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WO2001070975A1 - Nouveau recepteur d'hormone concentrant la melanine - Google Patents

Nouveau recepteur d'hormone concentrant la melanine Download PDF

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Publication number
WO2001070975A1
WO2001070975A1 PCT/JP2001/002343 JP0102343W WO0170975A1 WO 2001070975 A1 WO2001070975 A1 WO 2001070975A1 JP 0102343 W JP0102343 W JP 0102343W WO 0170975 A1 WO0170975 A1 WO 0170975A1
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Prior art keywords
protein
seq
dna
amino acid
acid sequence
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PCT/JP2001/002343
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English (en)
Japanese (ja)
Inventor
Takeshi Kurama
Shunichiro Matsumoto
Jun Takasaki
Mitsuyuki Matsumoto
Masazumi Kamohara
Tetsu Saito
Tamaki Oda
Youko Saito
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Helix Research Institute
Yamanouchi Pharmaceutical Co Ltd
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Helix Research Institute
Yamanouchi Pharmaceutical Co Ltd
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Priority to AU2001239562A priority Critical patent/AU2001239562A1/en
Publication of WO2001070975A1 publication Critical patent/WO2001070975A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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

Definitions

  • the present invention relates to a novel melanin concentrating hormone receptor, a gene encoding the receptor, a vector containing the gene, a host cell containing the vector, and a drug screening method using the receptor.
  • the present invention also relates to a substance that modulates the receptor activity, a method for preventing and / or treating obesity and / or eating disorders using the substance, and a pharmaceutical composition.
  • MCH Melanin-concentrating-hormone
  • MCH is mainly expressed in the lateral hypothalamus and the uncertain zone in rats and humans (Bittencourt, JC et al. (1992) J. Comp. Neurol. 319, 218-245, Viale 5 A. et. al. (1997) Brain Res Mol Brain Res 46, 243-255) 0
  • the lateral hypothalamus has long been known to function as a feeding center. (1) Suppression and disappearance of eating behavior due to destruction of the lateral hypothalamus. (Anand, BK & Brobeck, JR (1951) Yale J Biol Med 24, 123-140)
  • MCH-deficient mice showed reduced body weight due to reduced body fat, decreased food intake, and increased oxygen consumption per body weight compared to wild-type mice (Shimada, M. et al.). al. (1998) Nature 396, 670-674). It is also known that MCH has a regulatory relationship with other factors involved in energy homeostasis.
  • MSH melanocyte stimulating hormone
  • M4R melanocortin-4 receptor
  • MCH and MSH Similar to the antagonism in fish color change, MCH and MSH also have opposing effects on feeding behavior in mammals. MCH-induced hyperphagia is suppressed by spike-MSH, and conversely, spite-MSH suppresses the antifeedant effect by MCH (Ludwig, DS et al. (1998) Am. J. Physiol. 274 , E627-E633) 0
  • MCH itself is not affinity MC4R, from the inability inhibit binding of "-MSH and MC4R, has been assumed that exerts its action through its own receptor (Ludwig , DS et al. (199 8) Am. J. Physiol. 274, E627-E633) 0
  • MCH is related to various energy homeostasis regulators, but itself exhibits an effect of increasing food intake and suppresses energy consumption, and is responsible for regulating energy balance.
  • MCH has reported the following effects in addition to the above effects.
  • SLC-1 has been reported as an MCH receptor in humans (Saito, Y. et al. (1999) Nature 400, 265-269). SLC-1 is a gene originally isolated on the basis of homology with the somatosustin receptor, one of the G protein-coupled receptors (Kolakowski, LF et al. (1996) FEBS Lett. 398, 253-258). The homology with the somatosustin receptor gene was about 40%, and no binding activity with somatosustin was found, but subsequent studies revealed that the ligand was MCH. Disclosure of the invention
  • the present inventors have succeeded in isolating a gene encoding a novel human MCH receptor, and have found that the amino acid sequence of human MCH receptor protein and human MCH receptor The nucleotide sequence of DNA encoding was determined. Further, the expression of the MCH receptor, the production of a recombinant protein is enabled, and the vector containing the gene, the vector We have established a method for producing host cells, including Michigan, MCH receptor protein using the host cells, and antibodies against the MCH receptor.
  • a substance that modifies the activity of the receptor in particular, a substance having angiogonist activity was obtained, and the present invention was completed.
  • the present inventors further studied the isolation of a homolog of the MCH receptor in monkeys. Succeeded and revealed that it is located in the feeding center.
  • the present invention relates to the following proteins, genes encoding the same, and a method for screening a substance that enhances the activity of an MCH receptor, particularly a substance that has angiogonist activity, using the protein.
  • the present invention also relates to a substance that modulates the receptor activity, particularly a substance having angiogonist activity, a method for preventing and / or treating obesity and / or eating disorders using the substance, and a pharmaceutical composition. I will consider things.
  • a method for screening for a substance having angiogonist activity of melanin concentrating hormone receptor comprising the following steps:
  • step (3) selecting a test drug that reduces the binding activity measured in step (2) as compared with the binding activity of the melanin concentrating hormone to the protein measured in the absence of the test drug
  • a method for screening for a substance having an angiotensin activity of melanin concentrating hormone receptor comprising the following steps:
  • step (3) selecting a test drug that suppresses the cell change measured in step (2) compared to the cell change measured in the absence of the test drug
  • the change in the cell is any change selected from the group consisting of a change in GTP binding activity, a change in intracellular Ca ion concentration, and a change in intracellular cAMP concentration.
  • the substance having antagonist activity of melanin concentrating hormone receptor is a substance for preventing and / or treating obesity and / or eating disorders (13) or (14) Screening method.
  • a pharmaceutical composition for preventing and / or treating an eating disorder is provided.
  • a melanin concentrating hormone receptor gene comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA complementary to its complementary strand and having a chain length of at least 15 nucleotides Reagent for detection.
  • DNA containing the base sequence of SEQ ID NO: 1 or DNA complementary to the complementary strand thereof and having a chain length of at least 15 nucleotides is brought into contact with the sample, and the DNA is placed in the sample.
  • a method for detecting a melanin concentrating hormone receptor gene comprising a step of hybridizing to a DNA of the present invention.
  • the present invention also relates to a method for preventing and / or treating obesity, and Z or an eating disorder, which comprises a step of administering the antagonist according to [17].
  • the present invention relates to the use of the angonist according to [17] in the manufacture of a pharmaceutical composition for preventing and / or treating obesity and / or eating disorders.
  • WO 00/49046 published after the priority date describes the base sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 of the present invention. However, the gene is cloned, the nucleotide sequence is determined, and only the deduced amino acid sequence encoded by the gene is described. A specific method for producing a protein having the amino acid sequence of SEQ ID NO: 2 There is no description of the production of the protein, the specific function and use of the protein, and the description that the protein is an MCH receptor.
  • melanin concentrating hormone (MCH) receptor refers to “melanin concentrating hormone (MCH) receptor protein I. Represents
  • the MCH receptor protein of the present invention includes
  • hybridizing homolog of a protein having the amino acid sequence of SEQ ID NO: 2
  • the “equivalent substance” of the protein having the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 10, more preferably 1 to 7, particularly preferably 1 to 10 in the amino acid sequence of SEQ ID NO: 2. And has the same activity as a protein having an amino acid sequence having amino acid substitutions, deletions and / or insertions of up to 5 amino acids and having the amino acid sequence of SEQ ID NO: 2, that is, MCH receptor activity.
  • the proteins shown can be mentioned.
  • MCH receptor activity means that it can be confirmed, for example, by the method described in Example 3. That is, it can be confirmed that the protein exhibits the same activity as that of the protein using the MCH-dependent increase in intracellular Ca concentration as an index.
  • the conditions for hybridization are as follows: 5xSSPE, 5xDenhanfs solution, 0.5% SDS, 40% formamide, 200 g / ml salmon sperm DNA, 37 ° C overnight.
  • the conditions were about 5xSSPE, 5xDenhard, s solution, 0.5% SDS, 50% formamide, 200 g / ml salmon sperm DNA, 42 ° C overnight.
  • the conditions for washing are as follows: loose conditions are about 5xSSC, 1% SDS, 42 ° C, and usually about 0.5xSSC, 0.1% SDS, 42 ° C. The condition is about “0.2xSSC, 0.1% SDS, 65 ° C!”.
  • the homology between the amino acid sequence of the homologous protein j and the amino acid sequence of SEQ ID NO: 2 of the protein having the amino acid sequence of SEQ ID NO: 2 is at least 95% or more.
  • the homology of the amino acid sequence can be identified by the following conditions (parameters) using a BLAST search.
  • “same drug” in the present invention for example, a protein having the amino acid sequence described in SEQ ID NO: 8 can be shown.
  • the amino acid sequence set forth in SEQ ID NO: 8 is encoded by the base sequence set forth in SEQ ID NO: 7.
  • the cDNA consisting of the nucleotide sequence of SEQ ID NO: 7 was isolated by the present inventors as a homolog of the MCH receptor in cynomolgus monkeys.
  • the amino acid sequence described in SEQ ID NO: 8 (Rinus cynomolgus monkey) has 97% homology with the amino acid sequence described in SEQ ID NO: 2 (human), and a protein comprising this amino acid sequence has MCH receptor activity. It was confirmed that it had.
  • the origin of the MCH receptor of the present invention is not limited to humans and monkeys. As long as it corresponds to any of the receptors of the present invention described in the above (1) to (5), for example, a receptor derived from an organism other than humans and monkeys is also a sequence represented by SEQ ID NO: 2 or SEQ ID NO: 8.
  • the receptor of the present invention also includes a receptor artificially modified by genetic engineering based on the above. Further, the receptor of the present invention is preferably a recombinant receptor.
  • a gene having a nucleotide sequence encoding the protein of the present invention that is, a MCH receptor represented by the amino acid sequence of SEQ ID NO: 2, or a gene having a nucleotide sequence encoding the “same drug” in the present invention
  • it is included in the present invention.
  • it is a gene having a base sequence encoding the amino acid sequence of SEQ ID NO: 2.
  • it is a gene having the 1st to 1023rd of SEQ ID NO: 1.
  • "Genes" of the present invention are DNA and RNA, preferably DNA.
  • the medicaments are described in 1) to 5) below.
  • MRNA is extracted from cells or tissues capable of producing the MCH receptor protein of the present invention.
  • two types of primers sandwiching the receptor mRNA or a part of the mRNA region are prepared using this mRNA as type III.
  • the MCH receptor cDNA or a part thereof can be obtained by performing a reverse transcriptase-polymerase chain reaction (hereinafter, referred to as RT-PCR). Further, by incorporating the obtained MCH receptor cDNA or a part thereof into an appropriate expression vector, it can be expressed in a host cell to produce the receptor protein.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • mRNAs including those encoding the protein are extracted from cells or tissues capable of producing the MCH receptor of the present invention, for example, human brain, by a known method.
  • the extraction method include a guanidine-thiocyanate 'hot' phenol method and a guanidine-thiocyanate-guanidine 'hydrochloric acid method, and preferably a guanidine-thiocyanate cesium chloride method.
  • Cells or tissues having the ability to produce the protein can be obtained by Northern blotting using a gene or a part of a gene having a nucleotide sequence encoding the protein, or Western plotting using an antibody specific to the protein. It can be specified by such as.
  • the mRNA may be purified according to a conventional method.
  • the mRNA can be adsorbed and eluted to an oligo (dT) cell source column and purified.
  • mRNA can be further fractionated by sucrose density gradient centrifugation or the like.
  • a commercially available extracted mRNA may be used without extracting the mRNA.
  • the purified mRNA is subjected to a reverse transcriptase reaction in the presence of a random primer or an oligo dT primer to synthesize a first-strand cDNA.
  • This synthesis can be performed by a conventional method.
  • the obtained first-strand cDNA is subjected to PCR using two types of primers sandwiching a partial region of the target gene to amplify the target MCH receptor DNA.
  • the obtained DNA is fractionated by agarose gel electrophoresis or the like. If desired, cut the above DNA with restricted oxygen or the like and connect The target DNA fragment can also be obtained.
  • the gene of the present invention can be produced by a conventional genetic engineering technique in addition to the above-mentioned production method.
  • a single-stranded cDNA is synthesized using reverse transcriptase using the mRNA obtained by the above-mentioned method as a type II, and then a double-stranded cDNA is synthesized from the single-stranded cDNA.
  • the S1 nuclease method Esfstratiadis, A. et al. (1976) Cell, 7, 279-288) ⁇ Land method (Land, H. et al. (1981) Nucleic Acids Res., 9) , 2251-2266) s 0. Joon Yoo method (Yoo, 0. J. et al.
  • the recombinant plasmid obtained by the above-described method is introduced into Escherichia coli, for example, DH5 strain, and transformed, and a recombinant can be selected using tetracycline resistance or ampicillin resistance as an indicator. Transformation of host cells can be performed, for example, by the method of Hanahan (Hanahan, D. (1983) J. Mol. Biol., 166, 557-580) when the host cell is Escherichia coli, ie, CaCl 2 , MgCl 2 or RbCl 2. Can be carried out by a method of adding the recombinant DNA to a recombinant cell prepared in the presence of the recombinant DNA. 'As a vector, a phage vector such as a lambda system can be used in addition to a plasmid.
  • An oligonucleotide corresponding to all or a part of the MCH receptor of the present invention is synthesized (in this case, a plurality of nucleotide sequences obtained by combining a nucleotide sequence derived using codon usage or a possible nucleotide sequence). well, and if the latter, it is also possible to reduce the type by including inosine), this professional one-flop (32 labeled with P or 33 P), nitrate obtained by modifying fix the DNA of transformants Hybridize with Rocellulose Fill Yuichi, search for the obtained positive strain, and select it.
  • the cDNA is integrated into the expression vector, a protein is produced on the surface of the transformant, and a desired MCH receptor-producing strain is prepared using the antibody against the MCH receptor of the present invention and a secondary antibody against the antibody. And select the target strain.
  • a method for collecting DNA encoding the MCH receptor of the present invention from the obtained transformant of interest can be obtained by a known method (Maniatis, T. et al. (1982): “Molecular Cloning-A Laboratory Manual”). Cold Spring Harbor Laboratory, NY). An example For example, it can be carried out by separating a fraction corresponding to the plasmid DNA from the cells and cutting out the cDNA region from the plasmid DNA.
  • a gene having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 8 can also be produced by joining a DNA fragment produced by a chemical synthesis method.
  • Each DNA can be synthesized using a DNA synthesizer (for example, Oligo 1000M DNA Synthesizer (Beckman) or 394 DNA / RNA Synthesizer (Applied Biosystems)).
  • the substance In order for a substance obtained by a genetic engineering technique using the gene of the present invention to express the function of the MCH receptor of the present invention, the substance necessarily has all of the amino acid sequence shown in SEQ ID NO: 2. No need to be. For example, even if a part of the sequence or another amino acid sequence is added, as long as it shows “the same activity” as the MCH receptor represented by the amino acid sequence shown in SEQ ID NO: 2, However, those proteins are also included in the protein of the present invention.
  • eukaryotic genes are considered to exhibit polymorphism, as is known in the case of the inferior ferron gene (eg, NishiJ. Et al. (1985) J. Biochem. , 97, 153-159), and one or more amino acids may be replaced by this polymorphism. Therefore, even in a protein in which one or more amino acid residues are substituted, deleted, and / or inserted at one or more sites in the amino acid sequence represented by SEQ ID NO: 2, : Highly likely to have “the same activity” as the MCH receptor represented by the amino acid sequence described in 2. As described above, these proteins are “equivalents” of the protein having the amino acid sequence of SEQ ID NO: 2, and are included in the present invention.
  • genes having the nucleotide sequences encoding these “similar substances” are also included in the present invention.
  • Such various genes of the present invention are, for example, phosphite-based based on the above-mentioned information of the MCH receptor of the present invention. It can also be produced by chemical synthesis of nucleic acids according to a conventional method such as the ester method (Hunkapiller, M. et al. (1984) Nature, 10, 105-111).
  • the codon for the desired amino acid is known per se, and its choice may be arbitrary. For example, it can be determined according to a conventional method in consideration of the codon usage frequency of the host to be used (Crantham, IL et al.
  • partial modification of the codons of these base sequences can be performed by a conventional method using a primer comprising a synthetic oligonucleotide encoding the desired modification.
  • the site-specific mutag enesis Mark, DF et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 5662-5666) can be used.
  • the method for preparing the “same substance” in the present invention there is a method using a hybridization technique or a method utilizing a gene amplification technique. That is, those skilled in the art can use the hybridization technology (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4) to synthesize the protein of the present invention.
  • a DNA highly homologous thereto is isolated from a DNA sample derived from a homologous or heterologous organism. It is usually necessary to obtain a protein functionally equivalent to the protein having the amino acid sequence described in No. 3 in No.
  • the DNA and the protein are preferably a DNA that hybridizes under “stringent conditions” and a protein encoded thereby, and a DNA that hybridizes with the DNA of SEQ ID NO: 1, And the protein encoded thereby is preferred.
  • Non-human organisms can be used to isolate DNA encoding such proteins.
  • the stringent hybridization conditions for isolating the DNA encoding the “hybridization equivalent” of the protein having the amino acid sequence of SEQ ID NO: 2 include the following conditions for the hybridization. 5xSSPE, 5xD enhanf s solution, 0.5% SDS, 40% formamide, 200 g / ml salmon sperm DNA, 37 ° C single burn, and more severe conditions are “5xSSPE, 5 Denhard, Solution, 0.5% SDS, 50% formamide, 200 / g / ml salmon sperm DNA, 42 ° C overnight.
  • a protein encoded by DNA isolated using such a hybridization technique usually has a high homology in the amino acid sequence with the protein having the amino acid sequence of SEQ ID NO: 2.
  • High homology refers to sequence homology of at least 80% or more, preferably 90% or more, more preferably 95% or more, and more preferably 97% or more.
  • Amino acid sequence homology can be determined using the BLAST search algorithm. Specifically, the bl2seq program (Tatiana A. Tatusova, Thomas L. Madden (1999), FEMS Microbiol Lett. 174: 247-250) of the BLAST package (sgi32bit version, Purge Yong 2.0.12, obtained from NCBI) It can be used and calculated according to the default parameters. Pairwise alignment Parame overnight First, use the program name blastp, Gap input Cost value of 0, Gap extension Cost value of 0, SEG as a filter for the query array, and BL0SUM62 as a matrix.
  • a primer was designed based on a portion of 1), and a DNA fragment containing a nucleotide sequence highly homologous to the nucleotide sequence of the DNA encoding the protein having the amino acid sequence of SEQ ID NO: 2 was isolated. It is also possible to obtain a protein functionally equivalent to the MCH receptor protein having the amino acid sequence of SEQ ID NO: 2 based on DNA. For example, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10 is mentioned as a preferred primer. Example 6 is illustrated as an acquisition method using this primer.
  • the sequencing of the DNA obtained by a) to d) is carried out, for example, by the chemical modification method of Maxam / Gilbert (Maxam, AM and Gilbert, W. (1980): “Methods in Enzymology” 65, 499). -559) or the dideoxynucleotide chain termination method using M13 (Messing, J. and “Vieira, J (1982)" Gene, 19, 269-276).
  • the vector of the present invention, the host cell of the present invention, and the MCH receptor of the present invention can be obtained by the following methods.
  • the isolated fragment containing the gene encoding the protein of the present invention can be transformed into another eukaryotic host cell by re-incorporation into an appropriate vector DNA. Furthermore, by introducing appropriate promoters and sequences relating to expression into these vectors, the gene can be expressed in each host cell.
  • Eukaryotic host cells include cells such as vertebrates, insects, and yeast.
  • vertebrate cells include COS cells, which are monkey cells (Gluzman, Y. (1981) Cell, 2 3, 175-182) and Chinese 'Hams ovary cell (CHO) deficient in dihydrofolate reductase (Urlaub, G. and Chasin 5 LA (1980) Proc. Atl. Acad. Sci. USA, 77 HEK 293 cells derived from human fetal kidney and 293-EBNA cells (Invitrogen) with the Epstein Barr Virus EBNA-1 gene introduced into the cells are commonly used, but are not limited to these. Not necessarily.
  • RNA splice site As a vertebrate cell expression vector, one having a promoter located upstream of the gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like can be used. May further have a replication origin if necessary.
  • expression vectors include pSV2dhfr (Subbandi ni, S. et al. (1981) Mol. Cell. Biol., 1, 854-864), which has an initial promoter of SV40, and human el.
  • pEF-BOS Mizushima, S. and Nagata 5 S.
  • the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcriptional promoter and a transcription termination signal. And those having an RNA splice site can be used.
  • pME18S Maruyama, K. and Takebe 3 Y. (1990) Med. Immunol., 20, 27-32
  • pEF-BOS Mizushima , S. and Nagata 3 S. (1990) Nucleic Acids Res., 18, 5322
  • pCDM8 Seed, B. (1987) Nature, 329, 840-842
  • the expression vector is a DEAE-dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res., 11, 1295-1308), a calcium phosphate-DNA coprecipitation method (G ⁇ aham, FL and van der Ed). , AJ (1973) Virology, 52, 456-457), method using FuGENE6 (Boeringer Mannheim), and electric pulse perforation method (Neumann, E. et al. (1982) EMBO J Li 1, 841-845)
  • the cells can be incorporated into C0S cells, and thus desired transformed cells can be obtained.
  • G A vector capable of expressing the neo gene that functions as a resistance marker such as pRSVneo (Sambrook, J. et al. (1989): “Molecular Cloning-A Laboratory Manua 1" Cold Spring Harbor Laboratory, ⁇ ) ⁇ ⁇ pSV2 -neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet., 1, 327-341), etc., and select G4 18 resistant colonies for MCH.
  • a transformed cell stably producing the receptor can be obtained.
  • a vector capable of expressing a zeocin resistance gene that functions as a zeocin resistance gene together with an expression vector for example, pcDNA3.1 / Zeo (+) (Invitrogen ) Can be used to transfect and select zeocin-resistant cells to obtain transformed cells that stably produce the MCH receptor.
  • expression vectors such as pCEP4 (Invitrogen), which has an Epstein Barr Virus replication origin and is capable of self-propagation in 293-EBNA cells, are used.
  • the desired transformed cells can be obtained using the kit.
  • the desired transformant obtained above can be cultured according to a conventional method, and the culture produces the protein of the present invention in the cell or on the cell surface.
  • the medium used for the culture can be appropriately selected from various conventional ones according to the host cell used.
  • RPMI-1640 medium or Dulbecco's modified single cell A medium obtained by adding a serum component such as fetal bovine serum (FBS) to a medium such as small essential medium (DMEM) as necessary can be used.
  • FBS fetal bovine serum
  • DMEM small essential medium
  • 293-EBNA cells use a medium such as Dulbecco's modified minimum essential medium (DMEM) supplemented with serum components such as fetal bovine serum (FBS) plus G418. it can.
  • the protein of the present invention produced in the cells of the transformant or on the cell surface is separated and purified by various known separation procedures utilizing the physical properties, chemical properties, and the like of the protein. be able to.
  • a membrane fraction containing a receptor protein treatment with a usual protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ionization
  • liquid chromatography such as exchanger chromatography, affinity take chromatography, high performance liquid chromatography (HPLC), dialysis methods, and combinations thereof.
  • the membrane fraction can be obtained according to a conventional method. For example, it can be obtained by culturing cells expressing the MCH receptor of the present invention on the surface thereof, suspending them in a buffer, homogenizing, and centrifuging. In addition, by solubilizing the MCH receptor with a solubilizing agent that is as mild as possible (CHAPS, Triton X-100, dichitonin, etc.), the characteristics of the receptor can be maintained after solubilization.
  • a solubilizing agent that is as mild as possible (CHAPS, Triton X-100, dichitonin, etc.
  • the fusion of the protein of the present invention with the primary sequence in frame allows expression of the MCH receptor, confirmation of intracellular localization, purification, and the like.
  • Marker sequences include, for example, FLAG epitope ⁇ Hexa-Histidine tag, Hemagglutinin tag, myc epitope and the like.
  • proteases such as enterokinase, factor-1Xa, and thrombin
  • the present invention includes a method for screening a substance (compound, peptide, and antibody) that modifies the activity of the MCH receptor.
  • a substance compound, peptide, and antibody
  • Substances that modify the activity of the protein of the present invention include artificially synthesized substances and naturally occurring substances. In addition, it can be an inorganic substance as well as an organic substance.
  • Peptides include peptides composed of more amino acids, such as enzymes, in addition to peptides composed of relatively few amino acids.
  • the screening method comprises the steps of: using the MCH receptor constructed as described above, a system for measuring an index of modification of the receptor protein in accordance with the physiological characteristics of the receptor protein. A means for adding the test drug and measuring the index.
  • MCH receptor a cell in which the receptor has been expressed, a membrane fraction of the cell, a purified preparation of the receptor protein, or the like can also be used.
  • Specific examples of the measurement system include the following screening methods.Test drugs include commercially available compounds, various known compounds and peptides registered in the chemical file, combinatorial chemistry (Terrett technology). 5 NK, et al. (1995) Tetrahedron, 51, 8135-8137) Compounds and phage display method (Felici, F., et al. (1991) J. Mol. Biol., 222, 301 -310) can be used.
  • culture supernatants of microorganisms, natural components derived from plants and marine organisms, and animal tissue extracts are also targets for screening.
  • a compound or peptide obtained by chemically or biologically modifying the compound or peptide selected by the screening method of the present invention may be used, but is not limited thereto.
  • Substances that bind to the MCH receptor of the present invention can be screened by the ligand binding assay.
  • a cell membrane expressing the receptor protein or a purified preparation of the receptor protein is prepared.
  • Optimizing the assay conditions such as buffer, ion and pH, the cell membrane expressing the receptor protein in the optimized buffer or the purified preparation of the receptor protein is used as a labeled ligand, for example.
  • [Phe 13 , [ 125 I] Tyr 19 ] ⁇ MCH is incubated with the test drug for a certain period of time. After the reaction, filter with a glass filter and wash with an appropriate amount of buffer, and measure the radioactivity remaining in the filter with an alcohol filter and the like.
  • a substance having an antagonistic activity of the receptor protein selected by the screening method of the present invention is useful for treating or preventing obesity or an eating disorder.
  • the screening method of the present invention is preferably performed under the conditions described in Example 2 or Example 4.
  • a substance having a binding inhibitory activity having a Ki of 10 ⁇ m or less and an IC50 of 16 ⁇ m or less, more preferably Ki or less and an IC50 of 1.6 zM or less can be selected.
  • Substances that modify the activity of the MCH receptor of the present invention can be screened by the GTP ⁇ S binding method (Lazareno, S. and Birdsall, NJM (1993) Br. J. Pharmacol. 109, 1120-1127).
  • Substances (compounds, peptides, and antibodies) having the agonist activity of the MCH receptor can be screened using an increase in specific GTPyS binding in the presence of the test drug as an index.
  • screening for substances (compounds, peptides, and antibodies) having an agonistic activity of the receptor protein based on the suppression of increase in GTPaS binding by MCH 'in the presence of the test drug is used as an index. Can be.
  • the substances (compounds, peptides, and antibodies) that modify the activity of the MCH receptor of the present invention can be screened by utilizing the fluctuation of intracellular Ca ++ or cAMP concentration of cells expressing the MCH receptor. It is possible to The intracellular Ca ++ concentration can be measured using fura2, uo3, or the like. The cAMP concentration can be measured using a commercially available cAMP measurement kit (Amersham, etc.).
  • the concentration of Ca ++ and cAMP can be measured indirectly by detecting the transcriptional activity of a gene whose transcription level is regulated depending on the concentration of Ca ++ or cAMP. is there.
  • Ca ++ and cAMP concentrations can be measured by the following method. First, a repo overnight gene linked to a serum responsive element or a cAMP responsive element is introduced into a cell in which the receptor protein has been expressed, and a test drug is added to a culture solution of the cell. Any gene capable of producing a detectable signal can be used as the repo overnight gene. For example, the luciferase gene is desirable as the repo overnight gene.
  • substances (compounds, peptides, and antibodies) having the agonist activity of the receptor protein can be screened using the induction of lucif Xase activity upon addition of the test drug as an index.
  • MCH at a final concentration of 0.4 nM is added, and the luciferase activity is measured in the same manner.
  • substances (compounds, peptides, and antibodies) having angiogonist activity of the receptor protein can be screened using the inhibition of luciferase activity induction by MCH upon addition of the test drug as an index.
  • substances compounds, peptides, and antibodies
  • substances are allowed to act on cells expressing the protein and host cells not expressing the protein (control cells) for a certain period of time to obtain Ca ++ and cAMP. Measure the concentration directly or indirectly.
  • substances having agonist activity are indicated by an increase in cell-specific Ca ++ and / or an increase or decrease in cAMP concentration, which expresses the protein. Can be screened.
  • a substance (compound, peptide, and antibody) having an antagonistic gonist activity of the MCH receptor based on the inhibitory effect of MCH on Ca ++ increase and / or cAMP concentration increase or decrease in the presence of the test drug as an index ) can be screened.
  • the screening method of the present invention is preferably performed under the conditions described in Example 3 or Example 5.
  • a substance having an EC50 of 100 / M or less, more preferably a substance having an EC50 of 1 or less is selected as a substance having agonist activity.
  • a substance having an EC50 of 100 / M or less more preferably a substance having an EC50 of 1 or less is selected as a substance having agonist activity.
  • the test drug to the Athesay conditions described in Example 3, that is, a substance having an IC50 of 3 or less under the conditions of Example 5, preferably
  • a substance having 0 of 1 or less, and more preferably a substance having an IC50 of 1 / M or less, can be selected as a substance having angiogenesis activity.
  • the antibody that reacts with the MCH receptor of the present invention for example, a polyclonal antibody or a monoclonal antibody can be obtained by directly administering the MCH receptor ⁇ ⁇ the fragment of the MCH receptor to various animals.
  • a polyclonal antibody or a monoclonal antibody can be obtained by directly administering the MCH receptor ⁇ ⁇ the fragment of the MCH receptor to various animals.
  • the DNA vaccine method (Raz, E. et al. (1994) Proc. Natl. Acad.
  • the polyclonal antibody is prepared by emulsifying the protein or a fragment thereof in a suitable adjuvant such as Freund's complete adjuvant and immunizing the peritoneal cavity, subcutaneously or intravenously, and sensitizing animals such as serum of rabbits, rats, goats, chickens or the like. Manufactured from eggs.
  • the polyclonal antibody can be separated and purified from the serum or eggs thus produced by a conventional protein isolation and purification method. Examples of such methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatographic methods using DEAE-cell mouth, nose, needle mouth, xiapatite, and protein A agarose.
  • a preferred example for preparing an antibody is the method described in Example 8.
  • Monoclonal antibodies can be easily produced by those skilled in the art by the cell fusion method of Kerayl and Milstein (Kohler, G. and Milstein, C. (1975) Nature, 256, 495-497).
  • mice are immunized by inoculating the intraperitoneal, subcutaneous, or vein several times with an emulsion prepared by emulsifying the protein of the present invention or a fragment thereof in an appropriate adjuvant such as Freund's complete adjuvant every few weeks. After the final immunization, remove the spleen cells and A hybridoma is produced by fusing with an erotic cell.
  • Myeloma cells that have markers such as hypoxanthine, guanine, phosphoribosyltransferase deficiency, and thymidine kinase deficiency, such as Mau's Smieloma cells, Use strain P3X63Ag8.Ul.
  • polyethylene glycol is used as a fusion agent.
  • 10 to 30% fetal bovine serum is added as appropriate to commonly used mediums such as Eagle's minimum essential medium, Dullco's modified minimum essential medium, and RPMI-1640 as the medium for hybridoma production. Used. Fusion strains are selected by the HAT selection method.
  • Hybridoma screening is performed using the culture supernatant by a well-known method such as ELISA, immunohistochemical staining, or the above-mentioned screening method, and a clone of the hybridoma secreting the desired antibody is selected. Repeated subcloning by limiting dilution guarantees the monoclonality of the hybridoma.
  • the thus obtained hybridomas can be purified in an amount that can be purified by culturing them in the medium for 2-4 days or in the abdominal cavity of BALB / c mice pre-treated with prismin for 10-20 days. Produced.
  • the monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional protein isolation and purification method.
  • a conventional protein isolation and purification method examples include centrifugation, dialysis, salting out with ammonium sulfate, and chromatographic methods using DEAE-cellulose, hydroxyapatite, protein Aagaguchi, and the like.
  • an antibody fragment containing a monoclonal antibody or a part thereof can be produced by incorporating all or a part of a gene encoding the antibody into an expression vector and introducing the gene into E. coli, an enzyme, or an animal cell.
  • the antibody separated and purified as described above is digested with a protease such as pepsin, papain, etc. by a conventional method, and then separated and purified by a conventional protein isolation and purification method.
  • An antibody fragment containing, for example, F (ab ') 2, Fa Fab', and Fv can be obtained.
  • an antibody that reacts with the MCH receptor of the present invention can be obtained by the method of Clackson et al., Zebedee et al. (Clackson, T. et al. (1991) Nature, 352, 624-628; (1992) Proc. Natl. Acad. Sci. USA, 89, 3175-3179) to obtain a single chain Fv or Fab.
  • It is also possible to obtain a human antibody by immunizing a transgenic mouse (Lonberg. Et al. (1994) Nature, 368, 856-859) in which the mouse antibody gene is replaced with a human antibody gene.
  • the present invention includes a medicament comprising, as an active ingredient, an MCH receptor or a substance (compound, peptide, or antibody) selected by the above-mentioned screening method, which significantly modifies the activity of the protein.
  • the pharmaceutical composition of the present invention is preferably a pharmaceutical composition for preventing and / or treating obesity and eating disorders, which contains a substance having an agonist activity of the receptor as an active ingredient.
  • the present invention is a method for preventing and / or treating obesity and eating disorders, which comprises a step of administering the substance having the antagonist activity.
  • Parenteral administration such as tablets, pills, capsules, granules, fine granules, powders, oral solutions, injections such as intravenous and intramuscular injections, suppositories, transdermal preparations, and transmucosal Parenteral administration by administration agents and the like can be mentioned.
  • Parenteral administration such as intravenous injection, is particularly desirable for peptides digested in the stomach.
  • the solid compositions for oral administration according to the present invention may contain one or more active substances of at least one inert diluent, such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolid. Don, mixed with magnesium metasilicate and aluminate.
  • the composition may be prepared in a conventional manner using additives other than inert diluents, such as lubricants, disintegrants, stabilizers, It may contain a solubilizer or the like. Tablets and pills may be coated with a sugar coating or a film of a gastric or enteric substance, if necessary.
  • Liquid compositions for oral use include emulsions, solutions, suspensions, syrups, and elixirs, and include commonly used inert diluents, such as purified water, ethanol.
  • the compositions may contain additives other than inert diluents, such as wetting agents, suspending agents, sweetening agents, flavoring agents, preservatives.
  • Parenteral injections include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • Aqueous solutions and suspensions include, for example, distilled water for injection and physiological saline as diluents.
  • Diluents for non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80 and the like.
  • the composition may further contain a wetting agent, an emulsifying agent, a dispersing agent, a stabilizer, a solubilizing or solubilizing agent, a preservative and the like.
  • the composition may be sterilized, for example, by filtration through a nocturnal retention filter, combination with a bactericide, or irradiation.
  • a sterile solid composition can be produced and dissolved in sterile water or another sterile injectable medium before use.
  • the dose is appropriately determined in consideration of the activity intensity, symptoms, age, sex, and the like of the active ingredient selected by the screening method.
  • the dose in the case of oral administration, is generally about 0.1 to 100 mg, preferably 0.;! To 50 ing per day for an adult (assuming a body weight of 60 kg).
  • the dosage in the case of parenteral administration, is 0.01 to 50 mg / day, preferably 0.01 to 10 m / day in the form of injection.
  • the invention also encompasses the use of DNA encoding the MCH receptor as a diagnostic. Detection of variants of the MCH receptor gene associated with dysfunction can be used to diagnose diseases or susceptibility caused by under-, over- or altered expression of MCH receptors. That is, the present invention relates to a DNA that specifically hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and has a chain length of at least 15 nucleotides.
  • a DN consisting of the nucleotide sequence of SEQ ID NO: 1 A includes its complementary strand.
  • the DNA of the present invention includes a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or a DNA having a chain length of at least 15 nucleotides, which is complementary to the complementary strand thereof.
  • the term “specifically hybridize” with the DNA of the present invention means that the DNA hybridizes with the DNA of the present invention under ordinary hybridization conditions, preferably under strict conditions, and with other DNA. Does not hybridize.
  • Such a DNA can be used as a probe for detecting and isolating the DNA of the present invention and as a primer for amplifying the DNA of the present invention.
  • primers for PCR for amplifying DNA consisting of the nucleotide sequence of SEQ ID NO: 1 include, for example, SEQ ID NO: 3 (forward primer) and SEQ ID NO: 0.4 (river primer), SEQ ID NO: 5 (forward primer) and SEQ ID NO: 6 (reverse primer), SEQ ID NO: 11 (forward primer) and SEQ ID NO: 12 (reverse primer) can be used.
  • a DNA having at least a part or all of the DNA of the present invention or its complementary sequence) and a chain length of at least 15 'nucleotides is used.
  • a DNA having the 1st to 1023rd of the base sequence described in SEQ ID NO: 1 is used.
  • Diagnostic DNA can be obtained from a subject's cells, such as blood, urine, saliva, tissue biopsy or autopsy material. Deletion and insertion mutations can be detected by a change in the size of the amplified product when compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled MCH receptor nucleotides. Perfectly matched sequences can be distinguished from mismatched strands by RNase digestion or by differences in melting temperatures. In addition, a difference in the nucleotide sequence of DNA can be detected based on a change in the electrophoretic mobility of a DNA fragment in gel electrophoresis due to the presence or absence of a denaturing agent. Alternatively, you can detect nucleotide sequence differences by direct DNA sequencing.
  • an array of oligonucleotide probes containing the nucleotide sequence of the MCH receptor or a fragment thereof can be constructed.
  • This array technique is known and has been used to analyze gene expression, genetic linkage and genetic variability (Chee, M. et al. (1996) Science, 274, 610-613).
  • methods for measuring abnormally decreased or increased levels of MCH receptors from samples obtained from subjects can be used to determine whether a disease or susceptibility to disease resulting from under-, over- or altered expression of MCH receptors. Used for diagnosis. Decrease or increase in expression can be determined by any of the polynucleotide quantification methods known to those skilled in the art, for example, PCH,! ⁇ : ⁇ :? ⁇ ! ⁇ It can be measured at the RNA level by case protection, northern plot, and other hybridization methods.
  • a decrease or increase in the level of a protein such as MCH receptor in a sample obtained from a subject can be measured by an assay method known to those skilled in the art. Examples of such methods include radioimmunoassay, competitive binding assay, western plotting, and ELISA assay.
  • a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA complementary to a complementary strand thereof and having a chain length of at least 15 nucleotides includes expression of the protein of the present invention.
  • Antisense DNA for suppression is included.
  • the antisense DNA has a chain length of at least 15 nucleotides or more, preferably 100 nucleotides, more preferably 500 nucleotides or more, and usually has a length of not more than 3000 nucleotides, preferably not more than 2000 nucleotides in order to cause an antisense effect. Have a length.
  • Such antisense DNA is also considered to be applicable to gene therapy for diseases caused by abnormality (abnormal function or expression) of the protein of the present invention.
  • the antisense DNA is, for example, based on the sequence information of the DNA encoding the protein of the present invention (for example, the DNA described in SEQ ID NO: 1) based on the phosphorothioate method (Stein, 1988 Physicochemical properties of phosphorothioate). It can be prepared using oligodeox ynucleotides. Nucleic Acids Res 16, 3209-21 (1988)). By knocking out the MCH receptor gene using the antisense DNA according to the present invention, the elucidation of a disease involving the MCH receptor can be promoted.
  • a virus vector such as a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or a non-viral vector such as a ribosome is used. It is administered to patients by the e iw method or the i / 3 method.
  • FIG. 1 is a photograph showing the result of analyzing the distribution of GPRv17 protein in the monkey lateral hypothalamus by immunohistological staining.
  • LH lateral hypothalamus LH lateral hypothalamus
  • the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples. Unless otherwise specified, it can be carried out according to a known method (Maniatis, T. et a 1. (1982). "Molecular Cloning-A Laboratory Manual” Cold Spring Harbor Laboratory, NY).
  • Example 1 Isolation of Gene Encoding New G Protein-Coupled Receptor GPRv17
  • the full-length cDNA encoding the novel G protein-coupled receptor GPRv17 was obtained from PCI.
  • PCR was performed using Pyrobest DNA polymerase (Takara Shuzo) at 94 ° C (2.5 minutes).
  • the sequence has an open reading frame of 1023 bases (SEQ ID NO: 1 from 1st to 1023rd).
  • the amino acid sequence (340 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 2. Since the predicted amino acid sequence has seven transmembrane domains that are likely to be characteristic of G protein-coupled receptors, this gene should encode G protein-coupled receptors. There was found.
  • GPRvl7 showed the highest phase at 38% of HUMAN PROBABLE G PROTEIN-COUPLED RECEPTOR SLC-1 (Q99705, 402aa) among known G protein-coupled receptors. Showed the same sex. This proved that “GPRvl7” is a novel G protein-coupled receptor.
  • SLC-1 has been clarified to be a protein using MCH as a ligand similarly to the protein of the present invention (SaitoJ. Et al. (1999) Nature 400, 265-269). However, the homology in both amino acid sequences was only 38, indicating that the protein of the present invention was novel.
  • Example 2 The expression of GPRvl7 protein by 293 cells and the following experiment with [Phe i3 , [I] Tyr] -MCH confirmed the MCH receptor activity of the protein encoded by GPRvl7.
  • the cDNA was obtained by HT-PCR using human brain-derived poly (A) + RNA (Clontech) as a type III, and expressed in an expression vector. Incorporated.
  • the forward primer one as 5, -GGTCTAGAATGAATCCATTTCATGCATCTTGTT-3 5 (SEQ ID NO: 5), as a reverse Bly Ma one 5, -GGTCTAGACTAAAAGTGTGATTTCAGAGTGTTT-3 5 (SEQ ID No .: 6) was used (an Xbal site was added to each 5 'end).
  • RT--PCR was performed using Ex Taq DNA polymerase (Takara Shuzo) in the presence of 5% DMS0, and repeated 34 cycles of 94 ° C (30 seconds) Z55 ° C (30 seconds) / 72 ° C (2 minutes) 34 times .
  • a DNA fragment of about 1.0 kbp was amplified. After digesting this fragment with Xbal, it was cloned using pEF-BOS plasmid (Mizushima, S. and Nagata, S. (1990) Nucleic Acids Res., 18, 5322). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) according to the dideoxy-mine method. The obtained plasmid was designated as pEF-BOS-GPRv17.
  • 293 cells were seeded with lxlO 6 cells on a 10 cm culture dish (Asahi Techno Glass Co.) coated with Type I Collagen, cultured for 24 hours, and then 9 g of pEF-B0S-GPRvl7 and lg pcDNA3.1 / Zeo (+ ) (Invitrogen) using FuGENE6 (Boeringer Mannheim) The gene was introduced. 30 hours after transfection, the cells were newly seeded on a 10-cm culture dish, selected with 40 // g / ml zeocin (Invitrogen), and the cells that survived and formed colonies were collected, and the GPRvl7 protein of the present invention was recovered. 293 cells with stable expression were obtained.
  • the cells were collected, washed, suspended in 0.32M sucrose, and homogenized with a Dounce homogenizer. The mixture was centrifuged at 300 xg for 10 minutes to denucleate, and the supernatant was centrifuged again at 12, OOOxg for 15 minutes to obtain a precipitate fraction. This was suspended in 50 mM Tris.HCl (pH 7.4) containing 0.0075% Tron-X100, stirred slowly at 4 ° C for 30 minutes, and then centrifuged at 12,000 xg for 15 minutes to obtain a suspension.
  • the precipitate was washed once with 5 mM Tris.HCl (pH 7.4) and twice with 50 mM Tris.HCl (pH 7.4), 10 mM MgCl 2 , 2 mM EGTA, aprotinin, and pepstatin A, and this was used as a membrane fraction.
  • the membrane fraction 20 ⁇ G [Phe 13, [125 I] Tyr 19] -MCH (NEN Life Science Products, Inc) was added to a final concentration 0.07 ⁇ 5.7xl0- 9 M, 50mM Tris.HCl (pH7 .4), lOmM gCl 25 2mM E6TA, lmg / ml bacitracin, lmg / ml ovalbumin, 10KIU / ml aprotinin, l / g / ml pepstatin A It was collected at Vesu Yuichi at Grass Fill Yuichi. The radioactivity recovered in the glass filter was measured at the gamma level and the total amount bound to the membrane fraction was determined.
  • the amount of non-specific binding to the membrane fraction was determined by adding MCH (Bachem) at a final concentration of 1 ⁇ or 2 ⁇ to the above test. Specific binding was detected based on the total and non-specific binding of [Phe 13 , [ 125 I] Tyr 19 ] -MCH to the membrane fraction of 293 cells into which pEF-B0S-GPRvl7 was stably introduced. In each case, specific binding was observed at concentrations of 0.07 to 5.7xl ([Phe 13 , [ 125 I] Tyr 19 ] -MCH in T 9 M).
  • GPRvl7 protein stable expression 293 cells are seeded on 96-well Black / clear bottom plate, collagen I coated (manufactured by BECTON DICKINSON) at 2xl0 4 cells / well, cultured for 24 hours, and the medium is discarded.
  • AM manufactured by Molecular Probe
  • DMEM containing 0.004 acid pluronic acid and 10% FBS were added at 100 ⁇ per 1 ⁇ , and incubated at 37 ° C for 1 hour. After the incubation, the cells were washed four times with Hanks BSS (manufactured by GIBCO) containing 20 mM HEPES, and Hanks BSS containing 100 ⁇ 1 of 20 mM HEPES per well was added.
  • Hanks BSS manufactured by GIBCO
  • Example 2 To [lz5 I] Tyr 19] indicator activity to inhibit the binding of -MCH tested study drug.
  • As the test drug a commercially available compound was used. Basically, the method of Example 2 was used with some changes. The test drug was added to the solution 1001 described in Example 2 containing 15 ⁇ g of the membrane fraction, and lxlO- 9 M [Phe 13 , [ i25I ] Tyr 19 ] -MCH was added to perform the binding reaction. went. The recovered radioactivity was measured by adding a glass scintillator to a micro scintillator and using a top count (Packard).
  • Example 5 Screening of a substance that inhibits an increase in intracellular Ca ++ concentration by MCH using 293 cells stably expressing GPRvl7 protein
  • Example 3 The test drug was added to the conditions of Example 3, and after 5 minutes, 75 nM MCH was added, and the change in intracellular Ca ++ concentration was measured under the same conditions as in Example 3.
  • the IC50s of the compounds A, B, and C selected in Example 4 are 1.7 and 13, respectively, and the increase of the intracellular Ca ++ concentration due to MCH in 293 cells stably expressing GPRvl7 protein was dose-dependent. It was found to be a strong antagonist of the GPRvl7 protein, which suppresses it.
  • the elucidated sequence is shown in SEQ ID NO: 7.
  • the sequence has an open reading frame of 1023 bases (1st to 1023rd of SEQ ID NO: 7).
  • the amino acid sequence (340 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 8. This amino acid sequence had a high homology of 97% with the amino acid sequence of human GPRvl7 protein, and it was clarified that it encodes the monkey GPRvl7 protein.
  • Monkey GPRvl7 protein was transiently expressed in 293 cells, and the change of intracellular Ca ++ concentration by MCH was examined.
  • cDNA encoding monkey GPRv17 was inserted into pEF-BOS plasmid to obtain pEF-BOS-monkey GPRv17.
  • the gene was introduced using FuGENE6 (Boeringer Mannheim).
  • the medium was discarded 24 hours after gene introduction, and the change in intracellular Ca ++ concentration was measured under the conditions of Example 3. Similar to human GPRvl7-transient 293 cells, MCH capacity-dependent changes in intracellular Ca ++ concentration were observed in 293 cells of monkey GPRvl7 transiently expressing. The EC50 at that time was 171 nM. Thus, it was revealed that the monkey GPRvl7 gene is also a functional MCH receptor.
  • the distribution of human GPRv17 gene expression was analyzed by Northern blot hybridization.
  • a probe for the human GPRvl7 gene a cDNA fragment (first to 1023th of SEQ ID NO: 1) was used.
  • Membrane may be used once in a solution containing 2X SSPE, 0.1% SDS, once in a solution containing lx SSPE, 0.1% SDS, and finally twice in a solution containing 0.5x SSPE, 0.1% SDS. Also washed at 65 ° C for 15 minutes.
  • a cDNA derived from each part of the human brain is shown as SEQ ID NO.
  • Oligonucleotide 5, -TGC AATCCCAGTGTACCAAAACAGAGAG-3 ' was used as a forward primer and the oligonucleotide shown by SEQ ID NO: 12 was used as a 5'-CAGTGAGGCCACAGTGTGGAGGGCAAGG-3' reverse primer.
  • Ex Taq (Takara Shuzo) was used for PCR, and the cycle of 94 ° C (30 seconds) ⁇ 60 ° ⁇ (30 seconds) (1 minute) was repeated 40 times.
  • hypothalamus which controls the eating function, as one of the functions.
  • Expression was also observed in the cerebral cortex (frontal lobe), hippocampus, amygdala, caudate nucleus, and substantia nigra, but not in the cerebellum or pituitary.
  • the peptide CEKEINNMGNTLKSHF corresponding to the C-terminus of GPRvl7 was synthesized using a multi-item simultaneous solid phase method automated peptide synthesizer PSSM-8 (Shimadzu Corporation).
  • the synthesized peptide was purified using SepPakC18 (Waters).
  • Mosinine (KLH) was bound to 2 mg of purified peptide and 2 mg of keyhole phosphate by dry weight using an Inject Maleimide Activated mcKLH kit (Pierce) to obtain a KLH conjugate.
  • Emulsion was prepared by mixing 0.2 mg of KLH conjugate (0.25 ml) and 0.25 ml of TiterMax Gold (Funakoshi) in a launch tube by sonication.
  • the above-mentioned emulsion was administered subcutaneously to the back of two Japanese White Egrets several times to immunize. This immunization operation was performed eight times every two weeks. After eight immunizations, blood was collected from the ear vein to prepare antiserum.
  • the antiserum diluted 100- to 1000-fold gave a signal in COS cells transiently transformed with human GfPIlvl7 and monkey GPRvl7.
  • human GfPIlvl7 and monkey GPRvl7 similarly, no signal was given in COS cells transiently transformed with human SLC-1. Accordingly, antisera that specifically detect human and monkey GPRv17 proteins were obtained.
  • Example 9 Distribution analysis of GPRvl7 protein in monkey hypothalamus by anti-human GPRvl7 antiserum Using the anti-human GPRv17 antiserum prepared in Example 8, the distribution of GPRv17 protein in the monkey hypothalamus was analyzed.
  • cynomolgus monkeys Male cynomolgus monkeys (6.3 years old, weighing 9.65 kg, purchased from Hamley Co., Ltd.) were killed by exsanguinating the carotid artery under deep anesthesia with pentobarbi sodium sodium. Immediately thereafter, the brain was removed, sliced into 5 thighs, and infiltrated and fixed at 4 ° C for 2 days using lOOmM phosphate buffer (PH7.4) containing 4 paraformaldehyde. After soaking in lOOmM phosphate buffer (PH7.4) containing 16% sucrose for 2 days, the brain tissue including the hypothalamus was cut out, rapidly frozen with dry ice, and thickened with a microtome. Frozen sections of 20 / m2 were prepared.
  • the present invention provides a novel MCH receptor.
  • the receptor of the present invention is useful for the prevention of diseases associated with MCH, obesity and eating disorders such as keratia, anorexia nervosa, bulimia and the search and evaluation of drugs that modify the activity of the receptor as Z or therapeutic agents. It is useful and can provide a therapeutic agent for a disease associated with the receptor.
  • the DNA encoding the MCH receptor of the present invention is useful not only for producing the MCH receptor, but also for diagnosing a disease caused by mutation or abnormal fluctuation of the expression of the MCH receptor.
  • the polyclonal or monoclonal antibody of the receptor is useful as a drug acting on the receptor, a diagnostic agent or a means for separating and purifying a protein.

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Abstract

On a isolé un ADN complémentaire codant une protéine réceptrice d'hormone concentrant la mélanine. Grâce à cette nouvelle protéine réceptrice d'hormone concentrant la mélanine, des expériences de liaison sont possibles. En sélectionnant une substance modifiant l'activité du récepteur d'hormone concentrant la mélanine, sur la base de ces expériences de liaison, on peut développer des médicaments ayant pour cible cette protéine.
PCT/JP2001/002343 2000-03-24 2001-03-23 Nouveau recepteur d'hormone concentrant la melanine Ceased WO2001070975A1 (fr)

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AU2001239562A AU2001239562A1 (en) 2000-03-24 2001-03-23 Novel melanin concentrating hormone receptor

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JP2000088588A JP2005229804A (ja) 2000-03-24 2000-03-24 新規なメラニンコンセントレーティングホルモン受容体

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002003070A1 (fr) 2000-07-05 2002-01-10 Takeda Chemical Industries, Ltd. Procede de criblage d'antagoniste/agoniste du recepteur de l'hormone de concentration de la melanine (mch)
JP2002296277A (ja) * 2000-07-05 2002-10-09 Takeda Chem Ind Ltd Ｍｃｈ受容体アンタゴニスト・アゴニストのスクリーニング方法
EP1233981A4 (fr) * 1999-11-16 2003-02-12 Merck & Co Inc Recepteur couple a la proteine g
WO2003072780A1 (fr) * 2002-02-27 2003-09-04 Takeda Chemical Industries, Ltd. Nouvelles protéines, leurs adn et leur utilisation
WO2004023870A1 (fr) * 2002-09-10 2004-03-25 Takeda Pharmaceutical Company Limited Animal transgénique à slt humain
EP1588139A4 (fr) * 2001-05-31 2006-10-04 Merck & Co Inc Recepteur de type 2 a hormone de concentration de melanine de singe rhesus, chien et furet
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
US7141391B2 (en) 2001-11-13 2006-11-28 Neurogen Corporation Monkey and canine melanin concentrating hormone receptors
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100820095B1 (ko) 2006-12-14 2008-04-07 부경대학교 산학협력단 양식 넙치의 멜라닌 농축 호르몬 수용체

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049046A1 (fr) * 1999-02-19 2000-08-24 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couplee a la proteine g et adn

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049046A1 (fr) * 1999-02-19 2000-08-24 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couplee a la proteine g et adn

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAITO Y. ET AL.: "Molecular characterization of the melanin-concentrating-hormone receptor", NATURE, vol. 15, no. 400, 1999, pages 265 - 269, XP002942024 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1233981A4 (fr) * 1999-11-16 2003-02-12 Merck & Co Inc Recepteur couple a la proteine g
US6593108B1 (en) 1999-11-16 2003-07-15 Merck & Co., Inc. Nucleic acid molecule encoding a melanin-concentrating hormone receptor 2 polypeptide
EP1298439A4 (fr) * 2000-07-05 2005-11-23 Takeda Pharmaceutical Procede de criblage d'antagoniste/agoniste du recepteur de l'hormone de concentration de la melanine (mch)
JP2002296277A (ja) * 2000-07-05 2002-10-09 Takeda Chem Ind Ltd Ｍｃｈ受容体アンタゴニスト・アゴニストのスクリーニング方法
WO2002003070A1 (fr) 2000-07-05 2002-01-10 Takeda Chemical Industries, Ltd. Procede de criblage d'antagoniste/agoniste du recepteur de l'hormone de concentration de la melanine (mch)
US7273710B2 (en) 2000-07-05 2007-09-25 Takeda Pharmaceutical Company Limited Method for screening MCH receptor antagonist/agonist
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
EP1588139A4 (fr) * 2001-05-31 2006-10-04 Merck & Co Inc Recepteur de type 2 a hormone de concentration de melanine de singe rhesus, chien et furet
US7208282B2 (en) 2001-05-31 2007-04-24 Merck & Co., Inc. Rhesus monkey, dog and ferret melanin-concentrating hormone type 2 receptor
US7141391B2 (en) 2001-11-13 2006-11-28 Neurogen Corporation Monkey and canine melanin concentrating hormone receptors
WO2003072780A1 (fr) * 2002-02-27 2003-09-04 Takeda Chemical Industries, Ltd. Nouvelles protéines, leurs adn et leur utilisation
WO2004023870A1 (fr) * 2002-09-10 2004-03-25 Takeda Pharmaceutical Company Limited Animal transgénique à slt humain
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds

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