WO2001014883A1 - Methode de criblage - Google Patents

Methode de criblage Download PDF

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Publication number
WO2001014883A1
WO2001014883A1 PCT/JP2000/005639 JP0005639W WO0114883A1 WO 2001014883 A1 WO2001014883 A1 WO 2001014883A1 JP 0005639 W JP0005639 W JP 0005639W WO 0114883 A1 WO0114883 A1 WO 0114883A1
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Prior art keywords
receptor protein
orphan receptor
cell
cells
test compound
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PCT/JP2000/005639
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English (en)
Japanese (ja)
Inventor
Shuji Hinuma
Masaki Hosoya
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Takeda Chemical Industries, Ltd.
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Priority to AU67264/00A priority Critical patent/AU6726400A/en
Publication of WO2001014883A1 publication Critical patent/WO2001014883A1/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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention screens a high-concentration test compound using the activation of orphan receptor protein-expressing cells as an index, and utilizes the common structure of the test compound having agonist activity to produce an orphan receptor protein.
  • the present invention relates to a method for efficiently screening a compound that promotes or inhibits the function of a protein, and a method for determining a (endogenous) ligand of an orphan receptor protein using the common structure.
  • physiologically active substances such as hormones and neurotransmitters exert their effects by binding to receptor molecules present on the cell surface, and regulate various life phenomena. Searching for a substance that supplements, enhances, or inhibits the action of these physiologically active substances is one of the main means for the research and development of new pharmaceuticals. It is extremely important to understand the properties of body molecules. Recent advances in molecular biological techniques have made it possible to analyze the receptors of many physiologically active substances at the molecular level. A group of such receptor molecules with a common structural feature that penetrates the cell membrane seven times is known and is called a seven-transmembrane receptor. They are also called G protein-coupled receptors because they are coupled to intracellular signaling systems via GTP-binding proteins (G proteins). Ligands of the seven-transmembrane receptor are diverse, including proteins, peptides, amines, amino acids, nucleotides, nucleosides, eicosanoids, phospholipids, odorants, and light.
  • orphanin FQ which has studied the known ligands or their analogs as candidate ligands, has actually identified the endogenous ligand. / noc i cept in (Meunier, J. -C. Nature 393: 211-212, 1998), but the structure of the ligand is estimated only from the structural similarity of the known ligand or its analog. There is a limit to In many cases, endogenous ligands are identified by purification using the activation of the signal transduction system of orphan receptor protein-expressing cells as an index (Sakurai, T. et al. Cell 92: 573- 585, 1998; Hinuma, S.
  • the signal transduction system via the seven-transmembrane receptor is not unique, and it is necessary to screen several Atsui systems in parallel to detect the activity derived from endogenous ligands.
  • ligands for the seven-transmembrane receptor are diversified, and it has been difficult to rationally select a sample as a ligand candidate to be used for the assay.
  • the present inventors have conducted intensive studies in order to solve the above problems,
  • test compound preferably at a high concentration
  • test compound preferably at a high concentration
  • test compound (a) was brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction, and the test compound (a) was brought into contact with a cell that does not express the orphan receptor protein or its cell membrane fraction.
  • Test compound is added to orphan receptor protein-expressing cells or its cell membrane fraction.
  • test compound (a) was brought into contact with (a) and when the test compound (a) was brought into contact with cells not expressing the orphan receptor protein or its cell membrane fraction, the respective cell stimulating activities were measured.
  • FIG. 1 shows a comparison diagram of the commonality of amino acid sequences in Example 1.
  • Phenylalanine (F) and tyrosine (Y) have very similar steric structures, so they are similarly boxed.
  • orphan receptor protein means a protein whose ligand is unknown, and includes unknown proteins in addition to known proteins.
  • orphan receptor proteins include, for example, the FM-3 receptor protein (Tan, C. P et al, Genomi cs 52, 223-229) or the mas receptor used in Example 1 described below. Protein (Young D. et al., Proc. Natl. Acad. Sci. USA, 85, 5339-5342, 1988), etc., as well as those listed in the orphan receptor database of Swiss- plot And so on.
  • the orphan receptor protein used in the present invention may form a salt.
  • the salt of the “orphan receptor protein” include physiologically acceptable bases (eg, alkali metals) and acids (organic compounds). Acid, inorganic acid) Physiologically acceptable acid addition salts are preferred.
  • 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) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succin
  • the DNA encoding the orphan receptor protein used in the present invention may be any DNA containing the DNA encoding the orphan receptor protein.
  • genomic DNA genomic DNA libraries, human warm-blooded animals (eg, guinea pigs, rats, mice, chicks, egrets, bushes, sheep, swords, monkeys, etc.) (eg, retinal cells, hepatocytes, Spleen cells, neuronal cells, glial cells, kidney / 3 cells, bone marrow cells, mesangial cells, Langerens cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, Immune cells (eg, macrophage, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells Osteoblasts, osteoclasts, mamm
  • DNA encoding the orphan receptor protein used in the present invention can also be produced by the following genetic engineering techniques.
  • the DNA library was synthesized by a PCR method known per se using a synthetic DNA primer having a partial nucleotide sequence of orphan receptor protein. Amplification of the target DNA from the first place, or the DNA incorporated into an appropriate vector, for example, labeled using a DNA fragment or a synthetic DNA having a partial or entire region of the orphan receptor protein. Sorting can be performed by hybridization. The hybridization is performed according to, for example, the method described in Molecular Cloning (2nd ed .; J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using a commercially available library, follow the method described in the attached instruction manual.
  • the DNA encoding the cloned orphan receptor protein can be used as it is, or as desired, after digestion with a restriction enzyme or addition of a linker.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation start codon and translation stop codon can be added using an appropriate synthetic DNA adapter.
  • the DNA encoding the orphan receptor protein (A) can be expressed by the following method.
  • the expression vector of the orphan receptor protein is, for example, It can be produced by cutting out a DNA fragment of interest from DNA encoding an an receptor protein, and ligating the DNA fragment downstream of a promoter in an appropriate expression vector.
  • Escherichia coli-derived plasmids eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pCl94
  • yeast-derived plasmids eg, pSHl 9 pSHl 5
  • bacteriophage such as ⁇ phage
  • animal viruses such as retrovirus, vaccinia virus, and baculovirus are used.
  • the promoter used may be any promoter suitable for the host used for gene expression.
  • promoters derived from SV40, a retrovirus promoter, a metamouth thionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRa promoter, etc. Available. If the host is Escherichia, Trp promoter, T7 promoter overnight, lac promoter, recA promoter, APL promoter, 1 pp promoter, etc.If the host is Bacillus, SPO When the host is an enzyme such as 1 promoter, SP ⁇ 2 promoter, pen P promoter, etc., preferred are PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter and the like.
  • the host is an insect cell
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the host is preferably an animal cell or an insect cell in order to measure the cell stimulating activity via the orphan receptor protein.
  • the expression vector may further include 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, if desired. Can be used.
  • selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance] and ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ⁇ ) And a neomycin-resistant gene (hereinafter sometimes abbreviated as Neo, G418-resistant).
  • dh fr dihydrofolate reductase
  • MTX metalhotrexate
  • Amp ⁇ ampicillin resistance gene
  • Neo neomycin-resistant gene
  • a signal sequence suitable for the host is added to the N-terminal side of the polypeptide or its partial peptide.
  • the host is a bacterium belonging to the genus Escherichia
  • the phoA-signal sequence and the O-immediate A / signal sequence are included.
  • insulin signal sequence, a-in yellow ferron Signal sequence, antibody molecule, signal sequence, etc. can be used respectively.
  • a transformant can be produced using the vector containing DNA encoding the orphan receptor protein thus constructed.
  • a bacterium belonging to the genus Escherichia for example, a bacterium belonging to the genus Escherichia, a bacterium belonging to the genus Bacillus, a yeast, an insect or an insect cell, an animal cell, or the like is used.
  • the insect cell or the animal cell is preferably used.
  • Escherichia examples include Escherichia coli (Escherichia col 0 K12 • DH1 [Prosessing's of the National Academy of Sciences] of the United States (Pro Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal-Robb 'Molecular' Noology 1] (Journal of Molecular Biology)], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39] , 440 (1954)].
  • Bacillus bacteria examples include, for example, Bacillus subtilis M1114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95 , 87 (198 4)].
  • yeast examples include Saccharomyces cerevisiae) AH22, AH22R—, NA87-11A, DKD—5D, 20B-12 and the like are used.
  • insects for example, silkworm larvae are used [Maeda et al., Neycha (
  • insect cells for example, when the virus is Ac NPV, a cell line derived from a larva of night rob moth (Spodopter.a frugiperda cell; S f cell), and the midgut of Trichoplusia ni
  • MG1 cells derived therefrom High Five cells derived from Trichoplusia ni eggs, cells derived from Mamestra brasskae or cells derived from Estigmena acrea are used.
  • viruses When the virus is BmNPV, a cell line derived from silkworm (Bombyxmori N; BmN cell) is used.
  • Sf cells include, for example, Sf9 cells (ATCC CRL1711), Sf21 cells [Vaughn, J. L et al., In Vitro, Vol.
  • animal cells examples include monkey COS-7 cells, Vero cells, Chinese nose, muster cells CH ⁇ , DHFR gene deficient Chinese hamster cells CHO (dh fr-CH ⁇ cells), mouse L cells, mouse 3T3 cells, mouse Myeloma cells, human HEK293 cells, human FL cells, C127 cells, BALB 3T3 cells
  • Transformation of animal cells is performed, for example, according to the method described in Virology, 52, 456 (1973).
  • Methods for introducing orphan receptor protein expression vectors into cells include, for example, the lipofection method (Felgner, PL et al. Processing's 'the' National Academy of Sciences, Obs. U.S.A. (Proceedings of The National Academy of Sciences of The United States of America), 84, 7413 (1987)), calcium phosphate method [Graham, FL and van der Eb, AJ Virology, 52, 456- 46 p. 7 (1973)], electroporation [Nuemann, E. et al., Empo J. (EMBO J.), 1, 841-845 (1982)], and the like.
  • a method for stably expressing an orphan receptor protein using animal cells there is a method of selecting, by clonal selection, cells in which the expression vector introduced into the animal cell is integrated into the chromosome. . Specifically, a transformant is selected using the above selection abilities as an index. Furthermore, by repeatedly performing clonal selection on the animal cells obtained using the selectable marker in this manner, a stable animal cell line having high expression ability of orphan receptor protein can be obtained. You. When the dh fr gene or the dh fr gene is used as a selection marker, the MTX concentration is gradually increased, and the cells are cultured. By selecting resistant strains, the DNA encoding the orphan receptor protein together with the dh fr gene can be transferred to the cells. It can also be amplified in situ to obtain higher expressing animal cell lines.
  • a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nitrogen sources, inorganic substances and others. As a carbon source
  • Nitrogen sources such as glucose, dextrin, soluble starch, sucrose, etc.
  • Inorganic or organic substances such as ammonium salts, nitrates, corn chip liqueur, peptone, casein, meat extract, soybean meal, potato extract, etc.
  • Inorganic substances such as calcium chloride, sodium dihydrogen phosphate, and magnesium chloride And the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • Examples of a medium for culturing Escherichia sp. include, for example, M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, Journal of Experiments in Molecular Genetics, 431). -433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3) 3-indolylacrylic acid can be added in order to make the promoter work efficiently.
  • cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • culturing is usually performed at about 30 to 40 "C for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
  • the medium used is Grace's Insect.
  • a solution to which an additive such as 0% serum is appropriately added is used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4.
  • Culture is usually performed at about 27 ° C for about 3-5 days.
  • the pH is preferably about 6-8. Culture is usually performed at about 30 to 40 for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • DMEM medium containing dialysed fetal serum containing almost no thymidine.
  • the cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se.
  • Methods for crushing cells include crushing cells with a Potter-Elvehjem homogenizer, crushing with a Pelling Blender and Polytron (Kinematica), crushing with ultrasonic waves, and narrowing the cells while pressing with a French press. Crushing by ejecting from For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for 30 min. Centrifuge for 1 minute to 2 hours, and use the resulting precipitate as the membrane fraction.
  • the membrane fraction is rich in the expressed orphan receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the orphan receptor protein in the cell or membrane fraction containing the orphan receptor protein is preferably 10 to 10 molecules per cell, and more preferably 10 to 10 molecules per cell.
  • the cells that do not express the orphan receptor protein or the cell membrane fraction thereof are the cells listed as the above host cells, and do not express the orphan receptor protein.
  • Means As the cell membrane fraction those similar to the above can be used.
  • test compound or “test compound (a)” refers to, for example, natural / non-natural peptide, natural / non-natural protein, natural / non-natural non-peptide compound, synthetic Compounds, natural and non-natural fermented products.
  • test compound or the peptide, protein, compound or fermentation product used in the test compound (a) may form a salt, and these salts include a physiologically acceptable base (eg, an alkali metal). And the like, and salts with acids (organic acids and inorganic acids), and especially preferred are physiologically acceptable acid addition salts.
  • a physiologically acceptable base eg, an alkali metal
  • acids organic acids and inorganic acids
  • 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) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • 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
  • Tartaric acid citric acid, malic acid, oxalic acid
  • benzoic acid methanesulfonic acid, benzenesulfonic acid
  • test compound (a) When a test compound (specifically, test compound (a)) is brought into contact with an orphan receptor protein-expressing cell or its cell membrane fraction, the test compound is added to a cell that does not express an orphan receptor protein or its cell membrane fraction.
  • the respective cell stimulating activities include, for example, (a) fluctuation of extracellular pH, (b) release of arachidonic acid, (c) release of acetylcholine, ( d) intracellular Ca release, (e) intracellular cAMP fluctuation, (f) intracellular cGMP fluctuation, (g) inositol phosphate production, (h) cell membrane potential fluctuation, (i) cell phosphorylation of the inner proteins (] ') Gee 1: 0 5 activation,
  • (k) GTPTS binding, (1) reporter gene expression, and the like as indices it can be measured according to a known method or using a commercially available measurement kit.
  • a measurement method using the change in extracellular pH as an index is preferably used.
  • first, orphan receptor protein-expressing cells or their cell membrane fractions, and cells that do not express orphan receptor proteins or their cell membrane fractions The cultures are separately cultured in a multiwell plate or the like. Before measuring cell stimulating activity, replace the cells with fresh medium or an appropriate buffer that is not toxic to cells, and add the test compound (specifically, test compound (a)) to each cell. After incubation for a certain period of time, the cells or their cell membrane fractions are extracted or the supernatant is collected, and the resulting products are quantified according to the respective methods.
  • an inhibitor for the degrading enzyme may be added to perform the assay.
  • activities such as inhibition of cAMP production can be detected as an effect of inhibiting production of cells whose basal production has been increased by forskolin or the like or a cell membrane fraction thereof.
  • the transformant orphan receptor protein-expressing cell may be a stable expression cell or a transient expression cell.
  • the cell stimulating activity should be measured at the highest concentration as far as the specific reaction of the cell can be identified so that the weak agonist activity can be detected. Is preferred.
  • the high concentration of this case usually 1 0 one 8 M ⁇ 1M, preferably refers to 10- 6 M ⁇ 10- 2 M.
  • Examples of the test compound include those similar to those described in the above (C).
  • test compound specifically indicates “test compound (a)”.
  • the extracellular pH at which orphan receptor protein-expressing cells or their cell membrane fractions change in response to a test compound having agonist activity is measured using a cytosensor (Cy to sensor) device (Molecular Devices, etc.) To do Therefore, the cell stimulating activity can be measured.
  • cytosensor Cy to sensor
  • Molecular Devices, etc. Molecular Devices, etc.
  • the culture medium Stabilize pH.
  • an RPMI 1640 medium manufactured by Molecular Devices
  • serum albumin 0.1 serum albumin
  • test compound is brought into contact with the orphan receptor protein expressed in orphan receptor protein-expressing cells or its cell membrane fraction.
  • a method of perfusing a medium containing a test compound into orphan receptor protein-expressing cells or a cell membrane fraction thereof is generally used.
  • the stimulating activity of the test compound on orphan receptor protein-expressing cells and cells that do not express orphan receptor protein can be measured and compared.
  • This method expresses the orphan receptor protein and the orphan receptor protein.
  • This assay uses cell membrane fractions containing no cells, but measures cell stimulating activity.In this assay, it shows GT PrS binding promoting activity to orphan receptor protein membrane fraction, Substances that do not show GTP r S binding promoting activity to the membrane fraction that does not express protein receptor protein are candidate ligand substances.
  • the cell membrane fraction containing the orphan receptor protein is transferred to a membrane dilution buffer (for example,
  • the dilution ratio depends on the expression level of the receptor protein.
  • wash buffer for example, ice-cold 50 mM Tris, 5 mM MgCl 2 , 150 mM NaCl, 0.
  • wash buffer for example, ice-cold 50 mM Tris, 5 mM MgCl 2 , 150 mM NaCl, 0.
  • BSA 0.053 ⁇ 4 CHAPS pH 7.4 1.5 ml
  • filter eg, using glass fiber filter paper GF / F.
  • Cell stimulating activity measuring system characterized by measuring the fluctuation of intracellular cAMP.
  • the intracellular cAMP level fluctuates by agonist stimulation of a test compound having agonist activity.
  • the cell stimulating activity of the test compound on orphan receptor protein-expressing cells can be measured.
  • - Ofan amount of cAMP produced various animal cells receptor evening to express protein in mice, rats, Usagi, catcher formic, ⁇ and anti-cAMP antibody obtained by immunizing such '25 1-labeled cAMP ( Both can be measured by using TRIAS or other EIA system combining anti-cAMP antibody and labeled cAMP.
  • anti-cAMP antibody Is a quantitative possible by SPA method using a beads and iota 25 iota labeled cAMP containing scintillant was solid boss using such antibodies to such animal IgG used in A or anti-CAMP antibody production (e.g., Amashamufu Use Almatia Biotech kits
  • the activity of promoting cAMP production can be measured.
  • the intracellular cAMP level is increased by a substance that increases the intracellular cAMP level, such as forskolin, and the change in the intracellular cAMP level is observed by adding a test compound.
  • AMP production inhibitory activity can be measured.
  • the animal cells expressing the orphan receptor protein are buffered (eg, a Hanks buffer (PH7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES).
  • a Hanks buffer PH7.4 containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES.
  • a reaction buffer eg, a Hanks buffer (PH7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES.
  • the amount of cAMP in the extract can be determined using the cAMP EIA kit
  • CRE—DNA that contains a cell stimulating activity measurement system CRE (cAMP response element) characterized by introducing a reporter gene can be used to prepare a Pitka Gene Basic Vector or a Pitka Gene Enhancer Vector (Toyo Ink Manufacturing) And insert it into the multiple cloning site upstream of the luciferase gene, and use this as the CRE-reporter gene vector.
  • CRE cell stimulating activity measurement system
  • Cell stimulating activity can be measured using cells in which the CRE-reporter gene vector has been transfected into orphan receptor protein-expressing cells.
  • reaction buffer add an appropriate amount (eg, 0.25 ml) of the reaction buffer to the cells, and add an appropriate amount (eg, 1 nM) of the test compound and an appropriate amount (eg, 0.25 ml) of the reaction buffer (preferably containing forskolin when measuring cAMP production inhibitory activity) to the cells, and at about 37 ° C for about 24 hours.
  • an appropriate amount eg, 0.25 ml
  • an appropriate amount eg, 1 nM
  • an appropriate amount eg, 0.25 ml of the reaction buffer (preferably containing forskolin when measuring cAMP production inhibitory activity)
  • Luminescence from luciferase is measured with a luminometer, liquid scintillation counter or top counter.
  • repo overnight gene other than luciferase, for example, alkaline phosphatase, chloramphenicol acetyltransferase or 0-galactosidase can also be used.
  • alkaline phosphatase for example, chloramphenicol acetyltransferase or 0-galactosidase
  • the enzymatic activity of these repo overnight gene products can be easily measured using a commercially available assay kit as follows.
  • alkaline phosphatase activity can be measured, for example, by Lumi-Phos 530 manufactured by Wako Pure Chemical
  • chloramphenicol acetyltransferase activity can be measured, for example, by FAST CAT chrola manufactured by Wako Pure Chemical immediately henicol Acetyltransferase Assay KiT
  • 3-galactosidase activity can be measured, for example, by Aurora Gat XE (manufactured by Hako Pure Chemical Industries, Ltd.).
  • Cell stimulating activity measuring system characterized by measuring arachidonic acid release Orphan receptor protein-expressing cells release arachidonic acid metabolites extracellularly as a result of stimulation by agonist. By introducing arachidonic acid having radioactivity into cells in advance, the cell stimulating activity can be measured by measuring the radioactivity released outside the cells. At this time, the cell stimulating activity can be measured by adding the test compound and examining the arachidonic acid metabolite releasing activity of the test compound.
  • Inoculate orphan receptor protein-expressing cells for example, orphan receptor protein-expressing CH0 cells
  • an appropriate concentration for example, 5 ⁇ 10 4 cells / well
  • reaction buffer a buffer containing 20 mM HEPES (p ⁇ 7.4) and add a buffer (eg, Hanks buffer (PH7.4) containing 0.05% BSA and 20 mM HEPES: PH 7.4). Add the test compound dissolved in Hanks buffer (PH7.4) containing BSA and 20D1M HEPES (referred to as reaction buffer).
  • a buffer eg, Hanks buffer (PH7.4) containing 0.05% BSA and 20 mM HEPES: PH 7.4
  • reaction buffer eg, 400 l
  • the orphan receptor protein-expressing cells were seeded on a sterile microscope cover glass. After about 2 days, the culture solution was suspended in an appropriate amount (for example, 4 mM) of Fura-2 AM (Dojindo Laboratories). Replace with HBSS and leave at room temperature for about 2 hours and 30 minutes.
  • an appropriate amount for example, 4 mM
  • Fura-2 AM Diojindo Laboratories
  • FLIPR Fluo-3 AM (manufactured by Dojindo Laboratories) to the cell suspension, allow the cells to take up, wash the supernatant several times by centrifugation, and seed the cells on a 96-well plate. .
  • the orphan receptor protein-expressing cells are co-expressed with a gene for a protein such as aequor in, which emits light due to an increase in intracellular Ca ions, and aequor in is converted to Ca-binding by increasing intracellular Ca ion concentration.
  • aequor in which emits light due to an increase in intracellular Ca ions
  • aequor in is converted to Ca-binding by increasing intracellular Ca ion concentration.
  • a modified G protein such as a chimeric G protein
  • the chimeric G protein is a functional domain of a G protein that uses Ca 2+ as a signal transduction system, such as G 4 , a G protein that does not use Ca 2+ as a signal transduction system, such as Gi, Go, and Gs. Refers to the G protein replaced by.
  • the signaling system of any G protein can be monitored by changes in Ca 2+ .
  • Cell stimulating activity measuring system characterized by measuring inositol phosphate production When a test compound having agonist activity is added to a cell expressing a receptor receptor protein, the intracellular inositol triphosphate concentration increases. By observing this response in orphan receptor protein expressing cells produced by the test compound, the cell stimulating activity can be measured.
  • the “(test) compound having agonist activity” refers to the above ( Test compounds described in C) (eg, natural 'non-natural peptides, natural' non-natural proteins, natural and non-natural non-peptidic compounds, synthetic compounds, natural and non-natural fermentation products, etc.) or test Among the compounds (a), any one of the cell stimulating activity measuring systems described in the above (D) (preferably, a cell stimulating activity measuring system characterized by measuring fluctuation of extracellular pH (acidification rate), etc.) ), Cell stimulating activity was observed when using orphan receptor protein-expressing cells or their cell membrane fractions, and cells that did not express orphan receptor protein or cell membrane fractions when using orphan receptor proteins were used. Test compounds with no stimulatory activity (eg, natural and non-natural peptides, natural and non-natural proteins, natural and non-natural non-peptidic compounds, synthetic compounds, natural and non-natural sources) It refers to such products).
  • Test compounds with no stimulatory activity eg, natural and non-natural peptides, natural and non
  • a compound having an agonist activity when the orphan receptor protein is FM-3 is a C-terminal In the structure of R—X—NH 2 (X is Gly, Ala, Va1, Leu, Ile, Ser, Thr, Cys, Met ;, Glu, Asp, Lys, Ar g, His, Phe, Tyr, Trp, Pro, Asn, Gin, etc.), and more specifically, a peptide having SEQ ID NOs: 2, 6, and A peptide containing the amino acid sequence represented by 20; Peptides, proteins, compounds or fermentation products which are (test) compounds having these agonist activities may also form salts, and these salts may include physiologically acceptable bases (eg, alkali metals).
  • physiologically acceptable bases eg, alkali metals
  • salts with an acid 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) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • 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
  • Tartaric acid citric acid, malic acid, oxalic acid
  • benzoic acid methanesulfonic acid, benzenesulfonic acid
  • test compound (or test compound (a)) is a (test) compound having agonist activity
  • criteria for determining whether a test compound (or test compound (a)) is a (test) compound having agonist activity are shown below.
  • the following criteria are merely examples, and whether the test compound (or test compound (a)) has agonist activity is determined by the following criteria. It is not to be construed as limiting.
  • test compound When a cell stimulating activity measurement system characterized by measuring the fluctuation of extracellular pH (acidifi cat ion rate) described in (D) — (1) above is used, the test compound is If the extracellular pH at the peak of the reaction when the test compound is brought into contact with the cells exceeds 105%, assuming that the extracellular pH before contacting with the cells is 100%, the orphan receptor protein A test compound that does not show cell stimulating activity when cells that do not express it or its cell membrane fraction is used is selected as a (test) compound having agonist activity.
  • test compounds that do not have any agonist activity are selected as (test) compounds.
  • the amount of cAMP when the test compound is not added is 100%, and the amount of cAMP produced by the addition of the test compound is 105% or more.
  • a test compound that does not show cell stimulating activity when using cells that do not express a somatic protein or cell membrane fraction thereof is selected as a (test) compound having agonist activity.
  • test compound having agonist activity when used as an index, the amount of luminescence generated by forskolin stimulation is 100%, and the luminescence generated by the addition of the test compound is 95% or less.
  • a test compound that does not show cell stimulating activity when cells that do not appear or its cell membrane fraction is used is selected as a (test) compound having agonist activity.
  • the amount of luminescence without the addition of the test compound is 100%, and the amount of luminescence produced by the addition of the test compound is 105% or more.
  • a test compound that does not show cell stimulating activity when using cells that do not express the monofan receptor protein or a cell membrane fraction thereof is selected as a (test) compound having agonist activity.
  • test compound When a cell stimulating activity measuring system characterized by measuring intracellular Ca 2+ release described in (D) to (6) above is used, the fluorescence observed when no test compound is added is observed.
  • the fluorescence intensity when the test compound is added is 105% or more with the intensity set to 100%, and no cell stimulating activity is observed when cells that do not express the orphan receptor protein or their cell membrane fractions are used.
  • the test compound is selected as a (test) compound having agonist activity.
  • test compounds having agonist activity according to the above (E)
  • Test comparing the structures of (test) compounds having each agonist activity
  • Test Estimate (or determine) the common structure of a compound having a common activity, and prepare or obtain a ligand candidate substance having the common structure.
  • the ligand candidate substance has a structure common to the (test) compound having the agonist activity. Compared with each test compound (specifically, test compound (a)), Substances with strong cell stimulating activity (eg, natural peptides, natural proteins, natural non-peptide compounds, etc.).
  • test is a natural or non-natural peptide, natural-non-natural protein, etc.
  • amino acid sequences encoding those peptides or proteins are compared, and their partial sequences with high homology are compared. Or, a portion having a similar three-dimensional structure can be said to be a common structure.
  • the common structure includes SEQ ID NO: 2, 6, and By comparing the amino acid sequences represented by 20, a common structure can be derived that has an R—X —NH 2 structure (X represents an arbitrary amino acid residue) at the C-terminus.
  • the ligand candidate substance having the common structure, and further, the (endogenous) ligand of FM-3 is the C-terminal thereof. It is considered that the peptide has an R—X_NH 2 structure.
  • Peptides having an R—X—NH 2 structure at the C-terminus include A-18-F-NH, and F-8-F-NH 2 in mammals (Perry, SJ et al. FEBS Lett. 409: 426-430). , 1997) and prolactin-releasing peptide (Hinuma, S. et al. Nature 393: 272-276, 1998). In lower animals, it is ubiquitous as a family of RF amide peptide families. Furthermore, it is thought that unknown R—X—NH 2 ⁇ peptides are present in mammals as diverse as lower animals, and a new R _X—NH 2 peptide in mammals contains FM-3 (endogenous) ligand. Is considered to exist.
  • the (test) compound having agonist activity is a natural or non-natural non-peptidic compound or a synthetic compound
  • chemical structures of those compounds are compared, and a common basic skeleton (for example, specific Ring structures, eg, cyclo as “cycloaliphatic hydrocarbon” Saturated or unsaturated alicyclic hydrocarbons such as alkyl, cycloalkenyl, and cycloalkenyl, aromatic heterocycles as "heterocycles", saturated or unsaturated non-aromatic heterocycles (aliphatic heterocycles) Etc.)
  • a common basic skeleton for example, specific Ring structures, eg, cyclo as “cycloaliphatic hydrocarbon” Saturated or unsaturated alicyclic hydrocarbons such as alkyl, cycloalkenyl, and cycloalkenyl, aromatic heterocycles as "heterocycles", saturated or unsaturated non-aromatic heterocycles (aliphatic heterocycles) Etc.
  • the ligand determination method of the present invention determines ligands using the above-mentioned common structure as an index, so that obtaining a subtype of a ligand having a high structural similarity to the ligand can also be compared with the conventional method. It becomes much easier and more reliable.
  • test compound (a) Based on the common structure of the test compound described in (F) above (specifically, test compound (a)), search for natural peptides, natural proteins, and natural non-peptidic compounds having a common structure. Can be used to estimate the ligand or its subtype.
  • the cell stimulating activity of the putative ligand is measured using the cell stimulating activity measuring system described in (D) above, and is compared with the cell stimulating activity with the ligand candidate substance. Can be determined.
  • the (test) compound having agonist activity is a peptide, protein, or a salt thereof
  • a primer or probe containing a base sequence encoding a common structure is prepared. And encodes the ligand or its subtype c
  • the common structure of the (test) compound having the agonist activity described in (F) above that is, the base sequence encoding the highly homologous sequence portion between the amino acid sequences encoding the (test) compound having the 7 gonist activity, Create primers or probes to contain.
  • PCR is carried out in a manner known per se, and all tissues (eg, humans, warm-blooded animals (eg, guinea pigs, rats, mice, pigs, sheep, wedges, monkeys, etc.) and fish) are obtained.
  • tissues eg, humans, warm-blooded animals (eg, guinea pigs, rats, mice, pigs, sheep, wedges, monkeys, etc.) and fish
  • tissues eg, humans, warm-blooded animals (eg, guinea pigs, rats, mice, pigs, sheep, wedges, monkeys, etc.) and fish
  • tissues eg, humans, warm-blooded animals (eg, guinea pigs, rats, mice, pigs, sheep, wedges, monkeys, etc.) and fish) are obtained.
  • warm-blooded animals eg, guinea pigs, rats, mice, pigs, sheep, wedges, monkeys, etc.
  • fish
  • the cDNA containing the nucleotide sequence encoding the cloned ligand candidate substance can be used as it is depending on the purpose, or digested with a restriction enzyme or added with a linker if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and may have TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • a transformant containing DNA encoding a ligand candidate substance is cultured according to the method for producing cells expressing an orphan receptor protein described in (A) and (B) above, and the culture is performed.
  • the candidate ligand substance can be separated and purified from the product by, for example, the following method.
  • the buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark, sometimes abbreviated as TM hereinafter).
  • Ligand candidates contained in the culture supernatant or extract obtained in this way The substance can be purified by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • Method using difference in molecular weight Method using difference in charge such as ion exchange chromatography, Method using specific affinity such as affinity mouth chromatography, Hydrophobicity such as reverse phase high performance liquid chromatography
  • a method using the difference between isoelectric points, such as isoelectric focusing and chromatofocusing, is used.
  • the thus obtained ligand candidate substance When the thus obtained ligand candidate substance is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or The compound can be converted into a free form or another salt by an analogous method.
  • the target ligand candidate substance can be produced by condensing a partial peptide or amino acid that can constitute the ligand candidate substance with the remaining part, and if the product has a protective group, removing the protective group.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • the candidate ligand can be purified and isolated by a combination of laffy, liquid chromatography, and recrystallization.
  • the ligand candidate substance obtained by the above method is a free form, it can be converted to an appropriate salt by a known method. Conversely, when it is obtained as a salt, it is converted to a free form by a known method. be able to.
  • a candidate ligand substance can be obtained by culturing a transformant containing DNA encoding the candidate ligand substance according to the method for producing an orphan receptor protein described in (A) above.
  • test compound having agonist activity is a peptide, protein or a salt thereof
  • a peptide or protein having a common structure is sequenced to form a ligand by searching the database. How to search for candidate substances.
  • the compound having the agonist activity described in (F) above (test) should have a common structure, that is, a base sequence encoding a sequence with high homology between the amino acid sequences encoding the compounds having agonist activity (test). Peptides or proteins can be searched in sequence databases to determine candidate ligands.
  • sequence database examples include GenBank (registered trademark) file (National Institute of Health), VTS (Virtual Transcribed Sequence) and the like.
  • the candidate ligand substance can be obtained according to the method described in (G) ⁇ 2 above.
  • an antibody against the peptide is prepared according to the following method.
  • the antibody may be any of a polyclonal antibody and a monoclonal antibody as long as the antibody can recognize the peptide.
  • An antibody against the peptide can be produced by using the peptide as an antigen according to a method for producing an antibody or antiserum known per se.
  • the peptide is administered to a warm-blooded animal at a site where the antibody can be produced by administration, itself or together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • the warm-blooded animals used include, for example, monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens.
  • a warm-blooded animal immunized with an antigen for example, a mouse 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.
  • Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody producing cells contained with myeloma cells of the same or different species.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled peptide described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion procedure should be performed according to known methods, for example, the method of Köhler and Milstein [Nature, 256, 495 (1975)].
  • fusion promoter examples include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • myeloma cells examples include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is used at a concentration of about 10 to 80%.
  • Cell fusion can be carried out efficiently by adding and incubating at 20 to 40, preferably at 30 to 37 ° C for 1 to 10 minutes.
  • Various methods can be used to screen monoclonal antibody-producing hybridomas.
  • a hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a peptide antigen is adsorbed directly or together with a carrier.
  • An anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse) or protein A is added to the monoclonal antibody bound to the solid phase.
  • Detection method Add the hybridoma culture supernatant to the solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, add peptides labeled with radioactive substances, enzymes, etc., and detect the monoclonal antibody bound to the solid phase And the like.
  • Monoclonal antibodies can be selected according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as the hybridoma can grow.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or A serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used.
  • the culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 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 methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption and desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) Can do it.
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption and desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) Can do it.
  • the above-mentioned peptide antibody can be produced according to a method known per se or a method analogous thereto.
  • a immunizing antigen (peptide antigen) itself or a complex thereof with a carrier peptide is formed, and a warm-blooded animal is immunized in the same manner as in the above-described monoclonal antibody production method. It can be produced by collecting the substance and separating and purifying the antibody.
  • the type of carrier peptide and the mixing ratio of carrier and hapten depend on the efficiency of the antibody against hapten immunized by cross-linking with the carrier. It is possible to crosslink any material at any ratio, if possible.
  • serum serum albumin, thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 2 with respect to 1 hapten.
  • a method of pulling force at a rate of 0, preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The dose is usually given once every 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of polyclonal antibody Immunoglobulin separation and purification methods similar to those for antibody isolation and purification.
  • a candidate ligand is detected, and various immunoassays are used as indicators to combine various extraction methods and chromatography. By doing so, candidate ligand substances can be obtained.
  • a compound that promotes the function of an orphan receptor protein is referred to as a “highly active agonist”. “Highly active” refers to a “(test) compound having an agonist activity” described in the above (E). This means that the cell stimulating activity (specifically, the cell stimulating activity described in the above (D), etc.) is stronger than that of.
  • the above-mentioned (G) is obtained by using an orphan receptor protein or by constructing an expression system for a recombinant orphan receptor protein and using a receptor-binding atsey system using the expression system. Efficiently screens compounds that alter the binding between the selected ligand candidate and the orphan receptor protein (eg, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, etc.) or salts thereof be able to.
  • the orphan receptor protein eg, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, etc.
  • a ligand candidate substance is brought into contact with an orphan receptor protein-expressing cell or a cell membrane fraction thereof and a test compound ((i.e., the orphan receptor protein is added to the orphan receptor protein-expressing cell or its cell membrane fraction)
  • D (a) fluctuations in extracellular pH, (b) arachidonic acid release, (c) acetylcholine release,
  • test compound (b) i.e., the orphan receptor protein-expressing cell or a cell thereof
  • the test compound (b) is compared with the cell stimulating activity when the ligand candidate substance is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction.
  • the test compound (b) (or the candidate substance) ) Is likely to be a compound that promotes the function of the orphan receptor protein (so-called agonist).
  • test compound (b) or candidate substance
  • agonist a compound that promotes the function of the orphan receptor protein (selectively, specifically) depends on whether the orphan receptor protein and the The amount of specific binding to test compound (b) (or candidate substance) may be measured.
  • a labeled test compound (b) (or a candidate substance) is brought into contact with an orphan receptor protein
  • a labeled test compound (b) (or candidate And the like for example, a method of measuring the binding amount of the substance) to the orphan receptor protein.
  • the test compound (b) (or the candidate) Substance) is recognized as a compound that promotes the function of the orphan receptor protein (so-called agonist).
  • the orphan receptor protein used in the measurement method may be any as long as it contains the above-mentioned orphan receptor protein.
  • an orphan receptor protein or the like which is expressed in a large amount using a recombinant is suitable.
  • the above-mentioned method is used for producing the orphan receptor protein, but it is preferably carried out by expressing DNA encoding the orphan receptor protein in the above-mentioned animal cells or insect cells. D that encodes the desired protein part 639
  • Complementary DNA is used for the NA fragment, but is not necessarily limited to this.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment In order to introduce a DNA fragment encoding an orphan receptor protein into a host animal cell and express them efficiently, the DNA fragment should be a baculovirus belonging to a baculovirus using an insect as a host. (Nuclear poly edrosis virus; NPV) In the evening, it is preferable to incorporate it downstream such as the SR promoter. Inspection of the quantity and quality of the receptor that has occurred can be carried out in a manner known per se. For example, the method is carried out according to the method described in the literature [Nambi, P. et al., The Journal of Biological Chemistry, 267, 19555-19559, 1992]. be able to.
  • the orphan receptor protein may be an orphan receptor protein purified according to a method known per se, or a cell containing orphan receptor protein.
  • a membrane fraction of cells containing an orphan receptor protein may be used.
  • the cell containing the orphan receptor protein refers to a host cell that has expressed the receptor protein, and the host cell is preferably an insect cell, an animal cell, or the like.
  • the cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se.
  • Cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Pelling Blender ⁇ polytron (manufactured by Kinematica), crushing with ultrasonic waves, or narrowing the cells while pressing with a French press. Crushing by erupting from the ground.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for 30 min. Centrifuge for 1 minute to 2 hours, and use the resulting precipitate as the membrane fraction.
  • the membrane fraction the expressed orphan receptor protein and It contains a lot of membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the orphan receptor protein in the cell or membrane fraction containing the orphan receptor protein is preferably 10 to 10 molecules per cell,
  • it is 10 molecules.
  • labeled test compound (b) for example, [ 3 H], [ 125 ]
  • Test compounds (b) (or candidate substances) labeled with [I], [ 14 C], [ 35 S], etc. are used.
  • a receptor protein protein preparation is prepared by suspending a cell containing the o-phan receptor protein or a membrane fraction of the cell in a buffer suitable for screening.
  • the buffer may be any buffer as long as it does not inhibit the binding of the candidate substance such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or Tris-HCl buffer to the receptor protein.
  • a surfactant such as CHAPS, Tween-80 TM (Kaoichi Atlas), digitonin, or dexcholate can be added to the buffer.
  • protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptidyltin can be added for the purpose of suppressing the degradation of receptors and ligands by proteases.
  • an aliquot 5000 cpm to 500,000 cpm
  • the reaction is carried out 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 minutes to 3 hours.
  • the reaction solution is filtered through a glass fiber filter, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured using a liquid scintillation counter or a counter.
  • test compound (b) was added to the orphan receptor protein-expressing cell or its cell membrane fraction in comparison with the cell stimulating activity when the ligand candidate substance was brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction.
  • test compound (b) or Candidate substances are compounds that inhibit the function of the orphan receptor protein (so-called (Agonist).
  • test compound (b) (or the orphan receptor protein-expressing cell or the cell membrane fraction) was compared with the cell stimulating activity when the ligand candidate substance was brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction. If the cell membrane fraction is brought into contact with a candidate compound of a compound that promotes or inhibits the function of the orphan receptor protein) and a candidate ligand, if the cell stimulating activity is weak or not observed, the test is performed.
  • Compound (b) (or candidate substance) may be a compound (a so-called antagonist) that inhibits the function of the orphan receptor protein.
  • test compound or candidate substance
  • agonist a compound that inhibits the function of the orphan receptor protein (selectively to Specific) depends on whether the test compound (or the candidate substance) inhibits the function of the orphan receptor protein. What is necessary is just to measure the amount of specific binding.
  • a labeled test compound (b) (or a candidate substance) when a labeled test compound (b) (or a candidate substance) is brought into contact with an orphan receptor protein And the method of measuring the amount of binding to the orphan receptor protein.
  • the test compound (b) (or candidate) Substance) is recognized as a compound that inhibits the function of the orphan receptor protein (a so-called antagonist).
  • the same method as the above-mentioned method for screening a compound that promotes the function of the orphan receptor protein (a so-called highly active agonist) and the like are used.
  • a labeled ligand candidate when brought into contact with an orphan receptor protein, a labeled ligand candidate and a test compound (b) (ie, a candidate for a compound that inhibits the function of orphan receptor protein)
  • a test compound ie, a candidate for a compound that inhibits the function of orphan receptor protein
  • the amount of binding of the labeled ligand candidate substance to the orphan receptor protein is measured and compared to determine the compound that inhibits the function of the orphan receptor protein. It is also possible to screen.
  • the specific description of the screening method is as follows.
  • the orphan receptor protein used in the screening method of the present invention is Any one may be used as long as it contains the above-mentioned protein receptor protein.
  • an orphan receptor protein or the like which is expressed in large amounts using a recombinant is suitable.
  • the above-mentioned method is used to produce the orphan receptor protein, but it is preferably carried out by expressing DNA encoding the orphan receptor protein in the above-mentioned animal cells or insect cells.
  • the complementary DNA fragment is used for the DNA fragment encoding the protein portion of interest, but is not necessarily limited to this.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment In order to introduce a DNA fragment encoding an orphan receptor protein into a host animal cell and to express them efficiently, the DNA fragment must be converted to a nucleopolyhedron belonging to a baculovirus using an insect as a host.
  • Polyhedrin promoter of the disease virus (nul ear polyhedros is virus; NPV), promoter derived from SV40, retrovirus promoter, metamouth thionine promoter, human heat shock promoter, cytomegalovirus promoter
  • NPV neurotrophic virus
  • promoter derived from SV40 promoter derived from SV40
  • retrovirus promoter metamouth thionine promoter
  • human heat shock promoter cytomegalovirus promoter
  • cytomegalovirus promoter Preferably, it is incorporated downstream such as the SRa promoter. Inspection of the quantity and quality of the receptor that has occurred can be carried out in a manner known per se. For example, a method described in the literature [Nambi, P. et al., The 'Journal of Biological' Chemistry (J. Biol. Cem.), 267, 19555-19559, 1992] It can be done according to.
  • the orphan receptor protein may be an orphan receptor protein purified according to a method known per se, or a cell containing the orphan receptor protein may be used. Alternatively, a membrane fraction of cells containing orphan receptor protein may be used.
  • the cell containing the orphan receptor protein refers to a host cell that expresses the receptor protein or the like, and the host cell is preferably an insect cell, an animal cell, or the like.
  • the cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se.
  • Cell disruption methods include Potter— The method of crushing cells with an Elvehjem type homogenizer, crushing with a one-ring blender Polytron (manufactured by Kinematica), crushing by ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure with a French press, etc. can give.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for 30 minutes. Centrifuge for 1 minute to 2 hours, and use the resulting precipitate as the membrane fraction.
  • the membrane fraction is rich in the expressed orphan receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the orphan receptor protein in the cell or membrane fraction containing the orphan receptor protein is preferably 10 to 10 molecules per cell, and more preferably 10 to 10 molecules per cell.
  • an appropriate orphan receptor protein fraction and a labeled ligand or a subtype thereof are used.
  • an appropriate orphan receptor protein fraction and a labeled ligand or a subtype thereof are used.
  • the labeled ligand candidate substance for example, a ligand candidate substance labeled with [ 3 H], [ 125 I], [ 14 C], [ 35 S] or the like is used.
  • the membrane fraction of cells or cells containing orphan receptor protein is first suspended in a buffer suitable for screening.
  • a buffer suitable for screening may be any buffer that does not inhibit the binding of the ligand to the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer of tris-hydrochloride.
  • Surfactants such as CHAPS, Tween-80 TM (Kao-Atras), digitonin, and dexcholate can also be added to the buffer to reduce non-specific binding.
  • a protease inhibitor such as a protein may also be added.
  • M ( b) ie, a candidate compound that inhibits the function of orphan receptor protein.
  • the reaction is carried out at about 0 ° C to 50T, preferably at about 4 to 3, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • the solution is filtered through a glass fiber filter paper and the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a glass counter.
  • the specific binding amount (B-NSB) is, for example, and 50% or less of the test compound (b) (ie, a candidate compound that inhibits the function of the orphan receptor protein) is selected as the compound that inhibits the function of the orphan receptor protein (so-called angiogonist). can do.
  • test compound (b) described in ( ⁇ ) above that is, a candidate compound for promoting or inhibiting the function of orphan receptor protein
  • compound that promotes or inhibits the function of orphan receptor protein
  • test compound (b) ie, a candidate compound that promotes or inhibits the function of an orphan receptor protein
  • the test compound (b) includes a natural 'non-natural peptide, a natural and non-natural protein, a natural and non-natural Non-peptidic compounds, synthetic compounds, natural and non-natural fermentation products, etc.
  • the compound that promotes or inhibits the function of the orphan receptor protein includes the compound that promotes the function of the orphan receptor protein or the compound that inhibits the function of the orphan receptor protein in the screening method described in (H) above.
  • the salt of the compound include salts with a physiologically acceptable base (eg, an alkali metal or the like) and an acid (organic acid, inorganic acid), and particularly preferred are physiologically acceptable acid addition salts.
  • Such salts include, for example, inorganic acids (eg, hydrochloric acid T / JP00 / 05639
  • Phosphoric acid hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid
  • Methanesulfonic acid, benzenesulfonic acid Methanesulfonic acid, benzenesulfonic acid
  • compounds that promote (highly active agonist) or inhibit (antagonist) the function of orphan receptor protein obtainable by the above method (H) include the following (endogenous) ligands or biological activities possessed by the subtypes thereof. Since it has the same action as that described above, it is useful as a safe and low-toxic drug according to the ligand activity.
  • Antagonists against orphan receptor proteins can suppress the physiological activity of ligands or subtypes of orphan receptor proteins, and thus are useful as safe and low-toxic drugs that suppress the ligand activities. is there.
  • the highly active agonist against the orphan receptor protein is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the orphan receptor protein.
  • an agonist and a highly active agonist that can be obtained by the method of the present invention are used as a pharmaceutical composition, they can be administered in a conventional manner.
  • a pharmaceutical composition for example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used.
  • 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, egrets, sheep, pigs, pigs, cats, dogs, dogs, etc.). Can be administered.
  • the dosage of the ligand or its subgroup, antagonist, and agonist varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • oral administration for example, about 0. It is 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 mg.
  • parenteral administration the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.For example, in the case of an injection, it is usually, for example, about 0.0 per day. About 1 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg It is convenient to administer the degree by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the present invention not only provides a method for screening a compound that promotes or inhibits the function of the orphan receptor protein described in the above (H), but also uses, as an index, the common structure of (test) compounds having agonist activity. Methods for more efficiently and reliably determining ligands for orphan receptor proteins or subtypes thereof are also provided.
  • test compound bringing a test compound into contact with orphan receptor protein-expressing cells or a cell membrane fraction thereof, measuring the cell stimulating activity mediated by the orphan receptor protein, and (ii) comparing the cell stimulating activities of each test compound
  • the common structure of the test compound having agonist activity is determined
  • test compound (i) Contacting test compound (a) with orphan receptor protein-expressing cells or its cell membrane fraction, and contacting test compound (a) with cells not expressing orphan receptor protein or its cell membrane fraction In this case, each cell stimulating activity was measured,
  • test compound (a) an orphan receptor protein-expressing cell or a cell membrane thereof
  • test compound (a) is brought into contact with the fraction, and the cell stimulating activity mediated by the orphan receptor protein is measured.
  • test compound (a) By comparing the cell stimulating activity of each test compound (specifically, test compound (a)), a (test) compound having agonist activity was obtained, and the common structure of the compound having agonist activity was estimated.
  • test compound (a) By comparing the cell stimulating activity of each test compound (specifically, test compound (a)), a (test) compound having agonist activity was obtained, and the common structure of the compound having agonist activity was estimated.
  • the same method as described above is used.
  • Whether or not the ligand candidate substance is a specific ligand of orphan receptor protein can be determined by measuring the amount of specific binding of the candidate ligand to the orphan receptor protein. it can.
  • the candidate substance is identified as ( Endogenous) is recognized as a ligand.
  • the ligand candidate substance has a cell-stimulating activity but has a low specific binding ability to the orphan receptor protein, ie, a nonspecific agonist. Is likely to be a similar substance. A specific description of the determination method will be given below.
  • the orphan receptor protein used in the determination method may be any as long as it contains the above-described orphan receptor protein.
  • an orphan receptor protein or the like which is expressed in large amounts using a recombinant is suitable.
  • the above-mentioned method is used to produce the orphan receptor protein, but it is preferably carried out by expressing the DNA encoding the orphan receptor protein in the above-mentioned animal cells or insect cells. D that encodes the desired protein part 639
  • the complementary DNA is used for the NA fragment, but is not necessarily limited to this.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment In order to introduce a DNA fragment encoding an orphan receptor protein into a host animal cell and express them efficiently, the DNA fragment must be transferred to a nucleophilic polyhedrosis virus belonging to a baculovirus using an insect as a host.
  • nucl ear polyhedros is virus (NPV) polyhedrin promoter — Yuichi, SV40-derived promoter, retroviral promoter, metamouth thionine promoter, human heat shock promoter, cytomegalovirus promoter, SR ⁇
  • it is incorporated downstream, such as a promoter.
  • Inspection of the quantity and quality of the receptor that has occurred can be carried out in a manner known per se. For example, according to the method described in the literature [Najiibi, P. et al., The Journal of Biological Chemistry, 267, 19555-19559, 1992]. Can do it.
  • the substance containing the orphan receptor protein may be an orphan receptor protein purified according to a method known per se, or a cell containing the orphan receptor protein may be used. Alternatively, a membrane fraction of a cell containing an orphan receptor protein may be used.
  • the cell containing the orphan receptor protein refers to a host cell that has expressed the receptor protein, and the host cell is preferably an insect cell, an animal cell, or the like.
  • the cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se.
  • Cells can be crushed by crushing the cells with a Potter-Elvehj em-type homogenizer, crushing with a Perling Blender ⁇ Polytron (Kinematica), crushing with ultrasonic waves, or applying pressure with a French press, etc. And crushing by ejecting the gas from a thin nozzle.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 300 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at a high speed (1500 to 300 rpm). The mixture is centrifuged at 0,000 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as the membrane fraction.
  • the membrane fraction the expressed orphan receptor protein and It contains a lot of membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the orphan receptor protein in the cell or membrane fraction containing the orphan receptor protein is preferably 10 to 10 molecules per cell,
  • it is 10 molecules.
  • labeled ligand candidate substances include [ 3 H], C 125 I], [ 14 C],
  • a ligand candidate substance labeled with [ 35 s] or the like is used.
  • a cell containing the orphan receptor protein or a membrane fraction of the cell is suspended in a buffer suitable for screening to prepare a receptor protein sample.
  • a buffer suitable for screening to prepare a receptor protein sample.
  • Any buffer may be used as long as it does not inhibit the binding of the candidate protein to 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.
  • surfactants such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, and dexcholate can be added to the buffer for the purpose of reducing non-specific binding.
  • protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide suptin can be added for the purpose of suppressing receptor degradation and ligand degradation by proteases.
  • a fixed amount 5000 cpn! To 500,000 cm
  • the reaction is carried out at about 0 ° C. to 50 °, preferably about 4 ° to 37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • the solution is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured by a liquid scintillation counter or a ⁇ -counter.
  • DNA Deoxyribonucleic acid
  • cDNA Complementary deoxyribonucleic acid A adenine
  • RNA Messenger ribonucleic acid dATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate dGTP Deoxyguanosine triphosphate d CTP Deoxycytidine triphosphate.
  • ATP Adenosine triphosphate
  • Example 7 shows the base sequence of FM 3 R2 used in Reference Example 1 described later.
  • the human type FM-3 was obtained as follows. The following two types of synthetic DNAs were synthesized based on the arrangement U of human FM-3 reported in Genomics 52, 223-229 (1998).
  • F 3F2 5'-GTCGACCATGGCTTGCAATGGCAGTGCGGCCAGG-3 '(SEQ ID NO: 22)
  • FM3R2 5'-GCTAGCTCAGGATGGATCGGTCTCTTGCTG-3 '(SEQ ID NO: 23)
  • human fetal brain cDNA was obtained by PCR.
  • the PCR reaction solution was rat hypothalamus cDNA solution 1 ⁇ ⁇ (from 0.2 ng poly (A) + RNA), 1 ⁇ 2 (10 MM), 11 FM3R2 (10 M), 51 1x attached reaction solution, 51 dNTP (10 mM), 11 Ex Taq (evening color) and 36 1 distilled water were added to make a total of 501.
  • the reaction solution was subjected to a PCR reaction using a ThermalCycler 9600.
  • the PCR conditions were: denaturation at 95 ° C for 2 minutes, and a cycle of 98-10 seconds, 65 at 10 seconds, and 90 seconds was repeated 28 times.
  • the PCR product was subcloned into E. coli using a TA cloning kit (Invitrogen). Plasmids were extracted from the E. coli obtained by subcloning using a plasmid extractor (Kurapo Co., Ltd.), the base sequence of the inserted fragment was determined, and the sequence was the same as that reported in the above-mentioned literature.
  • the transformant E. coli JM109 / pAKK ⁇ FM3 was cultured to prepare a large amount of plasmid pAK KOFM3 DNA.
  • the amount of (251) ribosome solution corresponding to 0.5 DNA was added dropwise and incubated for 16 hours to introduce the plasmid DNA. Furthermore, after replacing the medium with fresh medium and culturing for 1 day, the medium is replaced with the selection medium and culturing is continued for 3 days. Finally, the cells dispersed by trypsin digestion are dispersed at a low density in the selection medium ( 0/05639
  • Deoxyr ibonucleosides (a minimum essential medium, alpha medium containing 10% dialyzed serum) containing no D ⁇ ribonucleosides) were selected for transformants. Only the transformants can grow in the selection medium, and the selection is repeated by repeating the passage, and CH ⁇ -FM3 cells are established.
  • Example 1 Detection of stimulatory activity on FM-3 expressing CH0 cells by cytosensor-Attach using various peptide samples
  • the FM- 3 expressing CHO cells were seeded in 2.7xl0 5 cells / capsule density sites for sensor power capsule of, was charged wearing site sensors one workstation after overnight culture.
  • a cell culture medium (low buffered RPMI1640 medium containing 0.1% serum albumin) set in the flow path of the site sensor is supplied to the cells in a cycle of pump ON (80 seconds) and pump OFF (40 seconds). In each cycle, the rate of change in extracellular ⁇ from 8 seconds after the pump was stopped for 30 seconds was calculated as the acidification rate. The time course of the acidification rate was monitored, and when the value became stable, the cells were exposed to the peptides shown in the table for 7 minutes and 2 seconds by switching the flow path. The values of the AcHcCationRate for each well were normalized to the values of the three cycles immediately before exposure to the peptide as 100%, and the cell responses were compared. The concentration of the exposed samples was 1-10.
  • Table 1 shows the results of detection of the stimulating activity of peptide samples on FM-3 expressing CH0 cells by cytosensory assay.
  • FIG. 1 shows a comparison of the similarity of the amino acid sequences.
  • the method of the present invention comprises contacting a test compound with orphan receptor protein-expressing cells or a cell membrane fraction thereof or orphan receptor protein expressed in orphan receptor protein-expressing cells or cell membrane fraction thereof.
  • a test compound By measuring the cell stimulating activity via the receptor protein and comparing the cell stimulating activities of each test compound, an agonist is selected, and the structure of each agonist is compared to determine the orphan receptor protein. It is possible to obtain ligands or their subtypes, angiogonists and highly active agonists efficiently and reliably.

Abstract

L'invention concerne un ligand d'une protéine réceptrice orpheline ou son sous-type, antagoniste, agoniste hautement actif, etc. qui peut être efficacement et sûrement obtenu en mettant des composés de test en contact avec des cellules montrant l'expression de la protéine réceptrice orpheline ou d'une fraction de membrane cellulaire de ladite protéine ou avec un produit d'expression de la protéine réceptrice orpheline exprimée dans les cellules montrant l'expression de la protéine réceptrice orpheline ou d'une fraction de membrane cellulaire de ladite protéine ; en mesurant l'activité de stimulation cellulaire de chaque composé de test induit par la protéine réceptrice orpheline ; en comparant les activités de stimulation cellulaire des composés de test ; en choisissant ainsi des antagonistes ; enfin en comparant les structures de ces agonistes.
PCT/JP2000/005639 1999-08-24 2000-08-23 Methode de criblage WO2001014883A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087366A1 (fr) * 2002-04-16 2003-10-23 Kyowa Hakko Kogyo Co., Ltd. Lignees de cellules endocriniennes et procede d'utilisation desdites lignees
KR20100060752A (ko) * 2008-11-28 2010-06-07 (주)아모레퍼시픽 꽃가루, 집먼지 진드기 또는 황사에 의한 피부 유해반응을 스크리닝하는 방법 및 이를 조절하는 피부 외용제 조성물

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0970289A (ja) * 1995-06-27 1997-03-18 Takeda Chem Ind Ltd ヒトｃｒｆ２レセプター蛋白質、その製造法および用途
JPH10127289A (ja) * 1996-10-29 1998-05-19 Takeda Chem Ind Ltd 新規ｇ蛋白質共役型レセプター蛋白質およびそのｄｎａ
JPH10313866A (ja) * 1997-05-15 1998-12-02 Shionogi & Co Ltd Ｄｏｃ２αとＭｕｎｃ１３との結合のアゴニストまたはアンタゴニストのスクリーニング方法
JPH11127872A (ja) * 1997-08-11 1999-05-18 Japan Tobacco Inc 新規核内レセプター蛋白質、遺伝子及びその用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0970289A (ja) * 1995-06-27 1997-03-18 Takeda Chem Ind Ltd ヒトｃｒｆ２レセプター蛋白質、その製造法および用途
JPH10127289A (ja) * 1996-10-29 1998-05-19 Takeda Chem Ind Ltd 新規ｇ蛋白質共役型レセプター蛋白質およびそのｄｎａ
JPH10313866A (ja) * 1997-05-15 1998-12-02 Shionogi & Co Ltd Ｄｏｃ２αとＭｕｎｃ１３との結合のアゴニストまたはアンタゴニストのスクリーニング方法
JPH11127872A (ja) * 1997-08-11 1999-05-18 Japan Tobacco Inc 新規核内レセプター蛋白質、遺伝子及びその用途

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087366A1 (fr) * 2002-04-16 2003-10-23 Kyowa Hakko Kogyo Co., Ltd. Lignees de cellules endocriniennes et procede d'utilisation desdites lignees
KR20100060752A (ko) * 2008-11-28 2010-06-07 (주)아모레퍼시픽 꽃가루, 집먼지 진드기 또는 황사에 의한 피부 유해반응을 스크리닝하는 방법 및 이를 조절하는 피부 외용제 조성물
KR101601865B1 (ko) 2008-11-28 2016-03-09 (주)아모레퍼시픽 꽃가루, 집먼지 진드기 또는 황사에 의한 피부 유해반응을 스크리닝하는 방법 및 이를 조절하는 피부 외용제 조성물

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