WO2007058336A1 - PROCEDE DE CRIBLAGE ET PROCEDE D’IDENTIFICATION D’UNE SUBSTANCE CAPABLE D’INHIBER UNE REACTION DE DEGRANULATION OU LA PRODUCTION DE PROSTAGLANDINE D2 AU SEIN D’UN MASTOCYTE A L’AIDE DE Rec168, ET AGENT THERAPEUTIQUE DESTINE A UNE PATHOLOGIE INFLAMMATOIRE COMPRENANT LA SUBSTANCE - Google Patents

PROCEDE DE CRIBLAGE ET PROCEDE D’IDENTIFICATION D’UNE SUBSTANCE CAPABLE D’INHIBER UNE REACTION DE DEGRANULATION OU LA PRODUCTION DE PROSTAGLANDINE D2 AU SEIN D’UN MASTOCYTE A L’AIDE DE Rec168, ET AGENT THERAPEUTIQUE DESTINE A UNE PATHOLOGIE INFLAMMATOIRE COMPRENANT LA SUBSTANCE Download PDF

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Publication number
WO2007058336A1
WO2007058336A1 PCT/JP2006/323063 JP2006323063W WO2007058336A1 WO 2007058336 A1 WO2007058336 A1 WO 2007058336A1 JP 2006323063 W JP2006323063 W JP 2006323063W WO 2007058336 A1 WO2007058336 A1 WO 2007058336A1
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WIPO (PCT)
Prior art keywords
recl68
substance
degranulation
cells
mast cells
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PCT/JP2006/323063
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English (en)
Japanese (ja)
Inventor
Kazuhiko Tatemoto
Takayuki Naito
Yuko Nozaki
Masahiro Furuno
Keiko Tomura
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Japan Tobacco Inc.
National University Corporation Gunma University
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Application filed by Japan Tobacco Inc., National University Corporation Gunma University filed Critical Japan Tobacco Inc.
Priority to JP2007545328A priority Critical patent/JPWO2007058336A1/ja
Publication of WO2007058336A1 publication Critical patent/WO2007058336A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
    • C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
    • C07D241/46—Phenazines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • 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
    • G01N33/5044—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 involving specific cell types
    • G01N33/5047—Cells of the immune system
    • 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
    • G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a substance having an inhibitory action on signal transduction ability mediated by G protein-coupled receptor (hereinafter referred to as GPCR) Rec l 68 protein, more specifically, mast cell detachment via Rec l68.
  • GPCR G protein-coupled receptor
  • the present invention relates to a screening method for a granule reaction inhibitor or a prostadalandin D 2 production inhibitor.
  • a method for identifying a mast cell degranulation inhibitor or a prostaglandin D 2 production inhibitor through Rec l 68, a degranulation inhibitor comprising a substance identified by the method, prostaglandin D 2 production inhibitor or mast cells is shall relates to a therapeutic agent for inflammatory diseases involving.
  • Background art
  • GPCRs exist on the surface of each functional cell in living cells and organs, and play a physiologically important role as targets for molecules that regulate cell and organ functions, such as hormones, neurotransmitters, and physiologically active substances. I'm in charge.
  • the receptor transmits a signal into the cell through binding with a physiologically active substance, and various signals such as inhibition of cell activation are induced by this signal.
  • GPCR has seven transmembrane sections and plays a role in transmitting ligand information to the Fecta system via GTP (guanosine 5'-triphosphate) binding regulatory protein (G protein). ing.
  • G protein is a trimer consisting of three subunits, ct, j3 and ⁇ , and is roughly divided into G s, G i, Go and G q depending on the type of ⁇ subunit.
  • the types of conjugated G protein receptors and effector systems are different.
  • glucagon-like peptide 1 receptor and] 3 2 adrenalin receptor are coupled to G s to promote the effector system, adenylate cyclase system -
  • c AMP Binary and increase cyclic AMP
  • the 2-adrenergic receptor coupled with G i, suppresses the adenylate cyclase system and decreases cAMP.
  • HI Hisuta Mi emissions receptors promote phospholipase C system is Efue Kuta one system conjugated with the G q, increases the di ⁇ sill glycerol and Lee Bruno shea torr 3-phosphate, intracellular C a 2 + increase.
  • the main types of effectors are adenylate cyclase system, phospholipase C system, and c GMP phosphodiesterase system.
  • the / 31 adrenalin receptor is localized in the heart, adipose tissue, cerebral cortex, etc., and G s promotes the adenylate cyclase system, resulting in increased heart rate, increased cardiac contractility, and lipolysis. Bring.
  • G D P guanosine 5, monophosphate
  • GD ⁇ bound to ⁇ is replaced with G ⁇ ⁇
  • GTP is bound to ct subunit ⁇ -G TP departs from / 3- ⁇ conjugate.
  • c -G TP and / 3- ⁇ act on the effector system and transmit signals.
  • GPCR biodistribution is often localized in specific organs, but G protein is widely distributed in the body. And as mentioned above, G protein triggers a series of intracellular phosphorylation reactions through GPCR, and controls cell and organ functions widely from transcriptional regulation to muscle contraction at the gene level. .
  • the information transmission system controlled by GPCRZ G protein is an essential mechanism for the control of the physiological functions of organisms.
  • the information transmission system is widely involved in the development of various diseases. It is. Therefore, once a GPCR involved in the development of a disease is identified, by developing drugs that regulate the physiological function of the receptor (agonists, antagonists, antagonists, etc.), The drug makes it possible to treat the disease.
  • GPCR may exist in the whole human genome in the range of 700 to 800, there are about 250 receptors with known ligands, and the rest are one-fan receptors with unknown ligands. is there. Therefore, most of the physiological phenomena involving GPCRs can be said to be undeveloped areas.
  • GPCRs are deeply involved in the control of physiological functions of the living body and the onset of various diseases, so clarifying the physiological function of the receptor elucidates the cause of the onset of various diseases. Is very important in doing.
  • GPCRs are receptors that receive extracellular stimuli. The close connection is also fully understood.
  • GPCR mutations in the secretion of hormones have been well reported, suggesting their importance in physiological roles.
  • Recl68 according to the present invention is a receptor belonging to the Mrg family
  • Nicola Robas et al. Have reported that cortistatin, which controls sleep and motor function, is a MrgX2 (Recl68) ligand (see Non-Patent Document 2 and Patent Document 8).
  • MrgX2 Recl68
  • MrgCll mouse MrgCll
  • MrgXl has 60-70% homology with MrgX2 (Recl68) at the protein level and belongs to the same family, but is a different receptor from MrgX2. The difference between the two is not only the sequence, but also the reactivity of the ligand. MrgXl reacts with BAM8-22, but MrgX2 does not react. MrgXl has high ligand specificity, but MrgX2 is also broad. Furthermore, the expression tissues of the two are also different. As described above, the N-terminal peptide fragment of ChaperoninlO (4-21), cortistatin, etc. have been disclosed as ligands for Recl68, but the physiological functions of Recl68 are not yet clear and are suggested functions.
  • Mast cells are widely distributed in systemic organs, especially in the skin and mucous membranes that are in contact with the outside world.
  • the cell has a large number of granules, and Fc f RI is expressed on the cell surface.
  • Fc ⁇ RI When antigen is bound via IgE molecule, Fc ⁇ RI is When activated, it releases histamine in the granules (degranulation) via various intracellular signal transduction mechanisms, and at the same time, substances such as prostaglandins and leucotrines are produced.
  • These substances called chemical mediators, contract the bronchi, increase the permeability of blood vessels, increase sputum secretion, and cause dyspnea in asthma. Since this reaction occurs in a short time, it is called an immediate reaction.
  • Cytokines such as tumor necrosis factor- ⁇ (TNF- ⁇ ) are produced and released, and it is known that the association stimulation of Fc £ RI is transmitted into the nucleus.
  • Chemical mediators such as histamine, enzymes, and lipid mediators cause smooth muscle contraction, increased vascular permeability, tissue edema, and various cytokines released after several hours. It has been revealed that it is involved in the development of clinical symptoms such as late-onset allergic reactions in the skin, lungs, nose, eyes, etc., i.e., measles, atopic dermatitis, bronchial asthma, allergic rhinitis, and allergic conjunctivitis It's getting on.
  • Substance P was known to cause degranulation at much higher concentrations (2-3 ⁇ ) than previously known receptors. Although there is a report that the mechanism may be via a receptor-independent route (for example, see Non-Patent Documents 10 to 11), details are unknown. There are no reports on specific receptors.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 9-2686
  • Patent Document 2 Japanese Patent Laid-Open No. 9-5 1 7 9 5
  • Patent Document 3 WO O 1/1 9 9 8 3 Pamphlet
  • Patent Document 4 wo o 1 Z 4 8 0 1 5 pamphlet ⁇
  • Patent Document 5 wo o 1/1 6 1 5 9 pamphlet
  • Patent Document 6 wo o 2/0 4 6 4 1 pamphlet
  • Patent Document 7 wo o 3/0 5 0 3 6 pamphlet ⁇
  • Patent Document 8 wo o 3/7 3 1 0 7 pamphlet ⁇
  • Patent Document 9 wo o 3/1 0 4 8 1 8 pamphlet ⁇
  • Non-Patent Document 1 Fl owe D. R., Bioch. Biophys. Acta (1999) 1422
  • Non-Patent Document 2 Robas, N. et al., J. Biol. Chem. (2003) 278, 44400-44404.
  • Non-Patent Literature 4 Piliponsky, A.M. et al., Molecular Immunology (2001) 38, 1369-1372.
  • Non-Patent Document 5 Buku, A., Peptides (1999) 20, 415-420.
  • Non-Patent Document 6 Erjavec, F. et a ⁇ ., Naunyn-Schmi edeberg's Arch
  • Non-Patent Document 8 Mori, T. et al., Arzneimi ttel f orschung (1994) 44, 1044-6.
  • Non-Patent Document 9 0dum, L. et al., Inflamm. Res. (1998) 47, 488-492.
  • Non-Patent Document 10 Aridor, M. et al, J. Cell Biol. (1990) 111 (3), 909-17.
  • Non-Patent Document 1 1 Peptides (2002) 23 (8), 1507-15.
  • Non-Patent Document 1 2 Lee, D. M. et al., Science (2002) 297, 1689-92.
  • Non-Patent Document 1 3 Mehlhop, P. D. et al, Proc. Natl. Acad. Sci. USA.
  • Non-Patent Document 1 4 Kamohara M. et al., Biochemical and Biophysical
  • An object of the present invention is to elucidate the physiological function of the G protein-coupled receptor Recl68, and to provide applications for Recl68 and its ligand that reflect the physiological function. More specifically, the object of the present invention is to provide a substance having an inhibitory action on signal transduction ability via G protein-coupled receptor Recl68 protein, more specifically, a substance that inhibits degranulation reaction or prostaglandin D 2 via Recl68. It is to provide a method for screening production inhibitory substances.
  • Another object of the present invention methods for identifying degranulation inhibitor or prostaglandin Aran Jin D 2 production inhibitors through Recl68, degranulation inhibiting agent comprising by connexion agent identified in the method, prostaglandins It is to provide a gin D 2 production inhibitor or a therapeutic agent for inflammatory diseases involving mast cells. Means for solving the problem
  • the present invention is as follows.
  • Using a cell expressing Recl68 including the step of measuring the signal transduction ability for a ligand having a Recl68 ligand action in the presence and absence of the test substance, and comparing the measured values.
  • a screening method for a substance that suppresses degranulation reaction of mast cells or prostaniesdin D 2 production via Recl68. 2. The method according to 1 above, further comprising measuring and comparing mast cell degranulation or prostaglandin D 2 production via Recl68 in the absence and coexistence of the test substance using mast cells. Screening method.
  • degranulation inhibitor or prostaglandin Aran Jin D 2 production suppression agent according to the 5 you containing as a least active ingredient one compound selected from the following.
  • the 3 or therapeutic agent for inflammatory diseases in which mast cell comprising a degranulation-inhibiting substance or prostaglandin D 2 production inhibitory substance identified by the method described in the above 4 as an active ingredient is involved.
  • the therapeutic agent for inflammatory diseases involving mast cells as described in 7 above which contains at least one compound selected from the following as an active ingredient.
  • Recl68 A mast cell degranulation inhibitor or prostaglandin D 2 production inhibitor comprising a substance having an antagonistic action as an active ingredient.
  • Recl68 Antagonis A therapeutic agent for inflammatory diseases involving mast cells, which contains a substance having a manicidal action as an active ingredient.
  • Mast cells characterized by containing as a reagent a Recl68 protein or a partial peptide thereof and a salt thereof, a polynucleotide encoding Recl68, an antibody against Recl68 or an antibody against Recl68 ligand A diagnostic agent for inflammatory diseases involving selenium.
  • a mast cell degranulation inhibitor comprising, as an active ingredient, a degranulation inhibitor identified by the method described in 1 or 4 above.
  • the degranulation inhibitor as described in 16 above which contains at least one compound selected from the following as an active ingredient.
  • a therapeutic agent for an inflammatory disease involving mast cells comprising as an active ingredient the degranulation inhibitor identified by the method described in 1 or 4 above.
  • the therapeutic agent for an inflammatory disease involving mast cells according to the above 18, comprising at least one compound selected from the following as an active ingredient.
  • a mast cell degranulation inhibitor comprising a substance having an antagonistic action as an active ingredient.
  • Rec 68 with a sequence characteristic of GPCR. Reverse transcription reaction was performed using human testis pol y (A) + RNA as a saddle, and cDNA was synthesized. The gene of interest was amplified by PCR and incorporated into an expression vector to produce Rec68-transient and stable expression cells.
  • N-terminal 20 amino acid peptide of porcine ChaperoninlO, porcine 2 ', 3'-cyclic nucleotide 3' phosphodiesterase (hereinafter abbreviated as pCNP) (DN-terminal 17 amino acid peptide, porcine tropomyosin N-terminal peptide) 12 Peptides consisting of amino acids, PACAP (6-36), VIP (3-27), Substance P, Somatostatin, Mast cell degranulating (hereinafter abbreviated as MCD) peptides, etc.-As mentioned above, Known as basic secretagogues such as MCD peptide, Substance P, VIP, Somatostatin, compound 48/80, etc., as non-immune-related mast cell degranulation inducers Substance P, VIP, Somatostat
  • the Recl68 agonist substance obtained from screening by the reporter gene assembly showed almost the same reactivity when measured in the intracellular free calcium assay system.
  • GTP binding activity As a result of examining GTP binding activity by selecting several types from those exhibiting agonist activity, it was found that all of the studied agonists showed GTP binding activity. It was strongly suggested that intracellular signal transduction occurs through G protein.
  • PGD 2 prostaglandin D 2
  • substance active substances such as Substance P, MBP fragment and ECP fragment induced PGD 2 production in a concentration-dependent manner.
  • the PGD 2 production activity by any stimulus was inhibited by 10 ⁇ M JTP-375854, which is a Recl68 inhibitory compound, and 445755, so that Recl68 activation was desensitized to human cultured mast cells. It was found to induce not only granules but also de novo synthesis of PGD 2 .
  • IgE is known to activate Syk through high-affinity FcRy epsilon and induce mast cell degranulation and de novo synthesis of PGD 2 , whereas Recl68 shares a signaling pathway with FcR ⁇ epsilon. It was guessed that the part was shared.
  • ECP eosinophil protective protein
  • MBP Major basic protein
  • Recl68 is a receptor that transmits degranulation signals is something that no one has ever anticipated, and is one of the mechanisms of non-immune degranulation without IgE involvement.
  • the present invention is extremely important in that it is clarified for the first time. Furthermore, not only degranulation, but also PGD 2 production that is known to occur simultaneously with degranulation. It should also be noted that it has become clear that Rec l 68 is also transmitted to Gunar. It is possible to proceed with the development of effective therapeutic agents in the future, targeting many chronic inflammations for which no effective therapeutic agent or method has been found since the onset mechanism was unknown. It became.
  • the present inventors have further researched and developed a screening method for a drug capable of suppressing the degranulation reaction or PGD 2 production from mast cells via Rec l 68, and actually screened. As a result, we succeeded in finding multiple effective compounds.
  • the present inventors have found a physiological function of Recl68 that has not been clarified so far, developed a screening method and a identification method for a new drug based on the function, and actually used a plurality of compounds that can be such a drug. Successfully acquired.
  • the method for screening a substance having an action of suppressing the signal transduction ability mediated by the Rec l 68 protein or the method for identifying a substance having an action of suppressing the signal transduction ability mediated by the Rec l 68 protein in the present invention it is simply linked to Rec l 68.
  • Rec l 68 Unlike the substance that regulates the binding ability of the substances to be combined and the substance that regulates the signal transduction ability from substances that transmit signals in artificial cell systems, the physiological function of Rec l 68 actually It is now possible to obtain substances that suppress degranulation or PGD 2 production. As a result, the development of therapeutic agents for inflammatory diseases, such as allergic diseases or autoimmune diseases, which have been impossible until now, such as inflammatory diseases involving mast cell immunity-free degranulation and PGD 2 production, etc. Became possible.
  • inflammatory diseases such as allergic diseases or autoimmune diseases
  • the present invention also provides a diagnostic agent for a therapeutic agent for inflammatory diseases involving mast cells such as allergic diseases or autoimmune diseases.
  • a diagnostic agent for a therapeutic agent for inflammatory diseases involving mast cells such as allergic diseases or autoimmune diseases.
  • Rec 68 induces degranulation of mast cells or PGD 2 production.
  • Increase or decrease in cell surface expression level or gene expression level of Rec l 68 protein, structural abnormality of expressed Rec l 68 protein, gene mutation may cause chronic inflammatory reaction through excessive degranulation or PGD 2 production
  • screening of substances capable of regulating the abnormal expression of Recl68 protein and Recl68 gene, which cause inflammatory diseases involving mast cells, in particular allergic diseases or autoimmune diseases is also possible.
  • the substance obtained by the method is capable of regulating the degranulation reaction or PGD 2 production via Recl68. This has made it possible to develop new types of therapeutic agents for inflammatory diseases.
  • Figure 1 shows ChaperoninlO ( ⁇ 20) in PC12h / zif reporter assembly (1)
  • FIG. 2 shows the results of 2,3-cyclic nucleotide 3 phosphodiesterase (1-17) Tropomyosin (1-12) CD.
  • the black circle is Chaperonin 10 ( ⁇ 20)
  • the black triangle is 2 ', 3'-cyclic nucleotide 3' phosphodiesterase (1-17) (pCNP ( ⁇ 17))
  • the black square is Tropomyosin (l-12).
  • Means. A shows the results with Recl68 expressing cells, and B shows the results with Mock.
  • FIG. 2 is a diagram showing the results of MCD-Peptide, Somatostatin 28, and Substance P in PC12h / zif reporter assembly (2).
  • the black circle means MCD-Peptide
  • the black triangle means Somatostatin 28, and the black square means Substance P.
  • A shows Recl68-expressing cells
  • B shows Mock results.
  • Figure 3 shows the results of Compound 48/80 in PC12h / zif reporter assembly (3).
  • the black circle means Compound 48/80.
  • A shows Recl68-expressing cells, B shows Mock results.
  • Figure 4 shows the expression vector pLHCX.
  • FIG. 5 shows pLHC—Recl68-14.
  • Figure 6 shows GTP-Eu binding (increase in GTP binding amount) to the membrane fraction dependent on Substance P concentration.
  • black circles indicate the results for the membrane fraction prepared from 293 cells stably expressing Recl68, and white circles indicate the results for the membrane fraction prepared from 293 (parent strain).
  • FIG. 7 is a graph showing GTP-Eu binding (increase in GTP binding amount) to the membrane fraction depending on Chaperoninl0 (l-20) concentration.
  • black circles indicate the results for the membrane fraction prepared from 293 cells stably expressing Recl68
  • white circles indicate the results for the membrane fraction prepared from 293 (parent strain).
  • FIG. 8 is a graph showing GTP-Eu binding (increase in GTP binding amount) to the membrane fraction depending on the pCNP (l-17) concentration.
  • the black circles indicate the results for the membrane fraction prepared from Recl68 stably expressing 293 cells
  • the white circles indicate the results for the membrane fraction prepared from 293 (parent strain).
  • FIG. 9 is a diagram showing GTP-Eu binding (increase in the amount of GTP binding) to the membrane fraction depending on the concentration of MCD peptide (Mast Cell Degranulating Peptide).
  • MCD peptide Mast Cell Degranulating Peptide
  • FIG. 10 is a diagram showing the degranulation reaction of human cultured mast cells by ligand (the degranulation reaction dependent on the concentration of the ligand substance). A23187 was used as a positive subject. The degranulation was shown as Mean ⁇ S. E. M of three experiments using 3 different donor-derived cultured mast cells.
  • Figure 11 shows the correlation between the degranulation reaction of human cultured mast cells (EC 25 ) and the activity (EC 5 ) of Ca Assay using Recl68-expressing cells.
  • Figure 1 2 shows Substance P ( Figure 1) in Donor 4-derived human cultured mast cells.
  • FIG. 12 B Compound 48/80
  • FIG. 12 C are diagrams showing the effect of FCS during culture on degranulation by stimulation.
  • Fig. 13 shows the effect of FCS during culture on degranulation induced by Substance P and Compound 48/80 in Donor 6-derived human cultured mast cells.
  • A shows the effect of serum added during culture on degranulation by Substance P stimulation and B by Compound 48/80 stimulation.
  • FIG. 14 is a graph showing a comparison of Recl68 mRNA expression levels by tissue.
  • FIG. 15 is a graph showing the inhibitory activity against mast cell degranulation of Recl68 inhibitory compounds (compound 1 (15-b), compound 2 (15_a), compound 3 (15-c)).
  • white squares Substance P (1 ⁇ M
  • Substance ⁇ (1- 11) (1 ⁇ ⁇
  • black circle means A23187 (0.3 ⁇ ).
  • FIG. 16 shows the fragment map of ECP and IV.
  • the ECP fragment map is shown divided into (A-1) to (A-3).
  • (B_l) through (B-3) are divided into three.
  • FIG. 17 shows the degranulation-inducing activity of fragmented ECP on human cultured mast cells (A) and the Ca response activity (B) on 293 cells stably expressing Recl68.
  • FIG. 18 shows the degranulation-inducing activity of fragmented MBP on human cultured mast cells (A) and the Ca-responsive activity (B) on 293 cells stably expressing Recl68.
  • FIG. 19 shows the recl68 inhibitory compounds (compound 1 (A), compound 2 (B), and compound 3 (C)) against degranulation induction of human cultured mast cells by MBP, ECP fragment RXl Substance P $ lj. It is a figure which shows an effect
  • FIG. 20 shows the effects of a Recl68 inhibitor compound and Cromolyn on the induction of degranulation of human mast cells by stimulation with Substance P (A) and Compound 48/80 (B).
  • black circles indicate Compound 1
  • white circles indicate Compound 3
  • black squares indicate Cromolyn.
  • FIG. 21 shows the degranulation induction of human cultured mast cells by IgE cross-linking (A) and the action of Recl68 inhibitory compound and Cromolyn (B).
  • the black circle means compound 1
  • the white circle means compound 3
  • the black square means Cromolyn.
  • FIG. 22 is a diagram showing PGD2 production in human cultured mast cells stimulated by Substance P (A), MBP fragment (B), and ECP fragment (C) and the action of a Recl68 inhibitor compound.
  • FIG. 23 shows immunostaining of mouse cultured mast cells using anti-Recl68 antibody (anti-MrgX2 peptide antibody), anti-human tryptase antibody and anti-human chymase antibody.
  • anti-Recl68 antibody anti-MrgX2 peptide antibody
  • anti-human tryptase antibody anti-human chymase antibody.
  • the bar in the figure indicates 20.
  • G protein-coupled receptor in the present invention is a receptor that exists on the cell membrane, and is received via a GTP (guanosine 5′-triphosphate) -binding regulatory protein (G protein). It means a protein that has the function of transmitting information on ligands that interact with the body to the effector system. Most G protein-coupled receptors identified so far have a structure that penetrates the cell membrane seven times.
  • GTP guanosine 5′-triphosphate
  • the “cell expressing Recl68” is not particularly limited as long as it is a cell expressing Recl68 protein on the cell surface.
  • a cell line into which the Recl68 gene has been introduced and forcibly expressed on the cell surface can also be used.
  • the parent strain can be used as a cell that does not express Recl68 for comparison.
  • the existing cell line used here is not particularly limited as long as it contains an expression vector and can maintain the replication or expression of the expression vector.
  • it can be a prokaryotic cell such as E. coli, or a eukaryotic cell such as yeast, insect or amphibian, or a mammalian cell such as CH0, HeLa, or 293.
  • a prokaryotic cell such as E. coli
  • a eukaryotic cell such as yeast, insect or amphibian
  • a mammalian cell such as CH0, HeLa, or 293.
  • the cells capable of efficiently expressing Recl68 293 cells are preferred.
  • Recl68 forced expression cell a cell in which Recl68 is transiently expressed or a cell in which stable expression is used may be used.
  • Recl68 forced expression cells can be prepared by methods known to those skilled in the art.
  • the cell expressing Recl68 a cell derived from a living body and expressing Rec 168 natively can also be used.
  • the cells are not particularly limited as long as they express Recl68, but among them, mast cells are preferable.
  • the mast cell in the present invention may be a primary cultured cell isolated from a living body or an existing cell line that retains the characteristics of a mast cell.
  • connective tissue type mast cells are preferable.
  • the primary cultured cells mast cells obtained by induction from human umbilical cord blood-derived CD34-positive hematopoietic stem cells according to a conventional method can be used.
  • Existing cell lines are not particularly limited, and examples include P815 and RBL-2H3 cells. Substance
  • a “substance” identified or screened by the method of the present invention is a natural substance existing in nature (protein, antibody, peptide, natural compound, etc.) or any artificially prepared substance (chemical synthesis). Means compound) To do.
  • any chemically synthesized “compound” can be mentioned.
  • the type and molecular weight of the compound are not particularly limited, but in terms of molecular weight, the molecular weight of a compound that may be used as a pharmaceutical is about 50 to about 3000 molecular weight, Has a molecular weight of about 100 to about 2000, and more typically a molecular weight of about 100 to about 1000.
  • the substance when the substance is a protein, antibody or peptide, it includes those isolated from living tissues and cells, and those prepared by genetic recombination or chemical synthesis. Furthermore, those chemical modifications are also included.
  • peptide examples include a peptide consisting of about 3 to about 500 amino acids, preferably about 3 to about 300 amino acids, more preferably about 3 to about 200 amino acids.
  • Recl68 used in the present invention is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.
  • Rec 68 is a known protein described in a plurality of documents (for example, W001 16159, W001 19983, W00172838, etc.).
  • Rec l68 is, for example, any cell of humans or other mammals (eg, guinea pigs, rats, mice, rabbits, pigs, hidges, mice, monkeys, etc.) (eg, retinal cells, spleen cells, neurons) , Dariya cells, knee / 3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibroblasts, muscle cells, adipocytes, immune cells (eg, macrophages, T Cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes, leukocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts , Breast cells, hepatocytes or stromal cells, or progenitors of these cells, stem cells or cancer cells (eg, breast cancer cell lines (GI
  • Cells or cultured cells thereof eg, ME L, Ml, CTLL 1, 2, HT-2, WEH I-3, HL-60.0, JOSK-1, 556-6, ML-1, MOLT-3, MO LT_4, MO LT—10, CCRF—C EM, TA LL-1, J ⁇ rkat, CCRT—HSB-2, KE—3 7, S KW—3, HUT—78, HUT—10 2, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG10, etc.), or synthetic proteins But let ’s do it.
  • the “substantially identical amino acid sequence” means, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more with respect to the amino acid sequences to be compared. Most preferably, it refers to an amino acid sequence having a homology of about 95% or more.
  • Recl68 is a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, for example, an amino acid sequence represented by SEQ ID NO: 1. And a protein having substantially the same amino acid sequence as that of the amino acid sequence represented by SEQ ID NO: 1, and having substantially the same activity as the protein.
  • the activity of substantially the same quality examples include a ligand binding activity and a signal signal transduction action. “Substantially homogeneous” means that their activities are homogeneous in nature. Therefore, the ligand binding activity is equivalent to the activity such as signal transmission (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5). The amount of these activities may be different in quantitative elements such as the molecular weight of the protein.
  • the activities such as ligand binding activity, receptor binding activity and signal information transmission activity can be measured according to a force that can be performed according to a known method, for example, a screening method described later.
  • Recl68 (1) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30 pieces, more preferably about 1 to 1 °, more preferably numbers) (1 to 5) amino acid sequences lacking amino acids, (2) SEQ ID NO: 1 or 2 or more (preferably about 1 to 30) in the amino acid sequence represented by 1 More preferably, about 1 to 10 amino acids, more preferably several (1 to 5)) amino acid sequences added, (3) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence in which the above (preferably about 1 to 30 pieces, more preferably about 1 to 10 pieces, and more preferably several (1 to 5 pieces)) are substituted with other amino acids, Or (4) Tan containing amino acid sequences in combination of them Click quality, such as can also be used.
  • Recl68 is N-terminal (amino terminal) at the left end and C-terminal (carboxyl terminal) at the right end according to the convention of peptide notation. Recl68 is, C-terminal, a carboxyl group (one CO OH), Karubokishire bets (- COO-), A Mi de (- CO NH 2) or an ester (- COOR) it may also be any of Rere.
  • R in the ester e.g., methyl, Echiru, n - propyl, C, such as isopropyl or n _ butyl - 6 alkyl group, for example, sik Ropenchiru, C 3, such as Kishinore the consequent Russia - 8 consequent Roanorekiru group, for example, phenyl, shed - C 6, such as naphthyl -, 2 Ariru group, for example, benzyl, phenyl _ C i-2 alkyl or c such phenethyl - such as naphthylmethyl alpha - Nafuchiru C, - 2 alkyl
  • aralkyl groups such as a group, a bivalyloxymethyl group that is widely used as an oral ester is used.
  • Recl68 has a carboxyl group (or carboxylate) in addition to the C-terminus
  • those in which the carboxyl group is amidated or esterified are also included in Recl68 in the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
  • Amino group protecting groups Mechio two emissions residues of N-terminal e.g., formyl groups, C such as C 2 _ fi Arukanoiru group such Asechiru, - such as 6 Ashiru group
  • N-terminal e.g., formyl groups, C such as C 2 _ fi Arukanoiru group such Asechiru, - such as 6 Ashiru group
  • a substituent on the side chain of an amino acid in the molecule eg, 1 OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.
  • a suitable protecting group eg, formyl group, C, such as 6 Arukanoi Le group, - - C 2 such Asechiru 6 Ashiru those protected by group, etc.
  • sugar chains are also conjugated proteins such as glycoproteins bound.
  • Recl68 examples include chick Recl68 consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • Recl68 or a salt thereof can be produced from the above-mentioned human mammalian cells or tissues by a known receptor protein purification method, and contains Recl68 or DNA encoding Recl68 described later. It can also be produced by culturing the transformant. Moreover, it can also be produced according to the protein synthesis method described later or this.
  • the human mammal tissue or cells are homogenized and extracted with acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. It can be purified and isolated by combining chromatography such as tomography.
  • the partial peptide of Recl68 (hereinafter sometimes simply referred to as “partial peptide”) is any peptide having the above-mentioned partial amino acid sequence of Recl68. However, for example, a portion of Recl68 that is exposed to the outside of the cell membrane and has a receptor-binding activity substantially the same as that of Recl68 is used.
  • the extracellular region (Hydrophi 1 ic) site in the hydrophobic plot analysis was used. It is a peptide containing a portion analyzed to be. In addition, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used. Peptides that contain individual domains can also be used, but peptides that contain multiple domains simultaneously may be used.
  • the number of amino acids of the partial peptide has at least 20 or more, preferably 50 or more, more preferably 100 or more amino acid sequences among the above-mentioned constituent amino acid sequences of Recl68. Peptides are preferred.
  • the partial peptide is 1 or 2 or more (preferably in the amino acid sequence).
  • about 1 to 10, more preferably several (1 to 5) amino acids are deleted, or 1 or more (preferably, the amino acid sequence).
  • the above amino acids (preferably about 1 to 10, more preferably several, and more preferably about 1 to 5) may be substituted with other amino acids.
  • the partial peptide has a carboxyl group at the C-terminus.
  • the amino group of the N-terminal methionine residue is protected with a protecting group, and the G 1 n produced by cleavage of the N-terminal side in vivo is pyrolyzed.
  • glutamine-oxidized ones substituents on the side chain of the amino acid in the molecule protected with appropriate protecting groups, or complex peptides such as so-called sugar peptides to which sugar chains are bound It is.
  • Examples of the salt of Recl68 or a partial peptide thereof include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable.
  • Examples of such salts include 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). Acid, succinic acid, tartaric acid, citrate, phosphonic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid.
  • a partial peptide of Recl68 or a salt thereof can be produced according to a known peptide synthesis method or by cleaving Recl68 with an appropriate peptidase.
  • a peptide synthesis method for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting Recl68 and the remaining part, and removing the protective group when the product has a protective group. Examples of known condensation methods and elimination of protecting groups include the methods described in the following a) to e).
  • the partial peptide of the present invention can be purified and isolated by combining ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography and recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method.
  • the partial peptide is obtained as a salt, the free form can be obtained by a known method.
  • the present invention relates to a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 1, and one or more amino acids of the amino acid sequence represented by SEQ ID NO: 1 substituted, deleted, inserted, and / or added.
  • a polynucleotide encoding the amino acid sequence of a polypeptide consisting of an amino acid sequence is also provided.
  • the polynucleotide of the present invention also includes both single-stranded and double-stranded DNA, and its RNA complement.
  • DNA includes, for example, naturally occurring DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. DNA is preferred as the polynucleotide of the present invention.
  • codons are degenerate and some amino acids have multiple base sequences that code for one amino acid. Those having the base sequence are also included in the scope of the present invention.
  • the base sequence of the cDNA actually cloned in Example 1 below is SEQ ID NO: 2. -
  • the polynucleotide of the present invention preferably has the base sequence of SEQ ID NO: 2.
  • the polynucleotide of the present invention can be prepared, for example, by the following method.
  • the base sequence characteristic of Recl68 is searched as a query, and Recl68 is found from the human genome.
  • the found genomic sequence or a part of the genomic sequence is used to obtain the present polynucleotide (for example, the present invention DNA) from one cDNA library or the like using basic techniques of genetic engineering such as hybridization or nucleic acid amplification reaction.
  • the base sequence characteristic of Recl68 is searched as a query, and Recl68 is found from the human genome.
  • the found genomic sequence or a part of the genomic sequence is used to obtain the present polynucleotide (for example, the present invention DNA) from one cDNA library or the like using basic techniques of genetic engineering such as hybridization or nucleic acid amplification reaction.
  • the nucleic acid amplification reaction is, for example, a polymerase chain reaction (PCR) [Saiki R. K. eta 1-, Science, 2 3 0, 1 3 5 0— 1 3 5 4 (1 9 8 5) ehrs chain reaction (LCR) [W u D. Y. etal., Genomics, 4, .5 6 0—5 6 9 (1 9 8 9); B arringer K. J- etal., Gene, 8 9, 1 1 7— 1 2 2 (1 9 9 0); Barrany F., Proc. Natl. A cad. S ci. USA, 8 8, 1 8 9— 1 9 3 (1 9 9 1)], and amplification based on transcription [Kw oh D. Y.
  • PCR polymerase chain reaction
  • nucleic acid sequence-based amplification (Nucleic A cid Sequence B ased Amplification: NASABA) reaction described in European Patent No. 0 5 2 5 8 8 2 is performed. Is available. The PCR method is preferred.
  • a DNA fragment of 1.0 kbp is obtained as a PCR product.
  • This can be obtained by, for example, agarose gel electrophoresis or the like.
  • DN depending on the molecular weight of The nucleic acid of the present invention can be obtained by separating A fragments by a method of sieving and isolating according to a conventional method such as a method of cutting out a specific band.
  • the homologous nucleic acid cloned using the above-mentioned hybridization, nucleic acid amplification reaction, etc. is at least 20% or more, preferably 30%, with respect to the base sequence described in SEQ ID NO: 1 in the Sequence Listing. % Or more, more preferably 50% or more, even more preferably 70% or more, and most preferably 90% or more.
  • the percent identity can be determined by visual inspection and mathematical calculations. Alternatively, the percent identity of two nucleic acid sequences can be found in Devereux et al., Nucl. A cids Res., 1, 3 8 7 (1 9 8 4), and the University of Wisconsin Genetics Computer It can be determined by comparing the sequence information using the GAP computer program, version 6.0, available from the group (UWG CG). Preferred default parameters for the GAP program include: (1) a single comparison matrix for nucleotides (including values of 1 for identical and 0 for nonidentical), and supervised by S chwartz and Daihoff , A tlasof P rotein Sequence and S compture, pp.
  • nucleotide complementary to the polynucleotide consisting of the nucleotide sequence described in 2 or a complementary strand thereof having a chain length of at least 15 nucleotides where “complementary strand” means A: T (U in the case of RNA) refers to the other strand of a double-stranded nucleic acid consisting of G: C base pairs.
  • “complementary” is not limited to a completely complementary sequence in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, more preferably 95% or more of the homology on the base sequence. As an algorithm for determining homology, those described in the present specification may be used.
  • Such a nucleotide can be used as a probe for detecting and isolating the polynucleotide of the present invention and as a primer for amplifying the nucleotide of the present invention. is there.
  • a primer When used as a primer, it usually has a chain length of 15 to 100 nucleotides, preferably 15 to 35 nucleotides.
  • Such a polynucleotide is preferably one that specifically hybridizes to a polynucleotide encoding the polypeptide of the present invention.
  • “Specifically hybridize” means a polynucleotide (SEQ ID NO: SEQ ID NO: 2) identified by the present inventors under normal hybridization conditions, preferably under stringent conditions. : 2) means that it hybridizes with DNA and does not hybridize with DNA encoding other polypeptides.
  • under stringent conditions means to hybridize under moderate or high stringent conditions. Specifically, moderate stringency conditions can be easily determined by a person having ordinary skill in the art based on the length of DNA, for example, fij. Basic conditions are: Sambrook, J. et al., Molecular C.
  • High stringency harsh conditions can also be easily determined by those skilled in the art, eg, based on DNA length. Is possible. In general, these conditions include hybridization and / or washing at higher temperatures and Z or lower salt concentrations than moderately stringent conditions, such as those described above. And approximately 68 ° C, 0.2 XSSC, with 0.1% SDS cleaning. One skilled in the art will recognize that the temperature and wash solution salt concentration can be adjusted as needed according to factors such as probe length.
  • This polynucleotide also includes a polynucleotide that suppresses the expression of the gene encoding Rec68.
  • a polynucleotide includes an antisense polynucleotide (antisense DNA / RNA; an antisense RNA complementary to the transcript of the gene encoding the polypeptide of the present invention, and the RNA.
  • Ribosomes DNA that encodes RNA having ribozyme activity that specifically cleaves the transcript of the gene encoding the polypeptide of the present invention).
  • the antisense polynucleotide used in the present invention may suppress the expression of a target gene by any of the above-mentioned actions.
  • designing an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of a gene is considered effective for inhibiting translation of the gene.
  • a sequence complementary to the coding region or the 3 'untranslated region can also be used.
  • a polynucleotide containing an antisense sequence of not only a translation region of a gene but also an untranslated region is also included in the antisense polynucleotide used in the present invention.
  • the antisense polynucleotide used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal on the 3 'side.
  • the antisense polynucleotide sequence is preferably a sequence complementary to the target gene or a part thereof, but may not be completely complementary as long as the gene expression can be effectively inhibited.
  • the transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene.
  • antisense polynucleotides must be at least 15 nucleotides or more, preferably 100 nucleotides or more, to cause an antisense effect. Preferably, it has a chain length of 500 nucleotides or more, and usually has a chain length of not more than 3000 nucleotides, preferably not more than 2000 nucleotides.
  • the antisense polynucleotide is, for example, based on the sequence information of a polynucleotide encoding Recl68 (eg, SEQ ID NO: 2) (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 16 3209-21 (1988)).
  • Ribozyme refers to an RNA molecule that has catalytic activity. Although some ribozymes have various activities, research on ribozymes as an enzyme that cleaves RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA. Ribozymes include group I introns and those with a size of 400 nucleotides or more, such as the M1RNA contained in RNaseP, and the hammerhead type is about 40 nucleotides called hairpin type. Some have active domains (Makoto Koizumi and Eiko Otsuka, (1990) Protein Nucleic Acid Fermentation Elemental, 35: 2191).
  • the self-cleaving domain of a hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to base-pair with A at position 9 for activity.
  • Has been shown to be cleaved by A or U in addition to C (M. Koizumi et al., (1988) FEBS
  • RNA cleavage ribozyme that recognizes the sequence UC, UU, or UA in the target RNA can be created.
  • Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin-type ribozymes are found, for example, in the minus strand of tobacco ring spot virus satellite RNA (J. M. Buzayan Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids Res. 19: 6751, Hiroshi Kikuchi, (1992) Organism 30: 112).
  • Polynucleotides that suppress the expression of the gene encoding Recl68 can be used for gene therapy, for example, viral vectors such as retrovirus vector, adenovirus vector, adeno-associated virus vector, etc. It may be possible to administer to patients by ex vivo or in vivo methods using a non-viral vector such as a liposome.
  • viral vectors such as retrovirus vector, adenovirus vector, adeno-associated virus vector, etc. It may be possible to administer to patients by ex vivo or in vivo methods using a non-viral vector such as a liposome.
  • antibodies that bind to Recl68.
  • antibodies include polyclonal and monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, and Fab fragments containing the products of Fab or other immunoglobulin expression libraries. included.
  • Recl68 or fragments or analogs thereof, or cells expressing them can also be used as an immunogen to produce antibodies that bind to Recl68.
  • the antibody is preferably immunospecific for Recl68. “Immunospecific” means that the antibody has a substantially higher affinity for Recl68 than its affinity for other polypeptides. Examples of the antibody in the present invention include those capable of detecting Rec 68 and having an activity that affects the function of Rec 68.
  • the function of Rec 68 means a function of binding a ligand substance and a function of inducing a cell degranulation reaction by binding of a ligand substance. Therefore, the antibody as a drug in the present invention inhibits part or all of the binding between Rec 68 and the ligand substance, and suppresses part or all of the cell degranulation reaction that occurs through Rec 168. And those that enhance.
  • Antibodies against Rec 68 can be prepared by methods known to those skilled in the art.
  • a polyclonal antibody can be obtained as follows. Serum is obtained by immunizing small animals such as rabbits with Rec 68 or a fragment thereof or a fusion protein thereof with GST. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column with the polypeptide of the present invention, or the like.
  • Rec 68 is immunized to a small animal such as a mouse, and the spleen is removed from the mouse, and this is ground to separate cells, such as mouse myeloma cells and polyethylene dallicol.
  • the fused cells (hybridomas) that are fused with the reagent select a clone that produces an antibody that binds to Recl68 and affects the function of Recl68.
  • the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is used for, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange. It can be prepared by purification using a chromatography column, an affinity column that is a copy of the polypeptide of the present invention, and the like.
  • the antibody used for detection of Rec 68 contained in the diagnostic agent for inflammatory disease and kit for diagnosing inflammatory disease in the present invention is not particularly limited as long as it is a detectable antibody. Can be prepared by methods known to those skilled in the art. Drug, drug
  • agent or “drug” or “pharmaceutical composition” of the present invention means “compound”, “peptide”, “polynucleotide” or “polynucleotide” of the present invention as defined above.
  • pharmaceutical composition comprising any of the “antibodies” and a pharmaceutically acceptable carrier.
  • the “compound” in these “agent” or “drug” and “pharmaceutical composition” means a natural or synthetic compound.
  • the “peptide” means a compound in which a plurality of amino acids are linked to an amino acid, and may be an oligopeptide or a polypeptide.
  • Polynucleotide means a polymer obtained by polymerizing about 30 or more nucleotides.
  • Antibody means so-called immunoglobulin (Ig).
  • compositions of the present invention are effective as therapeutic agents for inflammatory diseases described later, particularly inflammatory diseases involving mast cells.
  • “pharmaceutically acceptable carrier” refers to an excipient, a diluent, a bulking agent, a disintegrant, a stabilizer, a preservative, a buffer, an emulsifier, an fragrance, a coloring agent, a sweetener, and a thickener. , Flavoring agents, solubilizers or other additives.
  • a pharmaceutical composition in the form of can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include one or more active substances, externally formulated liquids formulated by conventional methods, suppositories for enteric administration, and pessaries.
  • the dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, or the type of active ingredient (such as the above polypeptide antibody) contained in the pharmaceutical composition. In general, it can be administered at a dose of 10 ⁇ g to l OOOmg (or 10 ⁇ g to 500 mg) per adult. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, and a dose exceeding the above range may be necessary.
  • the injection produced in this way is given to a human patient in need of treatment at a dose of 1 g / kg body weight per kg! OO mg, preferably 5 0 8 to 5 0 ⁇
  • the dose can be administered once to several times per day.
  • administration forms include medically suitable administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection. Intravenous injection is preferred.
  • injections are non-aqueous diluents (eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions or emulsions. It can also be prepared as an agent.
  • non-aqueous diluents eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol
  • suspensions or emulsions can also be prepared as an agent.
  • Such sterilization of injections can be performed by filtration sterilization through a bacteria-retaining filter, blending of bactericides, or irradiation.
  • An injection can be produced in a form prepared at the time of use. That is, it can be used as a sterile solid composition by lyophilization or the like, dissolved in sterile water for injection or other solvent before use.
  • the “agent” or “drug” to “pharmaceutical composition” of the present invention can be applied to the treatment and prevention of diseases involving degranulation of mast cells, such as inflammatory diseases. It is particularly effective for chronic inflammation such as allergic diseases and autoimmune diseases.
  • allergic inflammation involves the invasion of antigen into the body, macrophage phagocytosis and antigen presentation, Th antigen recognition and I production, B cell stimulation and IgE antibody production, binding to Fc f RI It develops through the process of progressing to degranulation.
  • allergic inflammations are pathologically as follows. (1) Atopic dermatitis that enhances IgE production.
  • Inflammatory cell function modification by cytokines from locally infiltrating T cells (Eosinophil migration and activation by Th2 cells, I-3, I-5, GM-CSF, etc.).
  • inflammations are local reactions exhibited by the living body when some harmful flame retardant acts on living tissue, and is a process of body defense. Increases vascular permeability, leach leukocytes locally, leaks protective factors such as plasma locally, promotes blood clotting, obstructs blood vessels, and reduces local oxygen concentration, thereby suppressing pathogen growth And prevent the spread of toxins.
  • the microvasculature temporarily contracts and then expands. As a result, the gap between the vascular endothelial cells spreads in the venule and the plasma infiltrates, and then the inflammatory cells migrate out of the blood vessel. Histamine and prostaglandins act on endothelial cells and inflammatory cells.
  • diseases that are particularly related to mast cells include the following diseases.
  • Cirrhosis Increased mass of mast cells around the bile ducts, increased liver fibrosis, intrahepatic microvascular regulation and angiogenesis.
  • Intrahepatic large bile ducts, fibrosis of the bile wall of the septal bile duct, and fibrosis around the bile ducts are observed.
  • Peri-biliary mast cells increase, and these mast cells are found to produce cytokines such as bFGF, TNF-a, I-1, and TGF- ⁇ , which are involved in fibrosis.
  • Mast cells are observed in human coronary artery lesions, and many activated mast cells are present in atherosclerotic vascular endothelial plaques.
  • Obese cells are present in the human heart, especially in the vicinity of blood vessels, in the vicinity of the sarcolemma, and in the atherosclerotic lesion. Mast cells are activated by type I allergies.
  • Mast cells increase in accordance with the location of collagen fiber deposition. The presence of bFGF-positive mast cells is confirmed.
  • Nephritis tubulointerstitial nephritis, glomerulonephritis
  • Enriched cells increase in the connective tissue around the blood vessels around the tubules and kidney stroma, or around the glomeruli. The presence of IL-4 positive mast cells is confirmed.
  • Atopic dermatitis Increased mast cells and production of TNF-chicken IL-4 are observed. Substance M secreted from the C-fiber nerve ending induces the activation and degranulation of new mast cells.
  • Membrane stabilizer Effective for treatment and prevention of tranilast.
  • Vascular proliferative disorder force positive sarcoma, malignant vascular endothelial cell type:
  • mast cell-related diseases include gastritis, gastric ulcer: cirrhosis: chronic biliary tract disease (intrahepatic stone disease, primary sclerosing cholangitis): ulcerative colitis: chronic inflammatory Vascular disease: Arteriosclerosis: Cardiac anaphylaxis: Pulmonary fibrosis: Bronchial asthma: Nephritis (tubulointerstitial nephritis, glomerulonephritis): Interstitial cystitis: Atopic dermatitis: Keloid: Vascular proliferative disorder (Positive positive sarcoma, Acute vascular endothelial cell type): Rheumatoid arthritis: Allergic rhinitis: Allergic conjunctivitis: Gastric cancer: Colorectal cancer, Breast cancer, Skin basal cell carcinoma, Soft tissue tumor, Melanoma, etc.
  • Malignant tumors non-small cell lung cancer: other urticaria, Systemic lupus erythematosus, multiple sclerosis, collagen disease, malignant tumor, B lymphoma, B cell leukemia, and the like.
  • the therapeutic effects of various drug symptoms of the “agent” or “drug” of the present invention can be tested and examined by administering to a known disease model animal according to a conventional method.
  • the “recl68 agonistic ligand” is a substance that can transmit a signal into a cell via Rec 168, and the kind of signal, the kind of substance, etc. are particularly limited. It is not a thing.
  • the “signal transduction ability” mentioned here is expressed by the degree of signal that a ligand substance having the action of Recl68 agonist can transmit into cells via Recl68. There are no particular limitations on the type of signal, the type of ligand substance, and the like.
  • the step of measuring the above signal transduction ability includes the following reporter gene reporter gene reporter gene reporter (International Patent Application Publication No. W092 / 02639 and JP 6-502527, etc.) Can be used.
  • the reporter gene assay refers to the amount of reporter protein expressed depending on the action of the compound by contacting a test substance with a cell and measuring the amount of fluorescence emitted by the protein indirectly. This is a method for analyzing the presence or absence of the interaction of the test substance with the G protein-coupled receptor by conducting a measurement.
  • the reporter gene assembly can be performed manually, but the so-called “Noise Knock Pupping” (High Throughput Screening) (Tissue Culture Engineering, Vol. 23) is performed automatically using a machine (robot). No. 13, p.521-524; U.S. Pat. No. 5,670,113) can be used more rapidly and easily.
  • the process of measuring the signal transduction ability of a test substance via Recl68 using a reporter gene assembly or selecting a ligand having a Recl68 agonistic action includes the following: Conceivable.
  • a method for screening a substance to identify a ligand for Recl68 comprising the following steps (a) to (c): Things to collect:
  • step (b) For each sample brought into contact with the substance in step (a), the detectable signal generated by the reporter gene expressed in each cell in the sample is not brought into contact with any substance. Quantitatively determining each of the detectable signals produced by the reporter gene expressed in each of the cells;
  • the promoter region of the gene that is induced by stimulation through Recl68 includes the promoter region of the immediate-early gene.
  • the early gene promoter used in the present invention include the c-fos promoter region and the zif268 promoter region ( proc . Natl. Acad. Sci. USA., Vol. 86, p. 377- 381, 1989; “EGR-1”, “NGF to A”, “Krox24j”, “Tis8” or “cef5”).
  • a particularly preferred embodiment is the zif268 (EGR-1) promoter region.
  • the promoter used in the present invention also includes the counterpart of any animal species of the promoter.
  • promoter region means an arbitrary region containing the minimum nucleotide sequence essential for expressing promoter activity. For example, it is possible to use part or all of the region of about 500 bp to about 2 kbp upstream from the transcription site of the gene. Furthermore, the title promoter region in the present invention has a transcription factor binding sequence O SRE k serum response e ⁇ ement and Z or 7 e ⁇ cAMP response element) upstream of the binary promoter. A promoter region.
  • the “reporter gene” in this step means a gene encoding a reporter protein that emits detectable fluorescence.
  • Examples include luciferase derived from fireflies or rumilla, or GFP (Green Fluorescence Protein) derived from jellyfish. Furthermore, for example, a gene encoding / 3 galactosidase, a gene encoding chloramphenicol acetyltransferase, and a gene encoding lactamase can be mentioned.
  • PC 12-derived cells in this process refers to rat adrenal pheochromocytoma-derived PC12 Hougetsu capsule (ATCC CRL-1721; Proc. Natl. Acad. Sci. USA., Vol. 73, p. 2424). -2426, 1976; Science, Vol. 229, p.393-395, 1985; EMBO J., Vol.2, p.643-648, 1983) or the PCI 2 cell line. Any cell line.
  • the subcloned cells have the following properties.
  • a gene encoding Recl68 (1) a gene encoding Recl68, and (2) a “reporter gene operably linked to the promoter region of a gene whose expression is induced by stimulation via a G protein” exogenously.
  • a recombinant cell obtained by introduction is contacted with a Recl68 ligand, the amount of the reporter gene product whose expression is induced is detected using the PC12 cell line as a host cell. The cell yields a value higher than the amount of the catalyst.
  • Preferred examples of the subcloned cells include PC12h cell line (Brain Research, Vol. 222, p.225-233, 1981) and various cells subcloned from the PC ⁇ 2h itoda cyst line. And can be illustrated.
  • a method of measuring the amount of intracellular free calcium may be used. Recl68 expressed on the cell surface is brought into contact with a test substance that is expected to contain a compound that binds to it, and the intracellular free calcium is measured according to a conventional method, and the signal transduction signal is transmitted through Recl68. A compound having can be selected.
  • a test substance is added to each of Recl68-expressing cells and Recl68 non-expressing cells, and the change in the concentration of free calcium in cells is measured.
  • the change in the concentration of free calcium in cells is measured.
  • only the cell line in which Recl68 was forcibly expressed was used to determine the substance with a large change in intracellular free calcium concentration.
  • Measurement of intracellular free calcium concentration can be performed by methods known to those skilled in the art.
  • seed cells in 96-well or 384-well multiwell plates load a fluorescent detection reagent for intracellular calcium (such as Fura-2), stimulate with a candidate ligand, It can be measured with a fluorescence spectrometer for measuring intracellular calcium.
  • a fluorescent detection reagent for intracellular calcium such as Fura-2
  • a GTP binding test can be used as the step of measuring the signal transduction ability.
  • the test substance was reacted with the cell membrane fraction of Recl68 expressing and non-expressing cells added to the filter plate, and the amount of GTP bound to the membrane fraction was measured and compared. It is possible to analyze whether or not a specific signal is transmitted through Recl68, a G protein-coupled receptor.
  • GTP binding activity can be measured by methods known to those skilled in the art and is not particularly limited. For example, GTP binding activity can be easily measured by using a kit such as DELFIA GTP-binding kit AD0167 manufactured by Parkin Elma. Can do.
  • Preferred examples of the “recl68 ligand having a ligand action” in the present invention include, for example, MCD-P, [D-Pro 2 , D-Trp 7 , 9 ′ 10 ] -Substance P ( 1-11), [D-Pro ⁇ D-Trp 7 - 9 - 10] -Substance P (l-ll), [D- Trp 7 9 '10] - Substance P (1-11), Somatostatin 28, ChaperoninlO (1-20) Platelet Factor-4, pCNP (1-17), Substance P, Indolicidin, Angiopeptin, PACAP (6-27), Somatostatin, Cortistatin ⁇ , ⁇ , MBP (99-110), ECP, ECP (29 -45).
  • the present invention uses a cell expressing Recl68 to measure the signal transduction ability of a ligand having a Recl68 agonist action in the presence and absence of a test substance, and compares both measured values. characterized that you comprising the step, providing a screening method of degranulation or prostaglandin D 2 production suppressing material through Recl68.
  • the present invention uses cells expressing Recl68 in the presence and absence of a test substance. Recl68-mediated degranulation or prostaglandin D 2 production, characterized in that it includes the steps of measuring the signal transduction ability of a ligand having Recl68 agonistic action, and comparing the measured values. It also provides a method for identifying substances to be suppressed.
  • the present inventors have found that a protein Recl68 induces degranulation and prostaglandin D 2 production of mast cells. That is, ligand substances having Recl68 ⁇ Gonisu preparative activity Ri by the a coupling child to Recl68, through the intracellular signaling processes, degranulation and prostaglandin D 2 production is induced.
  • the “substance that suppresses the degranulation reaction or prostaglandin D 2 production mediated by Recl68” refers to the cell uncondylation caused by binding of a ligand substance having Recl68 agonist activity to Recl68. it is that it is possible for substances that react or inhibit prostaglandin D 2 production, as long as it has such an action, it is not particularly limited. In other words, the substance having such an action is “Recl68 substance having an antagonistic action”.
  • Recl68 because degranulation or prostaglandin D 2 production in mast cells, a protein that induction, using cells expressing Recl68, Recl68 ⁇ Gonisu Bok effect in the presence and non-presence of the test substance If the substance that suppresses the signal transduction ability selected by the step of measuring the signal transduction ability of the ligand having the above and the step of comparing the two measured values is considered to be a degranulation reaction via Recl68 or prostaglandin D 2 It is thought to be a substance that suppresses production.
  • subject may include only the process of measuring the signal transduction ability of a ligand that has a Rec l 68 ligand action in the presence and absence of substances, and the comparison of both measurements. .
  • the method which combined the following processes with the said process is also preferable.
  • the screening method or identification method of a substance that suppresses degranulation reaction or prostaglandin D 2 production via Rec l 68 may be performed only by the following steps.
  • a substance that suppresses the binding between Rec l 68 and a ligand substance having Rec l 68 agonist activity is considered to be a substance that suppresses the degranulation reaction or prostaglandin D 2 production via Rec l 68. It is done.
  • a screening method or identification method for such a substance it is possible to compare the binding ability of the ligand substance to Rec 68 in the presence or absence of the test substance.
  • the detection and comparison of ligand substance binding can be performed as follows.
  • Rec 68 may be a recombinant polypeptide or a naturally derived polypeptide. It may also be a partial peptide. Further, it may be a form expressed on the cell surface or a form as a membrane fraction.
  • the test substance is not particularly limited. For example, cell extracts, cell culture supernatants, fermented microbial organisms, marine organism extracts, plant extracts, purified or crude polypeptides, non-peptide compounds, synthesis Examples include low-molecular compounds and natural compounds.
  • the polypeptide of the present invention to be contacted with a test substance is, for example, a purified polypeptide, a soluble polypeptide, a form bound to a carrier, and another polypeptide. As a fusion polypeptide, it can be brought into contact with a test substance as a membrane fraction as a form expressed on a cell membrane.
  • the ligand substance is a polypeptide as follows. This can be done by many methods known to those skilled in the art.
  • the gene encoding Rec68 is inserted into a vector for expressing a foreign gene such as pSV2neo, pcDNAI, or pCD8 to express the gene in animal cells.
  • a foreign gene such as pSV2neo, pcDNAI, or pCD8
  • SV 4 0 earlypromoter Rigby I n W i 1 1 iamson (ed.), Genetic Engineering, Vol. 3. A cademic Press, London, p. (1 9 8 2)
  • EF— 1 apromoter Karl et al. Gene 9 1, p. 2 1 7-2 2 3 (1 9 9 9
  • epitope-antibody systems can be used (Experimental Medicine 13, 85-90 (199)).
  • Commercially available vectors that can express fusion polypeptides with 3-trigalactosidase, maltose binding protein, dartathione S-transferase, green fluorescent protein (GFP), etc. Has been.
  • a fusion polypeptide is prepared by introducing only a small epitope consisting of several to a dozen amino acids. The method is also reported.
  • polyhistidine His—tag
  • influenza agglutinin HA influenza agglutinin HA
  • human c—myc FL AG
  • VSV—GP Vesicu 1 arstomatitis virus glycoprotein
  • T7—tag T7 gene 10 protein
  • Polypeptides that bind Ecl tags such as human herpesvirus glycoprotein (HSV-tag) and E-tag (epoxy groups on monoclonal phage) and monoclonal antibodies that recognize them to Recl68. It can be used as an epitope-antibody system for peptide screening (Experimental Medicine 1 3 8 5-9 0 (1 9 9 5)).
  • these antibodies are added to a cell lysate prepared using an appropriate surfactant in the presence and absence of a test substance to form an immune complex.
  • This immune complex consists of Recl68, a polypeptide capable of binding to it, and an antibody. If the test substance has an activity that suppresses the binding between ecl68 and the ligand substance, the amount of ligand substance in these immune complexes should be reduced compared to the control mouth. It becomes.
  • immunoprecipitation can also be performed using the antibody against the polypeptide of the present invention.
  • the antibody against the polypeptide of the present invention can be expressed, for example, by introducing a gene encoding Recl68 into an appropriate E.
  • E. coli expression vector and expressing it in E. coli, purifying the expressed polypeptide can be prepared by immunizing rats, goats, chickens and the like. In addition, by immunizing the above animals with the synthesized partial peptide of Recl68 It can also be prepared.
  • the immune complex can be precipitated using P ro t e 1 n A S p h a r o s e or P r o t e n G o p h a r o se.
  • Rec 168 is prepared, for example, as a fusion polypeptide with an epitope such as GST, Recl68 using a substance that specifically binds to such an epitope such as gnorethione Sepharose 4 B.
  • an immune complex can be formed.
  • a method for measuring the amount of a polypeptide that is a ligand substance binding to Recl68 for example, the West Western blotting method (S kolnik, E. Y. etal., Cell (1 9 9 1) 6 5, 8 3-9 0).
  • a cDNA library using a phage vector eg, gt 11 or ZAP was prepared from cells, tissues, or organs (eg, testis) expressing a ligand polypeptide that binds to Rec 168.
  • the polypeptide expressed on LB-agarose and immobilized on the filter is immobilized, and the purified and labeled polypeptide of the present invention and the above filter in the presence and absence of the test substance.
  • a plaque expressing a polypeptide bound to Recl68 may be detected by labeling. If the test substance has an activity that inhibits the binding between Recl68 and the ligand substance, the count will be detected low.
  • a method for labeling Recl68 a method using the binding property of piotin and avidin, a method using an antibody that specifically binds to Recl68 or a polypeptide fused to Recl68 (for example, GST), radioisotope, etc. Examples include a method using a torp or a method using fluorescence.
  • Rec 168 is immobilized on a carrier of an affinity column and has Recl68 gonist activity in the presence or absence of the test substance. After the ligand substance to be bound is bound, the column can be washed and the amount of ligand substance bound to Recl68 can be compared.
  • a biosensor using the surface plasmon resonance phenomenon can also be used as a means for detecting or measuring the bound substance.
  • Biosensors using the surface plasmon resonance phenomenon observe the interaction between Recl68 and the test substance in real time as a surface plasmon resonance signal without labeling with a small amount of polypeptide. It is possible (eg BIA core, made by P harmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the present invention and the test substance by using a biosensor such as BIACore.
  • mast cells which do not express the mast cells that expressed Recl68, although In its contact to mast cells expressing Recl68 induces degranulation or prostaglandin D 2 production It is conceivable to select a substance that does not induce in non-expressing cells.
  • Mast cells can be induced to differentiate from hematopoietic stem cells according to conventional methods, and Recl68-expressing cells and non-expressing cells can be induced depending on the culture conditions.
  • mast cells those directly separated from blood or tissue can be used.
  • Recl68-expressing cells and non-expressing cells can be separated by sorting using antibodies against Recl68.
  • mast cells cultured cell lines can also be used.
  • cells with ligand substances via Recl68 in the presence or absence of the test substance can be used.
  • degranulation reaction is not particularly limited if it contains the step of comparing the degree of prostaglandin D 2 production.
  • the present invention can also provide a method for screening a substance that regulates the expression of Recl68 protein or a polynucleotide encoding Recl68 in cells.
  • One embodiment of the present invention can be performed by comparing the level of Recl68 protein expression or Recl68 gene expression on the cell surface before and after the test substance is allowed to act on the cells.
  • a method for detecting a change in the expression of Recl68 in cells a method known to those skilled in the art can be used. Examples of such methods include, but are not limited to, Northern blotting and RT-PCR.
  • Northern blotting the RNA of the cells to be tested is purified, electrophoresed in an agarose gel, A probe labeled with the gene of the present invention with radioactivity or the like is hybridized with what is blocked on the membrane, and the presence or absence of a specifically appearing band is measured, and the expression of the polynucleotide of the present invention is measured by shading.
  • the RNA of the cells to be tested is purified and converted into cDNA using reverse transcriptase, and this is used as a template, using a sequence characteristic of Recl68 as a primer.
  • the cDNA derived from the transcript of the gene of the invention is amplified by PCR. Since the amount of cDNA amplified by this method is considered to be proportional to the amount of cDNA in the cocoon-like form, and hence the amount of the transcript of Recl68, the amount of DNA fragment amplified by this PCR was electrophoresed.
  • the expression level of Recl68 can be measured by measuring using a method.
  • Rec 168 or a cell containing the polypeptide can be immunologically detected by performing an antigen-antibody reaction using the antibody of the present invention.
  • This detection method can also be used for diagnosis of diseases that can be caused by mutations or abnormal expression of genes encoding Recl68 (such as genomic DNA).
  • Recl68 quantification methods include ELISA methods using a microtiter plate, fluorescent antibody methods, Western plot methods, immunohistochemical staining methods, etc. as methods of immunological detection. .
  • the polypeptide of the present invention labeled with a radioactive isotope such as a sandwich ELISA method using two kinds of monoclonal antibodies with different epitopes among antibodies that react with Rec 168 in the liquid phase and a radioactive peptide.
  • a radioimmunoassay method using a peptide and an antibody recognizing the polypeptide of the present invention is a radioactive isotope such as a sandwich ELISA method using two kinds of monoclonal antibodies with different epitopes among antibodies that react with Rec 168 in the liquid phase and a radioactive peptide.
  • a radioimmunoassay method using a peptide and an antibody recognizing the polypeptide of the present invention such as a sandwich ELISA method using two kinds of monoclonal antibodies with different epitopes among antibodies that react with Rec 168 in the liquid phase and a radioactive peptide.
  • a transformant transformed with a plasmid containing DNA linked to a reporter gene downstream of a region that controls transcription of Recl68 is contacted with a test substance, and Recl68 It can also be carried out by selecting a compound that regulates expression.
  • the reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lac Z gene, a luciferase gene, / 3-Dalcronidase commonly used in the art. Gene (GUS) and GFP gene.
  • the reporter gene also includes DNA encoding Recl68.
  • the expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of reporter gene used.
  • the reporter gene is a CAT gene
  • the expression level of the reporter gene can be measured by detecting the acetylation of kuramfum felicol by the gene product.
  • the reporter gene is a 1 ac Z gene
  • the catalytic action of the gene expression product By detecting the fluorescence of the fluorescent compound, and in the case of] 3-Daluronidase gene (GUS), G 1 ucuron (ICN) luminescence and 5-bromo 1 4 _ Cloguchi 1-Indolinole]
  • GUS 3-Daluronidase gene
  • ICN G 1 ucuron
  • the expression level of the gene can be measured by methods known to those skilled in the art. For example, the mRNA level of the gene is extracted according to a standard method, and the transcription level of the gene is measured by carrying out a Northern hybridization method or RT-PCR method using this mRNA as a cage. It can be carried out. Furthermore, the expression level of the gene can be measured using a DNA array technique.
  • a fraction containing Recl68 encoded by the gene is collected according to a standard method, and the expression level of the polynucleotide is detected by an electrophoresis method such as SDS-PAGE to measure the translation level of the gene. You can also. Moreover, it is also possible to measure the translation level of a gene by performing Western blotting using an antibody against Recl68 and detecting the expression of the polypeptide.
  • the substance obtained by the screening method or identification method of the present invention has the ability to control the expression of Recl68 protein or a polynucleotide encoding Recl68, or the activity of Recl68 protein (for example, signal transduction ability through Recl68 protein). It is a candidate for a drug for regulation (for example, suppression), and can be applied to the treatment of diseases caused by abnormal expression or functional abnormality of Recl68 and diseases that can be treated by controlling the activity of Recl68. Examples of the diseases to be treated and prevented include inflammatory diseases, particularly chronic inflammation such as allergic diseases and autoimmune diseases.
  • the present invention Substances in which a part of the structure of the substance obtained by the refining method or identification method is converted by 0, deletion and / or substitution are also included in the scope of the present invention. Inflammatory disease
  • Inflammatory diseases mean inflammatory diseases involving mast cells in particular, which means chronic inflammations such as allergic diseases, autoimmune diseases, etc., particularly involving fertile cells, and these allergic diseases or autoimmune diseases
  • gastritis gastric ulcer: cirrhosis: chronic biliary tract disease (intrahepatic calculi, primary sclerosing cholangitis): ulcerative colitis: chronic inflammatory vascular disease: arteries Sclerosis: cardiac anaphylaxis: pulmonary fibrosis: bronchial asthma: nephritis (tubulointerstitial nephritis, glomerulonephritis): interstitial cystitis: atopic dermatitis: keloid: vascular proliferative disorder (force positive) Sarcoma, malignant vascular endothelial cell type): rheumatoid arthritis: allergic rhinitis: allergic conjunctivitis: gastric cancer: colon cancer, breast cancer, basal cell carcinoma of the skin, soft
  • the present invention provides a diagnostic agent for a disease associated with abnormal expression of a gene encoding Rec 1 68 or abnormal activity of Rec 1 68. Since Rec l 68 has a function of inducing degranulation of mast cells, abnormal expression or function thereof can cause various diseases such as chronic inflammation. Therefore, it is possible to diagnose such diseases by using inappropriate activity or expression of Rec 68 or the amount of ligand molecule expressed in the body as an index.
  • the term “diagnostic agent” refers not only to a diagnostic agent for establishing a treatment strategy for a subject presenting with a symptom of a disease, but also for prevention used to determine whether or not a subject is susceptible to a disease. Diagnostic agents are also included.
  • One embodiment of the diagnostic agent of the present invention is a diagnostic agent containing Rec l 68 protein or a partial peptide thereof used for measuring the in vivo expression level of a Rec l 68 ligand substance in a subject.
  • One embodiment of the diagnostic agent of the present invention is capable of detecting a mutation in the gene encoding Rec 68 or its expression control region in a subject.
  • One is a diagnostic agent for testing by directly determining the base sequence of the gene encoding Rec l 68 or its expression control region in a subject, for example, encoding Rec l 68. It contains a probe or primer that hybridizes to a gene or DNA containing its expression control region.
  • the method of use is not particularly limited as long as it can detect a mutation in the gene encoding Recl68 or its expression control region in a subject. For example, first prepare a DNA sample from a subject.
  • the DNA sample can be prepared based on chromosomal DNA or RNA extracted from a subject's cells, such as blood, urine, saliva and the like.
  • a chromosomal DNA can be cleaved with an appropriate restriction enzyme and cloned into a vector to prepare a genomic library.
  • a cDNA library can be prepared from RNA using reverse transcriptase.
  • a DNA encoding Rec l68 or a DNA containing its expression control region is isolated. Isolation of the DNA should be performed by screening a genomic library or cDNA library using a probe that hybridizes to the gene encoding Recl 68 or DNA containing its expression control region. Can do.
  • a primer that hybridizes to a gene encoding the polypeptide of the present invention or a DNA containing its expression control region is used. It can also be separated.
  • the base sequence of the isolated DNA is determined.
  • the base sequence of the selected DNA can be determined by a method known to those skilled in the art.
  • the determined DNA base sequence is then compared with a control. “Control” refers to the base sequence of DNA containing the normal (wild type) Rec 68 encoding gene or its expression control region. As a result of such comparison, if the subject's DNA base sequence is different from the control, the subject is determined to be affected or at risk of developing the disease.
  • One embodiment of the diagnostic agent of the present invention is Rec l 68 or a partial peptide thereof used for measuring the expression level or activity of Rec l 68 protein in a subject. It is a diagnostic agent containing an antibody against a cadmium or a ligand substance of Recl68.
  • a mast cell is isolated from a blood sample prepared from a subject, reacted with an antibody against Recl68 or its partial peptide, or a ligand substance, and the expression level of Recl68 molecule on the obesity cell and the ligand binding ability are measured by a flow cytometer. It can be measured by If the expression level or ligand binding capacity is increased, it is determined that there is a risk of onset.
  • a reverse transcription reaction was carried out using rabbit testis poly (A) + RNA as a saddle type to synthesize cDNA. Reagents attached to High Fidelity RNA PCR Kit (TaKaRa) were used for the reaction.
  • the reaction system was human testis poly (A) + RNA (1 g // z L) 0.01 L, 2 X Bca 1st buffer 2 ⁇ , MgS0 4 (25 mM) 0.8 ⁇ , dNTP Mixture (10 mM ) 0.2 ⁇ L, RNase Inhibitor (40 units / ⁇ L) 0.1 ⁇ L, Bca PLUS RTase (42 U / ⁇ L) 0.2 and Random Hexamer (50 ⁇ M) 0.2 ⁇ L in sterile water To make up to 4 ⁇ L. This was reacted at 65 ° C for 1 minute, 30 ° C for 1 minute, 30-65 ° C for 15 minutes, 65 ° C for 30 minutes, 98 ° C for 5 minutes
  • reagents were attached to Pyrobest DNA Polymerase (TaKaRa).
  • TaKaRa Pyrobest DNA Polymerase
  • the volume was made up with sterile water to 20 uL.
  • the primers are: 168—PCR—F2 (IJ number: 5), 168—PCR—R2 (IJ number: 6), 168—SEQ—F1 (IJ number: 7), 168—SEQ—F2 (Self ID number: 8), 168—SEQ—R 1 (Self number: 9), and 168—SEQ—R2 (Sequence number: 10) were used.
  • the reaction was performed by a dye terminator method using DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences Co., Ltd.) and analyzed using Sequencer MegaBacelOOO (Amersham Biosciences Co., Ltd.).
  • the plasmid DNA with the desired sequence inserted was named “pENTR-Recl68”.
  • pENTR-Recl68 cloned into pENTRIA is inserted into the target expression vector (destination vector) using Gateway System technology (Invitrogen Corporation). Replaced.
  • Enzyme Mix (Invitrogene Corporation 11791-019) was used. Reagents are attached to the enzyme.
  • pME18S.rfA which is the destination of pME18S, was used as the expression vector.
  • entry vector 100 ng 100 ng of the destination vector was made up to 6 ⁇ L with TE buffer, mixed with 5 ⁇ L of RCI onase Reaction Buffer 2 ⁇ . 2 ⁇ L of LR CI onase Enzyme Mix was added and incubated for 1 hour at room temperature (25 ° C).
  • 1 ⁇ L of Proteinase K Solution (2 ⁇ g / ⁇ I) was added and incubated at 37 ° C for 10 minutes.
  • D-MEM High-glucose
  • Iwaki Glass 4143-010 the rat kidney PC12h cells cultured, 225 cm 2 of the flask (1 X 10 7 cells cells) PBS (-) was re license by (Nikken organism CM6201), trypsin / EDTA solution (Invitrogen 5300- 054 ) Containing 0.5% horse serum (Invitrogen 26050-088), 0.25% fetus serum (JRH 12103-78P) and 0.5% penicillin / streptomycin (Sigma P0906).
  • D-EM the (High-glucose) Fuenoruretsu Zadoff Lee (Nikken bio CM4410) medium test substance prepared with added 50 ⁇ L, C0 2 Inkyubeta one 37 °. For 6 hours. All of the culture supernatant was removed, and Luciferase Cell Culture Lysis Reagent (Promega E1531) was added with 25; u L / wenore to dissolve the Itoda cells.
  • PEI Polyethyleneimine
  • Bok Bok
  • the cell suspension transflector We click Chillon Like the 96 Uweru method, PEI (Polyethyleneimine) Koti link , the 384 Uenore white play Bok (Falcon 3988) in dispensed at 40 mu L, with C0 2 Inkyubeta one 37
  • the cells were cultured at ° C for 48 hours.
  • PEI coating PEI (Sigma P3143) was diluted 1000-fold with sterilized water, added 25 // L / ul and allowed to stand for more than 1 hour, and then washed twice with 70; u L sterilized water.
  • test substance prepared using D-MEM (High-glucose) (Nikken Biological CM4402) medium to a 5-fold concentrated solution.
  • the cells were cultured for 6 hours at 37 ° C in an incubator.
  • steady luciferase assay reagent Promega E2550 was added to 25 ⁇ ! Create a concentration-dependent curve with luciferase activity plotted on the Y-axis and test substance concentration plotted on the X-axis, EC 5 . The value was derived.
  • Test substances include Mast Cell Degranulating Peptide (MCD-P) (American Peptide 41-9-30), Somatostatin 28 (American Peptide 68-1-50), Atrial Natriuretic Peptide (1-28) N rat (American Peptide 14-5-41) ⁇ Bradykinin (American Peptide 18-1-10) Neurotensin (American Peptide 60-1-12) x Magainin 2 (American Peptide 72-2-26), Granuliberin R (American Peptide 87-0-72), Substance P (Peptide Institute 4014-v), Histatin5 (Peptide Institute 4270- s;), Platelet Factor- 4 (Peptide Laboratory 4305-v), c-Def ensin-1 (HNP-1) (Peptide Laboratory 4271-s), Compound 48/80 (Sigma C2313), Poly- and lysine (Sigma P9404) , Chaperonin GroES (Takara Shuzo 7331), chaperonin 10 (1-20) (Compound), 2
  • the peptide sequence of the test substance used is shown in Table 1 below.
  • PC12h_17 cells in which Recl68 was transiently expressed were stimulated with various test substances.
  • Substance P antagonist ⁇ substance P antagonist, Substance P, pCNP (l-17) Indolicidin, Angiopepsin, PACAP (6-27) histamine releasing peptide, human HRP-1 showed an increase in luciferase activity in PC12h Itodatsuki-pack with Recl68 introduced.
  • FIG. 1 An increase in luciferase activity was observed in all PC12h cells into which Recl68 had been introduced.
  • A shows the results in Recl68 expressing cells
  • B shows the results in Mock.
  • Recl68 cDNA was subcloned into the expression vector pLHCXI4 (see Fig. 4) to construct pLHC-Recl68-14 (see Fig. 5). After culturing E. coli DH5a transformed with this pLHC-Rec 168-14, DNA of pLHC-Recl68-I4 was prepared using EndoFree Plasmid Mega Kit (Kiagen).
  • D-MEM medium supplemented with 293 cells (ATCC CRL-1573) and 10% fetal calf serum
  • CD4 positive cells were recovered with MACSelect 4 transfected cell selection kit (Miltenyi Biotec).
  • the hygromycin-resistant cells obtained above were selected using a selective medium [200 ⁇ g / mL hygromycin B (Invitrogen 10687-010) and 10% fetal calf serum in D-MEM medium (Nikken Biomedical Research Institute CM4402)]
  • the seeds were seeded in 75 cm 2 flasks and cultured to 1 ⁇ 10 7 pieces. The medium was discarded, and the cells were washed with 10 mL of D-PBS (Nikken Biomedical Research Institute CM6201).
  • Ateno-Kuffer (20 mM HEPES (Nacalai 17514-15); (pH 7.4; prepared with 1 M K0H (Nacalai 286-16)), 115 mM NaCl (Sigma S5150), 5.4 mM KC1 (Nacalai 285-14), 0.8 mM MgCl 2 (Naka Lai 209 - 09), 1.8 mM CaCl 2 ( Nacalai 067-29), 13.8 mM Glucose (Naka Lai 168 - 06), 0.2% BSA (Sigma A8806), 2.5 After washing once with mM Probenicid (Sigma P8761), the above Ca 2+ containing Fura2 loading buffer (2 M Fura2-AM (Dojin 343-05401), 0.02% Pluronic F127 (Molecular Probe P6866)) Suspended in 10 mL of Atsey Buffer) and incubated for 1 hour at room temperature Washed twice with Ca 2+ Atsy Buffer
  • the intracellular free Ca 2+ concentration was measured over time using FDSS3000 (Hamamatsu Photonics). Specifically, the fluorescence intensity of 384 wall plates containing cells was measured every 1.5 seconds for 3.5 minutes. The ligand prepared using Ca 2+ buffer in a 5-fold concentrated solution was added 10 ⁇ l 30 seconds after the start of measurement, and stirred for 20 seconds.
  • the measured values are the ratio of the fluorescence intensity at a wavelength of 500 nm obtained for two types of excitation wavelengths of 340 nm (Ca 2+ -bonded ) and 380 nm (Ca 2+ -unbonded) [340 nm. Excitation (Ca 2+ bond) ⁇ 380 nm excitation (Ca 2+ non-bonded type)] Plotting this Ratio value on the Y-axis and time on the X-axis, changes in intracellular free Ca 2 'concentration with respect to various ligands were measured.
  • a concentration-dependent curve was created by plotting the maximum ratio value for various concentration stimuli of various test substances on the Y axis and the test substance concentration on the X axis, EC 5 . The value was derived. Atsy was conducted 4 times, each EC 5 . The value was calculated using the calibration software 'rxLfitj', and the average value was calculated.
  • test substances were Angiopeptin (American Peptide 68-45), Bradykinin (American Peptide 18-1-10), Granulberin R (American Peptide 87-0-72), Indolicidin (American Peptide 72 -2-28), Magainin 2 (American Peptide 72-2-26), PACAP (6-27) (American Peptide 34-0-45), Somatostatin 28 (American Peptide 68-1-50), VIP (1- 28) (American Peptide 48 + 10), Compound 48/80 (Sigma C2313), Atrial Natriuretic Peptide (1-28), human (Peptide Laboratory 4135-v), cortistatin 14 (Peptide Laboratory 4329-v), Histatin 5 (Peptide Laboratory 4270-s), MCD-P (Peptide Laboratory 4258-v) Neurotensin (Peptide Laboratory 4029-v), Platelet Factor-4 (Peptide Laboratory 4305-v), Somatostat i ⁇ (Peptide Laboratory 4023- [nu), Substance [rho (
  • the above peptide was synthesized using an automatic peptide synthesizer (Model 431A, manufactured by Applied Biosystems). Peptides were synthesized and protected according to the best manual. Impurities contained in the obtained peptide preparation were analyzed using a high-performance liquid chromatograph (Waters), reversed-phase C 1 8 column (5 m, 2 X 2 500 mm, YMC-Pac 0DS-A , YMC) and separated with a gradient of 0.1% trill; 'e acid (liquid A) and acetonitrile (solution B). The structure and purity of the obtained synthetic peptide preparation were confirmed by molecular weight measurement using a laser ionization mass spectrometer (K0MPACT MALDI III, manufactured by Shimadzu Corporation).
  • K0MPACT MALDI III laser ionization mass spectrometer
  • Recl68 stable expression Preparation of membrane fraction of 293 cells Recl68 stably expressed 293 cells were cultured in a cell culture flask (culture area: 175 cm 2 ) in 50 mL medium (10 ° /. Fetal bovine serum, 50 units / mL penicillin, 0.05 mg / mL Cultured at 37 ° C 5% C0 2 in Dulbecco's Modified Eagle Medium (D-MEM; high glucose) (manufactured by Nikken Biomedical Research Laboratories) containing syn.
  • D-MEM Dulbecco's Modified Eagle Medium
  • the disrupted solution was centrifuged (50, 000 X g, 30 minutes, 4 ° C), and lysis buffer was added to the resulting precipitate, and this was centrifuged (50, 000 X g, 30 minutes, 4 minutes). ° C)
  • the resulting precipitate preservative solution 75 mM Tris-HCl (pH7.6 ), 12.5 mM MgCl 2, 0.3 mM EDTA, 1 mM EGTA, 250 mM Sucrose, pH7.5
  • Recl68 stably expressing 293 was obtained.
  • the GTP binding experiment was performed using DELFIA GTP-binding kit AD0167 (PerkinElmer).
  • a 96-well filter plate (AcroWell Filter Plate) was loaded with reaction buffer (0.03 M GDP, 1 mM MgCl 2 , 10 mM NaCl, 300 ⁇ g / mL Saponin, final concentration) per well.
  • a test substance diluted to an appropriate concentration with 50 mM HEPES buf fer was added to this at 20 ⁇ I per well.
  • the membrane fraction prepared as described above was added to 20 ⁇ L with 50 mM HEPES buffer so as to obtain 3.858 ⁇ g per well.
  • Nonspecific binding was measured in the presence of 5 ⁇ ⁇ (final concentration) of GTP-Y-S per well.
  • GTP binding activity was observed under the same conditions using a membrane fraction prepared in the same manner as described above from 293 cells (parent strain) into which Recl68 had not been introduced.
  • MCD Peptide Peptide Institute
  • Substance P American Peptide
  • human Arial Natriuretic Peptide 1-28
  • ChaperoninlO Peptide Institute
  • Heparinized umbilical cord blood 10-60 mL was diluted with an equal volume of Buffer (0.5% bovine serum albumin (BSA, 2 methylenediaminetetraacetic acid / Phosphate Buffered Saline (-)), then Ficoll-Paque (Amershani Pharmacia Biotech) (Ficoll-Paque: Blood ⁇ -'2)), and centrifuge at 400 X g (1350 rpm) at 4 ° C for 30 minutes to obtain the mononuclear cell fraction.
  • BSA bovine serum albumin
  • Ficoll-Paque Anamershani Pharmacia Biotech
  • Reagent Al Fc blocking CD34 Progenitor Cell Isolation Kit for 1 X 10 8 cells
  • 0.1 mL Reagent A2 (CD34 ant i body—hapten) was prepared (final volume 0.5 itiL / 1 X 10 8 cell) and further stirred. Incubated for 15 minutes at 9 ° C.
  • methylcellulose-containing I country (Methocult, SFBIT, StemCell technology) was added 0.9m to start culture. After 1 week, the above medium except 0.9% methylcellulose was added at 100 ⁇ L / well, and then the medium except for I 3 was added at 100 ⁇ L / well once a week. .
  • the cells were diluted while observing the degree of proliferation and differentiated into human mast cells by culturing for about 8 weeks or more while maintaining a level of about 10 5 cells / mL / well. Effect of fetal calf serum (FCS) during culture
  • FCS fetal calf serum
  • the following degranulation test all 1% Insulin- Transferrin, 5 x 10- 5 M 2_ME, were performed with 0. 1% BSA-containing IMDM medium.
  • the obtained human cultured mast cells were washed with a medium and then seeded on a 96 well U bottom culture plate at 6 ⁇ 10 3 cells / 180 ⁇ L / well.
  • Compound 48/80 and various peptides were placed at 20 L to a concentration of 0.1 – 10 ⁇ M and stimulated at 37 ° C for 30 minutes.
  • 20 ⁇ L of A-23187 (Sigma) dissolved in DMS0 was added to a concentration of 0.1 ⁇ 10 M (DMS0 final concentration 0.1%) and stimulated at 37 ° C. for 30 minutes.
  • the degranulation reaction by Substance P and Compound 48/80 is up to 10 ⁇ M in Donor 4 Tsukiyomi Mitsuda Tsutsugu cultured in the presence of FCS from day 57 to day 92 of cord blood collection. It was not recognized at all. Cells in the absence of FCS exhibited approximately 20-25% degranulation at 10 ⁇ (see Figure 12). On the other hand, 23-23187 showed about 45-55% degranulation at 10 ⁇ regardless of the presence or absence of FCS. In addition, as a result of the same study with Donor 6, no study using A-23187 was conducted, but as with Donor 4, the degranulation reaction with Substance P and Compound 48/80 was cultured in the presence of FCS. It was not observed in the treated cells (see Fig. 13). Example 8
  • the tissue distribution of Recl68 expression was analyzed by a quantitative PCR method (TaqMan method) using real-time monitoring.
  • the TaqMan method is based on the principle of detecting and quantifying a PCR-amplified specific PCR strand using a real time reagent (this 7900HT Sequence Detection System (Applied Biosystems)) based on the fluorescence intensity of a fluorescent probe called TaqMan probe.
  • Assays-on-Demand registered trademark
  • Gene Expression Products was used assays-on-Demand (registered trademark) was used.
  • the type in the TaqMan PCR reaction is dorsal root ganglion S3 ⁇ 4 total
  • RNA and 41 types of human tissue-derived poly (A) + RNA (CL0NTECH) was used as a cage.
  • cDNA was synthesized from 6.5 ⁇ g of total RNA or poly (A) + RNA 1 / g, using Superscript II as the reverse transcriptase.
  • the composition of the reaction mixture was the amount of RNA that was synthesized from total RNA as a vertical cDNA.
  • GAPDH expression level is measured simultaneously with Recl68 expression level to normalize
  • the tissue distribution of Recl68 was compared by using pME-Recl68 prepared as standard DNA in 2.3x 101—2.4 X 108 copies / 4 ⁇ L PCR reaction after 95 ° C for 10 minutes , 95 ° C ⁇ 15 seconds, 60 ° C ⁇ 1 minute cycle was performed 40 times, and at the same time as the reaction was completed, PCR was quantitatively analyzed automatically. Shown in 4.
  • Recl68 and related receptors in cord blood-derived human cultured mast cells were performed by a quantitative PCR method (TaqMan method) using real-time monitoring.
  • TaqMan probes and primers that specifically recognize Recl68 and related receptors were prepared using Assays-on-Demand® Gene Expression Products (see Table 5).
  • the reaction composition in TaqMan PCR reaction is derived from cord blood-derived human mast cell-derived total RNA (Donor 4, Donor 5) and cord blood-derived human cultured mast cells that have been induced to differentiate with or without serum.
  • superscript II as a reverse transcriptase from total RNA (FCS +, FCS—)
  • FCS +, FCS— total RNA
  • random hexamer as a primer
  • 5 ng of cDNA synthesized at 42 ° C according to the attached instruction manual in terms of total RNA Use as a cage
  • Assays-on-Demand registered trademark
  • Gene Expression Products 0.5 ⁇ L TaqMan GAPDH Control Reagents (Applied Biosystems 3 ⁇ 4 :) 0.5 ⁇ L was added to make a total liquid volume of 10.
  • Recl68 expression level or related receptor expression level We sometimes compared the expression of Recl68 and related receptors in cord blood-derived rabbit cultured mast cells by measuring and normalizing GAPDH expression levels.
  • a vector holding each gene cDNA fragment was prepared to 2.3 ⁇ 10′-8.9 ⁇ 10 6 copies / 4 L and used.
  • the PCR reaction was performed at 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute, and a quantitative automatic analysis of PCR was performed at the end of the reaction.
  • the results (copy number / 5 ng total RNA) are shown in Table 6.
  • Recl68 inhibitory expression In 293 cells, Recl68 inhibitors were screened using as an index the suppression of the increase in intracellular free Ca concentration by substance P. Recl68 stably expressing 293 cells cultured in D-MEM medium (Nikken Biomedical Research Institute CM4402) supplemented with 200 ⁇ g / mL hygromycin B (Invitrogen 10687-010) and 10% fetal bovine serum, ⁇ Includes Fura-2 / ⁇ (Dojindo Laboratories) Mu Ca Atsuno Cuffer (20 mM HEPES (Nacalai 17514-15), 115 mM NaCl (Sigma S5150), 5.4 mM KC1 (Nacalai 285-14), 0.8 mM MgCl, (Nacalai 209-09), 1.8 mM CaCl " Suspended in Nacalai 067-29), 13.8 mM Glucose (Nacalai 168-06), 0.
  • the fluorescence intensity ratio (fluorescence intensity at excitation light wavelength 340 nm; fluorescence intensity at excitation light wavelength 380 nm; Ratio value) is calculated, and the maximum direct force of Ratio after substance P is added.
  • the inhibition rate of intracellular Ca 2+ release by the test substance was calculated by the following formula.
  • a and B have the following meanings.
  • Recl68 stably expressing 293 cells cultured in D-MEM medium (Nikken Biomedical Research Institute CM4402) supplemented with 200 ⁇ g / mL of nouromycin B (Invitrogen 10687-010) and 10% fetal calf serum , 2 ⁇ ⁇ Fura-2 / ⁇ (Dojindo Laboratories) Ca 2+ Atseino Quafer (20 mM HEPES (Nacalai 17514-15), 115 mM NaCl (Sigma S5150), 5.4 mM KC1 (Nacalai 285- 14), 0.8 mM MgCl 2 (Nacalai 209-09), 1.8 mM CaCl, (Nacalai 067-29), 13.8 mM Glucose (Nacalai 168-06), 0.2% BSA (Sigma A8806), 2.5 mM Probenicid (Sigma P8761) , Adjusted to pH 7.4 with
  • FDSS3000 Hamamatsu Photonics Co., Ltd.
  • 25 ⁇ M Substance P Peptide Laboratories, Inc.
  • 2.5 ⁇ M PACAP 67)
  • 7.5 ⁇ M [D-Pro 4 , D_Trp 7 , 9 ''.]-Substance P (1-11) (Synthetic product)
  • 7.5 ⁇ M Ionomycin (BI0M0L CA-201) added in 16 L
  • the change in fluorescence intensity at excitation light wavelengths of 340 nm and 380 nm was measured, and the fluorescence intensity was calculated from the measured fluorescence intensity.
  • the MethocuUSF B added 10 volumes, and culturing was started at C0 2 incubator base one coater. 100 ng / mL Recombinant Human SCF, 50 ng / mL Recombinant Human IL-6, 1 ng / mL Recombinant once a week Cells were diluted with a basic medium containing Nantohi 3 to about lxlO 5 cells / mL, and when cell growth was no longer observed, half of the cells were exchanged. Human cultured mast cells were obtained by maintaining the culture for 8 weeks or more.
  • the obtained human cultured fertilized cells were collected and 0.1% BSA, 1%
  • the culture supernatant 50 was supplemented with an equal volume of 4 mM pN-GlcNAc (0.1 M citrate buffer, pH 4.5) and incubated at 37 ° C. for 2 hours. The reaction was stopped by adding an equal volume of 0.1 M carbonate buffer (pH 10), and the absorbance at a wavelength of 405 nm was measured with a plate reader.
  • the degree of degranulation by each stimulus was calculated assuming that the cells were disrupted with water and the 3-Hexosamidase activity was 100%.
  • the degranulation inhibitory activity of the compound is Substance P, [D—Pro 4 , D—Trp 7 , 9 , ' 0 ] —Substance ⁇ (1-11), PACAP (6-27), and 0.1% DMS0.
  • the degranulation rate after stimulation with A23187 was increased to 100%.
  • Substance P [D-Pro 4 , D-Trp 7 , 9 , 10 ] -Substance P (g 11) and PACAP (6-27) stimulated degranulation induction of human cultured mast cells Compound 1, Compound 2, and Compound 3 inhibited in a concentration-dependent manner (see Table 7).
  • Substance P Compound 1 to the stimulus the compound inhibition of 2 and compound 3 (IC 5. Value) waso respectively 1.5, 0.6 and 1.0 ⁇ M, [D_Pro 4, D- Trp 7, 9 '10] -Substance P ( ⁇ 11) IC 5 for stimulation.
  • the values were 3.0, 1.4, and 4.4 ⁇ 1, respectively, and the IC 5Q values for PACAP (6-27) stimulation were 1.2, 0.4, and 1.0 ⁇ M, respectively.
  • none of the compounds inhibited the degranulation induction of mast cells in response to A23187 stimulation. Inhibition of human mast cell degranulation by Recl68 inhibitor compounds
  • Cord Blood CD34 + (CAMBREX, 2C-101A), Cord Blood MNC (STEMCYTE, CAI 001), Recl68 expression HEK293 Itodao 0 rhSCF (PeproTech, 300—07), rhIL-6 (PeproTech, 200-06), rhIL- 3 (PeproTech, 200-03), 55 mM 2-ME (GIBC0, 21985-023), Iscove's Modified Dulbecco's Medium (IMDM, GIBC0, 12440-053), Insul in-Transferrin (GIBC0, 41400-045 ), 35% BSA
  • the above peptides were synthesized using an automatic peptide synthesizer (Model 431A, manufactured by Applied Biosystems). Peptides were synthesized and protected according to the best manual. Impurities contained in the obtained peptide preparations were analyzed using a high-speed liquid chromatograph (Waters) with reversed-phase C 1 8 column (5 ⁇ ⁇ , 2 X 2 50 mm, YMC-Pac ODS—A, YMC) and separated with a gradient of 0.1% trifluoroacetic acid (solution A) and acetonitrile (solution B). The structure and purity of the obtained synthetic peptide preparation were confirmed by molecular weight measurement using a laser ionization mass spectrometer (KOMPACT MALDI III, manufactured by Shimadzu Corporation).
  • the medium was prepared and human mast cells were cultured as follows.
  • IMDM To 100 mL of IMDM, 286 ⁇ L of 35% BSA and 1 mL of Insul in-Transferrin were added, and 100 ⁇ L of 2-ME was added to serve as a basic medium for human culture. 20 ⁇ L of 10 mM acetic acid aqueous solution was added to 10 ⁇ g of rhSCF, dissolved, and after light centrifugation, 1 mL of basic medium was added (10 ⁇ g / mL rhSCF). To 40 ⁇ z of rhIL-6, 10 mM aqueous acetic acid solution 40 / zL was added and dissolved. After light centrifugation, 1 mL of basic medium was added (20 ⁇ g / mL rhIL-6). Diluted water was added to rhIL-3 10 ⁇ g for 40, and after light centrifugation, 1 mL of basic medium was added (10 ⁇ g / mL rh-3).
  • Cord Blood CD34 + cells (lxlO 5 cells) are added to 500 ⁇ L of basic medium containing 1 g / mL rhSCF, 0.5 ⁇ g / mL rhIL-6, 0.01 ⁇ g / mL rhIL-3, and cultured in a 24-well culture plate. (Falcon, 3047) was seeded at 100 ⁇ L each. The MethocultSF BIT was added in 0.9 mL, and culturing was started at C0 2 Inkyubeta scratch.
  • CD34 positive cells were prepared from Cord Blood MNC as follows.
  • the measurement method of degranulation is as follows.
  • the resulting human cultured mast cells were collected, washed with basal medium, seeded into culture plates 96-well U-bottomed to be about 1.5xl0 4 cells / well, for each concentration Substance P, MBP fragment , Add 1/10 volume of ECP fragment or A23187, and incubate for 30 minutes at 37 ° C using a 0 2 incubator (final volume 200 ⁇ L / well) After centrifugation (4 ° (: , 3,000 rpm, 5 minutes), and collected 50 ⁇ L of the supernatant each time The amount of degranulation was measured using -Hexosamidase activity as an index.
  • the Ca assay was performed as follows.
  • Recl68 ligands are 8-30 amino acid residues residue peptides
  • ECP and MBP tryptase cleavage sites were estimated (Fig. 16), and ECP and 8-25 amino acid residues were fragmented.
  • MBP was synthesized.
  • ECP and MBP fragments (10 M) was searched using the degranulation-inducing activity of human cultured mast cells as an index, and the 29th force of ECP, the 45th region, and the 99th force of MBP, the 110th region, etc. The contained peptide fragment was found to be active ( Figures 17 and 18).
  • ECP and MBP fragments (30 / M) that showed degranulation-inducing activity showed Ca responsive activity to 293 cells stably expressing Recl68, and degranulation-inducing activity of human cultured mast cells and Ca to 293 cells stably expressing Recl68. Response activity was in phase W ( Figures 17 and 18).
  • the 9th to 25th amino acid residue peptide (Fr. No. 23) of MBP showed degranulation-inducing activity against human cultured mast cells, but the MBP fragment (Fr. 22 and 24) were not active, so it was judged that the agonist action was not in the 9th to 25th region of MBP. Therefore, among the ECP and MBP fragments, each fragement having the strongest activity was used in the subsequent experiments.
  • this fragment (30 ⁇ 30) showed not only activation of human cultured mast cells but also Ca responsive activity to Rec 168 stably expressing 293 cells (Figs. 16 and 17).
  • the activation of mast cells induced by these fragments was suppressed by Recl68 inhibitor compounds; ⁇ , the ability to stimulate MBP fragments and ECP fragments via the Reel 68 receptor did.
  • both MBP fragment and ECP fragment were concentration-dependently compared to human cultured mast cells.
  • Degranulation was induced in a protein-dependent manner. Whether these fragments have the same activity as the full-length protein, the fragment activity is comparable to the activity reported by Patella, and, as reported by Piliponsky et al., Recl68-mediated human mast cells. Since degranulation induction was Gi sensitive, MBP fragment and ECP fragment were considered to have obesity cell degranulation-inducing activity similar to that of each full-length protein. Furthermore, it was found that the Recl68 P and the harmful compound suppressed the mast cell degranulation inducing force of MBP fragment and ECP fragment stimulation, thereby mediated by the MBP fragment and ECP fragment stimulation Recl68 receptor.
  • Recl68 may be involved in the activation of neurogenic mast cells and the activation of Eosinophil-rich cells.
  • a stress is applied to a model mouse (NC / Na mouse) for phototoxic dermatitis, it shows atopic dermatitis-like symptoms.
  • the amount of substance P in the blood of NC / Na mice which showed atopic dermatitis-like symptoms due to stress, increased, causing increased mast cells and Eosinophil, and mania.
  • Recl68 is likely to play a central role in the vicious cycle during chronic inflammation, and Recl68 inhibitors were considered to be effective therapeutic agents during chronic inflammation.
  • Example 1 4 the neuropeptides are secreted by the hypersensitivity state caused by stress and the like, and thus cytokines that activate mast cells via Recl68 Eosinoid grows and activates Eosinophil, proliferated and activated Eosinophil It was speculated that it would be an important role for activating mast cells via Recl68 again by secreted ECP. Thus, Recl68 is likely to play a central role in the vicious cycle during chronic inflammation, and Recl68 inhibitors were considered to be effective therapeutic agents during chronic inflammation.
  • Example 1 4 the neuropeptides are secreted by the hypersensitivity state caused by stress and the like, and thus cytokines that activate mast cells via Recl68 Eosinoid grows and activates Eosinophil, proliferated and activated Eosinophil It was speculated that it would be an important role for activating mast cells via Rec
  • the degranulation was measured using Substance P or BP fragment instead of Substance P, BP fragment, ECP fragment or A23187. Except for using Compound 48/80, the ECP and Was performed by the same method as the measurement method of the degranulation activity "against the human cultured mast cells MBP fragments. From the above experiment, the following became clear.
  • Recl68 inhibitor compound neither the Recl68 inhibitor compound nor romolyn showed an inhibitory effect on the degranulation induction of human mast cells by IgE cross-linking (see FIG. 21 (B)).
  • Recl68 contributes to the activation of mast cells in a complex network of inflammation, because the neuropeptide Supstance P and the basic peptides derived from MBP and ECP involved in inflammation show Recl68 agonist activity there is a possibility. Therefore, we compared the inhibitory effects of Cromolyn, a mast cell stabilizer known as a wide-ranging allergy remedy, and human cultured mast cells. Although degranulation of mast cells by P stimulation was suppressed, Cromolyn did not (Fig. 20).
  • the Recl68 inhibitor compound inhibited the uncondylar response induced by Compound 48/80 stimulation by about 50% at 10 ⁇ M, but Cromolyn completely suppressed at a high concentration of lOmM (Fig. 20 (B)). Since Cromolyn does not suppress the Ca response of 293 cells that stably express Recl68, it does not suppress the degranulation reaction of human cultured mast cells via Recl68 but via Compound 48/80 action points other than Recl68. It was thought that this was suppression of degranulation reaction.
  • Recl68 inhibitory compounds show a higher inhibitory effect on in vivo agonists than Cromolyn, and Recl68 inhibitory compounds are more useful than Cromolyn from the viewpoint of identifying receptors as molecular targets. it was thought.
  • Example 1 5
  • Cord Blood CD34 1 (CAMBREX, 2C-101A), Cord Blood MNC (STEMCYTE, CAI 001) 0 rhSCF (PeproTech, 300-07), rhIL-6 (PeproTech, 200-06), rhIL-3 (PeproTech, 200- 03), 55 mM 2-ME (GIBC0, 21985-023), Iscove 's Modified Dulbecco' s Medium (IMDM, GIBC0, 12440-053), Insulin-Transferrin (GIBC0, 41400-045), 35% BSA (SIGMA , A-7409), MethocultSF B, T (StemCell technology, 04236), acetic acid (JUNSEI, 31010-0330), PBS— (National Institute of Science, CM6201), DMSO (SIGMA, D— 2660), Compound 1, Compound 1 ( Reel 68 molecule inhibitor), c-MEM (GIBC0, 41061-029), FBS (JR
  • the PGD 2 production activity by any stimulus was inhibited by 10 M of compound 1 or compound 3 which is a Recl68 inhibitory compound (FIG. 22).
  • Substance P, MBP fragment, and ECP fragment induced PGD 2 production in a concentration-dependent manner in rabbit cultured moon cake.
  • PGD 2 production activity by any stimulus was inhibited by 10 M Compound 1 or Compound 3 (Fig. 2 2), which is a Recl68 inhibitory compound (Fig. 2 2), activation of Recl68 is desensitized to human cultured mast cells. It was found to induce de novo synthesis of PGD 2 as well as granules.
  • MC T cells is MC T cells and Tryptase and Chymase positive cells are Tryptase positive cells.
  • MC T cells are called mucosal mast cells, mainly much to the gastrointestinal mucosa, whereas, MC K cells are called connective tissue type, common in subcutaneous tissue.
  • Tsukiyomi Manitoba which reacts to SubstanceP and compound 48/80, is the latter type.
  • Recl68-positive mast cells were Tryptase and Chymase positive cells, indicating that they were connective tissue mast cells.
  • the screening method for a degranulation regulator and the identification method for a degranulation inhibitor in mast cells based on the new mechanism via the G protein coupled receptor Rec68 of the present invention include: It is useful as a method for developing new therapeutic agents for allergic diseases or autoimmune diseases, and is particularly suitable as a method for developing therapeutic agents for allergic diseases or autoimmune diseases that involve mast cells. .

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Abstract

La présente invention concerne un procédé de criblage d’une substance en mesure de réguler une dégranulation ou d’inhiber une production de prostaglandine D2 et un procédé d’identification d’une substance en mesure d’inhiber la dégranulation ou la production de prostaglandine D2 au sein d’un mastocyte, ces procédés étant utiles au développement d’un agent thérapeutique destiné à une pathologie allergique ou à une pathologie auto-immune et s’appuyant sur un nouveau mécanisme à l’aide d’un récepteur conjugué à la protéine G Rec168. L’invention concerne un procédé de criblage d’une substance en mesure d’inhiber la capacité de transduction du signal ou d’une substance en mesure de réguler la dégranulation ou d’inhiber la production de prostaglandine D2 au sein d’un mastocyte à l’aide de Rec168. Le procédé comprend les étapes consistant à déterminer la capacité de transduction du signal d'un ligand présentant une activité agoniste envers Rec168 en présence ou en l’absence d’une substance à tester ; et consistant à comparer une valeur de mesure obtenue en présence de la substance à une valeur de mesure obtenue en l’absence de la substance. L’invention concerne en outre un procédé d’identification d’une substance en mesure d’inhiber la dégranulation ou une production de prostaglandine D2 au sein d'un mastocyte.
PCT/JP2006/323063 2005-11-15 2006-11-14 PROCEDE DE CRIBLAGE ET PROCEDE D’IDENTIFICATION D’UNE SUBSTANCE CAPABLE D’INHIBER UNE REACTION DE DEGRANULATION OU LA PRODUCTION DE PROSTAGLANDINE D2 AU SEIN D’UN MASTOCYTE A L’AIDE DE Rec168, ET AGENT THERAPEUTIQUE DESTINE A UNE PATHOLOGIE INFLAMMATOIRE COMPRENANT LA SUBSTANCE WO2007058336A1 (fr)

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