WO2008041665A1 - Procédé de criblage pour un agent prophylactique et/ou thérapeutique pour une maladie accompagnée de l'hépatite c - Google Patents
Procédé de criblage pour un agent prophylactique et/ou thérapeutique pour une maladie accompagnée de l'hépatite c Download PDFInfo
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
- C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
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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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
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- 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
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Definitions
- the present invention relates to a method for screening a preventive and / or therapeutic agent for diseases such as fatty liver and liver cancer associated with hepatitis C, and a prophylactic and / or therapeutic agent obtained by the method.
- HCV Hepatitis C virus
- HCV RNA power A huge precursor protein with approximately 3000 amino acid powers translated is a core protein, envelope protein, and other non-structural proteins that compose virus particles by proteolytic enzymes derived from host cells and viruses. Disconnected.
- J. Exp. Med. 196: 641-653 (2002) discloses that STAT3 is activated by phosphorylation of the JAK-independent pathway by directly binding to core protein SSTAT3. Yes. It is reported in the same document that cell force anchorage-independent growth and tumor formation in which HCV core protein and STAT3 are forcibly expressed are shown. J. Virol., 77, 19, 10237-10249 (2003) show that the core protein interacts with ⁇ 28 ⁇ and localizes in the nucleus. ⁇ 28 ⁇ is known as a proteasome regulatory protein that is localized in the cell nucleus and interacts with the 20S proteasome to improve its beptidase activity. Furthermore, the above-mentioned document reports that the 44-71 amino acid region of the core protein is involved in both binding to ⁇ 28 ⁇ and nuclear localization, and that the core protein undergoes ⁇ 28 ⁇ -dependent degradation.
- Non-Patent Document 1 Nature Medicine 4, 1065-1067 (1998)
- Non-Patent Document 2 J. Virol. 73: 2841-28453 (1999)
- Non-Patent Document 3 J. Biol. Chem. 275: 34122-34130 (2000)
- Non-Patent Document 4 J. Exp. Med. 196: 641-653 (2002)
- Non-Patent Document 5 J. Virol., 77, 19, 10237-10249 (2003)
- Non-Patent Document 6 EMBO J. 19: 729-740 (2000)
- Non-Patent Document 7 Oncogene 22 (17): 2573-80 (2003)
- Non-Patent Document 8 Mol Cell Biol. 23 (21): 7498-509 (2003)
- Non-Patent Document 9 Virology 328 (1): 120-30 (2004)
- Non-Patent Document 10 Oncogene Jan 19; 25 (3): 448-62 (2006)
- An object of the present invention is to provide a drug useful for prevention and Z or treatment of hepatitis C virus-related diseases It is an object of the present invention to provide a screening method capable of easily and efficiently selecting agents, and a preventive and therapeutic agent for hepatitis C virus-related diseases obtained by the method.
- the present inventors have determined that hepatitis C via lipid metabolism control by inhibiting the interaction between hepatitis C virus core protein and ⁇ 28 ⁇ . It was found that the progression of symptoms such as fatty liver and cirrhosis associated with the disease can be suppressed and the incidence of liver cancer can be significantly reduced, and the present invention has been completed.
- the present invention is a method of screening for a prophylactic and / or therapeutic agent for at least one disease selected from fatty liver associated with hepatitis C, cirrhosis, and liver cancer, comprising:
- a method comprising the step of evaluating the inhibitory activity of the protein-protein interaction between hepatitis C virus core protein and ⁇ 28 ⁇ (hereinafter simply referred to as “method 1J of the present invention” in some cases) is provided.
- method 1J of the present invention for example, the transcriptional activity of a lipid metabolism controlling factor may be evaluated as an index.
- ⁇ 28 y gene expression or function inhibitory activity may be evaluated as an index.
- the present invention provides prevention of at least one disease selected from fatty liver, cirrhosis, and liver cancer associated with hepatitis C, comprising as an active ingredient the substance obtained by the screening method of the present invention.
- Z or a therapeutic agent hereinafter referred to simply as “the prophylactic and therapeutic agent of the present invention”.
- the substance that inhibits the protein-protein interaction may be a substance having an activity of inhibiting the expression or function of ⁇ 28 ⁇ gene.
- the present invention provides a method for screening a test substance for a lipid synthesis inhibitor comprising a step of evaluating an inhibitory activity of a protein-protein interaction between hepatitis C virus core protein and ⁇ 28 ⁇ .
- method 2 of the present invention The inhibitory activity of the protein-protein interaction can be evaluated by using the expression of a reporter gene linked under the control of a promoter of a lipid metabolism regulator as an index, or using SREBP-lc as the lipid metabolism regulator. Good.
- the present invention provides a lipid synthesis inhibitor comprising a substance obtained by the screening method of the present invention as an active ingredient.
- the lipid synthesis inhibitor of the present invention may comprise a substance having an activity of inhibiting the expression or function of PA28 y gene as an active ingredient. The invention's effect
- a substance capable of controlling the expression of lipid synthase through transcriptional activity such as a lipid metabolism control factor induced by the interaction. It can.
- the substance thus obtained can be used as an active ingredient of a fat synthesis inhibitor, and in particular, a preventive or therapeutic drug that prevents the onset of fatty liver, cirrhosis, liver cancer, etc. associated with hepatitis C and suppresses the progression of symptoms. It is extremely useful as an active ingredient.
- the prophylactic / therapeutic agent of the present invention provides a substance capable of exerting an excellent therapeutic effect on an already developed hepatitis C virus-related disease, and is further administered to HCV carrier patients and the like before the onset of the disease. By doing so, it is possible to obtain a substance useful as a prophylactic agent that suppresses the onset of HCV-related diseases.
- FIG. 1 A schematic diagram of the sequence structure that the mouse in Example 1 has genotype-specifically on the chromosome, (i) is the wild type ( ⁇ 28 ⁇ + / + ), (ii) is the mutation Types ⁇ 28 ⁇ ( ⁇ 28 ⁇ — ⁇ ) and (iii) are schematic diagrams of the sequence structure of the HCV core protein-introduced type (core Tg).
- FIG. 2 (a) is a photograph of stained tissue stained with anti-HCV core protein antibody from a frozen section of mouse liver, (b) is a photograph of tissue stained with hematoxylin'eosin, and (c) is an oil red O stain. It is a tissue dyeing
- FIG. 3 is a graph showing the amount of transcription of each adipogenesis control gene in mouse livers having four genotypes.
- (A) to (C) are SREBP-la (A), SR in 2-month-old mouse liver.
- (E)-(I) are SREBP-lc (E) in 6 month old mouse liver
- Transcription of gene encoding fatty acid synthase (F), facetyl CoA carboxylase (G), stearoyl CoA desaturase (H), HMG CoA reductase (I), and HMGCoA synthase (J) 6 is a graph showing the relative value of the transfer amount standard.
- FIG. 4 Reporter assembly results for investigating the effects of HCV core protein and PA28 ⁇ on SREBP-lc promoter activity.
- A shows a conceptual diagram of the assembly.
- B Is a graph showing reporter activity for each combination of genes expressed in PA28 y MEF cells (left) or PA28 y _ / — MEF cells (right),
- c) is the core protein, LXR a, RXR a Graph showing reporter activity when the corresponding ligand is added to HEK293T cells in which the combination is expressed exogeneous,
- (d) compares the reporter activity of the core protein and the mutant core protein lacking the C-terminal region. It is a graph.
- FIG. 5 (i) is a schematic diagram showing a lipid synthesis control pathway by SREBP-lc, and (mouth) is a schematic diagram showing a lipid synthesis control pathway by SREBP-2.
- Method 1 of the present invention is characterized in that it comprises a step of evaluating the inhibitory activity of the interaction between hepatitis C virus core protein and ⁇ 28 ⁇ for the test substance.
- the hepatitis C virus (HCV) core protein is a structural protein produced by the degradation of a large precursor protein translated from the RNA of hepatitis C virus.
- the amino acid sequence of the HCV core protein and the base sequence encoding the protein are known, and examples of the amino acid sequence include a sequence represented by Swiss-Prot accession No. P26662.
- the HCV core protein used in the screening method of the present invention is not limited to the wild-type protein having the above-mentioned sequence.
- the amino acid of the wild-type protein can be used to induce the onset of HCV-related diseases. It is also possible to use a mutant protein consisting of a part of which is deleted, substituted, or added with other amino acid sequences.
- mutant protein examples include a mutant protein having at least an interaction activity with PA2 8 ⁇ or a nuclear localization activity, and specifically, 44a.a of the amino acid sequence of the wild-type protein.
- examples include proteins containing at least the region of ⁇ 7 la.a.
- the HCV core protein was expressed in cells using a recombinant vector containing a base sequence encoding the same protein, whether it is a natural protein or a synthetic protein derived from hepatitis C virus. It may be a thing.
- PA28 ⁇ is a nuclear localization protein known as a proteasome regulatory protein, and has an action of specifically binding to the HC V core protein to promote degradation of the protein in the nucleus.
- the amino acid sequence of PA28 ⁇ and the base sequence encoding the protein are known.
- human-derived PA28 ⁇ having the sequence represented by Swiss-Prot accession number P61289 is known as the amino acid sequence.
- ⁇ 28 ⁇ includes monkeys, mice, The presence of homologues such as pigs is known, and these amino acid sequences are published in Swiss-Prot, and any of them can be used as ⁇ 28 ⁇ in the present invention.
- the ⁇ 28 ⁇ used in the screening method of the present invention is not limited to the wild-type protein having the above-mentioned sequence ability.
- one amino acid of the wild-type protein is used.
- a mutant protein consisting of a sequence with a deletion, substitution, or other amino acid sequence added.
- a mutant protein for example, a mutant protein having at least proteanom regulatory activity or nuclear translocation activity can be used.
- the interaction between the HCV core protein derived from Winores and ⁇ 28 ⁇ derived from the host is considered to be required in the onset of HCV-related diseases and the onset of various symptoms.
- the mechanism was not always clear.
- the present inventors transferred the core protein from the nucleus to the cytoplasm due to HCV core protein or ⁇ 28 ⁇ deficient in the interaction region with ⁇ 28 ⁇ , and overexpression of ⁇ 28 y. It has already been reported that the proteolysis of the core protein is enhanced.
- the present inventors further examined that, when PA28y is deleted, the core protein is accumulated in the nucleus, and has the effect of suppressing the onset and progression of fatty liver, liver cirrhosis, liver cancer, etc.
- Method 1 of the present invention is a method for screening for a prophylactic and / or therapeutic drug for at least one disease selected from fatty liver associated with hepatitis C, cirrhosis, and liver cancer.
- test substance used in the method 1 of the present invention may be any known or novel compound, for example, synthesis of nucleic acids, carbohydrates, lipids, proteins, peptides, organic low molecular weight compounds, etc. Examples include natural organic compounds.
- the evaluation of the inhibitory activity of the protein-protein interaction between hepatitis C virus core protein and ⁇ ⁇ 28y can be performed either in a cell or in a test tube.
- labeling is performed in a test tube.
- a method of evaluating the inhibitory activity of protein-protein interaction using the PA2 8 ⁇ gene expression or function inhibitory activity as an index is also preferably used.
- Examples of the method for detecting the interaction in the cell include a method using a prokaryotic or eukaryotic host cell such as Yeast two hybrid system, reporter assembly.
- Examples of the method for detecting the interaction between proteins outside the cell include a method using a purified protein labeled with fluorescence or radiation. Among them, a method for detecting protein-protein interaction in a cell is preferable because it is excellent in reproducibility of a virus infection state and simple, and reporter assembly is particularly preferably used.
- Reporter assembly uses, for example, the expression of a reporter gene linked under the control of the promoter region of a gene that is directly or indirectly subjected to transcriptional control by the interaction of HCV core protein and PA28 ⁇ .
- An evaluation method is used.
- a lipid metabolism control factor preferably a promoter region of a gene encoding a sterol regulatory sequence binding protein (SREBPs) such as SREBP-lc or SREBP-2 is used. it can. It is known that the transcriptional activity (promoter activity) of these lipid metabolism control factors regulates lipid synthesis through regulation of the expression of lipid synthase.
- SREBPs sterol regulatory sequence binding protein
- SREBP-lc can control lipid synthesis through regulation of lipid synthase expression as shown in FIG. 5 (i).
- SREBP-lc is a saturated fatty acid, a monounsaturated fatty acid, through the expression control of lipid synthases such as acetyl CoA force lupoxylase, fatty acid synthase, and stearoyl CoA desaturase.
- lipid synthases such as acetyl CoA force lupoxylase, fatty acid synthase, and stearoyl CoA desaturase.
- Tridaricelide and Phosphorus melamine Controls the quality and lipid synthesis.
- Fig. 5 (mouth) is a schematic diagram showing the same pathway by SREBP-2.
- SREBP-2 is a lipid metabolism regulator composed of HMG CoA synthase, HMG CoA reductase and the like. It regulates the synthesis of saturated fatty acids, monounsaturated fatty acids, triglycerides and phospholipids through the regulation of expression of lipid metabolism regulators such as fatty acid synthase and stearoyl CoA desaturase.
- lipid metabolism regulators such as fatty acid synthase and stearoyl CoA desaturase.
- the transcriptional activity of lipid metabolism regulators can control lipid synthesis. Therefore, according to the method for evaluating the inhibitory activity of protein-protein interaction using transcription of lipid metabolism regulator as an index, cholesterol synthesis is controlled, and thus, for example, prevention of fatty liver induced by lipid accumulation, etc.
- a fat synthesis inhibitor containing as an active ingredient a substance that inhibits the interaction between hepatitis C virus core protein and ⁇ 28 ⁇ .
- the substance as the active ingredient preferably has an inhibitory activity on the expression or function of the 28y gene.
- the reporter gene is not particularly limited, and a luciferase such as firefly luciferase and renilla luciferase is preferably used in that it can be used with known force detection sensitivity and operability.
- a luciferase such as firefly luciferase and renilla luciferase
- the dual luciferase reporter assay method using firefly luciferase and renilla luciferase in combination is preferably used in terms of excellent reproducibility, high reliability, and results.
- the expression vector, the cells and the like used in the screening method of the present invention known or conventional ones can be used depending on the screening method. Specifically, in the example of Yeast two hybrid sy stem, pGBKT7, pACT2, GADT7 etc. can be used as the expression vector, and Saccharomyces cerevisiae AH 109 strain etc. can be used as the cell.
- pGL3 Promega
- pGL3 Promega
- pGL3 Promega
- cells it is excellent in transformation efficiency
- embryonic fibroblasts (MEFs) embryonic fibroblasts
- HEK293T human embryos Established cell lines
- Live cells collected from liver strength of HCV-infected animals are preferably used.
- cells having the ability to produce PA28 y such as human embryonic kidney cells (HEK293T) and liver cells are preferably used because they are suitable for reproducibility of HCV infection.
- Transformation can be performed using a recombinant vector in which a desired sequence is incorporated into the above expression vector and appropriately selecting a known method according to the cell type.
- the resulting transformant is cultured under conventional culture conditions using a known medium according to the cell type. Can do.
- a preferred embodiment of the method (1, 2) of the present invention includes a reporter assay method using a promoter region of SREBP-lc as a lipid metabolism regulator.
- SREBP-lc binds to its promoter region a complex of liver X receptor (LXR) and retinoid X receptor (RXR) belonging to the nuclear hormone receptor family (RXR a / LXR ⁇ ). This is known to activate transcription.
- the RXR a / LXR ⁇ -dependent transcription is activated by co-expression of the HCV core protein.
- RXR a / LXR a heterodimer co-inhibitors eg, Spl lOb
- the screening method is based on a phenomenon controlled by the interaction between the transcriptional activity HCV core protein of SREBP-lc promoter region and HCV core protein and PA28 ⁇ .
- the reporter assay method using the promoter region of SREBP-lc is, for example, S
- genes encoding HCV core protein, PA28 ⁇ , RXR ⁇ , and LXR a proteins have been incorporated to allow expression.
- Cells were transformed with one or more recombinant vectors, and the resulting transformants were transformed into 9-cis-retinoic acid (9cisRA: RXR a ligand) and 22 (R) -hydroxycholesterol (22 ( R) HC: LXR ⁇ ligand) can be used in a culture medium in the presence or absence of a test substance to evaluate reporter activity.
- the test substance can be added as it is or after using a recombinant vector or a known carrier depending on the type of the test substance.
- test substance evaluated to have an inhibitory activity on the interaction between hepatitis C virus core protein and ⁇ 28 ⁇ by the above method has an inhibitory activity on the expression or function of ⁇ 28y gene. Is preferred. According to such a substance, the PA28 gamma-dependent proteasome pathway is blocked and the HCV core protein is accumulated in the nucleus without decomposing, resulting in further suppression of transcription of factors related to diseases, for example. It is thought that it can exert high preventive and therapeutic effects.
- the screening method of the present invention against the host after infection with hepatitis C virus, It is possible to efficiently obtain a drug excellent in the effect of suppressing the onset of HCV-related diseases by suppressing the onset of fatty liver and liver cancer.
- a lipid metabolism control factor when used as an indicator, it can be used as a simple screening method for obtaining a substance useful as a lipid synthesis inhibitor.
- the above-described reporter assay method is used to suppress the transcription of a lipid metabolism regulatory factor.
- Method of confirming the effect The test substance is applied to a non-human animal (HCV core Tg mouse, etc.) that has been infected with HCV or introduced with gene modification so that the gene encoding the HCV core protein can be expressed.
- a method for confirming a decrease in the amount of accumulated fat in liver cells and a decrease in the incidence of liver cancer can be used in the administered animal as compared to the non-administered animal.
- the preventive and therapeutic agent for hepatitis C virus-related diseases of the present invention is a hepatitis C virus core protein, PA28 ⁇ , It contains substances that inhibit these interactions as active ingredients.
- the substance that inhibits the protein-protein interaction is not particularly limited as long as it is a substance having an activity of preventing or preventing the interaction between hepatitis C virus core protein and ⁇ 28 ⁇ . Such a substance prevents or treats the disease by inhibiting transcription, translation, etc. of factors that induce the onset of HCV-related diseases and progression of symptoms through the interaction inhibitory activity described above. There is an effect.
- the substance that inhibits the protein-protein interaction can be obtained, for example, using the screening method of the present invention.
- the therapeutic agent for HCV of the present invention preferably has an activity of inhibiting the expression or function of the ⁇ 28 ⁇ gene.
- expression means that a protein is produced
- activity that inhibits expression means gene transcription, post-transcriptional regulation, translation into protein, post-translational modification, The action may be at any stage such as protein folding.
- activity inhibiting function means a substance that acts on functional sites such as protein-protein interaction sites and active sites in proteins to reduce or suppress signal transduction; ⁇ 28 ⁇ or a protein that interacts with core protein May be a dominant negative mutant.
- “dominant negative mutant” refers to the introduction of mutations in proteins (deletion of functional regions, etc.). Those whose physiological activity is further reduced. These mutants can compete with the wild type and indirectly inhibit its function.
- Substances that inhibit the expression of PA28 y include, for example, transcriptional repressors, RNA polymerase inhibitors, protein synthesis inhibitors, proteolytic enzymes, protein denaturing agents, splicing, and can inhibit mRNA cytoplasmic translocation.
- transcriptional repressors RNA polymerase inhibitors
- protein synthesis inhibitors protein synthesis inhibitors
- proteolytic enzymes protein denaturing agents
- splicing and can inhibit mRNA cytoplasmic translocation.
- Factors, mRNA-degrading enzymes, factors that inactivate by binding to mRNA, etc. but substances that can act specifically on the target molecule are preferable in order to minimize side effects on other genes and proteins. .
- Examples of such a substance that specifically inhibits the expression of PA28 ⁇ include, for example, functional nucleic acid such as ⁇ 28 ⁇ or its equivalent, siRNA, shRNA, miRNA, ribozyme, antisense nucleic acid, aptamer, decoy nucleic acid, etc.
- a functional protein such as an antibody is preferably used.
- These functional nucleic acids and proteins may be in the form produced within the administration subject after administration, and can be prepared by known methods using expression vectors, cells, etc., if necessary. Of these, functional nucleic acids are preferred, and in particular, butamonomers, antisense nucleic acids, siRNA, and the like are preferably used because they can easily impart the specificity of the target molecule and have excellent handleability.
- the length of the region complementary or identical to the target molecule is, for example, 15 to 30, preferably 18 to 25, more preferably about 20 to 23 nucleotides. It is.
- the prophylactic and Z or therapeutic agent for hepatitis C virus-related diseases containing the substance that inhibits the protein-protein interaction as an active ingredient is, for example, dissolved or suspended in an appropriate vehicle, and orally or parenterally. Be administered.
- parenteral administration for example, systemic administration via routes such as veins, arteries, muscles, abdominal cavity, and respiratory tract, or local administration to or near the liver can be used. Of these, oral administration is preferred.
- the HCV therapeutic agent of the present invention allows the substance that inhibits the protein-protein interaction to stably reach the liver or cells in the vicinity thereof, permeates the cell membrane, and promotes the release of the drug from the lysosomal nodosome. It is preferable that the drug delivery system is designed.
- methods for improving nuclease resistance, intracellular translocation, and drug release from lysosomes / endosomes methods such as various chemical modifications to the functional nucleic acids and proteins can be used.
- oligos such as antisense nucleic acids can be used.
- PN In the case of nucleic acid molecules, an oligonucleic acid with a morphine skeleton instead of a phosphate skeleton, PN A known method such as a method of chemically modifying such as a method using A or the like can be applied.
- the dosage form may be either a liquid agent or a solid agent.
- a liquid agent in which an effective amount of an inhibitor is dissolved, dispersed, or emulsified in a dilute solution or dispersion medium such as water or physiological saline.
- solid agents such as capsules, sachets and tablets containing an effective amount of the inhibitor as a solid or granule.
- the HCV therapeutic agent of the present invention is also encapsulated in an active ingredient strength S ribosome, sustained-release material, etc., which may be added with the substance that inhibits the protein-protein interaction as an active ingredient. It may be a sealed body or a carrier supported on a carrier. Encapsulation in ribosomes is advantageous in that the active ingredient is protected from degradation by nucleases and proteases, and the liposome membrane binds to the cell surface and easily reaches the cell by endocytosis. Encapsulation in sustained-release materials such as collagen provides long-term sustainability of the active ingredient.
- the HCV therapeutic agent of the present invention can be added with known pharmaceutically acceptable additives.
- the dose of the HCV therapeutic agent varies depending on the type of active ingredient constituting the therapeutic agent, the method of administration, the symptoms, the type, size, drug characteristics, etc. of the subject of administration.
- the amount of the active ingredient is, for example, about 0.0001 to about LOmgZkg, preferably about 0.005 to 5 mg / kg, and can be administered once or divided into several times.
- the fat synthesis inhibitor of the present invention contains a substance that inhibits the interaction between hepatitis C virus core protein and ⁇ 28 ⁇ as an active ingredient.
- the screening method of the present invention can be used, and among them, the reporter assay method using transcription activity of a lipid metabolism control factor as an index is preferably used.
- the activity of inhibiting the interaction between the hepatitis C virus-derived core protein and the host-derived ⁇ 28 ⁇ , and suppressing the transcription of lipid metabolism control factors and the like induced by the interaction can be obtained by a simple method.
- Such a substance can suppress and avoid the accumulation of fat, and exert an excellent preventive and therapeutic effect on hepatitis C virus-related diseases such as fatty liver and liver cancer.
- ⁇ 28 ⁇ cDNA was isolated from human fetal brain library (K. Moriishi et al., J. Virol.
- HCV protein was amplified from HCV3 ⁇ 4J1 (genotype lb; H. Aizaki et al., Hepatology 27, 621 (1998)) and P CAG-GS (H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)).
- RXR a and RXR a mouse cDNAs were amplified by PCR from all mouse liver cDNAs. The obtained RXR a and RXR ⁇ genes were introduced into pEFFlagGsp GBK (DC Huang, S. Cory, A.
- Mouse anti-Flag (M2) antibody and mouse anti-J3-actin antibody are manufactured by Sigma; Rabbit polyclonal antibodies against synthetic peptides corresponding to 70-85 amino acids of PA28 ⁇ are manufactured by Affinity; Horseradish Peroxide Ze-bound goat anti-mouse IgG and anti-rabbit IgG were manufactured by ICN Pharmaceuticals, respectively.
- Rabbit anti-HCV core protein was prepared by immunization with recombinant HCV core protein (1-71 amino acid sequence) according to the method described in R. Suzuki et al., J. Virol. 79, 1 271 (2005). did.
- a mouse monoclonal antibody against HCV core protein was provided with an antibody described in K. Aoyagi et al., J Clin Microbiol 37, 1802 (1999).
- the characteristic components on these mouse chromosomes are shown in Figs. La (i) to (iii).
- (I) is a wild type ( ⁇ 28 y + / + ), and (ii) is a recombination of exons 2-8 of the PA28 y gene into a marker-one gene (neo), resulting in a PA28 ⁇ gene
- the mutant ⁇ 28 y (PA28 y ' ⁇ , (iii)) lacking the function is an HCV core protein-introduced type (core Tg) in which the gene encoding the HCV core protein is introduced under the control of the HBV X promoter.
- Mouse dienotyping is a process that amplifies 0.75 kb of DNA from wild-type ( ⁇ 28 ⁇ + / + ).
- PA28 ⁇ _ ⁇ mouse and the core Tg mouse were crossed, and the PA2 8 ⁇ +/- core Tg mice obtained in the next step were crossed to produce a PA28 ⁇ core Tg mouse.
- the identification of PA28 ⁇ — ⁇ core Tg was carried out according to the method described in K. Moriya et al ”J Gen Virol 78, 1527 (1997) and S. urata et al., J Biol Chem 274, 38211 (1999). PCR analysis was performed targeting ⁇ or HCV core genes, specifically, synthetic oligonucleotide sense primer PA28-3 (SEQ ID NO: 1) and anti-antibody were detected against 1 ⁇ g of genomic DNA extracted from the mouse tail.
- SEQ ID NO: 1 5 '-AGGTGGATCAGGAAGTGAAGCTCAA-3' (PA28-3)
- Fig. Lb shows an electrophoretogram of the PCR amplification product used for genotyping.
- the wild type mouse is “28 / + / + ”
- the core protein introduction mouse is “core 8 ” or “? 2 8 7 + / + core Tg”
- the PA28 y gene mutant PA28 y mice with “PA28 y” mice with the PA28 y gene deleted and core protein introduced ⁇ 28 ⁇ — Core Tg Called J.
- the molecular weight and expression level of HCV core protein were similar in both ⁇ 28 ⁇ core Tg mice and ⁇ 28 ⁇ + / + mice.
- the expression level of ⁇ 28 ⁇ in ⁇ 28 + / + core Tg mice was similar to that of the wild type ( ⁇ 28 + / + ).
- Figure lc shows Western blotting for analyzing expressed proteins in mouse liver tissue. Specifically, SDS-PAGE was performed using 6-month-old mouse liver tissue supernatant (200 / g protein nolane) as a sample, HCV core protein, ⁇ 28 ⁇ , and 3) monoclonal antibody against Ichinchu. Western blotting analysis was performed using The mice were treated according to the guidelines, fed with CRF-1 (manufactured by Charles River Laboratories) commercially available for breeding mice, and stored under special pathogen-free conditions.
- CRF-1 manufactured by Charles River Laboratories
- mice with various genotypes were fixed in formalin according to standard methods, embedded in paraffin, and the prepared frozen sections (formalin-fixed tissue sections) were confirmed for HCV core protein expression by the following method. Yejin dyeing and oil red O dyeing were performed. The results are shown in Figures 2a-c.
- Figure 2a is a photograph showing visualization of HCV core protein expression in mouse liver sections of three different genotypes. More specifically, a formalin-fixed tissue section prepared from 2-month-old mouse liver with genotypes of PA28 ⁇ + / PA28 y + / + core Tg and PA28 y— core Tg was treated with 3% hydrogen peroxide, Wash twice with phosphate buffer (PBS), 5% sera serum After blocking with PBS containing bumine and incubating with anti-HCV core protein rabbit antibody, the second antibody was incubated with horseradish 'peroxidase-conjugated anti-rabbit IgG antibody (ICN) and 3, 3'-diamino. The immunoreactive antigen was visualized using benzidine as a substrate.
- PBS phosphate buffer
- ICN horseradish 'peroxidase-conjugated anti-rabbit IgG antibody
- HCV core protein As a result, the accumulation of HCV core protein was clearly observed in the nucleus of liver cells of ⁇ 28 ⁇ -core Tg mice. This clearly shows that at least a fraction of the HCV core protein has translocated to the nucleus and is undergoing degradation by the PA28y-dependent pathway.
- FIG. 2b is a photograph of hematoxylin and eosin staining of mouse liver sections of two genotypes. Specifically, formalin-fixed tissue sections prepared from 6-month-old mouse force-collected livers having the PA28 y + / + core Tg and PA28 ⁇ core Tg genotypes were stained with hematoxylin and eosin.
- FIG. 2c is a photograph in which fat accumulated in mouse liver sections of two different genotypes is visualized by Euenore Red O staining. Specifically, formalin-fixed tissue sections prepared from 6-month-old mouse livers having the PA28 y + / + core Tg and PA28 y + Tg genotypes were stained with oil red O.
- FIG. 2d is a graph showing the area of fat droplets accumulated in mouse liver sections of four different genotypes in comparison with males and females. Specifically, formalin-fixed tissue sections produced by 6-month-old mouse liver strains with the PA28 y + / + , PA28 ⁇ + / + core Tg, PA 28 y _ / —core Tg, and PA28 ⁇ chi genotypes. The oil-red stain is used to image the area of the stained lipid droplets (area ratio to the section). ). In the graph, the lipid droplet area (Y-axis) shows an average of 10 bodies per genotype, measuring 3 different sites for 5 randomly selected sections.
- the area of lipid droplets in the liver of ⁇ 28 ⁇ + / + core Tg mice was about 10 times and 2 to 4 times the size of wild-type male and female mice, respectively. Therefore, it was shown that ⁇ 28 ⁇ is necessary for induction of fatty liver by HCV core protein in mice.
- Fat synthesis regulators include SREBP-la, SREBP_lc, SREBP-2 belonging to the SREBP family, and the production of saturated and monounsaturated fatty acids and triglycerides that are known to be transcriptionally controlled by the SREBP-lc.
- Acetyl CoA carboxylase, fatty acid synthase, and stearoyl CoA desaturase were used.
- Trizol LS (manufactured by Invitrogen) was obtained from the livers of 2- month and 6- month- old mice having the PA28 y + / + , PA28 y + / + core Tg, PA28 7 _ ⁇ core Tg, and PA28 ⁇ 1 / _ gene types. ) was used to prepare total RNA.
- First strand cDNA was synthesized using a first strand cDNA synthesis kit (Amersham Pharmacia Biotech). The amount of each cDNA was estimated using Platinum SYBR Green pPCR Supermix UDG (Invitrogen) according to the attached protocol. The fluorescence signal was measured using an ABI prism 7000 (manufactured by Applied Biosystems).
- Fig. 3 shows SREBP-la (A), SREBP-lc (B), SREBP-2 (C), and stearoyl CoA desaturase (2) in 2-month-old mouse liver.
- D Transcription of gene encoding, SREBP-lc (E), fatty acid synthase (F), acetylyl CoA carboxylase (G), stearoyl CoA desaturase (H) in 6-month-old mouse liver
- SREBP-lc E
- F fatty acid synthase
- G acetylyl CoA carboxylase
- H stearoyl CoA desaturase
- liver cancer was examined in males and females over the age of 16-18 months having genotypes of PA28 y + ⁇ core Tg, PA28 y + ', PA28 y h, and PA28 y core Tg. Specifically, the liver was removed from each mouse and the presence or absence of liver cancer was confirmed. These results are shown in Table 2.
- a genomic DNA fragment encoding the SREBP-lc promoter region (located between 410 and +24 residues) is amplified from the mouse genome, and the resulting fragment is the Kpnl and Hindlll sites of pGL3-Basic (Promega).
- PGL3-SREBP-lcPro was constructed (Fig. 4a).
- Embryonic fibroblasts derived from mice having the PA28 y + / + and PA28 ⁇ genotypes were obtained by the method described in S. Murata et al., J Biol Chem 274, 38211 (1999). Prepare a medium containing 10% urinary fetal serum, penicillin, streptomycin, sodium pyruvate and non-essential amino acids in DMEM (Sigma) at 37 ° C (5% carbon dioxide).
- PA28 o / + / + MEFs cells or PA2 8 ⁇ - 'MEFs cells were combined with the control plasmids (Promega) encoding the pGL3-srebp-lcPro and Renilla luciferase in the following combinations: (I) empty plasmid, (ii) plasmid encoding only RXR a, (ii i) plasmid encoding only LXR ⁇ , (iv) encoding RXR ⁇ and LXR a
- transformation was performed with (v) the plasmid encoding the HCV core protein (white bar in the graph of FIG.
- Human embryonic kidney cells (HEK293T: cells expressing endogenous PA28 y) were combined with the above control plasmid (Promega) encoding pGL3-srebp-lcPro and Renilla luciferase, (i) empty plasmid, (ii ) Transformation using one of the plasmids encoding HCV core protein, (iii) Plasmid encoding RXR a and LXR a, (iv) RXR a, LXR c and plasmid encoding HCV core protein did.
- HEK293T cells expressing endogenous PA28 y
- the resulting transformants were supplemented with 9-cis-retinoic acid (9cisRA; Sigma), a ligand for RXR a, and 22 (R) -hydroxycholesterol (22 (R) HC, a ligand for LXR a, as additives.
- 9cisRA 9-cis-retinoic acid
- 22 (R) HC 22 (R) -hydroxycholesterol
- LXR a ligand for LXR a
- a mutant core protein in which the C-terminal transmembrane region and the ER anchor region (amino acid sequence 174-191) were deleted was prepared from the HCV core protein.
- PA28 y + / + MEFs or 293T cells copy an empty plasmid, a plasmid encoding HCV core protein, and a mutant core protein.
- One of the plasmids to be loaded was used for ribosome-mediated transfection with LipofectAMINE2000 (Invitrogen).
- the amount of HCV core protein in liver tissue was measured by ELISA according to the description of K. Aoyagi et al "J Clin Microbiol 37, 1802 (1999). 12.5% SDS-PAGE was performed using cell supernatant.
- the protein on the membrane was treated with a specific antibody and SuperSignal Femto (Pierce) and visualized with the LAS3000 Imaging System (Fuji Photo Film Co., Ltd.).
- Human embryonic kidney cells ( ⁇ 293 ⁇ ) were combined with a control plasmid (Promega) encoding pGL3-srebp-lcPro, Renilla luciferase, and a plasmid encoding RXR ⁇ , LXRa and HCV core protein.
- the activity is expressed as a multiple of the luciferase unit RLUs, with the Firefly luciferase activity as the index, Renilla luciferase activity (RLUs).
- RLUs Renilla luciferase activity
- RNA Double-stranded oligo RNA (siRNA) complementary to partial mRNA sequence of PA28 ⁇ gene Made.
- Human embryonic kidney cells HEK293T
- the pGL3- srebp- lcPro a combination comprising Renilla Noreshifera control encoding peptidase plasmid (Promega)
- Transformants transformed with 9-cis-retinoic acid 9cisRA; manufactured by Sigma
- 9-cisRA 9-cis-retinoic acid
- RXR ⁇ and 22 (R) -hydroxycholesterol (22 (R) HC) which is a ligand for LXR ⁇
- the cells are collected, and the luciferase activity is measured using a dual luciferase reporter assembly system (manufactured by Promega) in the same manner as in Example 3, and the activity is converted to a multiple of RLUs.
- siRNA significantly reduces luciferase activity, which is useful as a preventive and / or therapeutic agent for HCV-related diseases.
- the method of the present invention by evaluating the activity of inhibiting the interaction between the hepatitis C virus-derived core protein and host-derived PA28 y, the lipid metabolism control factor induced by the interaction, etc.
- a substance capable of controlling the expression of lipid synthase through its transcriptional activity can be used as an active ingredient of a fat synthesis inhibitor, and in particular, a preventive or therapeutic drug that prevents the onset of fatty liver, cirrhosis, liver cancer, etc. associated with hepatitis C and suppresses the progression of symptoms. It is extremely useful as an active ingredient.
- the preventive and / or therapeutic agent of the present invention it is possible to exert an excellent therapeutic effect on a disease related to hepatitis C virus that has already developed, and further administered to an HCV carrier patient or the like before the onset of the disease. By doing so, it is possible to obtain a substance useful as a prophylactic agent that suppresses the onset of HCV-related diseases.
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WO2010038796A1 (ja) * | 2008-09-30 | 2010-04-08 | 持田製薬株式会社 | C型肝炎治療剤 |
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CN113801893A (zh) * | 2020-06-12 | 2021-12-17 | 华东师范大学 | 一种Psme3条件性基因敲除小鼠模型的构建方法及其应用 |
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Cited By (6)
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WO2010024384A1 (ja) | 2008-08-29 | 2010-03-04 | 国立大学法人大阪大学 | 抗c型肝炎ウイルス組成物 |
US8580759B2 (en) | 2008-08-29 | 2013-11-12 | Osaka University | Anti-hepatitis C virus composition |
JP5409636B2 (ja) * | 2008-08-29 | 2014-02-05 | 国立大学法人大阪大学 | 抗c型肝炎ウイルス組成物 |
WO2010038796A1 (ja) * | 2008-09-30 | 2010-04-08 | 持田製薬株式会社 | C型肝炎治療剤 |
JPWO2010038796A1 (ja) * | 2008-09-30 | 2012-03-01 | 持田製薬株式会社 | C型肝炎治療剤 |
US9006285B2 (en) | 2008-09-30 | 2015-04-14 | Mochida Pharmaceutical Co., Ltd. | Therapeutic agent for hepatitis C |
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