WO2002059295A1 - Nouvelle serine protease, la mp493 - Google Patents

Nouvelle serine protease, la mp493 Download PDF

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Publication number
WO2002059295A1
WO2002059295A1 PCT/JP2002/000465 JP0200465W WO02059295A1 WO 2002059295 A1 WO2002059295 A1 WO 2002059295A1 JP 0200465 W JP0200465 W JP 0200465W WO 02059295 A1 WO02059295 A1 WO 02059295A1
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coordinate
protein
dna
activity
amino acid
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PCT/JP2002/000465
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English (en)
Japanese (ja)
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Yusuke Nakamura
Sumio Sugano
Tomokazu Matsusue
Atsushi Okamoto
Kazufumi Okawa
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Mochida Pharmaceutical Co., Ltd.
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Publication of WO2002059295A1 publication Critical patent/WO2002059295A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to a novel transmembrane type relating to diseases such as various cancers, tumors, various lung diseases, asthma, allergy, bronchitis, emphysema, viral diseases, shock, multiple organ failure, Tengitis, various nephritis, etc.
  • An antisense nucleic acid that suppresses the expression of the serine protease; and screening of a diagnostic agent, an inhibitor, a pharmaceutical, etc., using the serine protease or a DNA encoding the same.
  • the method relates to the spatial coordinates of the serine protease domain of MP493 and its use.
  • Prior Art 'Serine proteases are a group of proteases with Ser residues essential for activity at the catalytic site.
  • the Ser residue in this active site is highly nucleophilic and activated. This nucleophilicity is Asp- It is maintained by three kinds of catalytic groups consisting of His-Ser.
  • Serine proteases are broadly classified into four types. That is, they are chymoto's ribosin type, spurtlysin type, serine carboxypeptidase type and site megalovirus protease type. Chymotrypsin, trypsin, and elastase have high homology in the amino acid sequence. However, their functions are different due to differences in the structure of substrate-binding pockets such as specificity pockets (S1 pockets). Although the primary and tertiary structures of the sptylysine form and the serine carboxy form are different, the arrangement of the active three residues is the same. On the other hand, the cytomegalovirus protease type has a similar arrangement of three active residues, but the Asp residue is replaced by the His residue.
  • Serine proteases are involved in a variety of cellular processes, from proprotein processing to intracellular degradation. In higher animals, it is involved in a large number of biological reactions such as food digestion, blood coagulation and fibrinolysis, complement activation, hormone production, ovulation and fertilization, phagocytosis, cell proliferation, development, differentiation, aging, and cancer metastasis. (Neurath, H. Science., Vol. 224, pp. 350-357, (1984)). Therefore, many researchers have been paying attention to this, but this is This is because the theatase is deeply involved in the pathogenesis and progression of various malignant diseases, including cancer and allergies.
  • proteases which are expressed in cancer cells and play important physiological functions, exist mainly in the group of proteases.
  • plasmin thrombin is a matrix phenomenon. It activates the oral protease group and promotes the secretion of murine macrophage elastase, and the PAR-I activation peptide activates murine macrophage elastase. It is known to induce the release of zeolites.
  • MMP 9 is activated by trypsin (Duncan ME et al. 'Eur J Biochem Vol. 258, pp. 37-43, (1998)).
  • TMPRS S2 serine protease that is specifically expressed in the prostate has been selected and cloned using the cDNA microarray technology in prostate cancer cells.
  • This serine protease named TMPRS S2
  • TMPRS S2 has been implicated in cancer invasion through the degradation of extracellular matrix ⁇ basement membrane components and activation of type IV collagenase.
  • serine proteases which have been May be involved in canceration by a mechanism that can be controlled
  • hepsin a protein discovered in 1998, has been used in liver and hepatocellular carcinoma. High expression has been reported. Knockout mice lacking this hepsin-encoding gene have also been produced, and are noted in blood coagulation due to the presence of factor VIII upregulation. Due to the rise of the medium repel, it has been suggested that hepsin may be one of the most effective tumors.
  • a ligand for protease-activated receptor-2 which enhances fibroblast proliferation and collagen production in vitro, and thus is a new source of asthma and pulmonary fibrosis. It is attracting attention as a therapeutic drug.
  • trypsin-zeclara expressed in the lung H. Kido et al., J. Biol. Chem., Vol. 267, p. 13573-13579 (1992); M. Tashiro et al., J. Virol., Vol. 66, pp. 7211-7216 (1992)
  • Serine proteases expressed in the viscera include the digestive enzyme trypsin. Trypsin is known to be derived from the gut of various animals, such as porcupine, pusin, human, and egret, and is used as the name of an enzyme that has a proteolytic activity contained in Teng's solution. It is known to be an enzyme of the endopeptidase group that is produced as inactive trypsinogen in acinous cells of the kidney. Tribosine is autocatalytically activated from trypsinogen by enterokinase, mold kinase, and cathepsin B to form tribosine.
  • the purified enzyme has been used in clinical practice because of its blood coagulation, lowering blood pressure, and anti-inflammatory effects. On the other hand, it is also known as a target for diseases such as inflammation. Development of enzymes with trypsin-like substrate specificity, development of trypsin-like proteases, and serine proteases that have trypsin-like activity and are targets for diseases such as inflammation There is an eager need for isolation.
  • Nephritis is a glomerular inflammatory disease of the kidney (spleen), and is minimally altered. Intraproliferative nephritis, crescentic nephritis, glomerulosclerosis, mesangial proliferative nephritis, membranous nephropathy, Membranous proliferative nephritis, end-stage sclerosing nephritis,
  • nephritis A nephritis is known. For treatment of nephritis, adrenal cortical hormone (steroids), immunosuppressants, anticoagulants, antiplatelets, diuretics, etc. are used. In recent years, cases where glomerulonephritis patients lack Factor I, a membrane-bound serine protease, have been reported. Factor I is an enzyme that inactivates C4b / C3b in the complement system, and its association has been noted (Sadallah S et al. 'Am J Kidney Dis., Vol. 33, pp. 1153.7, (1999)).
  • trombin a type of trypsin-type serine protease, regulates hexokinase activity and is associated with mesangial cells. It has also been reported to be involved in renal impairment (Robey RB et al. Kidney Int., Vol. 57, pp. 2308-18 (2000)).
  • endogenous inhibitor Megsin an inhibitor of serine protease, is expressed in a wide variety of nephritis, including IgA nephritis. It has been speculated that certain types of serine protease may also contribute to senility disorders.
  • the present invention relates to a novel gene isolated from a human gastric signet-ring cell carcinoma cell line.
  • the present invention provides a host cell transformed with the gene, and a method for producing recombinant MP493 using the transformed cell.
  • the present invention provides a method for evaluating a protease activity regulator for MP493, using a spatially coordinated partial structure model of MP493.
  • the present invention provides a gene, mp493, which encodes MP493 (described in detail below).
  • the gene can be isolated and identified from gastric signet ring carcinoma cells, but based on the sequence disclosed herein, a general engineering technique such as hybridization was used. It may be DNA prepared by a chemical synthesis method such as the cloning-phosphoramidite method. Examples of the form include cDNA, genomic DNA, and chemically synthesized DNA, but are not particularly limited. Further, the DNA of the present invention may be single-stranded or may form a double-stranded or triple-stranded chain by binding to DNA or RNA having a sequence complementary thereto. In addition, the DNA is used for enzymes such as horseradish peroxidase (HRPO), radioisotopes, fluorescent substances, and chemical sources. It may be labeled with a light substance or the like.
  • HRPO horseradish peroxidase
  • radioisotopes radioisotopes
  • fluorescent substances and chemical sources. It
  • the base sequence of mp493 is provided, the sequence of the RNA derived therefrom and the sequence of the complementary DNA and RNA are uniquely determined, so that the present invention corresponds to the DNA of the present invention. It should be understood that DNA or RNA having a sequence complementary to the RNA or DNA of the present invention is also provided.
  • the DNA of the present invention also includes a DNA that hybridizes with a DNA consisting of the base sequence of SEQ ID NO: 1 or 4 under stringent conditions.
  • the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 4 hybridizes with the DNA under stringent conditions, and the protein encoded by the DNA is a serine protein. Variation of the nucleotide sequence is allowed as long as the enzyme activity is maintained. For example, the presence of multiple codons that code for the same amino acid residue due to so-called codon degeneracy, or the use of various artificial treatments, such as site-directed mutagenesis or treatment with a mutagen Mutation of DNA fragments due to mutation or restriction enzyme cleavage Even if the DNA sequence is partially changed due to ligation, etc., these DNA mutants may be replaced with the DNA described in SEQ ID NO: 1 or 4. High under stringent conditions As long as the DNA is a pre-sidized DNA encoding serine protease, it is within the scope of the present invention, regardless of the difference from the DNA sequence shown in SEQ ID NO: 1 or 4.
  • the extent of the above DNA mutation is acceptable as long as it has 70% or more, preferably 80% or more, more preferably 90% or more homology with the DNA sequence shown in SEQ ID NO: 1 or 4.
  • the degree of high pre-hybridization can be measured under normal conditions, for example, when the probe is lapel with the DIG DNA Labeling kit (Chain No. 1175033 manufactured by Lininger, Mannheim). 3 Neutralize in DIG Easy Hy solution (Behringer's Mannheim Cat No. 1603558) at 22 ° C, and then add 0.5 XSSC solution at 50 ° C.
  • the recombinant vector having the DNA of the present invention may be in any form, such as circular or linear. Such a recombinant vector may have another nucleotide sequence, if necessary, in addition to the DNA of the present invention. Good.
  • Other nucleotide sequences include the sequences of geno, nucleotide sequence, promoter sequence, ribosome binding sequence, nucleotide sequence used for amplification of copy number, and signal peptide. This refers to the nucleotide sequence to be transcribed, the nucleotide sequence encoding other polypeptides, the polyA-added sequence, the splicing sequence, the replication origin, the nucleotide sequence of the gene that plays a key role in selection, and the like.
  • a translation initiation codon and a translation termination codon are added to the DNA of the present invention using an appropriate synthetic DNA adapter, or an appropriate restriction enzyme cleavage sequence is added to the nucleotide sequence. It can be newly generated or eliminated. These are within the range of the work normally performed by those skilled in the art, and can be arbitrarily and easily processed based on the DNA of the present invention.
  • the vector carrying the DNA of the present invention may be used by selecting an appropriate vector according to the host to be used.
  • a vector such as a nacteriophage, a baculovirus, or a baculovirus.
  • viruses such as Troilei les and Vaccinia viruses, and there is no particular limitation.
  • the protein encoded by mp493 is the amino acid shown in SEQ ID NO: 2.
  • MP493 consisting of an array.
  • MP493 has catalytic residues, Asp304, Ser400, and His257, which are conserved in serine protease.
  • a serine protein that preferentially cleaves a substrate having a basic residue (L-Asp, L-Lys) at the P1 site of the substrate was analyzed. It turned out to be one. It also demonstrated that MP493, expressed and purified in recombinant cells, hydrolyzes the expected synthetic substrate and has the validity of the model structure and serine protease activity.
  • MP493 was found to have approximately 60% homology with TMPRSS2, Factor I, trypsin, hepsin, and ⁇ -trib-unicase in the primary structure. From the results of the motif analysis, ⁇ 493 showed an LDL receptor domain, a scavenger receptor domain and a transmembrane domain. Based on the above facts, it is determined that # 493 is a serine protease having a transmembrane structure.
  • MP 493 is thought to be involved in various lung diseases such as emphysema and pulmonary fibrosis./5 Tribuse, or a transmembrane cell that is thought to be involved in the development of glomerulonephritis. It has a structure similar to that of protein protease.
  • the gene mp493 in the living body was confirmed using cDNA libraries derived from various organs, the gene was expressed in the lung, lentil, kidney, and placenta. This was confirmed.
  • MP493 is closely involved in the development of these various diseases.
  • hydrolysis of the target protein by serine protease activity is considered to be significant in the development of these diseases, compounds that can specifically inhibit MP493 are useful as pharmaceuticals. Is expected to have.
  • the search for inhibitors of the protease activity using MP493 is expected to have important significance in drug development.
  • the protein has serine protease activity, one or more amino acids are substituted in the amino acid sequence of the protein shown in SEQ ID NO: 2; Polypeptides or proteins consisting of amino acid sequences that have been deleted and / or added are also within the scope of the present invention.
  • the protein consisting of the amino acid sequence shown in SEQ ID NO: 5 is the serine protease domain of MP493, and has serine protease activity.
  • the protein comprising the amino acid sequence shown in SEQ ID NO: 5 can be easily produced by genetic engineering as compared with MP493, and is suitable for use in screening for an activity regulating substance of the protein. .
  • Amino acid residue side chains which are constituents of proteins, differ in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein
  • several highly conservative partners are known. For example, for substitution of aminoic acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Al a) or norin (Val), leucine (Le u) And isoleucine (lie), glutamic acid (Glu) and glutamine (Gin), ascraginate (Asp) and asparagine (Asn), cystine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala; lysine (Lys) and arginine (Arg);
  • A1a Val, Leu, Ile, Pro, methionine (Met), phenylalanine (Phe), tripfan (Trp), Gly, C Since both ys are classified
  • Examples of uncharged electrode-type amino acids include Ser, Thr, tyrosine (Tyr), Asn, and Gin.
  • Examples of acidic amino acids include Asp and G1u.
  • Examples of the basic amino acid include Lys, Arg, and histidine (His).
  • substitutions on the amino acid sequence shown in SEQ ID NO: 2 or 5 Even if the protein is a mutant protein due to insertion, deletion, or the like, if the mutation is a protein having serine protease activity, which is an essential function of the present invention, these are within the scope of the present invention.
  • the protein specified by the spatial coordinates in FIG. 9 also has serine protease activity as MP493, and is therefore within the scope of the present invention. .
  • the substitution, deletion, and / or addition of amino acids is allowed for the protein specified by the spatial coordinates in Fig. 9 as long as it retains serine protease activity. It is.
  • Such modification of amino acid is found in nature, such as a mutation caused by a genetic polymorphism, etc., as well as by methods known to those skilled in the art, for example, mutagenesis using a mutagenic agent such as NTG. It can be performed artificially by using an induction method or a site-specific mutagenesis method using various recombinant gene techniques.
  • the amino acid mutation site and number are not particularly limited as long as the mutant protein retains serine protease activity, but the number of mutations is usually within several tens of amino acids, and preferably 10 amino acids. Within the amino acid, more preferably within one or several.
  • the present invention further provides an antibody that binds to MP493 or a protein comprising the amino acid sequence of SEQ ID NO: 5.
  • the antibody of the present invention An antibody that specifically recognizes the whole protein or its partial peptide as an antigen, and includes a monoclonal antibody and / or a polyclonal antibody.
  • there are five classes (IgG IgA, IgM, IgD, IgE) classified as immunoglobulin structure, physicochemical properties and immunological properties, or subclasses depending on the type of H chain. It may belong to it.
  • fragments such as F (ab,) 2 when immunoglobulin is decomposed with pepsin, Fab when decomposed with pappine, and chimeric antibodies are also used. You may. These antibodies are useful for research or clinical detection of MP493, clinical treatment of diseases that can be caused by MP493, and the like.
  • the present invention provides a so-called antisense nucleic acid capable of suppressing MP493 biosynthesis at the nucleic acid level in a living body.
  • the antisense nucleic acid is a transcription step from the genomic region necessary for producing MP493-encoding mRNA to pre-mRNA, a processing step from pre_mRNA to mature mRNA, and a passage through the nuclear membrane.
  • Step-At one of the translation steps into protein, it binds to DNA or RNA, which carries the genetic information, and affects the normal flow of genetic information. It means a substance that regulates the expression of a protein, and may consist of a sequence complementary to the whole or any part of the nucleic acid sequence of gene mp493.
  • nucleic acid including DNA and RNA
  • it is a nucleic acid (including DNA and RNA) consisting of a sequence corresponding to or complementary to the nucleotide sequence of SEQ ID NO: 1 or 3.
  • the mRNA transcribed from the genomic region has an intron structure or a form containing a translated region at the 5'-end, 3'-end or 3'-end, the mRNA is equivalent to or complementary to the sequence of the untranslated portion.
  • the antisense nucleic acid used will also have a function equivalent to that of the antisense nucleic acid of the present invention.
  • the antisense nucleic acid of the present invention includes, in addition to DNA and RNA, all derivatives of three-dimensional structure and function similar to DNA and RNA. For example, substitution or modification of at least one of the nucleic acid, oligonucleotide base, sugar, and phosphoric acid with another substance bound to the 3'-end or 5'-end And nucleic acids having a skeleton (backbone) other than the sugar-monophosphate skeleton, such as a nucleic acid having a non-naturally occurring base, a sugar or phosphoric acid, and the like. These nucleic acids are suitable as inducers with at least one increase in nuclease resistance, tissue selectivity, cell permeability, and avidity. That is, it is possible to suppress the expression of MP493 activity. There is no limitation on the form of the nucleic acid as long as it has the function of
  • a base sequence that hybridizes to a loop portion of an inRNA forming a stem-loop that is, a base complementary to a base sequence of a region forming a stem-loop.
  • Antisense nucleic acids having a sequence are preferred.
  • an antisense nucleic acid that binds to the vicinity of the translation initiation codon, ribosome binding site, cabling site, or splice site that is, an antisense nucleic acid having a sequence complementary to the sequence of these sites is also generally used. It is preferable because a high expression suppression effect can be expected.
  • the antisense nucleic acid of the present invention has a chain length of 15 bases or more and 30 bases or less, preferably 15 bases or more. Those comprising a base sequence having a base number of 25 bases or less, more preferably 18 bases or more and 22 bases or less are preferred.
  • the effect of suppressing the expression of the antisense nucleic acid of the present invention can be determined by a known method, such as anti-in vitro transcription, promeca Chito: Ribo max system, etc., and in vitro translation reaction (promega, Rabbit Reticulocyte Lysate System, etc.). 5 'untranslated region, region near translation initiation site, 5' translation It can be evaluated by measuring the expression level of a reporter gene using an expression plasmid in which a DNA containing the region and a reporter gene such as luciferase are linked.
  • the antisense nucleic acid of the present invention can suppress the expression of MP493 in a living body, it is useful as an agent for preventing and treating MP493-related diseases.
  • the present invention also provides a method for screening a compound having an activity of regulating the activity of MP493, using all or a part of the spatial coordinates of the serine protease domain of MP493.
  • MP493 has a characteristic domain of serine protease at a portion corresponding to amino acid residues 217 to 453 shown in SEQ ID NO: 2.
  • the spatial coordinates of the MP493 serine proteinase domain in the present invention are derived from the serine proteinase domain that is three-dimensionally structured based on this partial sequence using an appropriate protein modeling program. It is a thing that can be done.
  • the term “spatial coordinates” means a spatial arrangement substantially determined by each distance between molecules (atoms) constituting a chemical structure. This spatial arrangement is —In the case of processing as information above, the relative arrangement is converted into numerical information as specific coordinates in a certain coordinate system (referred to as coordinate conversion).
  • the essence of spatial coordinates is the arrangement determined by the mutual distance between each molecule (atom) as described above, and it is a temporary process at the time of computer processing. It should be understood that the coordinate values are not specified coordinate values.c Also, in the present specification, the atomic coordinates are the spatial coordinates of individual atoms constituting a substance (protein, amino acid, etc.). Means
  • a C-terminal amino acid residue newly generated by cleavage is called P1, and extends from that residue to the N-terminal side.
  • S 1 The site on the protease side to which P 1 binds is called S 1, and the sites to which P 2, P 3, P 4,... Bind are called S 2, S 3, S 4,.
  • chymotoribsin-type serine protease is given a sequence number corresponding to the three-dimensional structure of chymotoribosine. This number is generally called the chymotrypsin number.
  • the chymotrypsin number By using the chymotrypsin number, it is possible to associate the sequence with the three-dimensional structure.
  • Sl, S2, and S4 also referred to as SI pocket, S2 pocket, and S4 pocket
  • Sl formed from residues 394 to 400, 418 to 425, 429 to 433 (Kimotoribsin numbers 189 to 195, 213 to 221, 224 to 228).
  • S4 It is composed of residues 299 to 301, 378 and 420 (chymotrypsin numbers 97 to 99, 175 and 215).
  • the 300th, 378th, and 420th amino acid residues (98, 175, and 215 residues of chymotrypsin number) that form the S4 bottle have Thr and Leu in TMPRSS2.
  • MP493 is Arg, Ile, and Phe. Therefore, MP493 cleaves a trypsin-type substrate having Lys and Arg at the P1 site (S1 pocket binding site), as in TMPRSS2 and trypsin, but cleaves TMPRSS2 at other S2 and S4 subsites.
  • the S4 subsite has a different substrate specificity from that of trypsin and TMPRSS2. This means that targeting the S2 and S4 subsites enables the molecular design of compounds that selectively inhibit MP493.
  • the method for screening a compound using the spatial coordinates of the MP493 serine protein domain is a method for screening all the spatial coordinates of the above-mentioned MP493 serine protein domain and any compound.
  • the spatial coordinates representing the body structure are fitted on a computer, and the binding state is quantified using, for example, an empirical scoring function as an index to evaluate the binding ability of the compound to MP493. It is a way to do it.
  • residues that can come into contact with small molecule inhibitors of serine protease that are not the original substrates, that is, target binding sites It is advantageous to identify the site and pay attention to the state of connection with this site.
  • the screening method of the present invention uses the whole or a part of the space coordinates of the MP493 Serine Protease Domain and Converter ⁇ Accelrys Inc., San Francisco. Diego Co., CA), Concord (Tripos, Inc. St. Louis, MO), etc., and the spatial coordinates representing the three-dimensional structure of the desired compound obtained by molecular docking. (University of California, San Francisco, CA), etc., superimposed on a computer using a suitable program, and quantified the conformity using the scoring function etc. included in the program as an index. This is performed by evaluating the binding ability of the compound.
  • the above-mentioned method allows the search for an inhibitor to be started without waiting for the expression and purification of the protein MP493, thereby promoting the efficiency of drug discovery development.
  • the MP493 can be obtained from a large number of compounds. This makes it possible to efficiently select an activity-controlling substance that is specific to the enzyme.
  • the number of candidate substances selected by screening at all times in the combi depends on the time and cost required for experimental candidate substance evaluation. For example, it is preferable to determine the number in consideration of the number of substances obtained as a result of the experimental evaluation.
  • the present invention is not limited to the present invention, and a program for performing screening on a computer includes an evaluation system.
  • an evaluation system uses a unique method tailored to each algorithm of a corresponding program. And many. If the activity value of a candidate substance can be obtained as an evaluation system, it is possible to select a compound to be subjected to experimental evaluation based on that value, but it is not an activity value but an empirical value. There are many evaluation systems for which only numerical values can be obtained.
  • computer screening has the main advantage of narrowing down the selection of compounds to be subjected to experimental evaluation, as already mentioned, that is, the so-called narrowing down, the evaluation system ranks It is also meaningful to select as many assigned compounds as possible, which can be used for experiments from the top.
  • the top ranked compounds were evaluated for similarity in structure, physical properties, etc. It is also meaningful to select the number of compounds to be subjected to the experimental evaluation from each class evening after the class evening.
  • a candidate substance selected by screening on a computer has the expected activity is 5% to 30%, it is necessary to obtain 10 activity control substances. Approximately 30 to 200 candidate substances may be selected, and about 160 to 100 000 candidate substances may be selected to obtain 50 activity control substances.
  • the present invention further provides a compound based on the above-described screening method using a computer or a compound exhibiting an MP493 activity regulating effect obtained by an experimental screening method.
  • a pharmacophore is a physicochemical feature on a compound that is required to bind to a target protein.
  • the pharmacological agent expresses the structural features common to compounds exhibiting the activity of modulating MP493 activity as characteristic spheres, and can be defined by determining the relative distance between the characteristic spheres. It can be defined by determining the relative distance between the functional groups.
  • the pharmacophore can be defined by any method commonly used by those skilled in the art, and is not limited to the above method.
  • the pharmacological agent is defined as a set of characteristic spheres based on the properties of the characteristic sphere and the relative distance or coordinates. If so, it should be understood that it is included in the present invention. Substantially the same pharmacophore is considered to be the same compound obtained as a hit when searching for a compound that fits the pharmacophore against the same compound database. Can be done.
  • the characteristic sphere means a spatial region that retains various physicochemical properties such as hydrophobicity, chargeability, ability to form hydrogen bonds, and the like.
  • a pharmacophore-building program Catalyst (Accelrys Inc., San Diego, Calif.)
  • Hydrogen-bond Acceptor lipids can be classified.
  • Hy-arogen-bond Donorj ⁇ Hydrophobic
  • Hydrophobic Aromatic and Hydrophobic aliphatic aliphatic 'Negative Charge', 'Negative Ionizable'
  • the hydrophobic region, the hydrogen bond acceptor region, the cation region, the cyclic aromatic region, and the like are defined as physicochemical properties, and the radius of the unit A having these physicochemical properties is defined as The characteristic sphere is represented as a spherical area.
  • Examples of atoms and functional groups that are suitable for each characteristic sphere are defined in the manual attached to the program such as, for example, “Cat alyst” (Accelrys Inc., Catalyst D oc ume ntation Release 4.5). , 1999).
  • the MP493 activity modulator of the present invention is represented by a chemical structure that satisfies the spatial coordinates of characteristic spheres having certain physicochemical properties that are selectively adaptable to the substrate binding site specific to MP493. It is defined as a compound to be used.
  • the spatial arrangement between characteristic spheres means the relative spatial arrangement (relative distance) between characteristic spheres.
  • the arrangement of characteristic spheres is represented by using specific XYZ coordinate values.
  • the relative distance between each characteristic sphere can be compared. Since it is unambiguously determined by simple geometric calculation, only specific coordinates are used for convenience of description. Therefore, such coordinate values are limited except for the basis for calculating the relative distance between the characteristic spheres. It has no definite significance.
  • the spatial arrangement of the specific sphere is specified by the relative distance between the respective spheres, and in the claims, one coordinate for using this relative distance as a basis for deriving the calculation. It shows the value.
  • this spatial arrangement is processed as information on a computer, the relative arrangement is converted into numerical information as specific coordinates in a certain coordinate system (called coordinate conversion).
  • coordinate conversion is a process that is necessary for convenience in performing the integration process, and the essence of the spatial coordinates is the relative position determined by the distance between the characteristic spheres.
  • the coordinate values are not temporarily specified during the process of viewing the contents.
  • the MP493 activity modulator of the present invention has at least three arbitrary characteristic points among characteristic spheres when arranged at one coordinate on the XYZ coordinate system shown in each of Tables 1 to 3. It is a compound having a pharmacological agent that satisfies the relative configuration between spheres. table 1
  • Characteristic sphere X coordinate Y coordinate 2 Coordinate radius (A) Hydrophobic region 1 13.25 16.82 11.48 1.7 Hydrophobic region 2 7.00 7.07 7.66 1.7 Hydrogen bond acceptor region R 5.26 17.19 9.46 1.7 Hydrogen bond acceptor region T 3.93 16.71 6.80 2.3 Ring Aroma attribute region R 5.15 9.53 8.55 1.7 Cyclic aroma attribute region T 2.80 7.69 8.80 1.7
  • Characteristic sphere X coordinate Y coordinate Z coordinate Radius (A) Hydrophobic area 1 12.02 16.58 13.31 1.7 Hydrophobic area 2 7.34 14.92 16.73 1.7 Hydrophobic area 3 3.90 11.23 10.89 1.7 Cation area 3.96 10.44 7.46 1.7
  • R is defined as the starting point of the vector
  • T is defined as the ending point of the vector.
  • the hydrogen bond acceptor region is defined as the position and direction from the hydrogen bond acceptor atom to the non-covalent electron pair (present on the hydrogen bond acceptor atom) as a vector from R to T. It is a thing.
  • the plane of the ring in the cyclic aromatic attribute region is defined as a plane having a vector from R to T as a perpendicular. Also, graphs of the arrangement of characteristic spheres shown in each table are shown in Figs. 5, 6, and 7, respectively.
  • the spatial arrangement of the characteristic spheres derived from the coordinates in each table is divided into three classes according to the structural similarities of the compounds that actually exhibited the activity of MP493, and each class was classified into Structural features common to the steric structures of the compounds to which they belong are derived using appropriate programs, such as the aforementioned Catalys t. Therefore, by changing the selection and specific number of compounds used to derive the pharmacophore and the criteria for determining their binding ability, etc. Although changes can occur, it should be understood that the present invention substantially includes these changes.
  • the coordinate values of the characteristic spheres shown in Tables 1 to 3 represent the complex model of the compound used to determine each pharmacological group and the MP493 structure. Are based on the same coordinate system.
  • these characteristic spheres are considered to indicate a suitable site when the compound binds to MP493, all the characteristic spheres shown in Tables 1 to 3 are put together to obtain MP493. It can be considered as a pharmacological agent for an activity-modulating compound.
  • Fig. 8 shows a graphical representation of the arrangement of all the characteristic spheres shown in Tables 1 to 3.
  • a compound that satisfies the relative configuration of at least any three or more characteristic spheres is also expected to exert the activity of regulating the activity of MP493. Is done.
  • characteristic spheres specified in Tables 1 to 3 are superimposed on the same coordinate system based on the composite model with the MP493 structure, characteristic spheres with similar properties exist in the vicinity. In such a case, the compounds are classified as the same group, and by not using two or more characteristic spheres belonging to the same group at the same time, an efficient search for compounds becomes possible.
  • the present invention relates to a pharmacological agent consisting of the four types shown above (the pharmacological agent of Table 1, the pharmacological agent of Table 2, the pharmacological agent of Table 3 and all characteristic spheres of Tables 1 to 3
  • the present invention also provides a computerized numerical data for using the pharmacological groups of the group on a computer, and a recording medium for recording the data. (Method of searching for compounds that have MP493 activity regulating activity using pharmacological agents)
  • the search method using the pharmacological agent of the present invention means the four types shown above (the pharmacological agent in Table 1, the pharmacological agent in Table 2, the pharmacological agent in Table 3 and all of the pharmacological agents in Tables 1 to 3). Comparing the information of the pharmacological group of the pharmacological group consisting of characteristic spheres with the spatial arrangement of the steric structure of a compound, and calculating whether or not the compound satisfies the properties of the pharmacological group It is a method determined by For this calculation, a program that can be used by those skilled in the art, such as Catalyst and Unity (Tripos, Inc. St. Louis, MO), may be used as appropriate. This method allows for more efficient computer screening.
  • Catalyst and Unity Tripos, Inc. St. Louis, MO
  • the pharmacophore model obtained by the present invention can screen a large-scale compound database using only the pharmacophore model.
  • the pharmacophore model determined by superimposing the coordinated MP493 on the substrate binding site indicates that the compound that binds to the target binding site of MP493 is converted using DOCK or the like. It can be used as an effective filter for evening screening, or after each fragment is set to fit each pharmacophore, Binding with an appropriate functional group De Novo design use to build the compound is also possible.
  • a compound exhibiting an activity-regulating action on the protein of the present invention or an activity-regulating agent for the protein of the present invention refers to an action that enhances (agonist) or inhibits (agonist) the serine proteinase activity of protein MP493. Any of the compounds having It is preferably a compound having an inhibitory action.
  • the modulator of the activity of MP493, in particular, the inhibitor can be efficiently screened on a computer, so that the inhibitor of MP493 can be used without actually expressing MP493. Can be sorted out. Further, the compound screened and selected on the whole of the comb is further subjected to a biochemical assay using the protein of the present invention or a transformed cell that expresses the protein. This makes it possible to screen for inhibitors of MP493 more effectively.
  • a biochemical assay is used, whether or not a candidate compound exhibits the activity of regulating the activity of the protein of the present invention depends on the activity of the protein when the compound is added to a system in which the activity of the protein can be confirmed and when the compound is not added.
  • Having an activity regulating action means that the measured value of the protein activity is different between the group with the candidate compound added and the group without the candidate compound added.
  • Calculated inhibition (or suppression) or enhancement (or promotion) rate of 10% or more, preferably 30% or more, more preferably 50% or more, and even more preferably 70% As mentioned above, it is particularly preferably 90% or more.
  • Inhibition (suppression) rate or enhancement (acceleration) rate (%) (absolute value of (measured value of group without addition-measurement value of group with addition of candidate compound)) / measured value of group without addition * 100
  • the measured value is appropriately determined depending on the type of the system in which the activity of the protein can be confirmed.
  • the absorbance can be used, and if the measured value of the candidate substance-added group ⁇ the measured value of the candidate substance-free group, In this case, it can be said that the candidate substance has MP 493 protein activity inhibitory activity.
  • the measurement system includes background and noise values, the value obtained by subtracting such values can be used as the measured value.
  • the method for searching for a compound exhibiting the activity-regulating activity of the protein of the present invention is a technique that enables a prophylactic / therapeutic agent for a disease associated with MP493 to be provided to a medical site in a remarkably short period of time.
  • the present invention relates to a method for storing spatial coordinates of the serine proteinase domain of MP 493.
  • the spatial coordinates of a protein having a square root of the mean square deviation of the main chain atom of 2.0 A or less with respect to the main chain atom of the protein to be prepared are also within the scope of the present invention.
  • the "backbone atoms" of the protein are N, C, and covalently bonded to C and Ca of the amino acid constituting the protein and which form the peptide bond. Means 0.
  • the present invention provides (d) at least 257 (His), 30l (Leu), 304 (Asp), 378 (378) of the amino acid numbers in SEQ ID NO: 2 among the spatial coordinates shown in FIG.
  • a spatial coordinate comprising the atomic coordinates of amino acid residues corresponding to at least 257s 299-301, 378, 394-right, 418-425 and 429-433 in amino acid number;
  • Spatial coordinates of the target binding site where the square root of the mean square deviation of the main chain atom is less than or equal to 1.OA with respect to the main chain atom of the amino acid constituting the target binding site;
  • the substrate binding site It provides the space coordinates of the substrate binding site where the square root of the mean square deviation of the main chain atoms is 1.0 A or less for the main chain atoms of the constituent amino acids.
  • At least 'contains' means, in addition to the atomic coordinates of the amino acid residue specified by the amino acid number, one or more arbitrary amino acids in Figure 9. It means that the atomic coordinates of the residue may be included.
  • the amino acid residue which may be contained is not particularly limited, but, for example, an amino acid residue in the vicinity of an amino acid residue constituting a target binding site or a substrate binding site is preferable. No. “Nearby” means within 5A, preferably within 3A. The three-dimensional structure of the protein is tightly fixed Instead, it has some fluctuations.
  • the protein has a functionally equivalent structure. However, it is preferably 1.5 A or less, and more preferably 1.5 OA or less.
  • the functionally equivalent structure is provided in the case of the structure of the target binding site or substrate binding site. However, if the square root of the mean square deviation of the main chain atoms is 1.0 OA or less, it is considered that the functionally equivalent structure is provided. However, it is preferably 0.7 A or less, and more preferably 0.5 A or less.
  • the spatial coordinates including at least the target binding site or the substrate binding site As long as it includes the spatial coordinates of the target binding site or substrate binding site, it may also include the atomic coordinates of any one or more amino acid residues in FIG. 9 in addition to them.
  • all the spatial coordinates shown in FIG. 9 are given to the computer, and then a specific part is designated to specify the screen. It is also possible to perform the screening, or to perform the screening by giving only a part of the structure.
  • the minimum structure required for screening is specified. If so, adding the atomic coordinates of any one or more amino acid residues to them would improve the accuracy of the results without any decrease. Therefore, by focusing on the spatial coordinates of the target binding site or substrate binding site, the atomic coordinates of one or more amino acid residues in FIG. "Parts" are also within the scope of the present invention.
  • the three-dimensional structure of a protein is defined by the relative spatial arrangement of the atoms constituting the structure, and spatial coordinate conversion is necessary for treating the three-dimensional structure on a computer-by-computer basis. This is a convenient process. Therefore, the spatial coordinates obtained by rotating and / or translating the spatial coordinates (h), (a) to ( g ) also represent the same three-dimensional structure as the spatial coordinates before the operation. Things.
  • the present invention provides a computer-readable storage medium storing any of the above spatial coordinates (a) to (h).
  • a computer-readable storage medium> Is not particularly limited as long as the stored space coordinates can be led to various programs (for example, a program using space coordinates) on a computer.
  • an electric temporary storage medium called a memory may be a floppy disk, a hard disk,
  • a semi-permanent storage medium such as an optical disk, a magneto-optical disk, or a magnetic tape may be used.
  • the spatial coordinates and the storage medium storing the spatial coordinates of the present invention can be used for searching or designing a compound having an activity of regulating the activity of MP493, and are useful.
  • the gene mp493 can be isolated and identified from gastric signet carcinoma cells.
  • Examples of obtaining the DNA of the present invention from a DNA library include a screening method using a suitable genomic DNA library and a cDNA library by a hybridization method, and the like. There is a method of screening by an immunoscreening method or the like using an antibody, growing a clone having the target DNA, and cutting out the clone using a restriction enzyme or the like. Screening by the no-dip-digestion method involves labeling a DNA having the nucleotide sequence of SEQ ID NO: 1 or a part thereof with 32 P or the like to obtain a probe.
  • the cDNA library can be prepared by a known method (for example, Maniatis T.
  • the novel DNA of the present invention can also be obtained by PCR (Polymerase Chain Reaction) using a genomic DNA library or a cDNA library as a type II.
  • PCR Polymerase Chain Reaction
  • a sense primer and an antisense primer are prepared based on the nucleotide sequence shown in SEQ ID NO: 1, and any DNA library is prepared by a known method (for example, Michael AI, etc.).
  • PCR Protocols a Guide to Methods and Applications, Academic JPress, 1990) can be performed to obtain the DNA of the present invention.
  • a DNA library having the DNA of the present invention is selected and used.
  • any library having the DNA of the present invention can be used.
  • a commercially available DNA library can be used, or the DNA of the present invention can be used.
  • a cell suitable for preparing a cDNA library is selected from the cells having the DNA and a known method (J. Sambrook et al., Molecular Cloning, a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, New York, NY) According to 1989), a cDNA library can be prepared and used.
  • a phosphoamidite method It can also be prepared by a chemical synthesis method such as
  • the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 has been confirmed to have an altered expression in gastric seal ring cancer cells, lung, kidney, kidney, etc.
  • a partial fragment thereof is considered to be useful as a specific probe for cancer, lung, kidney, and disease.
  • the DNA of the present invention can be used to produce MP493 in large quantities.
  • the DNA can also be labeled with an enzyme or the like and used for examining the expression status of the protein of the present invention in a tissue. That is, by using the DNA as a probe and confirming the expression level of the protein of the present invention in cells using the mRNA expression level as an index, it is suitable for the production of the protein of the present invention. In addition to being able to determine the cells and their culture conditions, it is also possible to diagnose diseases associated with the protein of the present invention.
  • a method such as PCR-RFLP (Restriction fragment length polymorphism; method, PCR-SSCP (Single strand conformation polymorphism) method, sequencing, etc.) using a part of the DNA of the present invention as a primer, It can test and diagnose abnormalities or polymorphisms in nucleic acid sequences. Further, the DNA of the present invention can be introduced into cells in a living body, and used for gene therapy for diseases caused by impaired expression or activity of the protein of the present invention.
  • PCR-RFLP Restoral fragment length polymorphism
  • PCR-SSCP Single strand conformation polymorphism
  • the DNA of the present invention is extremely useful for the preparation of transformed cells, a method for producing the recombinant protein MP493 using the transformed cells, or the search for a compound that specifically suppresses the expression of mp493. is there.
  • the transformed cells of the present invention can be prepared by applying techniques known to those skilled in the art.For example, using various vectors that are commercially available or generally available to those skilled in the art, The DNA of the present invention can be incorporated into an appropriate host cell. At this time, the expression of the gene mp493 in the host cell was arbitrarily controlled by placing the gene mp493 under the influence of an expression control gene represented by the promoter. Is possible. This technique is suitable for use in the production of protein MP493 using transformed host cells, as well as for studying the mechanism of controlling the expression of gene mp493 or searching for a substance that can regulate the expression of the gene. It can be applied.
  • test substance is brought into contact with a cell transformed with a vector containing the gene mp493 under an appropriate condition, thereby obtaining a test substance. It is possible to search for or evaluate a substance having an action of promoting or suppressing the expression of the gene mp493 of the substance.
  • a transgenic animal can be prepared based on a mouse or other suitable animal by combining the DNA of the present invention with a known method. Even using such a transgenic animal, the same search or evaluation as in the above-described transformed cells can be performed.
  • the gene mp 493 or protein MP 493 of the present invention is associated with the onset of cancer, lung disease, renal disease and ⁇ disease, it was investigated through the above-mentioned search using the transformed cells or transgenic animals. It is expected that the compounds and the like obtained as described above will be effective therapeutic or prophylactic agents against cancer, lung, kidney or Teng disease.
  • the gene of the present invention can be expressed under the control of a promoter sequence unique to the gene.
  • a search for a substance that promotes or suppresses transcription of the gene of the present invention can be more advantageously performed.
  • another suitable expression promoter upstream of the gene of the present invention can be used by connecting or replacing it with a promoter sequence specific to the gene.
  • the promoter used in this case can be appropriately selected depending on the host and the purpose of expression.
  • the host is Escherichia coli, T7 promoter overnight, lac promoter overnight, trp promoter overnight, etc .: PL promoter overnight, etc., but if the host is yeast, If the host is an animal cell, PHO 5 Promote overnight, GAP Promote overnight, ADH Promote overnight, etc.
  • the first class can be exemplified, but of course the invention is not limited to these.
  • DNA and vector may be digested with appropriate restriction enzymes, and the obtained fragments may be ligated using DNA ligase.
  • the protein of the present invention can be prepared from various naturally expressed organs, and is a peptide synthesizer (for example, Peptide Synthesizer-1430A, manufactured by PerkinElmer Japan KK). Prepared by a chemical synthesis method using E. coli or by a recombinant method using an appropriate host cell selected from prokaryotes or eukaryotes. be able to. However, in terms of purity, production by genetic engineering techniques and recombinant proteins are preferred.
  • a peptide synthesizer for example, Peptide Synthesizer-1430A, manufactured by PerkinElmer Japan KK.
  • Methods for introducing a recombinant vector into a host cell include an electroporation method, a protoplast method, an alkali metal method, a calcium phosphate precipitation method, and a DEAE text.
  • Well-known methods such as the Run method, the Mycroinjection method, and the method using viral particles (see Extraordinary Special Issue on Experimental Medicine, Handbook of Genetic Engineering, published on March 20, 2001, Yodosha, etc.) Yes, any method may be used o
  • Transformants In order to produce the protein by genetic engineering, the above transformant is cultured to recover a culture mixture, and the protein is purified. Transformants can be cultured by a general method. Regarding the culture of transformants, various books (Taehae, “Microbial Experiment Method”, edited by The Japanese Biochemical Society, Tokyo Chemical Dojin, Ltd., 1992 ), So that they can be performed with reference to them.
  • an appropriate method can be appropriately selected from methods generally used for protein purification. That is, salting out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, hydrophobic chromatography, antibody chromatography, etc.
  • affinity chromatography such as chromatography, chromatofocusing, adsorption chromatography, and reversed-phase chromatography
  • An appropriate method can be appropriately selected from methods that can be generally used, such as filtration, and purification can be performed in an appropriate order using an HPLC system or the like as necessary.
  • the protein of the present invention can be combined with other proteins (eg, glutathione S-transferase, protein A, oxahistidine, FLAG tag, etc.). It can also be expressed as a fusion protein.
  • the expressed fusion form can be excised using an appropriate protease (eg, thrombin or the like), and in some cases, protein preparation can be performed more advantageously. It becomes possible.
  • Purification of the protein of the present invention may be carried out by appropriately combining general methods known to those skilled in the art, and particularly when expressed in the form of a fusion protein, It is preferable to use a purification method that is characteristic for the morphology.
  • the protein of the present invention can be prepared in its own form or in the form of a fusion protein with another kind of protein, but is not limited thereto. It is also possible to convert to various forms. For example, processing by various methods known to those skilled in the art, such as various chemical modifications to a protein, binding to a polymer such as polyethylene glycol, binding to an insoluble carrier, and the like can be considered. In addition, depending on the host used, the presence or absence of sugar chains and the degree of addition are also different. Even in such a case, as long as it functions as a serine protease, it should be considered as being under the concept of the present invention.
  • the protein of the present invention can be used as an antigen for producing an antibody, or can be used for screening a substance that binds to the protein or a substance that regulates the activity of the protein. It is.
  • the antibodies of the present invention include polyclonal antibodies and monoclonal antibodies. All of them can be obtained by referring to known methods (for example, immunization experiment procedures, edited by the Japanese Society of Immunology, published by the Japan Society of Immunology). This will be briefly described below.
  • an animal is first treated with the protein of the present invention as an immunizing antigen and, if necessary, a complete Freund's antigen.
  • the protein of the present invention used as an antigen may be obtained by any method as long as it has a degree of purification that can be used for producing an antibody.
  • the partial polypeptide of the protein of the present invention is also suitably used as an immunizing antigen. If the polypeptide used as the immunizing antigen is a low-molecular-weight polypeptide, that is, a polypeptide composed of about 10 to 20 amino acids, it is converted to a keyhole polypeptide. ⁇ To Hemochanin
  • the animal to be immunized can be any animal, but is preferably used in rats, mice, rabbits, sheep, sheep, dogs, and horses usually used in immunological experiments by those skilled in the art. ⁇ It is preferable to select and use an animal species that can produce the desired antibody from birds, goats, bushes, and sea bream.
  • the polyclonal antibody can be obtained by purifying the obtained antiserum. Purification may be carried out by appropriately combining known methods such as salting out, ion exchange chromatography, and affinity chromatography.
  • the procedure for obtaining a monoclonal antibody is as follows. That is, antibody-producing cells such as spleen cells or lymphocytes are collected from the immunized animal, and the cells are collected by a known method using polyethylene glycol, Sendai virus, electric pulse, or the like. And hybridis with myeloma cell lines to produce hybridomas. Thereafter, a clone producing an antibody that binds to the protein of the present invention is selected and cultured, and the culture supernatant of the selected clone is purified to obtain good results. For purification, a known method such as salting out, ion exchange chromatography, affinity chromatography, or the like may be appropriately used in combination.
  • the novel antibody can also be obtained by using a genetic engineering method.
  • a hybrid cell producing a monoclonal cell against a tile cell, a lymphocyte, or a protein of the present invention or a partial polypeptide thereof immunized with the protein of the present invention or a partial polypeptide thereof.
  • MRNA is collected from one mouse, and cDNA live Create a library.
  • a clone producing an antibody that reacts with an antigen is screened, the resulting clone is cultured, and the desired antibody can be purified from the culture mixture by a combination of known methods.
  • Antisense nucleic acids can be produced by known methods (eg, Stanley T. Crooke and BeRNAld Lebleu, eds. In Antisense Research and Applications, published by CRC, Florida, 1993). Natural DNA or RNA can be synthesized using a chemical synthesizer, or the antisense nucleic acid of the present invention can be obtained by a PCR method using mp493 as type III. Some derivatives, such as methyl phosphate type and phosphor type, use chemical synthesizers (for example, Model 394, manufactured by PerkinElmer Japan KK). Some can be synthesized. In this case, the operation is performed according to the manual attached to the chemical synthesizer, and the obtained synthetic product is purified by an HPLC method using a reversed-phase chromatography, for example. In addition, an antisense nucleic acid can be obtained.
  • the DNA or antisense nucleic acid of the present invention may be used as a radioprobe according to a known method. Label with sotopes, enzymes, fluorescent substances, or luminescent substances. Next, DNA or mRNA is prepared from the sample by a known method, and this is used as a test substance.Then, the labeled probe is added and reacted. Remove the probe. If the test substance contains the gene mp493 or RNA, the antisense nucleic acid binds to them. The presence or absence of bond formation can be determined by using, as an index, light emission, fluorescence, radioactivity, or the like caused by a labeled enzyme, fluorescent substance, luminescent substance, or radioisotope.
  • nucleic acid of the present invention in particular, an antisense nucleic acid is used for a pharmaceutical purpose
  • a nucleic acid having a purity suitable for use as a pharmaceutical should be used in a pharmacologically acceptable use method. Is preferred.
  • the nucleic acid or antisense nucleic acid of the present invention may be used by directly dissolving or suspending it in an appropriate solvent, or may be encapsulated in a liposome or incorporated into an appropriate vector. It may be used in the form of an ellipse.
  • pharmacologically acceptable auxiliary ingredients may be added, and injections, tablets, capsules, eye drops, creams, suppositories, suppositories, sprays and patches It may be used in an appropriate dosage form.
  • Pharmaceutically acceptable auxiliary ingredients include solvents, bases, stabilizers, preservatives, solubilizers, excipients, buffers and the like.
  • nucleic acid or antisense nucleic acid of the present invention When the nucleic acid or antisense nucleic acid of the present invention is in the above-mentioned dosage form, its administration method and dosage are set according to the patient's age, sex, disease type and degree. Can be used. That is, oral, or inhalation, transdermal, ophthalmic, intravaginal, intraarticular, rectal, intravenous, topical, intramuscular administration in an amount appropriate to improve the condition An appropriate method may be selected from subcutaneous administration, intraperitoneal administration and the like.
  • the present invention relates to a protein of the present invention, a transformed cell expressing the protein, a DNA of the present invention, a recombinant vector containing the DNA, or the recombinant vector.
  • the present invention relates to a method for screening a substance that regulates the function of the protein of the present invention, which comprises using the transformed cell transformed in (1). More specifically, (1) a method for evaluating the serine protease activity of MP493 in the presence / absence of a candidate substance; (2) a protein of the present invention in the presence / absence of a candidate substance; Examples include a method of comparing the expression levels of genes and screening a substance that regulates the expression of the protein of the present invention.
  • Example 5 in the system shown in Example 5, the substrate cleavage activity of MP493 in the absence of a test substance and in the absence of Z was measured. What is necessary is just to measure.
  • Examples of (2) include known methods, for example, in vitro transcription anti-d, (Prome-Riksha Co., Ltd .: Ribo max system) and in vitro translation anti-disease, and (from cane soil: RaDbit Reticulocyte Lysate System, etc.).
  • the DNA containing the 5 'untranslated region, the region near the translation initiation site, and the 5' translated region of the mp493 gene, and a repo overnight gene such as luciferase were ligated.
  • the expression level of the repo overnight gene using the expression plasmid can be measured and evaluated in the presence or absence of the candidate substance.
  • Candidate substances include proteins, peptides, oligonucleotides, synthetic compounds, naturally occurring compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. The substance is not limited to this, and may be a new substance or a known substance.
  • BLAST Altschul SF.'J Mol Evol., Vol. 36, pp. 290-300 (l993); Altschul SF et al., J. Mol. Biol., Vol. 215 , pp.40310 (1990)
  • ClustalW Thompson JD, Higgins DG, Gibson T J., Nucleic Acids Res., Vol.22, pp.4673-4680) 1994
  • PROSITE database http: www.expasy.ch/prosite/
  • Pfam http://www.Sanger.ac.uk
  • Pf am http://www.Sanger.ac.uk
  • E .LL Sonnhammer SR Eddy
  • R. Durbin Proteins., 28, pp. 405-420 (1997)
  • HMMER R. Durbin, S. Eddy
  • FAMS Ferta, K. et.al., J. Mol. Graph.Model., Vol. 18, pp. 258-272 (2000)
  • Modeler Accelelrys Inc., S andie o, CA
  • Homology Accelelrys Inc., S andie go, CA
  • protein structure evaluation programs include Profiles-3D (Accelrys Inc., Sandiego, CA) and Graphite. It is possible to use Insightll (Accelrys Inc., Sandiego, CA), Sybyl (ripos, Inc. St. Louis, MO), etc. as a box display program.
  • the compound for which the presence or absence of an activity regulating activity on MP493 is to be confirmed may be any of known and novel compounds, and there is no particular limitation on the structure, origin, physical properties, etc., but drug development in the future It is preferable that the compound is a low-molecular compound in performing the method.
  • compound information registered in the Available Chemicals Directory ⁇ ACD is useful.
  • Concord, Converter, etc. can be used as a program for converting the three-dimensional structure of such a low-molecular compound into a coordinate.
  • the binding between the coordinated MP493 and the low molecular weight compound can be automatically performed using the molecular docking package DOCK, etc., or molecular display software such as Insightll For It can also be done interactively.
  • the index for evaluating the conformity using these programs the free energy calculation value of the whole coupling body, the empirical scoring function, etc. are arbitrarily selected and used. can do. With this index, it is possible to objectively evaluate the quality of the connection.
  • a program such as Catalysts Unity (Tripos, Inc. St. Louis, MO) was used to determine the pharmacological agent from the compound exhibiting MP493 activity-modulating activity and to compare the determined pharmacological agent with the three-dimensional structure of the compound. It can be used. At this time, if a class ring of the compound is required, it is possible to use a program such as Daylight (Daylight Chemical Iniormation Systems, Inc., Mission "Vlejo, CA").
  • FIG. 1 shows the results of an alignment between MP493 and another protein having high homology on the amino acid sequence.
  • the left end shows the proteins by PDB accession numbers except for MP493.
  • Penetration type serine protection NP056590 indicates mouse transmembrane serine proteinase 2, respectively.
  • FIG. 2 shows the results of the alignment with a known serine protease.
  • TMS2 is transmembrane serine protease 2
  • HEPS is hepsin
  • TRP 1 is trypsin 1
  • TRP2 is trypsin 2
  • TRYB is /? Indicates Factor I, respectively.
  • FIG. 3 shows a schematic diagram of a domain structure with a known serine protease.
  • FIG. 4 shows the model structure of the MP493 serine protease domain.
  • FIG. 5 shows a pharmacological agent having the configuration of the characteristic spheres shown in Table 1.
  • H p 1 is the hydrophobic region 1
  • H p 2 is the hydrophobic region 2
  • HBA 1.R is the hydrogen bond receptor region R
  • HBA 1.T is the hydrogen bond receptor region ⁇
  • ⁇ I 1 is the hydrogen bond receptor region Represents the positive ion region.
  • FIG. 6 shows a pharmacological agent having the configuration of the characteristic spheres shown in Table 2.
  • ⁇ 3 is the hydrophobic region 1
  • ⁇ 4 is the hydrophobic region 2
  • R is the hydrogen bond acceptor region R
  • HBA 2.T is the hydrogen bond acceptor region T
  • RA 1 R is a cyclic aromatic region R
  • RA 1 T represents a cyclic aromatic region T.
  • FIG. 7 shows a pharmacological agent having the configuration of the characteristic spheres shown in Table 3.
  • ⁇ 5 represents the hydrophobic region 1
  • ⁇ 6 represents the hydrophobic region 2
  • ⁇ 7 represents the hydrophobic region 3
  • ⁇ I2 represents the cationic region.
  • FIG. 8 shows a pharmacological agent having all the characteristic sphere configurations shown in Tables 1 to 3.
  • Figure 9 shows the spatial coordinates of the ⁇ 493 serine protease domain.
  • the table is composed of eight columns. ATOM in the first column indicates that this row is a row of atomic coordinates, and the second column name indicates the order of the atoms (1 to 1728).
  • the third column shows the atom distinction in amino acid residues, etc., the fourth column shows the amino acid residues in three letters, and the fifth column shows the amino acid number corresponding to SEQ ID NO: 5.
  • Columns 6, 7, and 8 show the coordinates of the atom (X units on the X, Y, and ⁇ axes, respectively).
  • the last row indicates that this is the last row in the table.
  • the table is described in accordance with the format of Protein Data Bank, a method commonly used by those skilled in the art.
  • BAP Bacterial Alkaline Phosphatase
  • TiKaRa Bacterial Alkaline Phosphatase
  • the recovered poly (A) + RNA was used in a buffer containing 50 mM sodium acetate (pH 5.5), ImM EDTA, 5 mM 2-mercaptoethanol and 100 U RNasin in 20 U Tobacco.
  • oligopapori (A) + RNA was transferred to lOpmol dT adapter one primer (5, -GCG GCT GAA GAC GGC CTA TGT GGC CTT TTT TTT TTT TTT-3 ') And Superscript II I RNAse H "Reverse Transcriptase
  • the PCR was performed 5 to 10 times at 94 ° C for 1 minute, at 58 ° C for 1 minute, and at 72 ° C for 10 minutes.
  • a plasmid was prepared from the cDNA library prepared by the method (1) using a PI-100 port bottle (KURABO), the nucleotide sequence of each clone was confirmed, and a database was prepared.
  • Nucleotide sequencing Carry out the sequencing reaction according to the attached protocol using AutoCycle sequencing kit (Amersham Pharmacia) and ROB DNA processor (Amersham Pharmacia), and carry out using ALF DNA sequencer ⁇ Amersham Pharmacia).
  • the plasmid C-KAT00786 obtained by the method described in (1) encodes a novel protein consisting of 453 amino acids represented by SEQ ID NO: 2 and is represented by SEQ ID NO: 1. It contained a cDNA consisting of a 2418 base pair base sequence represented by SEQ ID NO: 3 including an open reading frame consisting of a base sequence of 1359 bases.
  • BLAST Altschul SF., J Mol Evol., Vol. 36, pp. 290-300 (1993); Altschul SF et al., J. Mol. Biol., Vol. 215, pp. 403) ⁇ 10 (1990)
  • BLAST Altschul SF., J Mol Evol., Vol. 36, pp. 290-300 (1993); Altschul SF et al., J. Mol. Biol., Vol. 215, pp. 403
  • search for local sequence matches For homology search, BLAST (Altschul SF., J Mol Evol., Vol. 36, pp. 290-300 (1993); Altschul SF et al., J. Mol. Biol., Vol. 215, pp. 403) ⁇ 10 (1990)
  • a BLAST homology search was performed on the Genbank protein database using the deduced protein sequence as a query sequence. Taking the methionine of the initiation codon as No. 1, it extends over 409 residues at the 448th position from 52 amino acids and has a transmembrane human serine protease 2 (GenBank Accession: NP— 005647) with 58% homology to the mouse serine protease TMPRSS2 (GenBank Accession: AAF97867) spanning 383 residues from the 72nd to the 447th.
  • FIG. 1 shows the results of multiple alignment using the reference program ClustalW (Thompson JD, Higgins DG, Gibson TJ., Nucleic Acids Res., Vol. 22, pp. 4673-4680 (1994)).
  • Human sequences with significant homology in the serine protein domain TMPRSS2; GenBank Accession 0015393; Factor I GenBank Accession P05156; Trypsin GenBank Accession P07477, P07478 Hepsin GenBank Accession Fig. 2 shows the results of multiple alignment using ClustalW in P05981; Tripichi GenBank Accession P20231).
  • the PR0SITE database http://www.expasy.ch/prosite/), which is a motivation database, is used to estimate the protein distribution.
  • a motif was found in which the 221st asparagine was a sugar chain binding site.
  • LDL-receptor class A (LDLRA) domain signature [PROSITE Accession: PS01209] (85-107) was observed. Furthermore, the motif [PROSITE Accession: PS00134] found in the histidine residue in the active center of the serine protease trypsin has amino acid residues 253 to 258. In the same manner, the motif [PROSITE Accession: PS 00135] found in the serine residue at the center of the activity of trypsin has amino acids 394 to 405. Residues were found.
  • a plasmid for expressing MP493 in mammalian cells was prepared by the following method.
  • Sense Primer 3 (5 '— ACA GAG GAT GGA GGT GAC GCC-3') and Antisense Primer 3 (5, -CCC A AG CTT GGT TTT TAG GTC TCT CTC CAT -3 ')
  • the C-KAT00786 plasmid designed and produced by the oligocap method was used as a type, and Pyrobest DNA Polymerase (TaKaRa) was used for 10 seconds at 98 ° C, 30 seconds at 55 ° C, 72 ° C. C was repeated 30 times for 1 minute cycle to perform PCR reaction.
  • the obtained DNA fragment of about 0.3 kb was digested with Bglll and Hindlll, and recovered by agarose gel electrophoresis.
  • mp493 plasmid prepared by the oligocap method was digested with XhoI and Bglll, A DNA fragment of about 1.3 kb was recovered.
  • pcDNA3.1 / Myc-His (-) A (Invitrogen) was digested with XhoI and Hindlll, and a DNA fragment of about 5.5 kb was recovered as a vector by agarose gel electrophoresis.
  • the two types of DNA fragments previously collected in the vector fragment were ligated by a standard method, and the JM109 competent cells (TaKaRA) were transformed to obtain the desired plasmid (pM5001). .
  • the expression plasmid pM5001 was introduced into COS-1 cells by the following method to express the protein. That is, COS-1 cells were inoculated at a concentration of 3.0 ⁇ 10 5 cells / well in 6 ⁇ l plates and cultured for 1 day and night under the conditions of 5% CO 2 and 37 ° C. The next day, FuGENEG (Roche's Diagnostics) per 1 ⁇ l and 1 g of pM5001 were mixed according to the attached protocol and supplemented to COS-1 cells.
  • Penta'His Antibody QIAGEN
  • HRP-labeled Egret anti-mouse immunoglobulin DAKO
  • Chemilmi was detected using an ECL detection reagent (Amersham-pharmacia). As a result, the expression of a protein of about 55 kDa was confirmed.
  • enterokinase recognition site In order to obtain the serine protease domain of MP493 as a protein, enterokinase recognition site (enterokinase recognition site) is located at the C-terminal side of the mouse Ig kappa chain signal peptide at the C-terminal side. Immediately after that, a plasmid for expressing chimera protein followed by MP493 serine protease domain, myc epitope and histidine tag was constructed by the following method.
  • Sense primer 4 (5'-CCA GAT ATA CGC GTT GAC ATT-3 ') and antisense primer 4 (5, -CTT ATC GTC ATC GTC GTC ACC AGT GGA ACC TGG AAC-3')
  • pSecTag2A (Invitrogen)
  • TaKaRa Pyrobest DNA Polymerase
  • Sense primer 5 (5'-ATC GTG GGT GGA AAC ATG Tecs ') and antisense primer 5 (5, -CCG CTC GAG TTT TTA GGT CTC TCT-3') were designed and PCR was performed in the same manner using the C-KAT00786 plasmid prepared by the step method as the type III.
  • the resulting PCR product is ligated with MEGALABEL kit (TaKaRa) at the 5 'end, the former PCR product is digested with SpeI and the latter PCR product is digested with XhoI and subjected to agarose gel electrophoresis. In both cases, a DNA fragment of about 0.7 kb was recovered.
  • pSecTag2 A 5'-ATC GTG GGT GGA AAC ATG Tecs '
  • antisense primer 5 5'-CCG CTC GAG TTT TTA GGT CTC TCT-3'
  • the expression plasmid pM5002 was introduced into COS-1 cells by the following method to express the protein.
  • the plasmid DNA was mixed with 12.5 g of the above plasmid DNA according to the attached protocol, and added to COS-1 cells grown on a semiconfluent in a 150 cm 2 flask. After culturing for 72 hours under the conditions of 5% C0 2, 37 ° C , the culture supernatant was recovered. The collected culture supernatant was dialyzed all day and night against PBS-to which 10 ⁇ M of pepsin-tin, oral dibutin, and EDTA were added, respectively. This was subjected to histidine column chromatography on a commercially available nickel column ProBond.
  • Activated MP493 serine proteinase domain lOmM CaCl 2, 130mM NaCl, 0.1 % final preparation of 0 to 1 g / ml concentration of 20 mM Tris-HCl buffer pH 7.5 was prepared containing BSA, B in earthenware pots by a final group protein concentration 4 mM z -L -Arg-pNA ⁇ HCl (3057, Protein Research Council) was added. After performing the reaction at 37 ° C for 30-60 minutes, the reaction was stopped with 50% acetic acid, and the substrate specificity was examined and analyzed by the absorbance at OD405nm. As a result, the serine protease domain of the present invention efficiently cleaved the trypsin-type substrate.
  • An expression plasmid for serine protease domain secretion of MP493 using E. coli as a host was constructed by the following method.
  • Sense primer 6 (5'-ACA GCT TAT CAT TGG GAG CTG-3 ') and antisense primer 6 (5, -TTT CCA CCC ACG ATC TTA TCA TCA TCA TCA CCC GAG CCT T-3') PM781 (Japanese Patent Laid-Open No. 6-315386) using Pyrobest DNA Polymerase (aKaRa) for 30 seconds at 98 ° C for 10 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute. The PCR reaction was repeated several times.
  • the collected culture supernatant was dialyzed for 48 hours against PBS containing 10 ⁇ M of pepsin, leuptin, and EDTA, respectively. The exchange was performed three times a day. Next, histidine gel column chromatography was performed on a commercial nickel-produced ProBond. Equilibration was carried out sufficiently with 20 mM phosphate buffer + 500 mM NaCl pH 7.8, and then the dialyzed culture supernatant was applied to a nickel column. Many of the proteins in the culture supernatant were passed through, washed thoroughly, and then eluted with 20 mM phosphate buffer + 500 mM NaCl pH 4.0.
  • an expression plasmid for expressing the cell domain of MP493 in the cell was prepared by the following method.
  • Sense primer 6 and anti-sense primer 9 (5, -ACG Pyrobest DNA Polymerase using ATC TTA TCG TCA TCG TCC ATT TTA TTT TAT ACC CTT T-3 ')
  • the collected Escherichia coli was suspended in PBS supplemented with 10 ⁇ M of peptide, leptin, and EDTA, and then disrupted by ultrasonication to dissolve the soluble fraction. And the insoluble fraction were separated by centrifugation. After that, the solubilization of the inclusion bodies was performed using 20 mM phosphoric acid phosphate + 500 mM NaCl + 8 M urea pH 7.8. Next, histidine tag chromatography was performed on a commercially available nickel column ProBond. The equilibrium was sufficiently performed with 20 mM phosphoric acid phosphate + 500 mM NaCl + 8 M urine, pH 7.8, and then the sample in which the aggregate was solubilized was applied to a nickel column.
  • This sample is reacted overnight at 20 ° C with a commercially available enterokinase cleavage capture kit (Novagen) to obtain MP493 serine proteinase domain, which is considered to be the active form.
  • Novagen enterokinase cleavage capture kit
  • the sample was applied to the above affinity column under the buffer conditions except for 8 M urea, and the permeated fraction was dialyzed against PBS-, concentrated, and activated MP493 serine protease domain. It was a purified sample. This sample showed a silver stain at a position with a molecular weight of about 30K. This purification system was suitable for preparing large quantities of MP493.
  • Bz-L-Arg-pNA'HCl (3057, the Society for the Promotion of White Matter Research) was prepared so that the final substrate concentration was 4 mM, and the reaction was carried out at 37 ° C for 30-60 minutes. After stopping the reaction with 50% acetic acid, the substrate specificity was examined and analyzed by OD405nm absorption.
  • the recombinant MP493 serine protein-zedomin which was produced and purified as a recombinant, was mixed with 20 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl 2 , 130 mM NaCl, and 0.1% BSA at 0 to 1 ⁇ l. It was prepared to a concentration of g / ml.
  • MeOSuc-Ala-Ala-Pro-Val-Pna.0.7H2O (EOS) was used as a substrate to be cleaved by human sputum-derived elasase (SE563, Elastin Products) so that the final substrate concentration was 4 mM.
  • Arg- ⁇ ⁇ HC1 (3057, Protein Research Initiative) is used as a base for Sue-Ala-Ala'Ala 'pNA (3071) as a substrate to be cut off by Buera Erasase (E-1250SIGUMA).
  • Figure 9 shows the obtained spatial coordinates.
  • amino acid residues at positions 300, 378, and 420 (residues at positions 98, 175, and 215 in chymotrypsin) forming the S4 pocket, and Thr, Leu, Trp (Thrips for trypsin, Lys ⁇ Trp)
  • Thr, Leu, Trp Thrips for trypsin, Lys ⁇ Trp
  • Agr, Ile, and Pe for MP493.
  • the target binding site of the MP 493 inhibitor was determined by referring to the crystal structure of the complex with the serine protease inhibitor (PDB code: 1EKB, 1A0L, IGCT, IBRU, 1A0J, 2TBS, IFXY, 1DAN).
  • PDB code 1EKB, 1A0L, IGCT, IBRU, 1A0J, 2TBS, IFXY, 1DAN.
  • the amino acid residue that constructs was estimated.
  • the representative compound uses the binding conformation of the complex model structure obtained above, and the compounds other than the representative are Catalyst Release 4.5 (Accelrys Inc., After generating up to 255 conformations using the Best method using CatConf from San Diego, CA), all compounds in the cluster were routinely generated using HipHop from Catalyst. Common pharmacophores were extracted.
  • the pharmacological agent of each class is superimposed on the model structure of the MP493 complex with the representative compound of that class, and the pharmacological action surface of all the classes is mapped to the MP493 structure. I pinged.
  • Catalyst's CatSearch ACD compounds were searched for compounds that simultaneously satisfied three or more points from all the characteristic spheres shown in Tables 1 to 3.
  • the obtained compounds were subjected to class ring according to the method described in (1), and the MP493 inhibitory activity of the 20 compounds was measured using the method described in Example 5. Showed more than 50% inhibition at a concentration of 10 g / mL.
  • a compound having a good docking state in the partial screening using DOCK in Example 6 three or more points of all the characteristic spheres shown in Tables 1 to 3 were simultaneously measured. Satisfactory compounds were extracted.
  • the compound obtained was obtained from the Hierachical Aggiomerrative Custering Package No.
  • the drug efficacy measurement for cancer cell invasion was performed according to the method of Isoai and Kumagaya using Boyden's chamber (Cancer Invasion and Metastasis Research Manual, Isoai, Kumagaya, pp. 125-131, Kinhodo, 1995). This can be done by examining the inhibitory effect of the test drug on the invasive potential of tumor-derived B16F10 cells. Coat a filter with a pore size of 8 M in the void chamber with matrigel, and add 2 ⁇ 10 5 B16F10 cells and the test drug to the upper chamber of the chamber.
  • BSA-DMEM 0.1% fetal serum albumin-Dalbeco modified Eagle's medium
  • the method is based on the method of Fuji Ino and Saiki (Cancer Invasion and Metastasis Research Manual, Fuji Ino and Saiki, pp. 7-11, Kinhodo, 1995).
  • Six-week-old male BDF1 mice are transplanted by injecting 1 ⁇ 10 5 mouse melanoma-derived B16F10 cells into the tail vein.
  • the appropriate dose of MP493 control substance, with a dose is injected into the tail vein together with B16F10 cells.
  • the samples were administered for the next two days, the mice were sacrificed 15 days after cell transplantation, and the number of cancer colonies in the lungs (ie, the number of metastatic nodules) was counted and compared with the control phosphate-buffered saline group.
  • MP493 inhibition The effect of the substance on suppressing lung metastasis of cancer cells can be evaluated.
  • An ARDS model is created by intraperitoneally or intravenously administering endotoxin to mice or rats.
  • the MP 493 activity modulator is administered intravenously, intraperitoneally, or orally immediately before or several hours after the endotoxin administration. Eighteen hours after administration of endotoxin, the lungs were removed, and the MP 493 activity modulator was determined by measuring the wet-dry weight ratio and the amount of pigment leaked into the lung tissue or the number of lymphocytes infiltrated into the lung 'tissue. Can be evaluated for its effect on lung injury.
  • glomerular basement membrane anti-sag antiserum was intravenously administered to SD rats weighing 180 g to 220 g (7 to 8 weeks), and the following day, Sagiria-guma Prince was treated with Freund's complete adjuvant. It is emulsified with knot and injected into both hind footprints intradermally.
  • the effect of MP493 activity modulator on renal impairment can be determined by collecting urine on the 19th day after administration and measuring urinary protein concentration and urine urea concentration or observing glomerular lesions by pathological observation. Can be evaluated.
  • Serine protease-related diseases in tissues where MP493 is predominantly expressed especially various cancers, various lung diseases, asthma, allergy, bronchitis, emphysema, viral diseases, shock, multiple organ failure, inflammatory disease It can be used for diagnosis, research, prevention and treatment of diseases related to various nephritis.

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Abstract

L'invention concerne un gène et une protéine qui se rapportent de manière générale à des infiltrats cancéreux, des maladies de reins, des pneumopathies, etc., et une méthode d'évaluation efficiente d'une substance régulant l'activité de cette protéine. L'invention concerne également un ADN présentant une séquence de bases représentée par la formule SEQ ID NO.1 ou 2; une sérine protéase codée par cet ADN; une méthode d'évaluation d'un composé régulant l'activité de ladite protéine par l'utilisation d'un acide nucléique antisens contre la séquence d'ADN de l'invention, la protéine de l'invention ou une structure modèle partielle de ladite protéine; l'utilisation d'un acide nucléique antisens contre l'ADN; et un anticorps spécifique de la protéine de l'invention.
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Title
LOWELL J. UNDERWOOD ET AL.: "Overian tumor cells express a novel multi-domain cell surface serine protease", BIOCHEMICA ET BIOPHYSICA ACTA, vol. 1502, June 2000 (2000-06-01), pages 337 - 350, XP001018948 *

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