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WO2001073023A1 - Nouveau recepteur gprv53 couple a une proteine se liant a la guanosine-triphosphate, gene correspondant et procede de production et d'utilisation associe - Google Patents

Nouveau recepteur gprv53 couple a une proteine se liant a la guanosine-triphosphate, gene correspondant et procede de production et d'utilisation associe Download PDF

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Publication number
WO2001073023A1
WO2001073023A1 PCT/JP2001/002767 JP0102767W WO0173023A1 WO 2001073023 A1 WO2001073023 A1 WO 2001073023A1 JP 0102767 W JP0102767 W JP 0102767W WO 0173023 A1 WO0173023 A1 WO 0173023A1
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protein
dna
present
ligand
compound
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PCT/JP2001/002767
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English (en)
Japanese (ja)
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Shun-Ichiro Matsumoto
Tamaki Oda
Youko Saito
Noriyuki Morikawa
Kenji Yoshida
Makiko Suwa
Tomoyasu Sugiyama
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Helix Research Institute
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Priority to AU44673/01A priority Critical patent/AU4467301A/en
Publication of WO2001073023A1 publication Critical patent/WO2001073023A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • 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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention relates to a novel G protein-coupled receptor and its gene, and their production and use.
  • G protein in-coupled receptors are a general term for a group of cell membrane receptors that transmit signals into cells through activation of trimeric GTP-binding proteins.
  • G protein-coupled receptors are also called “seven-transmembrane receptors” because of their structural characteristics of having seven transmembrane regions in the molecule.
  • G protein-coupled receptors transmit information on various physiologically active substances from the cell membrane into cells through the activation of trimeric GTP-binding proteins and the resulting changes in intracellular second messengers.
  • Intracellular second messengers which are controlled by trimeric GTP-binding proteins, cAMP which via adenylyl two cyclase, although such Ca 2 + which via Fosufuoriba one peptidase C is well known, via trimeric GTP-binding protein
  • cAMP which via adenylyl two cyclase, although such Ca 2 + which via Fosufuoriba one peptidase C is well known, via trimeric GTP-binding protein
  • Substrates (ligands) for G protein-coupled receptors are very diverse, including proteinaceous hormones, chemokines, peptides, amines, lipid-derived substances, and proteins such as thrombin. .
  • the number of G protein-coupled receptors for which genes have been identified is less than 300 in humans excluding sensory organ receptors, but the number of G proteins for which ligands have been identified ⁇ ⁇ There are only about 140 types and the ligand is unknown. There are more than 100 types of "fan G protein-coupled receptors". However, it has been assumed that there are at least 400, and in some cases as many as 1000, G-protein coupled receptors in the actual human genome (Trends Pharmacol. Sci. (97) 18: 430). This means that the number of orphan G protein-coupled receptors of unknown function will explode with the rapid progress of genome analysis in the future.
  • orphan G protein-coupled receptors have received a great deal of attention as targets that are likely to lead to the development of new drugs.
  • drugs targeting orphan G protein-coupled receptors by combining an extensive compound library and high-throughput screening (Trends Pharmacol. Sci. (97) 18 : 430, Br. J. Pharm. (98) 125: 1387).
  • the orphan G protein-coupled receptor identified by genetic manipulation is subjected to physiological agonism by functional screening using changes in cAMP and Ca, which are intracellular second messengers. And perform in vivo functional analysis.
  • Histamine receptor One of the G protein-coupled receptors that has attracted attention is the histamine receptor. Histamine is present in mast cells in all peripheral tissues and is one of the major mediators of inflammation-immunity. Histamine also plays an important role in gastric acid secretion of the gastric mucosa. The distribution of Hismin in the brain is in neuronal and non-neuronal cells. Histamine-positive neuronal traits are restricted to the posterior lobe of the hypothalamus, but they are projected to almost the entire brain, including the spinal cord and cerebral cortex. Histamine is thought to be involved in central nervous functions such as arousal response, sexual behavior, and analgesia. It is thought that the physiological activities of these Hismin-mins are expressed through three types of specific receptors, which are called Hl, H2, and H3 (Pharmacol. Rev. (1997) 49: 253). .
  • the HI receptor (GenBank acc. No. D14436) is expressed in brain, airway muscle, gastrointestinal and digestive organs, micturition-genital tract, circulatory organ, adrenal medulla, other endothelial cells, lymphocytes, etc. In the study of HI receptors, blood vessels and smooth muscle are often targeted. Histamine causes muscle contraction in smooth muscle. It is thought that this reaction is accompanied by an increase in intracellular Ca concentration via inosito 11,4,5_triphosphate by hismin (Eur. J Pharmacol. (1987) 135: 69).
  • histamine causes 1) changes in vascular permeability as a result of endothelial cell contraction, 2) proscine cyclin biosynthesis, 3) generation of platelet activating factor (PAT), 4) Von Willebrand factor (VWF) )), And 5) Nitric Oxide (NO) synthesis.
  • PAT platelet activating factor
  • VWF Von Willebrand factor
  • NO Nitric Oxide
  • the function of the HI receptor is mediated by a G-protein belonging to the pertussis toxin-insensitive Gq / 11 family, via increasing intracellular inositol phosphate calcium concentration (Br J Pharmacol. (1994) 112: 847). .
  • H2 receptor (GenBank acc. No. AB023486) is frequently expressed in the basal ganglion, amygdala, cerebral cortex, etc. in the brain, but is also expressed at low concentrations in the cerebellum and hypothalamus ( J Neurochem. (1992) 59: 290). H2 receptors are also expressed in the stomach and heart. The role of the H2 receptor in the stomach plays an important role in gastric acid secretion, which has been clarified by studies using H2 receptor-specific anginists (Br. J Pharmacol. 1985) 86: 571).
  • the H3 receptor was originally present in histamine-containing neurons and was suggested to exist as a pre-synaptic receptor that controls histamine release (Nature (198 3) 302: 832). Since histamine-containing neurons project to the entire cerebral cortex in the mammalian brain, the H3 receptor was assumed to play an important role in brain function (Trends Pharmacol. Sci. (1998) 19: 177). . In addition, the H3 receptor not only plays a role in histamine release, but also controls the release of various neurotransmitters such as acetylcholine, dopamine, gamma-aminobutyric acid, glutamic acid, noradrenaline, and serotonin at pre-synapse.
  • various neurotransmitters such as acetylcholine, dopamine, gamma-aminobutyric acid, glutamic acid, noradrenaline, and serotonin at pre-synapse.
  • H3 receptor The gene for the H3 receptor (GenBank acc.No.) 007232) was identified in 1999 (Mol. Pharmacol. (1999) 55: 1101). This receptor has only about 20% homology to the HI and H2 receptors in the entire molecule, but 27% and 33% homology in the transmembrane domain. (Trends Pharmacol. Sci. (2000) 21:11). Experimental properties of the H3 receptor suggest the existence of two receptor subtypes, H3a and H3b (Mol. Pharmacol. (1990) 38: 610). Furthermore, it has been reported that eosinophils have receptors with different properties from the above three types of histamine receptors and act on H3 receptor-specific agonists (Am. J Respir. Crit. Care Med. (1994) 14 9: 1506).
  • histamine receptor among G protein-coupled receptors is considered to be an important target for drug discovery research in combination with the wide range of physiological functions of ligand itself. Disclosure of the invention
  • the present invention has been made in view of the current situation surrounding such G protein-coupled receptors, and has as its object a novel G protein-coupled receptor, particularly a histamine receptor, and its gene, and production thereof. It is to provide a method and a use. Furthermore, the purpose is to provide these molecules as targets for drug development research.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by carrying out the polymerase chain reaction using human tissue cDNA as a ⁇ type, the characteristic of G protein-coupled receptor We succeeded in isolating a new gene that retains a hydrophobic region considered to be a transmembrane domain.
  • a search was made for a ligand for a protein encoded by the isolated gene using the change in intracellular calcium concentration as an index, it was found that histamine was a ligand for the protein.
  • These genes and their translation products can be used for screening for new ligands and for screening of agonists and gonists useful as pharmaceuticals.
  • the present invention relates to novel G protein-coupled receptors and their genes, and their production and use, and more specifically, (1) the DNA according to any one of the following (a) to (d), which encodes a guanosine triphosphate binding protein-coupled receptor;
  • a method for screening a compound having an activity of inhibiting the binding of the protein according to (5) to a ligand thereof (a) contacting a ligand with the protein or its partial peptide according to (5) in the presence of a test sample, and detecting the binding activity between the protein or its partial peptide and a ligand;
  • step (b) selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity in the absence of the test sample;
  • step (c) selecting a compound that suppresses or enhances the change in the cells detected in step (b), as compared to the change in the cells in the absence of the test sample,
  • a pharmaceutical composition comprising the compound according to (13) as an active ingredient, and
  • nucleotide having a chain length of at least 15 nucleotides which is complementary to DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary strand thereof,
  • the “G protein-coupled receptor” refers to a cell membrane receptor that transmits a signal into a cell through activation of a GTP-binding protein.
  • ligand refers to a physiological substance that binds to a G protein-coupled receptor and transmits a signal into a cell.
  • physiological substance refers to a compound that binds to a G protein-coupled receptor in a living body.
  • agonist refers to a compound capable of transmitting a signal into cells by binding to a G protein-coupled receptor, and includes physiological substances, artificially synthesized compounds, and naturally occurring compounds. Including.
  • angigonist refers to a compound that inhibits the binding of a ligand to a G protein-coupled receptor or the transmission of a signal into a cell, and is a physiological substance or an artificially synthesized substance.
  • the present invention provides a novel G protein-coupled receptor and a DNA encoding the protein.
  • the human-derived cDNA clone included in the present invention and isolated by the present inventors was named "GPRv53".
  • the nucleotide sequence of this cDNA is shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 1.
  • the protein encoded by GPRv53 cDNA showed significant amino acid sequence homology with a known G protein-coupled receptor.
  • “GPRv53” showed 31% homology to MUSCARINIC ACETYLCHOLINE RECEPTOR M3 (P49578, 639aa).
  • the protein encoded by the GPRv53 cDNA isolated by the present inventors retained a hydrophobic region, which is considered to be seven transmembrane domains characteristic of a G protein-coupled receptor. From these facts, GPRv53 cDNA was considered to encode a protein belonging to the G protein-coupled receptor family. Furthermore, when histamine was allowed to act on the GPRv53 protein expressed on the surface of HEK293 cells, a change in the intracellular calcium concentration was observed. From this, GPRv53 cDNA is a G protein-coupled receptor It turned out that it encodes the hissamine receptor in the family.
  • the GPRv53 protein of the present invention is an important target for drug discovery, because the Hisamine receptor has a wide range of physiological functions, depending on the properties of his ligand His, as described above. Becomes For example, as will be described later, it is possible to screen for the agonist and gonist of the GPRv53 protein, which is a drug candidate, using the change in intracellular calcium ion (Ca 2+ ) concentration due to its activation as an index. . In addition, the present inventors have found that clobenpropit acts not only on the histamine H3 receptor but also on the GPRv53 protein.
  • the GPRv53 protein is useful in the development of a drug specific to the Hismin H3 receptor.
  • the present invention also provides a protein functionally equivalent to the GPRv53 protein.
  • “functionally equivalent” means that the target protein has the same biological properties as the GPRv53 protein.
  • Biological properties of the GPRv53 protein include an activity of transmitting a signal into a cell through activation of a trimeric GTP-binding protein. The activity includes, for example, an activity of changing intracellular calcium concentration or cAMP concentration by activation thereof in response to histamine stimulation. Whether or not the target protein has such an activity is determined by applying histamine to cells expressing the target protein on its surface, and then changing the intracellular calcium concentration or cAMP concentration. It can be evaluated by detection (see Example 4).
  • One embodiment of a method for preparing a protein functionally equivalent to the GPRv53 protein includes a method of introducing a mutation into an amino acid sequence in a protein.
  • Such methods include, for example, site-directed mutagenesis (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 8. 1-8.5))) is included.
  • Amino acid mutations in proteins may also occur in nature.
  • one or more amino acids may be substituted, deleted, inserted and / or substituted in the amino acid sequence (SEQ ID NO: 1) of the GPRv53 protein, whether artificial or natural. Includes proteins mutated by addition or the like and functionally equivalent to the GPRv53 protein.
  • the number and location of the amino acid mutations in these proteins are not limited as long as the function of the GPRv53 protein is maintained.
  • the number of mutations will typically be within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids.
  • Another embodiment of a method for preparing a protein functionally equivalent to the GPRv53 protein includes a method using a hybridization technique and a method using a gene amplification technique. That is, if a person skilled in the art uses hybridization technology (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4), GPRv53 DNA sequence encoding the protein
  • a DNA highly homologous thereto is isolated from a DNA sample of the same or different species, and a protein functionally equivalent to the GPRv53 protein is isolated from the DNA. What you get is what you can usually do.
  • a protein functionally equivalent to a GPRv53 protein which is a protein encoded by a DNA that hybridizes with a DNA encoding the GPRv53 protein, is also included in the protein of the present invention.
  • Organisms for isolating such proteins include, but are not limited to, rats, mice, egrets, chicks, birds, birds, and the like, in addition to humans.
  • Stringent hybridization conditions for isolating DNA that encodes a protein functionally equivalent to the GPRv53 protein usually include conditions of ⁇ lxSSC, 0.13 ⁇ 4SDS, 37 ° C ''. Yes, more severe conditions are about 0.5xSSC, 0.13 ⁇ 4SDS, 42 ° C, and more severe conditions are 0.2xSSC, 0.13 ⁇ 4SDS, 6 5 ° C ”. Thus, the isolation of DNA having higher homology to the probe sequence can be expected as the hybridization conditions become more severe.
  • the combination of the SSC SDS and the temperature conditions described above is only an example, and those skilled in the art will recognize the above or other factors that determine the stringency of the hybridization (eg, probe concentration, probe length, The same stringency as described above can be achieved by appropriately combining the hybridization reaction times.
  • a protein encoded by DNA isolated using such a hybridization technique usually has a high homology in amino acid sequence with the GPRv53 protein.
  • High homology refers to sequence homology of at least 40% or more, preferably 60% or more, more preferably 80% or more (eg, 90% or more and 95% or more).
  • PCR Gene sequence amplification technology
  • the present invention also includes a partial peptide of the protein of the present invention.
  • This partial peptide includes a peptide which binds to hismin but does not transmit signals (for example, does not cause a change in intracellular calcium ion concentration).
  • a peptide can be a competitive inhibitor of the protein of the present invention.
  • the partial peptide of the protein of the present invention can also be used for producing antibodies.
  • the partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase.
  • the partial peptide of the present invention usually has at least 8 amino acid residues, preferably at least 12 amino acid residues (for example, at least 15 amino acid residues).
  • the protein of the present invention can be prepared as a recombinant protein or as a natural protein.
  • the recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention is inserted into an appropriate host cell as described below, and purifying the protein expressed in the transformant.
  • a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987)). Publish. John Wiley & Sons Section 16. 16.19).
  • the antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody.
  • the present invention also provides a DNA encoding the protein of the present invention.
  • the form of the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, etc. in addition to cMA.
  • the present invention DNAs having an arbitrary base sequence based on the degeneracy of the genetic code are included as long as they can encode the above protein.
  • the DNA of the present invention encodes a GPRv53 protein as described above! ) NA sequence (SEQ ID NO: 2) or a part thereof can be isolated by a conventional method such as a hybridization method using a probe or a PCR method using a primer synthesized based on these DNA sequences. .
  • the present invention also provides a vector into which the DNA of the present invention has been inserted.
  • the vector of the present invention is not particularly limited as long as it can stably maintain the inserted DNA.
  • a vector for cloning is a pBluescript vector (manufactured by Stratagene). Is preferred.
  • an expression vector is particularly useful.
  • a vector that expresses a protein in a test tube, in a bacterial cell (for example, Escherichia coli), in a cultured cell, or in an individual organism can be used.
  • pBEST for expression in a test tube Vector (Promega), E. coli for pET vector (In vitrogen), cultured cells for pME18S-FL3 vector (GenBank Accession No. AB009864), for living organisms, pME18S vector (Mol Cell l) Biol. 8: 466-472 (1988)).
  • Insertion of the DNA of the present invention into a vector can be carried out in a conventional manner, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley
  • the present invention also provides a transformant carrying the DNA of the present invention or the vector of the present invention.
  • the host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells may be used depending on the purpose. Examples of eukaryotic cells for highly expressing a protein include COS cells and CH0 cells. Vectors can be introduced into host cells by, for example, calcium phosphate precipitation, pulsed electroporation, etc.
  • the present invention provides a nucleotide having a chain length of at least 15 nucleotides, which is complementary to DNA encoding the protein of the present invention (DNA comprising the base sequence of SEQ ID NO: 2 or a complementary strand thereof).
  • DNA comprising the base sequence of SEQ ID NO: 2 or a complementary strand thereof.
  • complementary strand refers to one strand of a single-stranded nucleic acid composed of A: T (but U for RNA) and G: C base pairs with respect to the other strand.
  • the term "complementary" is not limited to a case where the sequence is completely complementary in at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably It suffices to have 95% or more homology on the base sequence.
  • the algorithm for determining homology may use the algorithm described in this specification.
  • Such nucleotides can be used as a probe for detecting and isolating the MA of the present invention, and as a primer for amplifying the DNA of the present invention. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp.
  • nucleotide having a chain length of at least 15 bp containing at least a part or the entire sequence of MA of the present invention is used.
  • Such nucleotides preferably hybridize specifically to DNA encoding the protein of the present invention.
  • “Specifically hybridize” refers to a DNA that hybridizes with a DNA encoding the protein of the present invention (SEQ ID NO: 2) under ordinary hybridization conditions, preferably under stringent conditions, and DNA encoding a protein means not hybridized.
  • nucleotides can be used for testing and diagnosing abnormalities of the protein of the present invention.
  • abnormal expression of the DNA encoding the protein of the present invention can be examined by Northern hybridization or RT-PCR using these nucleotides as probes or primers.
  • DNA encoding the protein of the present invention is obtained by polymerase chain reaction (PCR) using these nucleotides as primers.
  • PCR polymerase chain reaction
  • its expression control region can be amplified and DNA sequence abnormalities can be examined and diagnosed by methods such as RFLP analysis, SSCP, and sequencing.
  • these nucleotides include antisense DNA for suppressing the expression of the protein of the present invention.
  • the antisense DNA has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and usually has a chain length of 3000 bp or less, preferably 2000 bp or less in order to cause an antisense effect.
  • Such antisense DNA may also be applied to gene therapy for diseases caused by abnormalities (abnormal function or abnormal expression) of the protein of the present invention.
  • the antisense DNA is prepared, for example, by the phosphorothioate method (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucieotides. Nucleic Acids Res.) Based on the sequence information of the DNA encoding the protein of the present invention (eg, SEQ ID NO: 2). 16, 3209-21 (1988)).
  • the nucleotides of the present invention can be used, for example, by using exvl V, a viral vector such as a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector, or a non-viral vector such as a ribosome. It may be possible to administer to patients by the 0 method or the in vivo method.
  • the present invention also provides an antibody that binds to the protein of the present invention.
  • the form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. Also, all classes of antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.
  • the antibody of the present invention can be obtained by synthesizing an oligonucleotide corresponding to the amino acid sequence of the protein of the present invention according to a conventional method and immunizing a rabbit (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11. 12-11. 13).
  • a monoclonal antibody a mouse is immunized with a protein expressed and purified in Escherichia coli according to a conventional method, and a hybridoma cell obtained by fusing the spleen cell and myeloma cell of the mouse. A cell can be prepared and obtained from the hybridoma cell (Current protocols in Molecular Biology edit. Ausube 1 et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
  • Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of the protein of the present invention, in addition to purification of the protein of the present invention.
  • proteins are extracted from tissues, blood, or cells, and abnormalities in expression and structure are detected through detection of the proteins of the present invention by methods such as Western blotting, immunoprecipitation, and ELISA. Inspection / presence can be diagnosed.
  • an antibody that binds to the protein of the present invention for the purpose of treating a disease associated with the protein of the present invention.
  • the antibody of the present invention can act as an agonist of the protein of the present invention.
  • a human antibody or a humanized antibody is preferred because of its low immunogenicity.
  • Human antibodies include mice in which the immune system has been replaced with that of a human (e.g., "Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, endez, MJ et al. (1997) Nat. Genet. 15 : 146-156 ").
  • humanized antibodies can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).
  • the present invention also provides a method for screening for a new ligand that binds to the protein of the present invention, using the protein of the present invention. This screening method
  • test sample is not particularly limited.
  • known compounds and peptides whose ligand activities of various G protein-coupled receptors are unknown eg, those registered in chemical files
  • phage display Biol. (1991) 222, 301-310) can be.
  • culture supernatants of microorganisms and natural components derived from plants and marine organisms are also targets for screening.
  • Other examples include, but are not limited to, biological tissue extracts including the brain, cell extracts, and expression products of gene libraries.
  • the protein of the present invention used for screening may be, for example, a form expressed on a cell surface, a form as a cell membrane fraction of the cell, or a form bound to an affinity column.
  • Specific screening techniques include, for example, a method of contacting a test sample with an affinity column for the protein of the present invention to purify a compound that binds to the protein of the present invention, and a number of known methods such as a West Western plotting method. Methods are available. When these methods are used, the test sample is appropriately labeled, and the binding to the protein of the present invention can be detected using the label. In addition to these methods, a cell membrane expressing the protein of the present invention is prepared and immobilized on a chip, and the separation of the trimeric GTP-binding protein upon ligand binding is determined by surface plasmon resonance.
  • the binding activity between the test sample and the protein of the present invention can be detected by using, as an index, a change in cells caused by binding of the test sample to the protein of the present invention expressed on the cell surface.
  • changes include, but are not limited to, changes in intracellular Ca 2+ levels and changes in cAMP levels.
  • agonist activity for G protein-coupled receptors can be measured by the GTP-S binding method.
  • the cell membrane expressing the protein of the present invention was labeled with 35 S in a solution of 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl 2 , 50 / zM GDP. Mix with GTP yS 400 pM and incubate in the presence and absence of the test sample. After the filtration, filtration can be performed to compare the radioactivity of the bound GTPas.
  • the G protein of the present invention shares a system for transmitting a signal into a cell through activation of a trimeric GTP-binding protein.
  • Trimeric GTP-binding proteins are classified into three types, depending on the type of intracellular signaling system that activates, Gq type that increases Ca 2+ , Gs type that increases cAMP, and Gi type that suppresses cAMP. You. Applying this fact, Gq protein subunits are chimerized with other G protein subunits, and a positive signal during ligand screening results in an increase in Ca2 + , a Gq intracellular transduction pathway. Is possible.
  • the elevated Ca 2+ level can be detected using the repo overnight gene system having TRE (TP A response element) upstream, a staining indicator such as Fluor-3, and a change in the fluorescent protein aequorin as indicators.
  • TRE TP A response element
  • Gs protein subunits are chimerized with other G protein subunits, and a positive signal results in an increase in cAMP, a Gs intracellular transduction pathway, and a CRE (cAMP-responsive element) is upstream. It is also possible to use the change in the reporter gene system in
  • host cells that express the protein of the present invention in this screening system there are no particular restrictions on the host cells that express the protein of the present invention in this screening system, and various host cells may be used depending on the purpose.
  • examples include COS cells, CH0 cells, HEK293 cells, and the like. it can.
  • examples of the vector for expressing the protein of the present invention in vertebrate cells include a promoter located upstream of a gene encoding the protein of the present invention, an RNA splice site, a polyadenylation site, a transcription termination sequence, and an origin of replication. Those having the above can be preferably used.
  • pSV2dhfr Mol. Cell. Biol. (1981) 1, which has the SV40 early promoter
  • pEF-BOS Nucleic Acids Res. (1990) 18, 5322
  • pCDM8 Nature (1987) 329, 840-842
  • pCEP4 Invitrogen
  • G protein-coupled receptors Is a vector that is useful for expressing Insertion of the MA of the present invention into a vector can be carried out by a ligase reaction using a restriction enzyme site in a conventional manner (Curr Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.
  • Vector introduction into host cells can be performed, for example, by calcium phosphate precipitation, electropulse perforation (Current (1987) Publish. John Wiley & Sons. Section 9.1-9.9), ribofectamine method (GIBCO-BRL), FuGENE6 reagent (Behringer Mannheim), microinjection method. It can be performed by a known method such as
  • the present invention also provides a method for screening a compound having an activity of inhibiting the binding between the protein of the present invention and its ligand. This screening method
  • step (a) a step of contacting a ligand with the protein of the present invention or its partial peptide in the presence of a test sample and detecting the binding activity between the protein or its partial peptide and the ligand; (b) no test sample present Selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity below.
  • test sample is not particularly limited.
  • a compound group obtained by combinatorial chemistry—technology (Tetrahedron (1995) 51, 8135-8137), or a phage display method (J. Mol. Biol. (1991) 222, 301-310) can be used.
  • culture supernatants of microorganisms and natural components derived from plants and marine organisms are also targets for screening.
  • Other examples include, but are not limited to, brain and other biological tissue extracts, cell extracts, expression products of gene libraries, synthetic low molecular weight compounds, synthetic peptides, and natural compounds.
  • the protein of the present invention used for screening may be, for example, a form expressed on a cell surface, a form as a cell membrane fraction of the cell, or a form bound to an affinity column. Histamine can be used as the ligand.
  • a ligand is labeled with a radioisotope, and the ligand is contacted with the protein of the present invention in the presence of a test sample.
  • a method of detecting a compound that reduces the binding activity between the protein of the present invention and a ligand, based on a label attached to the ligand, as compared with the case where the compound is detected in the absence of a reagent, can be used.
  • screening can be performed using intracellular changes as an index: that is, the cells expressing the protein of the present invention can be screened.
  • the binding between the protein of the present invention and the ligand can be determined. It is possible to screen for compounds that inhibit.
  • Cells expressing the protein of the present invention can be prepared in the same manner as in the above-described screening for a ligand that binds to the protein of the present invention.
  • the compound isolated by this screening is a candidate for the agonist of the protein of the present invention.
  • the present invention also provides a method for screening a compound that inhibits or promotes the activity of the protein of the present invention.
  • This screening method comprises the steps of (a) contacting a cell expressing the protein of the present invention with a ligand of the protein in the presence of a test sample, and (b) a change in cells caused by binding of the ligand to the protein of the present invention. And (c) selecting a compound that suppresses or enhances the change in the cells detected in step (b) as compared to the change in the cells in the absence of the test sample.
  • a group of compounds obtained by combinatorial chemistry technology, a phage display method, etc. are applied in the same manner as in the above-described screening method of a compound that inhibits the binding of a protein to a ligand of the present invention.
  • a compound isolated by screening a compound that inhibits the binding between the protein of the present invention and a ligand can be used as a test sample. Use histamine as the ligand. Can be.
  • Cells expressing the protein of the present invention can be prepared in the same manner as in the above-described screening for a ligand that binds to the protein of the present invention. Changes in the cells after contact with the test sample can be detected using changes in intracellular Ca 2+ levels and cAP levels as indices, as in the screening method described above. When detecting intracellular signal transduction, it is also possible to detect using a measurement system such as a repo overnight assay system using luciferase or the like as a reporter gene.
  • a measurement system such as a repo overnight assay system using luciferase or the like as a reporter gene.
  • the sample is determined to be a compound that inhibits the activity of the protein of the present invention.
  • the test sample enhances the change in the cells, the compound can be determined to be a compound that promotes the activity of the protein of the present invention.
  • “promoting or inhibiting the activity of the protein of the present invention” means that the action of the protein of the present invention may be either direct or indirect. Indicates that the activity of the protein is promoted or inhibited.
  • compounds isolated by this screening include compounds that act on the protein or ligand of the present invention to inhibit or promote their binding and thereby inhibit or promote the activity of the protein of the present invention.
  • compounds that do not inhibit or promote these bindings per se but result in inhibiting or promoting the activity of the protein of the present invention are also included.
  • Such compounds include, for example, compounds that do not inhibit the binding of the protein of the present invention to the ligand, but inhibit or promote intracellular signaling pathways.
  • the present invention also relates to clozapine (8-clomouth-l- (4-methyl-1-piperazinyl) -5H-diben [b, e] [l, 4] -dazepine) or clobenpropit ([(4-clo
  • the present invention provides an agent for activating the protein of the present invention, which comprises, as an active ingredient, phenyl) methyl] _3- (13--imidazole-4-yl) propyl ester carbamide thionate.
  • the present inventors have found that clozapine, which has been reported to have affinity for the histamine H3 receptor, also has agonist activity for GPRv53.
  • drug includes both reagents used for test and research purposes and drugs used for the purpose of preventing or treating diseases.
  • a compound isolated by the screening method of the present invention or clozapine or clobenpropit is used as a drug
  • the isolated compound itself is directly administered to a patient, and a drug formulated by a known pharmaceutical method is used.
  • Administration can also be carried out as a composition.
  • a suitable combination specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspension and the like.
  • Administration to a patient can be generally performed by a method known to those skilled in the art such as, for example, intraarterial injection, intravenous injection, and subcutaneous injection.
  • the dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the DNA may be incorporated into a vector for gene therapy to perform gene therapy.
  • a viral vector such as a retrovirus vector or an adenovirus vector and a non-viral vector such as a ribosome can be used.
  • the vector can be administered to a living body by an ex vivo method or an in vivo method.
  • FIG. 1 is a view showing the results of performing a BLAST search on the entire sequence of SWISS-PR0T using “GPRv53” amino acid sequence as ⁇ Query j.
  • MUSCARINIC ACETYLCHOLINE RECEPTOR M3 (P49578) showed the highest homology at 31%.
  • FIG. 2 is a photograph showing the result of analyzing the expression distribution of the GPRv53 gene.
  • a DNA fragment of about 0.6 kbp was amplified in the thymus, small intestine, peripheral leukocytes, spleen, and large intestine.
  • FIG. 3 is a diagram showing the results of measuring changes in intracellular Ca 2+ concentration over time using FLIPR (Molecular Device). Fluorescence intensity was measured when HISTAMINES (-)-Hy-METHYL-, DIHYDR0CHL0iUDE (RaMeHA) (SIGMA), which is a specific histamine and H3 receptor agonist, was added.
  • the maximum value of the fluorescence intensity of the results obtained in the Y-axis is plotted ligand concentration in the X-axis, GPRv53 at a concentration of 10- 6 -10- 9 M against His evening Min, for R shed MeHA 10- 5 - concentration in dose-dependent changes in intracellular Ca z + concentration of 10 8 M was observed.
  • FIG. 4 is a diagram showing the results of changes in intracellular Ca 2+ concentration measured over time using FLIPR.
  • the fluorescence intensity was measured when clozapine, a neuroleptic drug, and clobenpropit (SIGMA), a specific antagonist of the H3 receptor, were added.
  • SIGMA clobenpropit
  • specific changes in the intracellular Ca 2+ concentration of GPRv53-expressing cells were observed by treatment with both drugs.
  • Example 1 Isolation of gene encoding novel G protein-coupled receptor GPRv53
  • Marathon Ready cDNA (Clontech) derived from human fetus was forwarded to ⁇ -type cDNA.
  • 5 -GAATTGTC TGGCTGGATTAATTTGCTAATTTG-3 '(SEQ ID NO: 3) was used as a primer
  • 5'-TTAAGAAGATACTGACCGACTGTGTTGT-3' (SEQ ID NO: 4) was used as a reverse primer.
  • PCR was performed using TaKaRa La Taq (Takara Shuzo) at 94 ° C (30 minutes) / 55 after 94 ° C (2.5 minutes).
  • This sequence has an open reading frame of 1173 bases (SEQ ID NO: 2).
  • the amino acid sequence (390 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 1. Since the predicted amino acid sequence has seven transmembrane domains that are likely to be characteristic of G protein-coupled receptors, the gene may encode a G protein-coupled receptor. found.
  • Example 2 BLAST search for SWISS-PR 0T in the amino acid sequence of GPRv53, a novel G protein-coupled receptor
  • GPRv53 is not the same among known G protein-coupled receptors, and showed the highest homology at 31% with MUSCARINIC ACETYLCHOLINE RECEPTOR M3 (P49578, 639a a). This proved that "GPRv53” was a novel G protein-coupled receptor.
  • the expression distribution of the GPRv53 gene was analyzed by PCR using Multiple Tissue cDNA Panels (Clontech). 5,-GMTTGTCTGGCTGGA TTAATTTGCTAATTTG-3 '(SEQ ID NO: 3) as the forward primer and 5,-MGMTG ATGTGATGGCAAGGATGTACC-3' (SEQ ID NO: 5) as the reverse primer, and PCR using TaKaRa La Taq (Takara Shuzo) After 94 ° C. (2.5 minutes), a cycle of 94 ° C. (30 seconds) / 55 ° C. (30 seconds) / 72 ° C. (30 seconds) was repeated 40 times.
  • a DNA fragment of about 0.6 kbp was amplified with cDNA derived from thymus, small intestine, peripheral leukocytes, spleen, and large intestine, and cDNA derived from other organs (lung, prostate, brain, heart, placenta, ovary, testis, Kidney, skeletal muscle, kidney, W
  • the following experiment confirmed the histamine receptor activity of the protein encoded by GPRv53.
  • the cDNA was obtained by PCR and incorporated into an expression vector.
  • clozapine has affinity for the histamine H3 receptor” (Rodrigues, A ⁇ , Jansen, FP, Leurs, R., Timmerman , H., and Prell, GD Br. J. Pharmacol. (1995) 114,
  • histamine H3 receptor was suggested as an "autoreceptor" in the central presynapse that inhibits the synthesis of histamine.
  • clobenpropit a histamine receptor-selective synthetic agonist, functioned as an agonist in GPRv53. Therefore, in creating a histamine-selective drug, it is useful to examine the effect of a candidate compound of the drug on GPRv53.
  • a histamine H3-selective drug candidate compound group is allowed to act on GPRv53-expressing cells or a cell membrane fraction expressing GPRv53, and the activation of GPRv53-expressing cells is detected, and then GPRv53 is selected from the candidate compound group.
  • Activity of expressing cells By removing the compound that induces the oxidization (this compound is also considered to bind to histamine H3), negative selection of a compound selective for histamine H3 can be performed.
  • the present invention provides a novel histamine receptor, a gene encoding the protein, a vector containing the gene, a host cell containing the vector, and a method for producing the protein. Furthermore, a method for screening a compound that modifies the activity of the protein was provided.
  • the protein of the present invention, its gene, or a compound that modifies the activity of the protein of the present invention is expected to be used for the development of new preventive or therapeutic agents for diseases involving the histamine receptor of the present invention.

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Abstract

L'invention concerne un nouveau gène soutenant des domaines hydrophobes se présentant apparemment sous forme de sept domaines transmembranaires caractéristiques des récepteurs couplés à la protéine G. On a réussi à isoler ce gène par criblage d'ADN complémentaires de tissu humain. Une protéine codée par cet ADN complémentaire est un récepteur d'histamine présentant la capacité de modifier la concentration de calcium intracellulaire en réponse à un stimulus avec de l'histamine. Ce gène et cette protéine, qui est son produit de traduction, peuvent être utilisés dans le criblage d'un nouveau ligand et d'un agoniste ou d'un antagoniste utiles comme médicament.
PCT/JP2001/002767 2000-03-31 2001-03-30 Nouveau recepteur gprv53 couple a une proteine se liant a la guanosine-triphosphate, gene correspondant et procede de production et d'utilisation associe WO2001073023A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999033978A1 (fr) * 1997-12-26 1999-07-08 Banyu Pharmaceutical Co., Ltd. Nouvelles proteines receptrices du type conjugue guanosine triphosphate (gtp)-proteine de liaison
WO2000022131A2 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive
WO2000031258A2 (fr) * 1998-11-20 2000-06-02 Arena Pharmaceuticals, Inc. Recepteurs humains couples a la proteine g orphan

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999033978A1 (fr) * 1997-12-26 1999-07-08 Banyu Pharmaceutical Co., Ltd. Nouvelles proteines receptrices du type conjugue guanosine triphosphate (gtp)-proteine de liaison
WO2000022131A2 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive
WO2000031258A2 (fr) * 1998-11-20 2000-06-02 Arena Pharmaceuticals, Inc. Recepteurs humains couples a la proteine g orphan

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LEURS R. ET AL.: "H3 receptor gene is cloned at last", TRENDS PHARMACOL. SCI., vol. 21, no. 1, January 2000 (2000-01-01), pages 11 - 12, XP002942019 *
LOVENBERG T.W. ET AL.: "Cloning and functional expression of the human histamine H3 receptor", MOLECULAR PHARMACOLOGY, vol. 55, 1999, pages 1101 - 1107, XP002942017 *
TAMAKI ODA ET AL.: "Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 47, 2000, pages 36781 - 36786, XP002942018 *

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