably, it is G aoA.
  • the “G protein / 3 subunit” includes, for example, G; 31, G ⁇ 32, G ⁇ 33, G5 and the like. Preferably, it is G31 or the like.
  • the “G protein ⁇ subunit” includes, for example, Gyl, Gy2, G73, Gy4, Gy5, Gy7, Gy8, Gy9, Gyl0, Gyl, Gyl2, Gyl3, and the like. Preferably it is 2.
  • the cells are seeded on a 96-well plate, and cultured once in, for example, DMEM containing 10% fetal bovine serum.
  • DMEM fetal bovine serum
  • a plasmid in which DNA encoding the reporter protein is ligated downstream of the promoter protein expression plasmid and the promoter containing the serum response element is simultaneously introduced into the cells, and the cells are further cultured in culture, whereby the receptor protein is intracellularly expressed. The quality is transiently expressed.
  • the test compound is added. After a certain period of incubation, the cells are lysed and the activity of the reporter protein is measured.
  • G protein-coupled receptor protein cDNAs prepared by known methods, namely Gi-coupled (RF amide-related peptide (RFRP) receptor (0T7T022), melatonin receptor 1 (MelRl), Peptide Y receptor 5 (NPY5), apelin receptor (APJ), neuropeptide FF receptor (NPGPR), cannabinoid receptor 1 (CB1), dopamine receptor 2 (D2), Gi / q conjugated long-chain fatty acid receptor ( GPR40), a DNA encoding the Gq-coupled norepinephrine receptor ⁇ ⁇ ⁇ ( ⁇ ⁇ ) was converted to ⁇ 0_111 ⁇ (Biochem.
  • RFRP RF amide-related peptide
  • MelRl melatonin receptor 1
  • NPY5 Peptide Y receptor 5
  • AJ apelin receptor
  • NPGPR neuropeptide FF receptor
  • CB1 cannabinoid receptor 1
  • D2 dopamine receptor 2
  • G protein-coupled receptor protein expression plasmid and reporter HeLa cells were used as the host cells into which rasmid was to be introduced.
  • the cells were seeded on a 96-well Atsushi plate (C0STAR 3904) at 16,000 cells / well at a culture volume of 100 ⁇ l and cultured once.
  • the medium used was DMEM (Invitrogen) supplemented with 10% fetal calf serum and 1% MEM nonessential amino acids solution (DMEM / NEM).
  • Plasmid of the vector pAKKO-111H was added to 240 ⁇ of Opti-MEM-1 (GibcoBRL) at a ratio of 6 ⁇ l and 4 ⁇ l of the reporter plasmid. Equally mix this with 240 1 Opti-MEM-1 plus 10 ⁇ l of Lipofectamine TM 2000 Reagent (Invitrogen) and mix according to the method described in the manual attached to Lipofectamine TM 2000. And a complex of plasmid were formed.
  • the ligand substance dissolved in the same medium was added to the cells in each well. Incubation was performed for 6 hours after the addition of the sample to induce the transcription and translation of a reporter gene derived from intracellular signal transduction induced by agonist activity of the ligand via the receptor. After completion of the incubation, the medium in each well was removed, and Pitka Gene LT2.0 (Toyo Ink), a substrate for measuring luciferase activity, was added in 50 portions.
  • Pitka Gene LT2.0 Toyo Ink
  • GPR40 a long-chain fatty acid receptor, contains gamma-linolenic acid ( ⁇ -LA), RFamide-related peptide (RFRP) receptor ⁇ 7-022, RFRP; melatonin receptor 1 contains melatonin, Neuropeptide Y ( ⁇ ) is used for the peptide Y receptor NPY5, apelin (apelin) is used for the aperin receptor APJ, and a new mouth peptide FF (F-8-F) is used for the NPGPR receptor.
  • Gy2 expression vector Plasmids were converted to G aoA alone or a combination of human G j31 and human G y2, or a combination of human G aoA and human G) 31 and human G 0/2 in HeLa cells, respectively. Introduced. Next, according to the method of Example 2, neuropeptide Y (100 nM) was added as a ligand substance to the cells, and the same operation as in Example 2 was performed, and then the luciferase activity was measured.
  • the method for determining the ligand of a receptor protein for which a ligand has not been determined according to the present invention can use various cell lines that express the receptor protein, does not require a special cell line, and enables signal detection.
  • the ligand can be determined easily, quickly and with high sensitivity.

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Abstract

La présente invention concerne un procédé permettant de déterminer un ligand pour une protéine réceptrice pour laquelle le ligand n'est toujours pas déterminé. Plus spécifiquement, cette invention concerne un procédé permettant de déterminer un ligand pour une protéine réceptrice, qui se caractérise par des étapes consistant à doser l'activité d'une protéine rapporteuse d'une cellule contenant un plasmide présentant un ADN codant pour la protéine rapporteuse reliée en aval d'un promoteur présentant une séquence sensible au sérum et exprimant la protéine réceptrice, dont le ligand est toujours indéterminé, en présence d'un composé d'essai.
PCT/JP2004/017519 2003-11-21 2004-11-18 Procede permettant de determiner un ligand Ceased WO2005049855A1 (fr)

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JP2003393056 2003-11-21
JP2003-393056 2003-11-21

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WO2005049855A1 true WO2005049855A1 (fr) 2005-06-02

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002084286A1 (fr) * 2001-04-12 2002-10-24 Takeda Chemical Industries, Ltd. Procede de criblage

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002084286A1 (fr) * 2001-04-12 2002-10-24 Takeda Chemical Industries, Ltd. Procede de criblage

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DUTT P. ET AL: "Activated Galphaq family members induce Rho GTPase activation and Rho-dependent actin filament assembly", FEBS LETT., vol. 531, 2002, pages 565 - 569, XP004392705 *
NIU J. ET AL: "G Protein betagamma Subunits Stimulate p114RhoGEF Guanine Nucleotide Exchange Factor for RhoA and Rac1", CIRC. RES., vol. 93, no. 9, October 2003 (2003-10-01), pages 848 - 856, XP002985942 *
ZHU K. ET AL: "Sphingosylphosphorylcholine and Lysophosphatidylcholine Are Ligands for the G Protein-coupled Receptor GPR4", J. BIOL. CHEM., vol. 276, no. 44, 2001, pages 41325 - 41335, XP002974915 *

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