WO2012029792A1 - TGF-β受容体の活性化を抑制する活性を有する化合物、そのスクリーニング方法、並びにC型肝炎ウィルスに起因する疾患の予防又は治療のための組成物 - Google Patents
TGF-β受容体の活性化を抑制する活性を有する化合物、そのスクリーニング方法、並びにC型肝炎ウィルスに起因する疾患の予防又は治療のための組成物 Download PDFInfo
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- 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
- A61P1/16—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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- 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
- A61P31/14—Antivirals for RNA viruses
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- 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
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- 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
- C12N9/99—Enzyme inactivation by chemical treatment
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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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 compound having an activity of inhibiting the activation of TGF- ⁇ receptor, and a screening method thereof, and more particularly, by inhibiting the binding of NS3 protease and type I TGF- ⁇ receptor,
- the present invention relates to a compound having an activity of suppressing activation of TGF- ⁇ receptor by a protease, and a screening method thereof.
- Liver cirrhosis is the ninth most common cause of death in Japan (Ministry of Health, Labor and Welfare “2009, Summary of Monthly Demographic Statistics”), with approximately 300,000 patients and approximately 3.5 million hepatitis patients.
- This disease is an intractable disease in which liver tissue hardens due to abnormal accumulation of extracellular matrix proteins.
- Liver sclerosis (liver fibrosis) occurs in the process of repeated liver damage and regeneration, and as a result, hepatocytes fall into apoptosis. It exhibits a series of pathological conditions that lead to liver failure.
- Non-patent Document 1 Activating TGF- ⁇ , which is a kind of cytokine, is known as a main factor causing this liver fibrosis.
- TGF- ⁇ a kind of cytokine
- hepatic stellate cells that exist between the liver sinusoids and hepatic parenchymal cells are activated, resulting in excessive production of extracellular matrix such as collagen.
- TGF- ⁇ promotes the overproduction of collagen and the like, and it has been shown in animal models that cirrhosis can be prevented if the function of TGF- ⁇ is stopped by gene therapy or the like
- Non-patent Document 2 Furthermore, liver cancer develops and dies from the liver with cirrhosis at an annual rate of 5-7%. Again, it is said that TGF- ⁇ induces EMT (epithelial-mesenchymal transition) and a decrease in cancer immunity via regulatory T cell induction (Non-patent Document 3).
- Non-Patent Documents 4-5 76% of cirrhosis in Japan is caused by hepatitis C virus (HCV) infection, and it is said that there are an estimated 2 million people infected with HCV in Japan alone and about 200 million people worldwide. .
- HCV hepatitis C virus
- cirrhosis develops after 10 to 30 years, and further shifts to liver cancer, which is a big social problem.
- a combination therapy of PEG-modified interferon and ribavirin is mainly applied at present, and the virus removal effect is recognized in 40 to 50% of patients.
- a protease activity inhibitor of serine protease NS3 essential for ripening of virus particles has been developed, and Phase II-III tests have been carried out (Non-Patent Documents 4-5).
- the present invention has been made in view of the above-described problems of the prior art, and is derived from a compound capable of suppressing the activation of TGF- ⁇ receptor by HCV, a screening method thereof, and hepatitis C virus.
- An object of the present invention is to provide a composition for preventing or treating a disease.
- NS3 protease derived from HCV binds to type I TGF- ⁇ receptor, and that binding activates TGF- ⁇ receptor. I found out. It has also been found that the activation promotes the production of hepatic stellate cells that cause hepatic fibrosis and collagen in the hepatocytes. Furthermore, the binding sites of NS3 protease and type I TGF- ⁇ receptor were identified, and it was found that activation of TGF- ⁇ receptor by NS3 protease is suppressed by antibodies that recognize these binding sites. The invention has been completed.
- the present invention provides the following inventions.
- the compound according to (1) which has binding activity to NS3 protease or type I TGF- ⁇ receptor.
- a composition for preventing or treating a disease caused by hepatitis C virus comprising the compound according to any one of (1) to (4) as an active ingredient.
- (6) A method for screening a compound having an activity of inhibiting the activation of TGF- ⁇ receptor by NS3 protease, comprising the following steps (a) to (c): (a) NS3 in the presence of a test compound Contacting a protease with a type I TGF- ⁇ receptor; (B) detecting the binding between NS3 protease and type I TGF- ⁇ receptor; (C) A step of selecting a compound that suppresses the binding.
- a method for screening a compound having the activity of inhibiting the activation of TGF- ⁇ receptor by NS3 protease comprising the following steps (a) to (c): (a) NS3 in the presence of a test compound Contacting a protease with a type I TGF- ⁇ receptor; (B) detecting activation of TGF- ⁇ receptor by NS3 protease; (C) A step of selecting a compound that suppresses the activation.
- a method for inhibiting activation of TGF- ⁇ receptor by NS3 protease which comprises inhibiting binding of NS3 protease to type I TGF- ⁇ receptor.
- a method for preventing or treating a disease caused by hepatitis C virus which comprises inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor.
- the present invention it becomes possible to provide a compound capable of suppressing the activation of TGF- ⁇ receptor by HCV, and a screening method thereof, thereby preventing or preventing diseases caused by hepatitis C virus. It becomes possible to provide a composition for treatment.
- FIG. 3 is an amino acid sequence diagram showing predicted binding sites and predicted contact residues with type I TGF- ⁇ receptor in NS3 protease.
- FIG. 3 is an amino acid sequence diagram showing a predicted binding site and a predicted contact residue with NS3 protease in a type I TGF- ⁇ receptor.
- FIG. 7 is a graph showing that activation of TGF- ⁇ signal by NS3 protease is suppressed in a dose-dependent manner by anti-NS3 antibody under the condition that each anti-NS3 antibody and NS3 protease are preincubated. Under the condition that each anti-type I TGF- ⁇ receptor antibody is preincubated with ⁇ 9CAGA / CCL64 cells, the anti-type I TGF- ⁇ receptor antibody suppresses activation of TGF- ⁇ signal by NS3 protease. It is a graph which shows that.
- each anti-type I TGF- ⁇ receptor antibody activates the TGF- ⁇ signal by NS3 protease.
- NS3 protease It is a graph which shows being suppressed in a dose-dependent manner.
- FIG. 6 is a graph showing that anti-TGF- ⁇ 2 antibody and the like that do not recognize a predicted binding site do not suppress activation of TGF- ⁇ signal by NS3 protease.
- FIG. 5 is a graph showing that activation of TGF- ⁇ signal by TGF- ⁇ 2 is not suppressed by anti-NS3 antibody under the condition of preincubating each anti-NS3 antibody and TGF- ⁇ 2.
- the anti-type I TGF- ⁇ receptor antibody activates TGF- ⁇ signal by TGF- ⁇ 2. It is a graph which shows being suppressed partially. It is a graph which shows that the production of collagen promoted by NS3 protease is suppressed by anti-NS3 monoclonal antibodies (e1211, e0458, s0647, P3-g0899, P3-g1390).
- the compound of the present invention is characterized by having an activity of inhibiting the activation of TGF- ⁇ receptor by NS3 protease by inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor. .
- NS3 protease is one of nonstructural proteins derived from hepatitis C virus (HCV) and consists of 631 amino acids. It contains a protease domain at the N-terminus and a helicase domain at the C-terminus. Typical examples of the protease domain and helicase domain are GenBank ACCESSION No. Examples thereof include a peptide consisting of 1056 to 1204 amino acids and a peptide consisting of 1224 to 1455 amino acids of the protein specified by AAB271277.1.
- type I TGF- ⁇ receptor forms a complex by binding to type II TGF- ⁇ receptor and TGF- ⁇ which is the ligand of these receptors, and is typical of human origin.
- GenBank ACCESSION No. The protein specified by BAG634344.1 is mentioned.
- the activation of the TGF- ⁇ receptor means that the TGF- ⁇ receptor forms a complex, and thus a downstream signal can be generated.
- a downstream signal can be generated.
- this signal transduction for example, phosphorylation of type I TGF- ⁇ receptor occurring after formation of the complex, phosphorylation of Smad2 / 3 by phosphorylated type I TGF- ⁇ receptor, phosphorylated Smad2 / 3 and Smad4 complex formation, translocation of the Smad complex into the nucleus, and transcriptional activation of the target gene by the Smad complex translocated into the nucleus can occur.
- suppression in the present invention includes both complete suppression (inhibition) and partial suppression.
- the compound of the present invention may be any substance that can inhibit the activation of TGF- ⁇ receptor by NS3 protease by inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor.
- those having a binding activity to NS3 protease or type I TGF- ⁇ receptor are preferable from the viewpoint of suppressing the binding, and are represented by the following SEQ ID NOs: 1 to 6 More preferred is an amino acid sequence described in any one, that is, one having a binding activity to a peptide comprising a binding site between NS3 protease and type I TGF- ⁇ receptor.
- a compound of the present invention from the viewpoint of specifically inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor without affecting the activation by TGF- ⁇ 2, the following SEQ ID NOs: 1 to 3 Those having a binding activity to a peptide comprising the amino acid sequence described in any of the above are particularly preferred.
- One embodiment of the compound of the present invention is an antibody.
- the “antibody” in the present invention includes all classes and subclasses of immunoglobulins.
- “Antibody” includes polyclonal antibodies and monoclonal antibodies, and also includes forms of functional fragments of antibodies.
- the antibodies of the present invention include chimeric antibodies, humanized antibodies, human antibodies, and functional fragments of these antibodies.
- Functional fragments of antibodies include Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific And the like, and polymers thereof.
- a chimeric antibody, a humanized antibody, or a human antibody is desirable from the viewpoint of reducing side effects.
- an immunized animal is immunized with an antigen (such as the inflammatory cytokine), and from its antiserum, conventional means (eg, salting out, centrifugation, dialysis, column chromatography, etc.) It can be obtained after purification.
- an antigen such as the inflammatory cytokine
- conventional means eg, salting out, centrifugation, dialysis, column chromatography, etc.
- Monoclonal antibodies can be prepared by a hybridoma method, a recombinant DNA method, or the like.
- a chimeric antibody for example, immunizes an antigen to a mouse, cuts out an antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, and binds to an antibody constant region (constant region) gene derived from human bone marrow.
- This can be obtained by incorporating it into an expression vector and introducing it into a host for production (for example, Japanese Patent Application Laid-Open No. 7-194384, Japanese Patent No. 3238049, US Pat. No. 4,816,397, US Pat. No. 4,816,567). Gazette, U.S. Pat. No. 5,807,715).
- a humanized antibody can be produced, for example, by grafting (CDR grafting) a gene sequence of an antigen-binding site (CDR) of a non-human-derived antibody to a human antibody gene (for example, Patent No. 292618, Patent No. 28828340 No. 3068507, European Patent 239400, European Patent 125023, International Publication No. 90/07861, International Publication No. 96/02576).
- CDR antigen-binding site
- Human antibodies can be produced using, for example, transgenic animals (eg, mice) capable of producing a repertoire of human antibodies (eg, Nature, 362: 255-258 (1992), Inter. Rev. Immunol, 13: 65-93 (1995), J. Mol. Biol, 222: 581-597 (1991), Nature Genetics, 15: 146-156 (1997), Proc. Natl. Acad. Sci. : 722-727 (2000), Japanese Patent Application Laid-Open No. 10-146194, Japanese Patent Application Laid-Open No. 10-155492, Japanese Patent No. 2938569, Japanese Patent Application Laid-Open No. 11-206387, Japanese Patent Application Laid-Open No. 8-509612, Japanese Patent Application Laid-Open No. 11 -50510 JP).
- transgenic animals eg, mice
- mice capable of producing a repertoire of human antibodies
- the antibody used in the present invention reduces desirable activity (activity of inhibiting the activation of TGF- ⁇ receptor by NS3 protease by inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor). Rather, antibodies with modified amino acid sequences are included. In addition, it can confirm using the evaluation system of the activity in the below-mentioned screening method that this desirable activity is not reducing in the modified antibody.
- An amino acid sequence variant of the antibody of the present invention can be prepared by introducing a mutation into DNA encoding the antibody chain of the present invention or by peptide synthesis. Such modifications include, for example, residue substitutions, deletions, additions and / or insertions within the amino acid sequences of the antibodies of the invention.
- the site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and the variable region (framework region and CDR). Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen, but at present, the amino acid of the CDR is modified to screen for an antibody having an increased affinity for the antigen.
- the modification of the antibody may be modification of a post-translational process of the antibody such as changing the number, position, and type of glycosylation sites.
- Antibody glycosylation is typically N-linked or O-linked.
- Antibody glycosylation is highly dependent on the host cell used to express the antibody.
- the glycosylation pattern can be modified by a known method such as introduction or deletion of a specific enzyme involved in sugar production (Japanese Patent Laid-Open No. 2008-113663, Japanese Patent No. 4368530, Japanese Patent No. 4290423, US Patent). No. 5,047,335, U.S. Pat. No. 5,510,261, U.S. Pat. No. 5,278,299, and International Publication No. 99/54342).
- deamidation is suppressed by substituting an amino acid adjacent to the amino acid deamidated or deamidated with another amino acid for the purpose of increasing the stability of the antibody. May be.
- glutamic acid can be substituted with other amino acids to increase antibody stability.
- the antibody used in the present invention also provides the antibody thus stabilized.
- the antibody used in the present invention has an activity of inhibiting the activation of TGF- ⁇ receptor by NS3 protease by inhibiting the binding of NS3 protease and type I TGF- ⁇ receptor.
- it is preferably an antibody against NS3 protease or type I TGF- ⁇ receptor, and more preferably from the viewpoint of specifically suppressing the binding between NS3 protease and type I TGF- ⁇ receptor,
- SEQ ID NOs: 1 to 3 are described from the viewpoint of specifically inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor without affecting the activation by TGF- ⁇ 2.
- An antibody that binds to a peptide consisting of an amino acid sequence is particularly preferred.
- NS3 protease, type I TGF- ⁇ receptor, etc. are used for binding of NS3 protease to type I TGF- ⁇ receptor and activation of TGF- ⁇ receptor by NS3 protease.
- it is a polypeptide having a dominant negative character. Examples of such a polypeptide include a screening method described later from a polypeptide or peptide library in which substitution, deletion, addition and / or insertion of the amino acid sequence described in any of SEQ ID NOs: 1 to 6 has been performed.
- a polypeptide selected by: Such a dominant negative body can be produced by a recombinant DNA method, chemical synthesis or the like.
- another embodiment of the compound of the present invention has an activity to suppress the activation of TGF- ⁇ receptor by NS3 protease, and can suppress the binding between NS3 protease and type I TGF- ⁇ receptor.
- a low molecular compound is mentioned.
- Such a low molecular weight compound is synthesized by designing and synthesizing based on the structure of a peptide site consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 6 in NS3 protease or type I TGF- ⁇ receptor, for example. Or it can obtain by selecting by the screening method mentioned later from a synthetic low molecular weight compound library.
- the present invention provides a composition containing the compound of the present invention.
- the form of the composition of the present invention includes a pharmaceutical composition, food and drink (including animal feed), or research purpose (for example, it may be a reagent used for in vitro and in vivo experiments.
- the composition of the present invention contains the compound of the present invention as an active ingredient, thereby inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor and inhibiting the activation of TGF- ⁇ receptor by NS3 protease. Therefore, it can be suitably used as a pharmaceutical composition to be administered for the prevention or treatment of diseases caused by hepatitis C virus, such as acute hepatitis, chronic hepatitis, liver cirrhosis, and liver cancer.
- diseases caused by hepatitis C virus such as acute hepatitis, chronic hepatitis, liver cirrhosis, and liver cancer.
- composition of the present invention can be suitably used as a food or drink that is taken on a daily basis for the prevention or amelioration of these diseases by containing the compound of the present invention.
- composition of the present invention contains the compound of the present invention, thereby inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor and inhibiting the activation of TGF- ⁇ receptor by NS3 protease. Can also be suitably used.
- composition in the present invention can be formulated by a known pharmaceutical method.
- a known pharmaceutical method for example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, To be used orally or parenterally as elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. Can do.
- carriers that are acceptable pharmacologically or as foods and beverages, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, Flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffering agent, coating agent, lubricant, colorant, sweetness Can be suitably combined with an agent, a thickening agent, a flavoring agent, a solubilizing agent or other additives.
- composition of the present invention When used as a pharmaceutical composition, it may be used in combination with a known pharmaceutical composition used for the prevention or treatment of diseases caused by hepatitis C virus.
- known pharmaceuticals include PEG interferon and ribavirin.
- NS3 protease activity inhibitors that are currently in phase III of clinical trials (Telaprevir manufactured by Vertex, Boseprevir manufactured by Schering-Plough, etc.) are also considered. These activity inhibitors are the same as the compounds of the present invention in that they target NS3 protease, but the target is only the activity of the protease necessary for the maturation of HCV virus particles and is attributed to hepatitis C virus.
- the point of action in the onset mechanism of the disease is different from the compound of the present invention. Therefore, the combined use of the compound of the present invention, PEG interferon, ribavirin, and NS3 protease activity inhibitor suppresses the progression of the pathological condition caused by hepatitis C virus and eliminates the virus while keeping the concentration of each drug used low. In addition, it can be expected to realize radical treatment of liver disease caused by hepatitis C virus.
- the food or drink is, for example, a health food, a functional food, a food for specified health use, a dietary supplement, a food for a sick person, a food additive, or an animal feed.
- the food / beverage products of this invention can be ingested as a composition as described above, and can also be ingested as various food / beverage products.
- Specific examples of food and drink products include edible oils, dressings, mayonnaise, margarine and other oils; soups, milk drinks, soft drinks, tea drinks, alcoholic drinks, drinks, jelly drinks, functional drinks, etc.
- Liquid foods Carbohydrate-containing foods such as rice, noodles, breads; Livestock processed foods such as ham and sausages; Fish processed foods such as kamaboko, dried fish, and salted vegetables; Vegetable processed foods such as pickles; Semis such as jelly and yogurt Solid foods; Fermented foods such as miso and fermented beverages; Various confectionery such as Western confectionery, Japanese confectionery, candy, gums, gummi, frozen confectionery, ice confectionery; Retort products such as curry, sauce, Chinese soup; Instant soup Examples include instant foods such as instant miso soup and foods for microwave ovens. Furthermore, health foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly are also included.
- composition of the present invention can be used for animals including humans, but is not particularly limited as animals other than humans, and may be used for various domestic animals, poultry, pets, laboratory animals, and the like. it can. Specific examples include but are not limited to pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like.
- the production of food and drink in the present invention can be performed by a production technique known in the technical field.
- the dose or intake is appropriately selected according to the age, weight, symptom, health condition of the subject, type of composition (pharmaceutical, food and drink, etc.), etc.
- the for example, the dose or intake of the composition of the present invention per dose is generally 0.01 mg / kg body weight to 100 mg / kg body weight.
- the product of the composition of the present invention may be labeled with an indication that it is used to inhibit activation of TGF- ⁇ receptor by NS3 protease.
- labeled product or instructions means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to.
- the indication that it is used to suppress the activation of TGF- ⁇ receptor by NS3 protease the activation of TGF- ⁇ receptor by NS3 protease is suppressed by administering or ingesting the composition of the present invention. Information about the mechanism to do can be included.
- TGF- ⁇ receptor for example, information on inhibiting the activation of TGF- ⁇ receptor by NS3 protease by inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor can be mentioned.
- it is used to suppress the binding between NS3 protease and type I TGF- ⁇ receptor, it is used for the prevention or treatment of diseases caused by hepatitis C virus. Can be included.
- the present invention suppresses the binding between NS3 protease and type I TGF- ⁇ receptor by administering or ingesting the compound or composition of the present invention to a subject, and TGF by NS3 protease.
- -It can suppress the activation of ⁇ receptor.
- diseases caused by hepatitis C virus can be prevented or treated.
- the present invention relates to a method for inhibiting activation of TGF- ⁇ receptor by NS3 protease, which comprises inhibiting the binding of NS3 protease to type I TGF- ⁇ receptor, and NS3 protease and I
- the present invention also provides a method for preventing or treating a disease caused by hepatitis C virus, characterized by suppressing the binding to type TGF- ⁇ receptor.
- the present invention also provides a method for screening a compound having an activity of inhibiting the activation of TGF- ⁇ receptor by NS3 protease, comprising the following steps (a) to (c). (A) contacting NS3 protease with a type I TGF- ⁇ receptor in the presence of a test compound; (B) detecting the binding between NS3 protease and type I TGF- ⁇ receptor; (C) A step of selecting a compound that suppresses the binding.
- test compound used in the screening method of the present invention is not particularly limited, and examples thereof include expression products of gene libraries, synthetic low molecular compound libraries, peptide libraries, antibodies, bacterial release substances, cells (microorganisms, plant cells, Animal cell) extracts and culture supernatants, purified or partially purified polypeptides, marine organisms, plant or animal extracts, soil, random phage peptide display libraries.
- the compound screened by this method has an activity of inhibiting the binding of NS3 protease and type I TGF- ⁇ receptor and inhibiting the activation of TGF- ⁇ receptor by NS3 protease. Therefore, as described above, such a compound can be a candidate for a prophylactic or therapeutic drug for a disease caused by hepatitis C virus.
- NS3 protease and type I TGF- ⁇ receptor used here are, for example, the above-mentioned GenBank ACCESSION No. Examples include peptides consisting of 1056 to 1204 amino acids and 1224-1455 amino acids of the protein specified by AAB271277.1. As a type I TGF- ⁇ receptor, GenBank ACCESSION No. The protein specified by BAG634344.1 is mentioned. Further, in addition to such natural proteins, fragments, modified forms, modified forms, etc. that maintain the binding activity to each can be used.
- marker proteins alkaline phosphatase (SEAP), ⁇ for easy detection
- An enzyme such as galactosidase, a protein obtained by expression as a fusion protein with a fluorescent protein such as glutathione-S-transferase (GST) or green fluorescent protein (GFP) can be used.
- GST glutathione-S-transferase
- GFP green fluorescent protein
- NS3 protease a fusion of a partial sequence of NS4A on the N-terminal side as described in the Examples below can also be used.
- NS4A is a protein derived from HCV, similar to NS3 protease, and functions as a cofactor that binds to the N-terminal site of NS3 protease to form a complex and enhances protease activity.
- NS3 protease and type I TGF- ⁇ receptor that bind to each other are brought into contact in the presence of a test compound. Such contact is performed in the absence of the test compound. What is necessary is just to perform on the conditions which the coupling
- the binding between NS3 protease and type I TGF- ⁇ receptor is detected, but the detection of such binding is not particularly limited, and a known method can be appropriately employed.
- a co-immunoprecipitation method yeast two-hybrid system, ELISA method, a method using a detection device using a surface plasmon resonance phenomenon (for example, BIAcore (manufactured by GE Healthcare)), using FRET (fluorescence resonance energy transfer) can be adopted.
- a co-immunoprecipitation method yeast two-hybrid system
- ELISA method a method using a detection device using a surface plasmon resonance phenomenon (for example, BIAcore (manufactured by GE Healthcare)
- FRET fluorescence resonance energy transfer
- a compound that suppresses the binding is selected.
- NS3 protease was precipitated by the specific antibody in the presence of the test compound. It can be evaluated by comparing the amount of type I TGF- ⁇ receptor co-precipitated with the amount of type I TGF- ⁇ receptor in the absence of the test compound (control value).
- the test compound when the amount of type I TGF- ⁇ receptor in the presence of the test compound is low compared to the amount in the absence of the test compound (for example, 80% or less, 50% or less, 30% or less of the control value) ),
- the test compound can be evaluated as a compound having an activity of suppressing activation of TGF- ⁇ receptor by NS3 protease.
- the degree of binding in the absence of the test compound can be used as a control value for evaluation.
- the present invention also provides a method for screening a compound having an activity of inhibiting the activation of TGF- ⁇ receptor by NS3 protease, comprising the following steps (a) to (c). (A) contacting NS3 protease with a type I TGF- ⁇ receptor in the presence of a test compound; (B) detecting activation of TGF- ⁇ receptor by NS3 protease; (C) A step of selecting a compound that suppresses the binding.
- test compound, NS3 protease, and type I TGF- ⁇ receptor used in this screening method, and the step (a) are the same as in the screening method.
- step (b) activation of the TGF- ⁇ receptor by NS3 protease is detected, but detection of such activation is not particularly limited, and a known method can be appropriately employed. For example, detection of phosphorylation of type I TGF- ⁇ receptor and Smad2 / 3 using a phosphorylation site-specific antibody, detection of translocation of Smad complex labeled with a fluorescent protein into the nucleus, described later Reporter assays such as those described in Examples 6-8 can be employed.
- a compound that suppresses the activation is selected.
- the value of the luciferase activity in the presence of the test compound and the value of the luciferase activity in the absence of the test compound can be evaluated by comparing with (control value). That is, when the value of luciferase activity in the presence of the test compound is low compared to the value in the absence of the test compound (for example, 80% or less, 50% or less, 30% or less of the control value), The test compound can be evaluated as a compound having an activity of suppressing the activation of TGF- ⁇ receptor by NS3 protease.
- the degree of activation in the absence of the test compound can be used as a control value for evaluation.
- the “purified recombinant NS3 protein” used in this example was prepared as follows.
- NS4A derived from HCV at the N-terminus (1027 to 1206 amino acids in the protein) of NS3 protease domain region derived from HCV (1027 to 1445 amino acids in the protein specified by GenBank ACCESSION No. AAB271277.1)
- a plasmid vector pE into which a gene encoding a protein encoding the amino acid sequence of SEQ ID NO: 7 has been inserted E. coli KRX strain transformed with T32a (+) (Novagen) was cultured. Then, IPTG (isopropyl- ⁇ -thiogalactopyranoside) induced the expression of the target protein to which the trx-His-S tag derived from the pET32a (+) vector was added, and then the cells were collected.
- the collected cells were suspended in a buffer (20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 4 mM MgCl 2 , 10% glycerol, 1 mM DTT, 0.1% n-ctyl- ⁇ -o-glucopyranoside). Thereafter, the mixture was crushed by ultrasonic waves and centrifuged, and the separated supernatant was filtered through a 0.45 ⁇ m pore size filter. The filtered supernatant was affinity purified with a HisTrap HP column (GE Healthcare) and buffered with a HiPrep26 / 60 Desalting column (GE Healthcare) (20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 0.1 mM).
- Example 1 Confirmation of TGF- ⁇ 2-like antigen activity of NS3 protease
- TGF- ⁇ 2-like antigen of purified recombinant NS3 protein The activity was examined by ELISA using TGF- ⁇ 2 Emax (R) ImmunoAssay System (Promega). Specifically, the TGF- ⁇ coated antibody attached to the kit was diluted 1/1000 with a carbonate buffer (pH 9.2), added to a 96-well ELISA plate (manufactured by NUNC) at a rate of 100 ⁇ l / well. The plate was coated by standing overnight.
- a carbonate buffer pH 9.2
- the coated plate was washed with a phosphate buffer containing 0.05% Tween 20 (hereinafter also referred to as “PBST”), and 270 ⁇ l / well of TGF- ⁇ blocking solution attached to the kit was added at 37 ° C. Blocking was carried out by standing for a minute. Next, the blocking-treated plate was washed with PBST, the prepared TGF- ⁇ 2 standard solution and the purified recombinant NS3 protein solution were added, and the plate was allowed to stand at 4 ° C. overnight.
- PBST phosphate buffer containing 0.05% Tween 20
- the purified recombinant NS3 protein is diluted to a final concentration of 2.5, 5, 10, 20 ⁇ g / ml with the TGF- ⁇ sample dilution solution attached to the kit, and each 100 ⁇ l / well is added as an ELISA sample. did. After the plate to which the specimen had been added was washed with PBST, 100 ⁇ l / well of an anti-TGF- ⁇ 2 polyclonal antibody diluted to 1/2000 in a TGF- ⁇ sample dilution solution was added and shaken at room temperature for 2 hours.
- the plate to which the polyclonal antibody was added was washed with PBST, and peroxidase-labeled TGF- ⁇ diluted to 1/100 with a diluted TGF- ⁇ sample solution was added at 100 ⁇ l / well and allowed to stand at room temperature for 2 hours. Furthermore, after washing the plate with TGF- ⁇ added with PBST, 100 ⁇ l / well of TMB (tetramethylbenzidine) solution was added as a chromogenic substrate for the peroxidase substrate, and the color was allowed to develop at room temperature for a maximum of 15 minutes. At the same time, an equal amount of 1 mol / l hydrochloric acid was added to stop the reaction.
- TMB tetramethylbenzidine
- NS3 protease derived from HCV exhibits TGF- ⁇ 2-like antigen activity and is involved in TGF- ⁇ signaling in hepatocytes during HCV infection.
- the strength of the antigen activity of NS3 protease was about 1 / 50,000 to 1/100000 of that of TGF- ⁇ 2.
- TGF- ⁇ signal Activation by NS3 Protease was examined by a reporter assay whether NS3 protease derived from HCV can activate TGF- ⁇ signal in cells. . That is, the TGF- ⁇ 2-like activity of the purified recombinant NS3 protein was determined by using a pGL reporter plasmid (Promega) in which nine DNA binding sequences (CAGA) of transcription factor Smad important for transcription of the TGF- ⁇ target gene were inserted upstream of the luciferase gene. And a cell line (hereinafter also referred to as “ ⁇ 9 CAGA / CCL64 cell”) established by introducing Mink lung epithelial cells (CCL64 cells).
- CAGA DNA binding sequences
- DMEM Dulbecco's modified Eagle medium
- EQUITECH-BIO fetal bovine serum
- preservative penicillin-streptomycin-glutamine solution, manufactured by Invitrogen
- TPP 96-well cell culture plate
- the culture supernatant is aspirated and removed from the plate, the cells are washed with a calcium-magnesium-containing phosphate buffer (hereinafter also referred to as “PBS (+)”), and then 0.1% bovine serum albumin (EQUITECH- BIO) and 1% preservative-containing DMEM (hereinafter also referred to as “treatment medium”) were added purified recombinant NS3 protein to final concentrations of 12.5, 25, 50, and 100 ⁇ g / ml. 100 ⁇ l of the mixture was added and cultured at 37 ° C. for another 20 hours in the presence of 5% CO 2 .
- PBS (+) calcium-magnesium-containing phosphate buffer
- EQUITECH- BIO 0.1% bovine serum albumin
- treatment medium 1% preservative-containing DMEM
- luciferase activity in the cells in the plate was measured using a luciferase assay system (Promega) according to the package insert of the kit. That is, after removing the culture supernatant by aspiration, the cells were washed with PBS (+), and 20 ⁇ l of 1-fold diluted Passive Lysis Buffer (5 ⁇ ) attached to the kit was added to each well. The cells were lysed by shaking for minutes.
- a luciferase assay system Promega
- Luciferase Assay Substrate is previously dissolved in 10 ml of Luciferase Assay Reagent II (hereinafter also referred to as “LARII solution”), and 100 ⁇ l / well is dispensed into a 96-well luciferase assay plate (Costar). Noted.
- 10 ⁇ l of the cell lysate was added to the LARII solution previously dispensed and mixed by pipetting, and the luminescence intensity was measured with a luminometer (product name: ARVO (TM), manufactured by PerkinElmer). The number of cases was 3 for each treatment, the reporter activity of untreated cells was 1, and the amount of activity of purified recombinant NS3 protein-treated cells was determined as a relative value.
- the purified recombinant NS3 protein increased luciferase activity in a concentration-dependent manner. This suggested that NS3 protease acts in some manner on the TGF- ⁇ receptor and mobilizes the TGF- ⁇ signal.
- Example 3 Examination of promotion of collagen production in hepatic stellate cells by NS3 protease TGF- ⁇ causes liver fibrosis by acting on liver stellate cells and promoting abnormal production of collagen from stellate cells It is known. Then, next, the relationship between NS3 protease and collagen production promotion was examined. That is, Wistar rats (bred under SPF, male, 15 weeks old) after laparotomy under pentobarbital anesthesia, a catheter was inserted into the portal vein, a blood removal washing solution, a 0.06% pronase solution (Carbiochem) ) And 0.03% collagenase (Wako Pure Chemical Industries) solution in this order.
- liver tissue lysate was filtered through a mesh, a buffer solution for hepatic stellate cell separation was added to make a total volume of 150 ml, dispensed into three 50 ml polypropylene tubes, and centrifuged at 4 ° C.
- the supernatant in the polypropylene tube is removed by aspiration, 0.2 ml of DNase I solution is added to each tube, gay neutral salt solution is added and mixed by pipetting, and the total volume is made up to 100 ml, and divided into two 50 ml polypropylene tubes. Note and centrifuged at 4 ° C. and 2000 rpm for 8 minutes. After removing the supernatant in the polypropylene tube by suction, 0.2 ml of DNase I solution was added to each tube, gay neutral salt solution was added and mixed by pipetting, and the total amount was transferred to a beaker with 67.5 ml.
- RNA purification kit RNeasy Micro Kit manufactured by QIAGEN
- the absorbance at 260 nm was measured using a spectrophotometer (Nano Drop)
- the concentration was determined.
- RT reaction was performed according to the package insert using PrimeScript (TM) RT reagent Kit (manufactured by TAKARA) using this mRNA as a template.
- NS3 protease acts like by binding to the TGF- ⁇ receptor, like TGF- ⁇ , and activates it to transmit the TGF- ⁇ signal, It was suggested that liver fibrosis is caused by promoting the production of.
- Example 4 Docking simulation of NS3 protease and type I TGF- ⁇ receptor TGF- ⁇ forms a complex with type I TGF- ⁇ receptor and type II TGF- ⁇ receptor to transmit signals (See “Joan Massague, Mol Cell, Vol. 29, No. 2, pp. 149-150, Feb. 1, 2008”). Therefore, since NS3 protease has TGF- ⁇ -like activity, molecular interactions cause intermolecular interaction between NS3 protease and type I TGF- ⁇ receptor, and these complexes are formed. It is suggested that Therefore, a protein-docking docking simulation was performed to examine the presence or absence of these protein-protein interactions. We also predicted which amino acid residues within the NS3 protease and within the TGF- ⁇ receptor are involved in such interactions.
- the surface was searched exhaustively, and the complementarity of the binding state was calculated from the score. That is, the higher the score (complementary score), the better the complementarity.
- the score can be calculated by converting protein coordinates to three-dimensional grid coordinates and applying fast Fourier transform, so the execution speed is high, so it is not part of the receptor.
- the complementarity of all receptor surfaces and ligand surfaces can be taken into account.
- Another advantage of this method is that the binding site can be predicted even when there is no external binding site designation.
- HCV NS3 protease is PDB code: 1NS3
- type I TGF- ⁇ receptor is PDB code 2PJY B chain (type-II), C chain (type-I) as protein coordinates.
- type-II PDB code 2PJY B chain
- C chain type-I
- the score distribution of this 20 structure is 760, 740, 731, 720, 708, 687, 687, 686, 684, 684, 682, 677, 669, 666, 663, 662, 662, 661, 660, 659, The average value was 435.5, the median was 428.0, and the standard deviation was 54.6.
- amino acid residues that frequently appear in the binding state higher in the complementarity score can be presumed to be residues that are likely to appear in the interaction with the actual receptor, it is possible to perform 3.
- An amino acid residue within a distance of 8 cm was defined as a contact residue, and was defined as a predicted contact residue between NS3 protease and type I TGF- ⁇ receptor.
- the obtained results are shown in FIGS.
- the amino acid sequence shown in FIG. 5 shows the amino acid sequence of the protease domain of NS3 protease (the amino acid sequence shown in SEQ ID NO: 8).
- the amino acid sequence shown in FIG. 6 shows the type I TGF- ⁇ receptor.
- 2 shows the amino acid sequence of the extracellular domain (the amino acid sequence described in SEQ ID NO: 9).
- the underlined amino acid residues indicate amino acid residues (predicted contact residues) that are within a distance of 3.8 cm between atoms in the predicted binding state
- the shaded site indicates a site (predicted binding site) involved in the binding between NS3 protease and type I TGF- ⁇ receptor.
- NS3 protease and type I TGF- ⁇ receptor bind to each other via the predicted binding site (amino acid sequences described in SEQ ID NOs: 1 to 6), and TGF- ⁇ by NS3 protease.
- a synthetic peptide or the like was prepared based on the amino acid sequence of the predicted binding site as described below, and a polyclonal antibody using such a synthetic peptide or the like as an antigen was prepared.
- Table 1 shows a list of synthetic peptide sequences used for polyclonal antibody production targeting the predicted binding site (amino acid sequence described in SEQ ID NOs: 1 to 6) in NS3 protease or type I TGF- ⁇ receptor.
- NH2 and “COOH” represent the N-terminal side and the C-terminal side of each synthetic peptide, respectively.
- C on the N-terminal side represents a cysteine residue necessary for binding mcKLH described later to each synthetic peptide.
- miniPEG refers to polyethylene glycol having an average molecular weight of 6,000, which is inserted as a spacer molecule and imparted for the purpose of improving steric hindrance between the antigen peptide and the carrier protein.
- the peptides shown in Table 1 were synthesized by Fmoc solid phase synthesis.
- the final peptide was prepared by deprotecting and cleaving from the resin with a cleave cocktail containing 95% TFA, and the synthesized peptide was purified by HPLC.
- mcKLH made by PIERCE was combined as carrier protein.
- PBS acid buffer
- Immunization was performed at intervals of 2 weeks when Freund's complete adjuvant was used, and at intervals of 1 week when PBS was used. And 7 days after the final immunization, whole blood was collected from the heart. The collected blood was allowed to stand at 4 ° C. overnight, and then the serum component was separated and collected by centrifugation to obtain antiserum. The obtained antiserum was added with sodium azide at a concentration of 0.1% and stored at 4 ° C. The antibody titer in the serum was determined using the ELISA method. In this assay, a microtiter plate was first coated with recombinant protein diluted in PBS.
- the obtained antiserum was added with sodium azide at a concentration of 0.1% and stored at 4 ° C.
- the antibody titer in the serum was determined using the ELISA method. In this assay, microtiter plates were first coated with a synthetic peptide diluted in PBS. Then serially diluted serum was added to the wells that had been washed with 0.2% Tween20 / PBS and blocked, and incubated. Antibodies against synthetic peptides were detected with peroxidase-conjugated antibodies against rabbit immunoglobulins.
- Antiserum against purified recombinant NS3 protein prepared as described above was diluted with an equal amount of binding buffer, and then filtered through a filter to remove insoluble matters. After adsorbing antibody components through a column packed with Protein A-sepharose 4B (manufactured by GE Healthcare) according to a conventional method, non-specifically adsorbed components are removed, and then free components are recovered by placing them in acidic conditions. A purified polyclonal antibody was obtained. The purified antibody thus obtained was dialyzed and replaced with a 100-fold volume of PBS buffer, and sodium azide was added to a final concentration of 0.1%.
- each synthetic peptide was bound to CNBr-activated Sepharose 4B (manufactured by GE Healthcare) according to a conventional method to obtain an antigen column.
- the antiserum diluted with an equal amount of binding buffer was filtered through a filter to remove insoluble matters, and then passed through an antigen column to adsorb a specific antibody. After removing non-specifically adsorbed components, the free components were recovered by placing them in acidic conditions and used as purified polyclonal antibodies.
- the purified antibody thus obtained was dialyzed and replaced with a 100-fold volume of PBS buffer, and sodium azide was added to a final concentration of 0.1%.
- Example 6 Inhibition of TGF- ⁇ -like activity by NS3 protease using an antibody against the binding site of NS3 protease and TGF- ⁇ receptor
- 2 ⁇ 9 CAGA / CCL64 cells were added to the culture medium.
- the suspension was suspended at a concentration of 10 5 cells / ml, seeded at 100 ⁇ l / well in a 96-well cell culture plate, and cultured overnight at 37 ° C. in the presence of 5% CO 2 . After culturing, the culture supernatant in the plate is removed by aspiration, and the cells are washed with PBS (+).
- control refers to the reporter activity of cells cultured under conditions in which proteins (purified recombinant NS3 protein and recombinant human TGF- ⁇ 2) and antibodies are not added to the medium and are not contacted.
- the values “ ⁇ 1” and “ ⁇ 2” indicate values that are polyclonal antibodies extracted from different rabbit individuals, although immunized with the same antigen.
- purified recombinant NS3 protein is added to the treatment medium so as to have a final concentration of 100 ⁇ g / ml, and further to the predicted binding site on the type I TGF- ⁇ receptor side.
- anti-type I receptor antibodies 6 types of antibodies
- anti-NS3 antibody 6 antibodies prepared against the NS3 protease predicted binding site on the NS3 protease side
- anti-recombinant NS3 antibody Two types of antibodies (hereinafter also referred to as “anti-recombinant NS3 antibody”) prepared against seeds or purified recombinant NS3 protein were added to a final concentration of 10 ⁇ g / ml.
- a “mixed solution of NS3 protease and antibody” purified recombinant NS3 protein is added to the treatment medium so that the final concentration is 100 ⁇ g / ml, and the anti-NS3 antibody 6 types are prepared so that the final concentration is 10 ⁇ g / ml.
- an anti-recombinant NS3 antibody or a non-immune mouse antibody was further added as a negative control and then pre-incubated at 4 ° C. for 1 hour was used. Reporter activity in the presence was determined. The obtained results are shown in FIG.
- a “mixed solution of NS3 protease and antibody” purified recombinant NS3 protein was added to the treatment medium so that the final concentration was 100 ⁇ g / ml, and the final concentrations were 1.25, 2.5, 5, 10 ⁇ g / ml.
- 2 types of anti-NS3 antibody so as to be ml, anti-recombinant NS3 antibody so that the final concentration is 3.1, 6.3, 12.5, 25, 50, 100 ⁇ g / ml, or the final concentration is 1.25 , 2.5, 5, and 10 ⁇ g / ml of non-immune mouse antibodies were added to each, followed by preincubation at 4 ° C. for 1 hour. Reporter activity in the presence of antibody was determined. The obtained results are shown in FIG.
- treatment medium supplemented with purified recombinant NS3 protein to 200 ⁇ g / ml is further added at 50 ⁇ l / well so that the final concentration of each antibody is 1.25, 2.5, 5, 10 ⁇ g / ml.
- the final concentration of the purified recombinant NS3 protein was adjusted to 100 ⁇ g / ml, and the cells were further cultured at 37 ° C. for 20 hours in the presence of 5% CO 2 .
- the reporter activity in the presence of each antibody was determined in the same manner as in Example 2. The obtained results are shown in FIG.
- NS3 protease and type I TGF- ⁇ receptor were actually bound via the predicted binding site, and activation of TGF- ⁇ receptor by NS3 protease occurred. Proved to be. It has also been shown that an antibody that recognizes a predicted binding site in NS3 protease or type I TGF- ⁇ receptor inhibits activation of TGF- ⁇ receptor by NS3 protease, and the results described in Example 3 above In addition, it was revealed that the antibody of the present invention is effective for the prevention or treatment of liver diseases caused by hepatitis C virus.
- Example 7 Examination of specificity for luciferase activity inhibitory effect by antibody against binding site of NS3 protease and TGF- ⁇ receptor Seed ⁇ 9CAGA / CCL64 cells in 96-well cell culture plate as in Example 2 And cultured overnight.
- a ⁇ 2 polyclonal antibody or two kinds of non-immune rabbit antibodies were added to a final concentration of about 10 ⁇ g / ml, respectively, and preincubated at 4 ° C. for 1 hour. Then, the culture supernatant is aspirated and removed from the plate after overnight culture, washed with PBS (+), and each pre-incubated treatment medium is added at 100 ⁇ l / well, at 37 ° C. in the presence of 5% CO 2. The culture was further continued for 20 hours. Thereafter, the reporter activity in the presence of each antibody was determined in the same manner as in Example 2. The obtained results are shown in FIG.
- the anti-NS3 antibody suppressed the increase in luciferase activity by NS3 protease almost 100%, whereas the anti-TGF- ⁇ 2 polyclonal antibody having affinity for NS3 protease and the nonimmune rabbit Such an inhibitory effect was not obtained with the antibody. Therefore, from these results, it was revealed that binding to the site predicted in Example 4 is effective in suppressing TGF- ⁇ signaling by NS3 protease (Example 8).
- ⁇ 9CAGA / CCL64 cells were seeded on a 96-well cell culture plate and cultured overnight. After removing the culture supernatant from the plate after overnight culture and washing the cells with PBS (+), anti-type I receptor antibody and anti-TGF- ⁇ 2 antibody (positive control) at a concentration of 20 ⁇ g / ml ) Or 50 ⁇ l / well of a treatment medium containing anti-TGF- ⁇ 2-LAP antibody (negative control), and treated at 37 ° C. in the presence of 5% CO 2 for 1 hour.
- the anti-TGF- ⁇ 2 antibody which is a positive control, inhibited TGF- ⁇ 2 activity almost 100%, whereas the antibody against NS3 protease against TGF- ⁇ 2 activity. The effect was not confirmed.
- the activity of TGF- ⁇ 2 was suppressed by 27% at the maximum by the antibody against the type I TGF- ⁇ receptor. Therefore, from these results, although the binding site between NS3 protease and TGF- ⁇ receptor is not the same as the binding site between TGF- ⁇ 2 and TGF- ⁇ receptor, there may be a site that shares a part. It was suggested.
- Example 9 Production of monoclonal antibody against binding site of NS3 protease and TGF- ⁇ receptor ⁇ Purification of NS3 protein for production of monoclonal antibody>
- a plasmid vector pET32a (+) into which a gene encoding scNS4A-NS3 protease was inserted was incorporated into an E. coli KRX strain into which a pMINOR plasmid had been introduced and cultured.
- scNS4A-NS3 protease see Protein Sci. 1998, Vol. 7, No. 10, pp. 2143-2149.
- pMINOR plasmid see J. Org. Struct. Func. Genom. 2006, volume 7, pages 31-36.
- the bacterial cells were collected by centrifuging the culture solution of E. coli that induced the expression of the protein.
- the collected bacterial cells were buffered (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 4 mM MgCl 2 , 10% glycerol, 20 ⁇ M ZnCl 2 , 1 mM DTT, 0.1% n-ctyl- ⁇ -o-glucopyroxide, The suspension was suspended in complete EDTA free (Roche Applied Science), and the cells were disrupted with ultrasonic waves, followed by centrifugation at 14,000 ⁇ g for 20 minutes to collect the supernatant.
- the collected supernatant was passed through a HisTrap HP column (manufactured by GE Healthcare), the target protein was once adsorbed to the resin packed in the column, and then the elution buffer (500 mM imidazole, 20 mM Tris-HCl pH 8.0, The target protein was eluted from the resin with 500 mM NaCl, 20 ⁇ M ZnCl 2 , 1 mM DTT).
- the elution buffer 500 mM imidazole, 20 mM Tris-HCl pH 8.0
- the eluate thus obtained was passed through a HiPrep 26/60 Desalting column (manufactured by GE Healthcare) to perform desalting operation, and a buffer containing the target protein (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 20 ⁇ M ZnCl 2 , 10 mM DTT, 100 mM CaCl 2 ).
- the solution filtered through an EndoTrap Blue column (manufactured by Hygros GmbH) for the purpose of removing endotoxin was collected, and subjected to the following experiment as NS3 protein for monoclonal antibody production (hereinafter also referred to as “recombinant NS3”). .
- recombinant NS3 was immunized by intraperitoneal injection into female BALB / c mice.
- 50 ⁇ g of recombinant NS3 in Freund's complete adjuvant was administered per animal, and for subsequent immunizations, 50 ⁇ g of recombinant NS3 in oil-in-water emulsion adjuvant was administered. Immunizations were performed at 3-week intervals. The final immunization was performed by administering a phosphate phosphate buffer (PBS) containing 50 ⁇ g of recombinant NS3.
- PBS phosphate phosphate buffer
- the spleen cells of individuals with high antibody titers in the serum were treated with P3.
- Cell fusion treatment with X63-Ag8.653 mouse myeloma cells was performed.
- Hybridoma cells were selected in HAT medium using known Kohler and Milstein techniques.
- the antibody titer in the serum was assayed using the ELISA method.
- a 96-well microplate was first coated with an antigen to which recombinant NS3 or the below-described Image OVA was bound (hereinafter also referred to as “OVA-binding synthetic peptide”).
- OVA-binding synthetic peptide an antigen to which recombinant NS3 or the below-described Image OVA was bound
- OVA-binding synthetic peptide an antigen to which recombinant NS3 or the below-described Image OVA was bound
- the plate was washed with TBS / 0.05% Tween 20, and a substrate solution (0.05% o-phenylenediamine / citrate buffer (pH 5) /0.03% H 2 O 2 ) was added to the well, and color was developed. Reaction was performed. Ten minutes after the addition of the substrate solution, the reaction was stopped by adding 2N sulfuric acid, and the absorbance at 490 nm was measured with a spectrophotometer.
- a substrate solution 0.05% o-phenylenediamine / citrate buffer (pH 5) /0.03% H 2 O 2
- peptides NS-1 to NS-3 (Biomatrix Laboratories, Inc.) shown in Table 1 were synthesized by the Fmoc solid phase synthesis method.
- the final peptide product was prepared by deprotection and excision from the resin using a cleave cocktail containing 95% TFA.
- Image OVA manufactured by Thermo SCIENTIFIC Pierce Protein Research Products
- Hybridomas that react with recombinant NS3 were selected using ELISA and immunoprecipitation ELISA utilizing immunoprecipitation. Specifically, recombinant NS3 (final concentration 1 ⁇ g / mL, prepared with 50 mM carbonate buffer) was first fixed on a sparse / hydrophilic molecule-adsorbing 96-well microplate at room temperature for 1 hour. The plate was then washed with TBS / 0.05% Tween 20, and the free adsorbed portion of the well surface was blocked with 1% skim milk / salt phosphate buffer for 1 hour at room temperature and again TBS / 0.05% Tween 20 Washed with.
- Hybridoma culture supernatant was added to the wells and incubated at room temperature for 1 hour.
- the plates were then washed with TBS / 0.05% Tween 20, then peroxidase-conjugated mouse anti-IgG antibody diluted in 1% skim milk / saline phosphate buffer was added to the wells and incubated for 1 hour at room temperature. After incubation, the plate was washed with TBS / 0.05% Tween 20, and a substrate solution (0.05% o-phenylenediamine / citrate buffer (pH 5) /0.03% H 2 O 2 ) was added to the well, and color was developed. Reaction was performed.
- anti-NS1 peptide polyclonal antibodies polyclonal antibodies that bind to NS-1 listed in Table 1 prepared in Example 5
- anti-NS3 peptide polyclonal antibodies prepared in Example 5; Table 1
- the described polyclonal antibody that binds to NS-3) and anti-riken NS3 peptide polyclonal antibody polyclonal antibody prepared in Example 5 using purified recombinant NS3 protein as an antigen
- ELISA is used with the reactivity to the recombinant trx-His-S tag protein prepared from the recombinant E. coli as an index. Screening was performed using an immunoprecipitation ELISA method using a method and immunoprecipitation. That is, trx-His-S tag protein (final concentration 1 ⁇ g / mL, prepared with 50 mM carbonate buffer) was first immobilized on a sparse / hydrophilic molecule-adsorbing 96-well microplate at room temperature for 1 hour.
- the plate was then washed with TBS / 0.05% Tween 20, and the free adsorbed portion of the well surface was blocked with 1% skim milk / salt phosphate buffer for 1 hour at room temperature and again TBS / 0.05% Tween 20 Washed with. Hybridoma culture supernatant was added to the wells and incubated at room temperature for 1 hour. The plates were then washed with TBS / 0.05% Tween 20, then peroxidase-conjugated mouse anti-IgG antibody diluted in 1% skim milk / saline phosphate buffer was added to the wells and incubated for 1 hour at room temperature.
- the plate was washed with TBS / 0.05% Tween 20, and a substrate solution (0.05% o-phenylenediamine / citrate buffer (pH 5) /0.03% H 2 O 2 ) was added to the well, and color was developed. Reaction was performed. Ten minutes after the addition of the substrate solution, the reaction was stopped with 2N sulfuric acid, and the absorbance at 490 nm was measured with a spectrophotometer.
- a substrate solution 0.05% o-phenylenediamine / citrate buffer (pH 5) /0.03% H 2 O 2
- the hybridoma recognized to be reactive with the Trx-His-S tag protein is an anti-trx-His-S tag protein. Since it was expected to be an antibody-producing clone, it was excluded from the study.
- the plate was then washed with TBS / 0.05% Tween 20, and the free adsorbed portion of the well surface was blocked with 1% skim milk / salt phosphate buffer for 1 hour at room temperature and again TBS / 0.05% Tween 20 Washed with.
- the hybridoma culture supernatant was then added to the wells and incubated for 1 hour at room temperature.
- the plates were then washed with TBS / 0.05% Tween 20, then peroxidase-conjugated mouse anti-IgG antibody diluted in 1% skim milk / saline phosphate buffer was added to the wells and incubated for 1 hour at room temperature.
- NS3-NS4A HCV
- His-tag manufactured by ALEXIS
- the plate was then washed with TBS / 0.05% Tween 20, and the free adsorbed portion of the well surface was blocked with 1% skim milk / salt phosphate buffer for 1 hour at room temperature and again TBS / 0.05% Tween 20 Washed with. Hybridoma culture supernatant was added to the wells and incubated at room temperature for 1 hour. The plates were then washed with TBS / 0.05% Tween 20, then peroxidase-conjugated mouse anti-IgG antibody diluted in 1% skim milk / saline phosphate buffer was added to the wells and incubated for 1 hour at room temperature.
- the culture supernatant of the hybridoma was filtered through a 0.22 ⁇ m filter to remove insoluble matters from the culture supernatant.
- the antibody components are adsorbed through the culture supernatant to a column packed with Protein G-sepharose 4B (manufactured by GE Helthecare), then the non-specific adsorbed components are removed by column washing, and then adsorbed under acidic conditions.
- the released IgG was released.
- the released IgG (monoclonal antibody) was recovered and used as a purified antibody.
- the purified antibody obtained was dialyzed against 100 times the amount of PBS to replace the buffer.
- the isotypes of the 35 anti-NS3 monoclonal antibodies listed in Tables 2 to 5 were identified using an isotype identification kit (Bethyl Laboratories) according to the attached protocol.
- the cells were washed with PBS and replaced with serum-free medium supplemented with recombinant NS3 so that the final concentration was 100 ⁇ g / mL.
- the hybridoma culture supernatant (serum free) containing anti-NS3 monoclonal antibody of known concentration was added to the serum-free medium supplemented with recombinant NS3 so that the final concentration would be 1 ⁇ g / mL or 0.1 ⁇ g / mL.
- a serum-free medium supplemented with recombinant NS3 without an anti-NS3 monoclonal antibody was also prepared.
- NS3 protease and type I by each anti-NS3 monoclonal antibody were converted by converting the obtained measurement values as 100 in the serum-free medium (medium not added with anti-NS3 monoclonal antibody) to which only recombinant NS3 was added.
- the degree of inhibition of binding to TGF- ⁇ receptor was calculated.
- the obtained results are shown in the A column of Tables 2 and 3.
- clones that showed activity (inhibition of binding) in this reporter assay or reactions to antigens by the immunoprecipitation ELISA method were used. It is a clone showing sex (IP is 10% or more).
- Example 10 Inhibition of collagen production promoting ability of NS3 protease by antibody against binding site between NS3 protease and TGF- ⁇ receptor Monoclonal antibody against binding site between NS3 protease and TGF- ⁇ receptor (anti-NS3 monoclonal antibody) Human normal hepatocyte-derived cell line Hc cell whether 5 types (clones described in Tables 2 to 5: e1211, e0458, s0647, P3-g0948, P3-g1390) suppress the ability to promote collagen production by NS3 protease It was examined using.
- DMEM Dulbecco's modified Eagle's medium
- EQUITECH-BIO fetal bovine serum
- preservative penicillin-streptomycin-glutamine solution, Invitrogen
- treatment medium supplemented with recombinant NS3 to 100 ⁇ g / ml is further added by 250 ⁇ l / well, and the final concentration of e1211 antibody is 0.01, 0.1, 1, 10 ⁇ g / ml, and other clones are finished.
- the concentration was adjusted to 10 ⁇ g / ml, and the final concentration of recombinant NS3 was adjusted to 50 ⁇ g / ml, and the cells were further cultured at 37 ° C. for 20 hours in the presence of 5% CO 2 .
- cells cultured in a treatment medium containing no anti-NS3 monoclonal antibody and purified recombinant NS3 protease were also prepared as negative controls.
- cells cultured in a treatment medium not containing an anti-NS3 monoclonal antibody and supplemented with purified recombinant NS3 protease to a final concentration of 50 ⁇ g / ml were also prepared as a positive control.
- RT reaction was performed according to the attached document using primeScript (TM) RT reagent Kit (manufactured by TAKARA).
- TM primeScript
- TAKARA Premix Ex Taq
- TM Premix Ex Taq
- the collagen expression level of Hc cells was increased about twice by NS3 protease (see negative control and positive control), but the ability of NS3 protease to promote collagen production is suppressed by anti-NS3 monoclonal antibody. It became clear. Of the five anti-NS3 monoclonal antibodies specifically examined, e1211 showed inhibitory activity from the lowest concentration.
- the present invention it is possible to provide a compound capable of suppressing the activation of TGF- ⁇ receptor by HCV, and a screening method thereof, and thus to hepatitis C virus. It becomes possible to provide a composition for preventing or treating a disease caused by the disease.
- the compound of the present invention and the pharmaceutical composition containing it as an active ingredient have an effect based on an action mechanism of inhibiting the binding between NS3 protease and type I TGF- ⁇ receptor, PEG interferon
- hepatitis C virus such as ribavirin, NS3 protease activity inhibitor, and the like
- the pharmaceutical composition of the present invention in combination with these current or developing therapeutic agents, the progression of the pathological condition caused by hepatitis C virus is suppressed and the virus is eliminated while the concentration of each pharmaceutical used is kept low. It is also possible to cure liver diseases caused by hepatitis C virus.
Abstract
Description
(1)NS3プロテアーゼとI型TGF-β受容体との結合を抑制することにより、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する活性を有する化合物。
(2)NS3プロテアーゼ又はI型TGF-β受容体に対して結合活性を有する、(1)に記載の化合物。
(3)配列番号:1~6のいずれかに記載のアミノ酸配列からなるペプチドに対して結合活性を有する、(2)に記載の化合物。
(4)NS3プロテアーゼ又はI型TGF-β受容体に対する抗体である、(1)~(3)のいずれかに記載の化合物。
(5)(1)~(4)のいずれかに記載の化合物を有効成分として含有する、C型肝炎ウィルスに起因する疾患の予防又は治療のための組成物。
(6)以下の(a)~(c)の工程を含む、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する活性を有する化合物のスクリーニング方法
(a)被検化合物の存在下で、NS3プロテアーゼとI型TGF-β受容体とを接触させる工程、
(b)NS3プロテアーゼとI型TGF-β受容体との結合を検出する工程、
(c)前記結合を抑制する化合物を選択する工程。
(7)以下の(a)~(c)の工程を含む、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する活性を有する化合物のスクリーニング方法
(a)被検化合物の存在下で、NS3プロテアーゼとI型TGF-β受容体とを接触させる工程、
(b)NS3プロテアーゼによるTGF-β受容体の活性化を検出する工程、
(c)前記活性化を抑制する化合物を選択する工程。
(8)NS3プロテアーゼとI型TGF-β受容体との結合を抑制することを特徴とする、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する方法。
(9)NS3プロテアーゼとI型TGF-β受容体との結合を抑制することを特徴とする、C型肝炎ウィルスに起因する疾患の予防又は治療のための方法。
TGRDKNQVEGEVQVVSTATQS(配列番号:1)
TNVDQDLVGWPAPPGARSLTP(配列番号:2)
RGDNRGSLLSPRPVSYLKGSS(配列番号:3)
FVSVTETTDKVIHNSM(配列番号:4)
IAEIDLIPRDRPFV(配列番号:5)
CAPSSKTGSVTTTY(配列番号:6)
なお、配列番号1~3に記載のアミノ酸配列はNS3プロテアーゼ側の結合サイトを示し、配列番号4~6に記載のアミノ酸配列はI型TGF-β受容体側の結合サイトを示すものである。
(a)被検化合物の存在下で、NS3プロテアーゼとI型TGF-β受容体とを接触させる工程、
(b)NS3プロテアーゼとI型TGF-β受容体との結合を検出する工程、
(c)前記結合を抑制する化合物を選択する工程。
(a)被検化合物の存在下で、NS3プロテアーゼとI型TGF-β受容体とを接触させる工程、
(b)NS3プロテアーゼによるTGF-β受容体の活性化を検出する工程、
(c)前記結合を抑制する化合物を選択する工程。
先ず、HCV由来のNS3プロテアーゼドメイン領域(GenBank ACCESSION No.AAB27127.1で特定されるタンパク質中の1027~1445アミノ酸)のN末端(当該タンパク質中の1027~1206アミノ酸)にHCV由来のNS4Aの一部配列(GenBank ACCESSION No.AAB27127.1で特定されるタンパク質中の1678~1690アミノ酸、GSVVIVGRIILSG)をリンカー(SGS)を介して融合させたタンパク質、scNS4A-NS3 protease(Protein Sci.、1998年、7巻、10号、2143~2149ページ 参照、配列番号:7に記載のアミノ酸配列からなるタンパク質)をコードする遺伝子を挿入したプラスミドベクターpET32a(+)(Novagen社製)でトランスフォームした大腸菌KRX株を培養した。そして、IPTG(イソプロピル-β-チオガラクトピラノシド)でpET32a(+)ベクター由来のtrx-His-Sタグが付加している目的タンパク質の発現を誘導後、菌体を回収した。回収した菌体は、バッファー(20mM Tris-HCl[pH8.0],300mM NaCl,4mM MgCl2,10% glycerol,1mM DTT,0.1% n-ctyl-β-o-glucopyranoside)に懸濁した後、超音波で破砕して遠心し、分離した上清を0.45μmポアサイズのフィルターで濾過した。濾過した上清をHisTrap HPカラム(GEヘルスケア社製)でアフィニティ精製し、HiPrep26/60Desaltingカラム(GEヘルスケア社製)でバッファー(20mM Tris-HCl[pH8.0],300mM NaCl,0.1mM CaCl2,2mM DTT)に交換後、LPSを除くために、EndoTrap Blueカラム(GEヘルスケア社製)に通した後、回収したフロースルーを精製組換えNS3タンパクとして本実施例に供した。
先ず、HCV由来のNS3プロテアーゼとTGF-βシグナル伝達との関連の有無を調べるために、精製組換えNS3タンパクのTGF-β2様抗原活性をTGF-β2 Emax(R) ImmunoAssay System(Promega社製)を用いてELISA法により検討した。すなわち、キットに添付のTGF-βコート抗体を炭酸緩衝液(pH9.2)にて1/1000に希釈し、96穴ELISA用プレート(NUNC社製)に100μl/穴ずつ加え、4℃で一晩静置してプレートをコートした。コートしたプレートを0.05% Tween20を含むリン酸緩衝液(以下、「PBST」とも称する)にて洗浄した後、キットに添付のTGF-βブロッキング溶液を270μl/穴ずつ加え、37℃で35分間静置してブロッキングを行った。次にブロッキング処理したプレートをPBSTにて洗浄し、調製したTGF-β2スタンダード溶液及び前記精製組換えNS3タンパク溶液を加え、4℃で一晩静置した。なお、精製組換えNS3タンパクはキットに添付のTGF-βサンプル希釈溶液により終濃度2.5、5、10、20μg/mlになるように希釈し、ELISAの検体として、各々100μl/穴ずつ添加した。検体を添加したプレートをPBSTにて洗浄した後、TGF-βサンプル希釈溶液にて1/2000に希釈した抗TGF-β2ポリクローナル抗体を100μl/穴ずつ加え、室温で2時間振盪した。そして、ポリクローナル抗体を添加したプレートをPBSTにて洗浄した後、TGF-βサンプル希釈溶液にて1/100に希釈したペルオキシダーゼ標識TGF-βを100μl/穴ずつ加え、室温で2時間静置した。さらにTGF-βを添加したプレートをPBSTにて洗浄後、ペルオキシダーゼ基質用発色基質としてTMB(テトラメチルベンチジン)溶液を100μl/穴ずつ加え、室温で最大15分間発色させ、十分な発色が得られた時点で1mol/lの塩酸を等量加えて反応を停止させた。その後、450nmの波長でプレート内における各穴の吸光度を測定した。そして、TGF-β2 Standardを用いて検量線を描き、精製組換えNS3タンパクのTGF-β2様抗原活性を活性型TGF-β2量に換算して求めた。得られた結果を図1に示す。
次に、HCV由来のNS3プロテアーゼが、細胞内においてTGF-βシグナルを活性化することが可能であるのかをリポーターアッセイにて調べた。すなわち、精製組換えNS3タンパクのTGF-β2様活性を、ルシフェラーゼ遺伝子の上流にTGF-β標的遺伝子の転写に重要な転写因子SmadのDNA結合配列(CAGA)を9つ挿入したpGLレポータープラスミド(Promega社製)をミンク肺上皮細胞(CCL64細胞)に導入し樹立した細胞株(以下、「×9CAGA/CCL64細胞」とも称する)を用いて検討した。具体的には、10%胎児ウシ血清(EQUITECH-BIO社製)と1%防腐剤(ペニシリン-ストレプトマイシン-グルタミン溶液、Invitrogen社製)を添加したダルベッコ変法イーグル培地(DMEM、Invitrogen社製)(以下、「培養培地」とも称する)に×9CAGA/CCL64細胞を2×105個/mlになるよう懸濁し、96穴の細胞培養用プレート(TPP社製)に100μl/穴ずつ播種し、5%CO2存在下、37℃で一晩培養した。そして、プレートから培養上清を吸引除去し、カルシウム-マグネシウム含有リン酸緩衝液(以下、「PBS(+)」とも称する)にて細胞を洗浄した後、0.1%ウシ血清アルブミン(EQUITECH-BIO社製)と前記1%防腐剤を含むDMEM(以下、「処理培地」とも称する)に終濃度が12.5、25、50、100μg/mlになるように精製組換えNS3タンパクを加えたものを100μl加え、5%CO2存在下、37℃で更に20時間培養した。その翌日、プレート内の細胞中のルシフェラーゼ活性をルシフェラーゼアッセイシステム(Promega社製)を用いて、そのキットの添付文書に従って測定した。すなわち、培養上清を吸引除去後、細胞をPBS(+)にて洗浄し、キットに添付のPassive Lysis Buffer(5×)を1倍に希釈した液を各穴に20μl加え、室温にて15分間振盪して細胞を溶解した。その間に、予め1バイアルのLuciferase Assay Substrateを10mlのLuciferase Assay Reagent II(以下、「LARII溶液」とも称する)にて溶解し、96穴のルシフェラーゼアッセイ用プレート(Costar社製)に100μl/穴ずつ分注した。次に、細胞溶解液を10μlずつ予め分注したLARII溶液に加え、ピペッティングにより混和した後、ルミノメーター(製品名:ARVO(TM)、PerkinElmer社製)にて発光強度を測定した。各処理につき例数は3とし、未処理の細胞のリポーター活性を1として、精製組換えNS3タンパク処理細胞の活性量を相対値として求めた。得られた結果を図2に示す。
TGF-βは、肝臓の星細胞に働きかけて、星細胞からのコラーゲンの異常産生を促すことで、肝線維化を引き起こすことが知られている。そこで、次にNS3プロテアーゼとコラーゲン産生促進との関連性について調べた。すなわち、Wistar系ラット(SPF下で飼育、雄性、15週齢)をペントバルビタール麻酔下にて開腹後、門脈にカテーテルを挿入し、脱血用洗浄液、0.06%プロナーゼ溶液(Carbiochem社製)、0.03%コラゲナーゼ(和光純薬社製)溶液の順に潅流を行った。その後、肝臓を摘出し、0.057%プロナーゼ、0.057%コラゲナーゼを含む肝星細胞分離用緩衝液に2mg/ml DNaseI(Roche Diagnostics社製)を1ml加えた溶液中にて、30分間、36℃の温浴中でインキュベーションした。なお、その間、1N NaOHにて溶解液中のpHは7.2~7.4に保った。次に、この肝組織溶解液をメッシュを通してろ過し、肝星細胞分離用緩衝液を加えて全量を150mlとして50mlポリプロピレンチューブ3本に分注し、4℃で2000rpm、8分間遠心した。さらにポリプロピレンチューブ中の上清を吸引除去し、各チューブにDNaseI溶液を0.2mlずつ加え、ゲイ平衝塩類溶液を加えてピペッティングにより混和し、全量を100mlとして50mlポリプロピレンチューブ2本に再度分注し、4℃で2000rpm、8分間遠心した。ポリプロピレンチューブ中の上清を吸引除去後、各チューブに0.2mlのDNaseI溶液を加え、ゲイ平衝塩類溶液を加えてピペッティングにより混和した後、全量を67.5mlとしてビーカーに移し、0.22μmフィルターを通して滅菌したNycodenz(SIGMA社製)溶液(終濃度7.75%)を27ml加えて混和した。この細胞溶液を15mlチューブ8本に分注し、1mlのゲイ平衝塩類溶液を重層し、4℃で3200rpm、15分間遠心して肝星細胞を分離した。分離した肝星細胞を10%胎児ウシ血清(EQUITECH-BIO社製)と1%防腐剤(Invitrogen社製)を含むDMEM培地中に1×105個/mlの濃度になるよう懸濁し、直径6cmの細胞培養用ディッシュ(CORNING社製)に3mlずつ播種し、37℃、5%CO2存在下にて一晩培養した。翌日、培地を交換すると同時に精製組換えNS3タンパクを終濃度20μg/mlになるように処理を開始し、隔日で培地を交換して7日間培養した。また、コントロールとして精製組換えNS3タンパク等で処理していない細胞(未処置細胞)も用意し、隔日で培地を交換して7日間培養した。7日後、これらの細胞からRNA精製キットRNeasy Micro Kit(QIAGEN社製)を用いてmRNAを抽出し、分光光度計(Nano Drop)を用いて260nmにおける吸光度を測定し、濃度を求めた。次いでこのmRNAをテンプレートとして、PrimeScript(TM) RT reagent Kit(TAKARA社製)を用い、添付文書に従ってRT反応を行った。さらにSYBR(R) Premix EX Taq(TM)II(TAKARA社製)を用い、添付文書に従って反応液を調製し、コラーゲン(Collagen1 α1)、α平滑筋アクチン(αSMA)、TGF-β1、及び内部標準であるGAPDHの各プライマー(インビトロジェン社製)を用いてPCR反応を行い、mRNA発現量を未処置細胞のそれらと比較した。得られた結果を図3に示す。
TGF-βは、I型TGF-β受容体及びII型TGF-β受容体と複合体を形成してシグナルを伝えることが知られている(「Joan Massague、Mol Cell、2008年2月1日、29巻、2号、149-150ページ」参照)。従って、NS3プロテアーゼはTGF-β様活性を有するということから、分子生物学的に、NS3プロテアーゼとI型TGF-β受容体との間において分子間相互作用が働き、これらの複合体が形成されていることが示唆される。そこで、タンパク質間ドッキングシミュレーションを実施し、これらのタンパク質間相互作用の有無を検討した。また、NS3プロテアーゼ内及びTGF-β受容体内のどのアミノ酸残基がかかる相互作用に関与しているのかも予測した。
前記予測結合サイト(配列番号1~6に記載のアミノ酸配列)を介して、NS3プロテアーゼとI型TGF-β受容体とが結合し、NS3プロテアーゼによるTGF-β受容体の活性化が生じていることを実証するため、以下の通り、前記予測結合サイトのアミノ酸配列に基づいて合成ペプチド等を調製し、かかる合成ペプチド等を抗原とするポリクローナル抗体を作製した。
NS3プロテアーゼ又はI型TGF-β受容体における予測結合サイト(配列番号1~6に記載のアミノ酸配列)をターゲットとしたポリクローナル抗体作製に用いた合成ペプチドの配列の一覧を表1に示す。なお、表1中、「NH2」及び「COOH」は各合成ペプチドのN末端側及びC末端側を各々示す。またN末端側の「C」は後述のmcKLHを各合成ペプチドに結合させるために必要なシステイン残基を示す。さらに「miniPEG」はスペーサー分子として挿入し、抗原ペプチドとキャリアータンパクとの立体障害を改善する目的で付与する平均分子量6,000のポリエチレングリコールを示す。
精製組換えNS3タンパクを用いて2把のウサギ(メス、日本白色種(ヘルシー))を免疫した。すなわち、初回は一匹あたりフロイント(Freund)の完全アジュバント中の150μgの精製組換えNS3タンパクを皮内投与し、以降はアジュバント中の300μgの精製組換えNS3タンパクを皮内に、または食塩加リン酸バッファー(PBS)中の50μgの精製組換えNS3タンパクを耳介静脈に投与した。免疫はフロイントの完全アジュバントを用いた場合は2週間隔、PBSを用いた場合では1週間間隔とした。そして、最終免疫の7日後に心臓より全採血を行った。回収した血液は4℃にて一晩静置後、遠心分離にて血清成分を分離回収し、抗血清とした。得られた抗血清はアジ化ナトリウムを0.1%濃度で添加し、4℃にて保管した。また、血清中の抗体価の決定はELISA法を用いて行った。この検定において、マイクロタイタープレートを先ずPBSで希釈したリコンビナント蛋白質でコートした。次いで0.2% Tween20/PBSで洗浄及びブロッキングしたウェルに連続希釈した血清を添加しインキュベートして、免疫原に対する抗体をウサギ免疫グロブリンに対するパーオキシターゼ結合抗体で検出した。
KLHと結合した各合成ペプチドを用いて、各々2把のウサギ(メス、日本白色種(ヘルシー))を皮内投与により免疫した。すなわち、初回は一匹あたりFreundの完全アジュバント中の300ugのKLH結合合成ペプチドを投与し、以降はアジュバント中の300ugのKLH結合合成ペプチドを投与した。免疫はそれぞれ2週間隔で行った。最終免疫の7日後に心臓より全採血を行った。回収した血液は4℃にて一晩静置後、遠心分離にて血清成分を分離回収し、抗血清とした。得られた抗血清はアジ化ナトリウムを0.1%濃度で添加し、4℃にて保管した。血清中の抗体価の決定はELISA法を用いて行った。この検定において、マイクロタイタープレートを先ずPBSで希釈した合成ペプチドでコートした。次いで0.2% Tween20/PBSで洗浄してブロッキングしたウェルに連続希釈した血清を添加しインキュベートした。合成ペプチドに対する抗体をウサギ免疫グロブリンに対するパーオキシターゼ結合抗体で検出した。
前記の通りに作製した精製組換えNS3タンパクに対する抗血清を等量の結合バッファーで希釈したのち、フィルターでろ過して不溶物を除去した。常法に従ってProteinA-sepharose4B(GEヘルスケア社製)を充填したカラムに通して抗体成分を吸着させたのち、非特異吸着分を除去してから酸性条件におくことで遊離した成分を回収し、精製ポリクローナル抗体とした。得られた精製抗体は100倍量のPBS緩衝液に透析し置換した後、終濃度0.1%となるようにアジ化ナトリウムを添加した。
実施例2と同様にして、培養培地に×9CAGA/CCL64細胞を2×105個/mlの濃度で懸濁し、96穴細胞培養用プレートに100μl/穴ずつ播種し、5%CO2存在下、37℃で一晩培養した。培養後プレート内の培養上清を吸引除去し、PBS(+)にて細胞を洗浄した後、「NS3プロテアーゼと抗体との混合溶液」又はNS3プロテアーゼのみ含有する溶液を100μl/穴ずつ添加し、5%CO2存在下、37℃で更に20時間培養した後、実施例2と同様にして、各抗体存在下におけるリポーター活性を求めた。得られた結果は図7に示す。なお、図7~14中、「コントロール」は、タンパク質(精製組換えNS3タンパク及び組換えヒトTGF-β2)及び抗体を培地中に添加せず、接触させない条件下で培養した細胞のリポーター活性の値を示し、「兎1」及び「兎2」という表記は、同一抗原によって免疫されているが、異なるウサギ個体から抽出されたポリクローナル抗体であることを示す。
実施例2と同様に×9CAGA/CCL64細胞を96穴細胞培養用プレートに播種し、一晩培養した。終濃度が100μg/mlになるように精製組換えNS3タンパクを加えた処理培地に、抗NS3抗体1種、TGF-β2 Emax(R) ImmunoAssay System(Promega社製)に添付されている抗TGF-β2ポリクローナル抗体、又は、2種類の無免疫ウサギ抗体を各々終濃度がおよそ10μg/mlなるように加えたものを調製し、4℃で1時間プレインキュベーションした。そして、一晩培養後のプレートから培養上清を吸引除去し、PBS(+)にて洗浄した後、プレインキュベーションした処理培地を各々100μl/穴ずつ加え、5%CO2存在下、37℃で更に20時間培養した。その後、実施例2と同様にして、各抗体存在下におけるリポーター活性を求めた。得られた結果は図12に示す。
(実施例8) NS3プロテアーゼとTGF-β受容体との結合部位に対する抗体による、TGF-β2の活性に及ぼす影響の検討
実施例2と同様にして、×9CAGA/CCL64細胞を96穴細胞培養用プレートに播種し、一晩培養した。終濃度500μg/mlになるように組換えヒトTGF-β2(PeproTech社製)を加えた処理培地に、抗NS3抗体、抗組換えNS3抗体、陽性コントロールとして抗TGF-β2抗体、又は、陰性コントロールとして抗TGF-β2-LAP抗体を各々終濃度が10μg/mlになるように加えたものを調製し、4℃で1時間プレインキュベーションした。そして、前記一晩培養後のプレートから培養上清を吸引除去し、細胞をPBS(+)にて洗浄した後、プレインキュベーションした処理培地を各々100μl/穴ずつ加え、5%CO2存在下、37℃で更に20時間培養した。その後、実施例2と同様にして、各抗体存在下におけるリポーター活性を求めた。得られた結果は図13に示す。
<モノクローナル抗体作製用NS3タンパクの精製>
先ず、scNS4A-NS3 proteaseをコードする遺伝子を挿入したプラスミドベクターpET32a(+)をpMINORプラスミドを導入した大腸菌KRX株に組み込み、培養した。なお、scNS4A-NS3 proteaseについては、Protein Sci.、1998年、7巻、10号、2143~2149ページ 参照のこと。またpMINORプラスミドについては、J.Struct.Func.Genom.、2006年、7巻、31~36ページ 参照のこと。
次に、リコンビナントNS3をメスのBALB/cマウスに腹腔注射して免疫を行った。初回免疫には一匹あたりFreundの完全アジュバント中の50μgのリコンビナントNS3を投与し、以降の免疫には水中油型エマルジョンアジュバント中の50μgのリコンビナントNS3を投与した。免疫はそれぞれ3週間隔で行った。最終免疫は50μgのリコンビナントNS3を含む食塩加リン酸バッファー(PBS)を投与して行った。最終免疫の3日後、血清中の抗体価が高い個体の脾臓細胞についてポリエチレングリコール4000を用いてP3.X63-Ag8.653マウス骨髄腫細胞との細胞融合処理を行った。ハイブリドーマ細胞は公知のKohler及びMilsteinの技術を用いてHAT培地中で選択した。
先ず、表1に示したペプチド NS-1~NS-3(株式会社バイオマトリックス研究所)をFmoc固相合成法にて合成した。ペプチド最終産物は95%TFAを含むクリベージカクテルにて脱保護及びレジンからの切り出しを行い調製した。また、合成ペプチドをスクリーニングに用いるために、Imject OVA(Thermo SCIENTIFIC Pierce Protein Research Products社製)をキャリアタンパク質として合成ペプチドに結合させた。
ELISA法及び免疫沈降を利用した免疫沈降ELISA法を用いて、リコンビナントNS3に反応するハイブリドーマを選別した。すなわち、先ずリコンビナントNS3(終濃度1μg/mL、50mM 炭酸バッファーにて調製)を疎/親水性分子吸着性96ウェルマイクロプレートに室温で1時間固定した。次いでプレートをTBS/0.05% Tween20で洗浄した後、ウェル表面のフリーの吸着部分を1% スキムミルク/食塩加リン酸バッファーを用いて室温で1時間ブロックし、再びTBS/0.05% Tween20で洗浄した。ハイブリドーマ培養上清をウェルに加え、室温で1時間インキュベートした。その後、プレートをTBS/0.05% Tween20で洗浄し、次いで1% スキムミルク/食塩加リン酸バッファーで希釈したパーオキシターゼ結合マウス抗IgG抗体をウェルに加え、室温で1時間インキュベートした。インキュベート後、プレートをTBS/0.05% Tween20で洗浄し、基質溶液(0.05% o-フェニレンジアミン/クエン酸バッファー(pH5)/0.03% H2O2)をウェルに加え、発色反応を行った。基質溶液添加の10分後、2N 硫酸で反応を停止し、分光光度計で490nmの吸光度を測定した。また、比較対象として、抗NS1ペプチドポリクローナル抗体(実施例5において調製した、表1に記載のNS-1に結合するポリクローナル抗体)、抗NS3ペプチドポリクローナル抗体(実施例5において調製した、表1に記載のNS-3に結合するポリクローナル抗体)、抗rikenNS3ペプチドポリクローナル抗体(実施例5において調製した、精製組換えNS3タンパクを抗原とするポリクローナル抗体)についても同様に評価した(以下、同様)。得られた結果を表2及び3のC欄に示す。
上記の方法にて選抜されたリコンビナントNS3に陽性を示すハイブリドーマについては、それらが産生する抗NS3モノクローナル抗体の前記合成ペプチド(NS-1~NS-3)に対する反応性をELISA法により調べ、これら抗体が認識するエピトープについて検討した。すなわち、先ずOVAを結合したNS-1、NS-2、NS-3の合成ペプチド(終濃度0.5μg/mL、50mM 炭酸バッファーにて調製)それぞれを疎/親水性分子吸着性96ウェルマイクロプレートに室温で1時間固定した。次いでプレートをTBS/0.05% Tween20で洗浄した後、ウェル表面のフリーの吸着部分を1% スキムミルク/食塩加リン酸バッファーを用いて室温で1時間ブロックし、再びTBS/0.05% Tween20で洗浄した。そして、ハイブリドーマ培養上清をウェルに加え、室温で1時間インキュベートした。その後、プレートをTBS/0.05% Tween20で洗浄し、次いで1% スキムミルク/食塩加リン酸バッファーで希釈したパーオキシターゼ結合マウス抗IgG抗体をウェルに加え、室温で1時間インキュベートした。インキュベート後、プレートをTBS/0.05% Tween20で洗浄し、基質溶液(0.05% o-フェニレンジアミン/クエン酸バッファー(pH5)/0.03% H2O2)をウェルに加え、発色反応を行った。基質溶液を添加してから10~15分後に、2N 硫酸を添加することにより反応を停止させ、分光光度計で490nmの吸光度を測定した。得られた結果を表4及び5のE~G欄に示す。
また、リコンビナントNS3に陽性を示すハイブリドーマについては、それらが産生する抗NS3モノクローナル抗体のNS3ペプチド(市販品)に対する反応性をもELISA法により調べた。すなわち、先ずNS3-NS4A(HCV),(recombinant)(His-tag)(ALEXIS社製)(終濃度0.5μg/mL、50mM 炭酸バッファーにて調製)を疎/親水性分子吸着性96ウェルマイクロプレートに室温で1時間固定した。次いでプレートをTBS/0.05% Tween20で洗浄した後、ウェル表面のフリーの吸着部分を1% スキムミルク/食塩加リン酸バッファーを用いて室温で1時間ブロックし、再びTBS/0.05% Tween20で洗浄した。ハイブリドーマ培養上清をウェルに加え、室温で1時間インキュベートした。その後、プレートをTBS/0.05% Tween20で洗浄し、次いで1% スキムミルク/食塩加リン酸バッファーで希釈したパーオキシターゼ結合マウス抗IgG抗体をウェルに加え、室温で1時間インキュベートした。インキュベート後、プレートをTBS/0.05% Tween20で洗浄し、基質溶液(0.05% o-フェニレンジアミン/クエン酸バッファー(pH5)/0.03% H2O2)をウェルに加え、発色反応を行った。基質溶液を添加してから10~15分後に、2N 硫酸を添加することにより反応を停止させ、分光光度計で490nmの吸光度を測定した。得られた結果を表2及び3のD欄に示す。
公知の方法に則り、前記ハイブリドーマの培養上清を0.22μmフィルターで濾過して培養上清から不溶物を除去した。次いで常法に則り、ProteinG-sepharose4B(GE Helthecare社製)を充填したカラムに培養上清を通して抗体成分を吸着させた後、カラム洗浄により非特異吸着成分を除去してから、酸性条件下で吸着したIgGを遊離させた。遊離したIgG(モノクローナル抗体)を回収し、精製抗体とした。また、得られた精製抗体は100倍量のPBSで透析し、バッファーを置換した。
リコンビナントNS3に陽性を示すハイブリドーマについては、それらが産生する抗NS3モノクローナル抗体による、NS3プロテアーゼとI型TGF-β受容体との結合(NS3プロテアーゼによるTGF-β様活性)の抑制について検討した。すなわち、先ずII型TGFβ受容体からのシグナル増強によりルシフェラーゼ遺伝子の発現が亢進するようにデザインされたリポーター細胞(×9CAGA/CCL64細胞)を96ウェルマイクロプレートにコンフルエントになるように播種し、37℃、5%CO2の条件で一晩培養した。細胞は培養上清除去後にPBSで洗浄し、終濃度が100μg/mLとなるようにリコンビナントNS3を加えた無血清培地に置換した。その際、リコンビナントNS3を添加した無血清培地には濃度既知の抗NS3モノクローナル抗体を含むハイブリドーマ培養上清(血清不含)を終濃度が1μg/mL又は0.1μg/mLとなるように加えた。また、リコンビナントNS3を添加した無血清培地に抗NS3モノクローナル抗体を添加しないものも用意した。そして、このように調製した細胞を37℃、5%CO2で一晩培養した後、培養上清の除去とPBSによる洗浄とを行った。次いで、洗浄後の細胞にPassive Lysis buffer(Promega社製)を30μL添加し、室温で15分間激しく撹拌して細胞を溶解させた。標準的なプロトコールに従って調製したLuciferase Assay substrate(Promega社製)をluciferase assay用96ウェルマイクロプレートに1ウェルあたり100μLずつ分注し、さらに、これらに細胞溶解液15μLを添加した。そして、細胞溶解液を添加したマイクロプレートは直ちに蛍光プレートリーダーにてluciferin蛍光シグナルを測定した。また、得られた各測定値をリコンビナントNS3のみを添加した無血清培地(抗NS3モノクローナル抗体を添加しない培地)における測定値を100として換算することにより、各抗NS3モノクローナル抗体によるNS3プロテアーゼとI型TGF-β受容体との結合の抑制の程度(結合抑制%)を算出した。得られた結果を表2及び3のA欄に示す。なお、表2~5に示した35種のクローンは、今回作製した抗NS3モノクローナル抗体のうち、このリポーターアッセイにて活性(結合抑制)を示したクローン又は前記免疫沈降ELISA法にて抗原に対する反応性(IPが10%以上)を示したクローンである。
NS3プロテアーゼとTGF-β受容体との結合部位に対するモノクローナル抗体(抗NS3モノクローナル抗体)5種(表2~5に記載のクローン:e1211、e0458、s0647、P3-g0948、P3-g1390)が、NS3プロテアーゼによるコラーゲン産生促進能を抑制するかどうかをヒト正常肝細胞由来細胞株Hc細胞を用いて検討した。10%胎児ウシ血清(EQUITECH-BIO社製)と1%防腐剤(ペニシリンーストレプトマイシンーグルタミン溶液、Invitrogen社製)を添加したダルベッコ変法イーグル培地(DMEM、Invitrogen社製)にHc細胞を4×105個/mlになるよう懸濁し、24穴の細胞培養用プレート(TPP社製)に500μl/穴ずつ播種し、5%CO2存在下、37℃で一晩培養した。そして、プレートから培養上清を吸引除去し、カルシウム-マグネシウム含有リン酸緩衝液にて細胞を洗浄した後、終濃度が0.02、0.2、2、20μg/mlになるように抗NS3モノクローナル抗体clone No.e1211、又は他のクローンについては20μg/mlになるように抗NS3モノクローナル抗体を添加した0.1%ウシ血清アルブミン(EQUITECH-BIO社製)と前記1%防腐剤とを含むDMEM(以下、「処理培地」とも称する)を250μl/穴ずつ加え、5%CO2存在下、37℃で1時間培養した。その後、100μg/mlになるようにリコンビナントNS3を添加した処理培地を更に250μl/穴ずつ加え、e1211抗体の終濃度が0.01、0.1、1、10μg/ml、他のクローンについては終濃度10μg/mlになるように、かつリコンビナントNS3の終濃度が50μg/mlになるようにし、5%CO2存在下、37℃で更に20時間培養した。また、抗NS3モノクローナル抗体及び精製組換えNS3プロテアーゼを含まない処理培地にて培養した細胞(無処理細胞)も陰性対照として調製した。さらに、抗NS3モノクローナル抗体を含まず、精製組換えNS3プロテアーゼを終濃度が50μg/mlになるように添加した処理培地にて培養した細胞も陽性対照として調製した。
<223> 組換えタンパク質の配列
Claims (9)
- NS3プロテアーゼとI型TGF-β受容体との結合を抑制することにより、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する活性を有する化合物。
- NS3プロテアーゼ又はI型TGF-β受容体に対して結合活性を有する、請求項1に記載の化合物。
- 配列番号:1~6のいずれかに記載のアミノ酸配列からなるペプチドに対して結合活性を有する、請求項2に記載の化合物。
- NS3プロテアーゼ又はI型TGF-β受容体に対する抗体である、請求項1~3のうちのいずれか一項に記載の化合物。
- 請求項1~4のうちのいずれか一項に記載の化合物を有効成分として含有する、C型肝炎ウィルスに起因する疾患の予防又は治療のための組成物。
- 以下の(a)~(c)の工程を含む、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する活性を有する化合物のスクリーニング方法
(a)被検化合物の存在下で、NS3プロテアーゼとI型TGF-β受容体とを接触させる工程、
(b)NS3プロテアーゼとI型TGF-β受容体との結合を検出する工程、
(c)前記結合を抑制する化合物を選択する工程。 - 以下の(a)~(c)の工程を含む、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する活性を有する化合物のスクリーニング方法
(a)被検化合物の存在下で、NS3プロテアーゼとI型TGF-β受容体とを接触させる工程、
(b)NS3プロテアーゼによるTGF-β受容体の活性化を検出する工程、
(c)前記活性化を抑制する化合物を選択する工程。 - NS3プロテアーゼとI型TGF-β受容体との結合を抑制することを特徴とする、NS3プロテアーゼによるTGF-β受容体の活性化を抑制する方法。
- NS3プロテアーゼとI型TGF-β受容体との結合を抑制することを特徴とする、C型肝炎ウィルスに起因する疾患の予防又は治療のための方法。
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CN201180042019.7A CN103119066B (zh) | 2010-08-30 | 2011-08-30 | 具有抑制TGF-β受体活化的活性的化合物、该化合物的筛选方法、以及用于预防或治疗由丙型肝炎病毒引起的疾病的组合物 |
JP2012531895A JP5975399B2 (ja) | 2010-08-30 | 2011-08-30 | TGF−β受容体の活性化を抑制する活性を有する化合物、そのスクリーニング方法、並びにC型肝炎ウィルスに起因する疾患の予防又は治療のための組成物 |
US13/819,979 US8951521B2 (en) | 2010-08-30 | 2011-08-30 | Compounds having activity of suppressing activation of TGF-β receptor, method for screening of the compounds, and composition for preventing or treating disease caused by hepatitis C virus |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11130802B2 (en) | 2018-10-10 | 2021-09-28 | Tilos Therapeutics, Inc. | Anti-lap antibody variants |
US11230601B2 (en) | 2017-10-10 | 2022-01-25 | Tilos Therapeutics, Inc. | Methods of using anti-lap antibodies |
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JPH11127861A (ja) * | 1997-10-29 | 1999-05-18 | Japan Energy Corp | C型肝炎ウイルス由来のセリンプロテアーゼに対する中和抗体部分ペプチド |
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2011
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- 2011-08-30 JP JP2012531895A patent/JP5975399B2/ja not_active Expired - Fee Related
- 2011-08-30 US US13/819,979 patent/US8951521B2/en not_active Expired - Fee Related
- 2011-08-30 CN CN201180042019.7A patent/CN103119066B/zh not_active Expired - Fee Related
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11230601B2 (en) | 2017-10-10 | 2022-01-25 | Tilos Therapeutics, Inc. | Methods of using anti-lap antibodies |
US11130802B2 (en) | 2018-10-10 | 2021-09-28 | Tilos Therapeutics, Inc. | Anti-lap antibody variants |
Also Published As
Publication number | Publication date |
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CN103119066B (zh) | 2016-04-20 |
US20130244253A1 (en) | 2013-09-19 |
JPWO2012029792A1 (ja) | 2013-10-31 |
US8951521B2 (en) | 2015-02-10 |
CN103119066A (zh) | 2013-05-22 |
JP5975399B2 (ja) | 2016-08-23 |
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