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WO2007043640A1 - Substance à utiliser pour traiter ou prévenir une infection à hcv - Google Patents

Substance à utiliser pour traiter ou prévenir une infection à hcv Download PDF

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Publication number
WO2007043640A1
WO2007043640A1 PCT/JP2006/320440 JP2006320440W WO2007043640A1 WO 2007043640 A1 WO2007043640 A1 WO 2007043640A1 JP 2006320440 W JP2006320440 W JP 2006320440W WO 2007043640 A1 WO2007043640 A1 WO 2007043640A1
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Prior art keywords
peptide
hcv
seq
amino acid
acid sequence
Prior art date
Application number
PCT/JP2006/320440
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English (en)
Japanese (ja)
Inventor
Masayuki Sudo
Hiroshi Sakamoto
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
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Publication date
Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to JP2007539995A priority Critical patent/JPWO2007043640A1/ja
Publication of WO2007043640A1 publication Critical patent/WO2007043640A1/fr

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • 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
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to a drug for treating or preventing HCV infection, which contains a compound that inhibits the binding of sphingomyelin and HCV protein as an active ingredient.
  • the present invention also relates to a method for screening a drug for treating or preventing HCV infection, and a kit for use in the method.
  • HCV was discovered in 1989 as a major causative virus for non-A non-B hepatitis after blood transfusion.
  • HCV is an enveloped RNA virus whose genome consists of single-stranded (+) RNA and is classified into the genus Hepacivirus of the Flaviviridae family.
  • HCV progresses to chronic hepatitis, liver cirrhosis, and liver cancer, where persistent infection is often established even when infected with an adult who has developed an immune system, in order to avoid the host's immune mechanism due to unclear reasons.
  • liver cancer recurrence due to inflammation that continues in non-cancerous areas even after surgery.
  • interferon treatment is known as the only effective treatment for HCV elimination.
  • about 1Z3 of all patients are effective for interferon.
  • the response rate of interferon against HCV genotype lb is very low. Therefore, the development of anti-HCV drugs that can replace or be used in combination with the interface is strongly desired.
  • ribavirin 1— ⁇ -D-ribofuranosyl 1 ⁇ — 1, 2, 4-triazol (Lu-3-Carboxamide) is marketed as a therapeutic agent for hepatitis C in combination with interferon, but there is a need for a new therapeutic agent for hepatitis C whose effectiveness is still low.
  • attempts have been made to eliminate viruses by enhancing the immunity of people such as interferon agonists and interleukin 12 agonists, but no effective drugs have been found yet.
  • Non-Patent Document 1 The mechanism of HCV RNA replication in this system is thought to be identical to the replication of the full-length HCV RNA genome infected with hepatocytes. Therefore, this system can be said to be a cell-based accessory system useful for identifying compounds that inhibit HCV replication.
  • Patent Document 1 International Publication No. W098 / 56755 Pamphlet
  • Patent Document 2 International Publication No. WO04 / 71503 Pamphlet
  • Patent Document 3 International Publication WO05 / 05372 Pamphlet
  • Non-Patent Document 1 Buoy Roman et al., Science, 1999, No. 285, 110-113
  • Patent Document 1 The inventors of the present application disclosed in International Publication No. W098 / 56755 (Patent Document 1), and a series of compounds derived from microorganisms such as the genus Aureobasidium, It was found by the Atsey method that it has high HCV replication inhibitory activity. Permissible literature 2). In addition, the present inventors have found that the compound has minimal in vitro cytotoxicity and is extremely useful as a prophylactic or therapeutic agent for HCV infection, and further constructed a method for synthesizing the compound and derivatives. (Patent Document 3).
  • the inventors of the present invention also found that sphingolipid biosynthesis is involved in HCV infection, and compounds that inhibit the activity and expression of enzymes involved in sphingolipid biosynthesis are extremely useful therapeutic or preventive agents for HCV infection. It was made clear (WO2006 / 16657).
  • Sphingomyelin a sphingolipid
  • rafts on cell membranes, and viruses such as influenza and HIV are replicated via rafts (Takeda M. et al. (2003) P NAS, 100 , 25, Lucero HA, et al. (2004) Archives of Biochemistry and Biophysics, 426, 208, Simons K. (1997) Nature, 387, 569, G.— Z. Leu et al. (2004)) 0 Therefore, the present inventors predicted that HCV is replicated via rafts and attempted to elucidate the detailed mechanism of HCV virus replication via rafts containing sphingolipids.
  • the present invention has been made in view of such a situation, and an object of the present invention is to treat or prevent HCV infectious diseases containing a compound that inhibits the binding of sphingomyelin and HCV protein as an active ingredient. It is to provide a drug to do. Another object of the present invention is to provide a method for screening a drug for treating or preventing HCV infection and a kit for use in the method.
  • the binding site of (sphingomyelin) was identified and the binding ability was examined.
  • the present inventors have succeeded in developing a drug for treating or preventing HCV infection containing a compound that inhibits the binding of sphingomyelin and HCV protein as an active ingredient, thus, the present invention has been completed.
  • the present invention provides the following [1] to [14].
  • a drug for treating or preventing HCV infection comprising as an active ingredient a compound that inhibits the binding of sphingomyelin and HCV protein.
  • [2] Compound ability to inhibit binding of sphingomyelin and HCV protein
  • the drug according to [1] which is a peptide according to the following (a) or (b).
  • [3] Compound ability to inhibit binding of sphingomyelin and HCV protein
  • the drug according to [1] which is an oligonucleotide encoding the peptide according to the following (a) or (b).
  • [4] Compound ability to inhibit binding of sphingomyelin to HCV protein
  • the drug according to [1] which is an antibody that recognizes the peptide according to (a) or (b) below.
  • [6] The drug according to any one of [1] to [5], wherein the HCV infection is hepatitis C, cirrhosis, liver fibrosis, or liver cancer.
  • a screening method for a drug for treating or preventing HCV infection comprising the following steps (A) to (C):
  • a method for screening a drug for treating or preventing HCV infection comprising the following steps (A) to (C):
  • SEQ ID NO: 1 in the amino acid sequence described in any one of 4, 10, or 11 Is a peptide with multiple amino acid substitutions, deletions, additions, and Z or inserted amino acid sequence
  • a method for evaluating the efficacy of a drug for treating or preventing HCV infection comprising the following steps (A) to (C):
  • a method for evaluating the efficacy of a drug for treating or preventing HCV infection comprising the following steps (A) and (B):
  • (B) A step of evaluating the efficacy of the treatment or prevention effect of HCV infection by measuring the binding ability of the peptide described in (a) or (b) above and sphingomyelin.
  • FIG. 1A is a view showing the amino acid sequences of HCV-NS5B sequences E230-G263 and V3 loop.
  • B is a diagram showing the structure of HCV-NS5B.
  • the vertical line is the predicted sphingolipid binding region identified by HCV-NS5B.
  • the diagonal line indicates the sphingolipid binding region of the HIV-1 V3 loop.
  • C shows the helix 'turn' helix motif in HCV-NS5B.
  • FIG. 2 is a diagram showing the results of a binding study of HCV-NS5B sphingo binding domain peptide (NS5B-SBD, SEQ ID NO: 1) and sphingomyelin (SM) using Biacore.
  • A is a diagram showing a sensorgram of NS5B-SB D peptide, and NS5B-SBD peptide was found to bind to sphingomyelin in a concentration-dependent manner.
  • B is a comparison of the binding ability of NS5B-SBD peptide and prion protein (PrP, SEQ ID NO: 2) to sphingomyelin.
  • Sensorgram Shows the RU value at 80 s.
  • FIG. 3 shows the results of measuring HCV replicon inhibitory activity of NS5B sphingo-binding domain peptides and their derivatives.
  • FIG. 4 is a diagram showing a sphingolipid synthetic pathway (synthetic pathway from palmitoyl CoA to sphingomyelin).
  • FIG. 5 is a photograph showing the HCV RNA replication inhibitory activity of myriocin by Northern plot analysis.
  • the horizontal axis represents the concentration of myriocin.
  • FIG. 6 is a photograph showing the HCV RNA replication inhibitory activity of the compound represented by formula (II) by Northern plot analysis.
  • the horizontal axis represents the concentration of the compound represented by the formula ( ⁇ ).
  • FIG. 7 A photograph showing the inhibitory activity of myriocin on HCV protein synthesis by Western plot analysis. The horizontal axis represents the concentration of myriocin.
  • FIG. 8 is a photograph showing the HCV protein synthesis inhibitory activity of the compound represented by formula ( ⁇ ) by Western plot analysis.
  • the horizontal axis represents the concentration of the compound represented by formula (II).
  • FIG. 9 is a graph showing the HCV replicon inhibitory activity of fumosin B1.
  • FIG. 10 is a photograph showing inhibition of protein expression of serine palmitoyltransferase (LCB1) by siRNA.
  • FIG. Ll A graph showing the effects of siRNA on the HCV replicon inhibitory activity and cytotoxicity.
  • FIG. 12 is a graph showing the inhibition of HCV levicon and the toxicity to host cells by the compound represented by formula (II).
  • FIG. 13 is a photograph showing inhibition of HCV-NS3 protein expression by a compound represented by formula (II). After immunostaining, it was observed with a fluorescence microscope. White indicates NS3 protein, gray indicates nuclei stained with hex 33342.
  • FIG. 14 is a photograph showing inhibition of NS3, NS5A, and NS-5B protein expression by a compound represented by formula (II). Each protein was expressed by Western plot analysis.
  • FIG. 15 is a graph showing SPT inhibitory activity of a compound represented by formula (II).
  • FIG. 16 is a photograph showing inhibition of de novo synthesis of ceramide and sphingomyelin by the compound represented by formula (II).
  • FIG. 17 is a photograph showing suppression of HCV replication inhibition of a compound represented by formula (II) by C2-ceramide.
  • FIG. 18 shows inhibition of HCV replication by a raft biosynthesis-related low molecular weight compound.
  • FIG. 19 is a photograph showing the influence of a compound represented by formula (II) on raft protein.
  • FIG. 20 is a diagram showing the influence of a compound represented by formula (II) on raft protein.
  • the present invention relates to a drug for treating or preventing HCV infection, which contains a compound that inhibits the binding of sphingomyelin and HCV protein as an active ingredient.
  • the compound that inhibits the binding of sphingomyelin and HCV protein is any compound that can directly or indirectly inhibit the binding reaction of sphingomyelin and HCV protein. It may be a compound. Further, it may be a compound that generates or increases these inhibitors and indirectly inhibits the binding reaction between sphingomyelin and HCV protein.
  • HCV proteins HCV-NS2, HCV-NS3, HCV-NS4A, HCV-NS4B, HCV-NS5A, HCV-NS5B can be mentioned, more preferably HCV-NS5B. it can.
  • Preferable examples of the compound that inhibits the binding of sphingomyelin and HCV protein of the present invention include the peptides described in the following (a) or (b).
  • These peptides also include peptides functionally equivalent to the peptides comprising the amino acid sequences described in any one of SEQ ID NOs: 1 to 4, 10 or 11, including the entered amino acid sequence.
  • the amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved.
  • amino acid side chain properties include hydrophobic amino acids (A, I, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl-containing side chains (S, T, ⁇ ), sulfur atoms
  • Amino acids with side chains C, M
  • amino acids with side chains containing carboxylic acids and amides D, N, E, Q
  • amino acids with side chains R, K, ⁇
  • aromatics Mention may be made of amino acids having a side chain (H, F, Y, W) (in parentheses all represent one letter of the amino acid).
  • SEQ ID NO: 1 to 4, 10 or 11 in the amino acid sequence described in any one or more of the amino acid substitution, deletion, addition and a peptide having an amino acid sequence ability that is Z or inserted, Examples thereof include peptides having amino acid strength described in any one of SEQ ID NOs: 5 to 7.
  • These peptides may specifically bind to the HCV protein binding site in sphingomyelin, and thus may reduce the binding activity of sphingomyelin and HCV protein. As a result, it is considered that replication of HCV virus via rafts is suppressed.
  • Preferred examples of the compound that inhibits the binding of sphingomyelin and HCV protein of the present invention include the oligonucleotides encoding the peptides described in (a) or (b) below. .
  • the oligonucleotide of the present invention can be used by incorporating it into an appropriate vector in a form capable of expressing the peptide.
  • the phrase "comprising an expressible form" of a polynucleotide encoding the peptide means that the polynucleotide is inserted into an expression vector and enters the animal cell. It means that a predetermined peptide can be expressed in a cell. That is, for example, it means that the coding DNA is arranged under the control of a promoter suitable for the species of animal to be administered and the administration site.
  • Preferable examples of the compound that inhibits the binding of sphingomyelin and HCV protein of the present invention include antibodies that recognize the peptides described in (a) or (b) below.
  • the origin of the antibody in the present invention is not particularly limited, but is preferably derived from a mammal, more preferably a human-derived antibody.
  • An antibody recognizing the peptide used in the present invention can be obtained as a polyclonal or monoclonal antibody using a known means.
  • a monoclonal antibody derived from a mammal is particularly preferable.
  • Monoclonal antibodies derived from mammals include those produced by hyperpridoma and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. This antibody inhibits the binding of HCV protein to sphingomyelin by binding to HCV protein. As a result, replication of HCV virus via rafts is thought to be suppressed.
  • An antibody-producing hybridoma that recognizes the peptide can be basically produced using a known technique as follows. That is, the peptide is used as a sensitizing antigen, and this is immunized according to a normal immunization method. The obtained immune cells are fused with a known parent cell by a normal cell fusion method, and a normal screening method is used. It can be produced by screening monoclonal antibody-producing cells.
  • an antibody that recognizes the peptide may be prepared as follows!
  • the peptide of interest is purified from the host cell or culture supernatant by a known method.
  • This purified peptide may be used as a sensitizing antigen.
  • a fusion protein of the peptide and another protein may be used as a sensitizing antigen.
  • treatment refers to the administration of the drug of the present invention to a subject to eliminate or reduce HCV, further suppress the spread of HCV, and symptoms caused by HCV infection. Means to reduce.
  • prevention refers to administration to a subject prior to HCV infection to prevent HCV infection or suppress proliferation. means.
  • Symptoms caused by HCV infection preferably include hepatitis C, cirrhosis, liver fibrosis, liver cancer and the like.
  • the compound of the present invention can be used in medicine.
  • the salt is not particularly limited as long as it is pharmacologically acceptable.
  • salts with mineral acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid; acetic acid, tartaric acid, lactic acid, ken Acid, fumaric acid, maleic acid, succinic acid, methane sulfonic acid, ethane sulfonic acid, benzene sulfonic acid, toluene sulfonic acid, naphthalene sulfonic acid, salt with organic acid such as camphor sulfonic acid; alkali such as sodium, potassium and calcium Examples thereof include salts with metals or alkaline earth metals.
  • the amount of the active ingredient-compound contained in the above-mentioned pharmaceutical preparation is not particularly limited and can be selected as appropriate over a wide range. For example, 0.1 to 99.5% by weight, preferably 0.5 to 90% by weight. .
  • the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc., with the compound of the present invention as the main agent according to conventional methods.
  • it can be formulated using known adjuvants that can be usually used.
  • conventionally known carriers can be widely used as carriers, such as lactose, sucrose, sodium chloride sodium salt, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and key acid.
  • Excipients such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, binders such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polybulurpyrrolidone; dry starch, sodium alginate Disintegrants such as lithium, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; white sugar, stearin, cocoa butter, hydrogenated oil, etc.
  • binders such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polybulurpyrrolidone
  • dry starch sodium alginate Disintegrants such as lithium, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid
  • Collapse inhibitor 4th Absorption accelerators such as ammonium salts and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal key acid; purified talc, stearate, boric acid powder, Examples include a lubricant such as polyethylene glycol.
  • the tablets can be made into tablets with ordinary coatings, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets as necessary.
  • the In molding into the form of a pill those conventionally known in this field can be widely used as a carrier.
  • excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and tar
  • binders such as tragacanth powder, gelatin and ethanol
  • disintegrants such as laminaran agar.
  • conventionally known carriers can be widely used as carriers, such as polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like. it can.
  • solutions and suspensions should be sterilized and used as diluents in the form of these solutions, emulsions and suspensions, which are preferably isotonic with blood. Any of those commonly used in this field can be used, and examples thereof include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.
  • a sufficient amount of sodium chloride, glucose, or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a normal solubilizing agent, buffering agent, soothing agent may be used.
  • An agent or the like may be added.
  • it may contain colorants, preservatives, fragrances, flavors, sweeteners, and other medicines as necessary.
  • the pharmaceutical composition is preferably administered in a dosage unit form for oral administration, tissue administration (subcutaneous administration, intramuscular administration, intravenous administration, etc.), topical administration (transdermal administration, etc.) Can be administered rectally.
  • tissue administration subcutaneous administration, intramuscular administration, intravenous administration, etc.
  • topical administration transdermal administration, etc.
  • the pharmaceutical composition is administered in a dosage form suitable for these administration methods.
  • the dose as an antiviral agent is preferably adjusted in consideration of the patient's condition such as age and weight, administration route, nature and degree of disease, etc.
  • the amount of the active ingredient of the present invention for adults is usually in the range of 0.1 to 2000 mg per day. In some cases, doses below the above range may be sufficient. Conversely, doses in excess of the above range may be required.
  • administering a large amount it is desirable to divide it into several times a day.
  • the oral administration can be carried out in solid, powder or liquid dosage units, for example, powders, powders, tablets, dragees, capsules, drops, sublingual agents, other dosage forms, etc. I can.
  • the intra-tissue administration can be performed, for example, by using a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection such as a solution or suspension. These are obtained by suspending or dissolving a certain amount of the compound of the present invention in a non-toxic liquid carrier suitable for injection purposes such as an aqueous or oily medium, and so on. It is manufactured by sterilizing.
  • the topical administration can be performed by using a form of external preparation such as a liquid, a cream, a powder, a paste, a gel, and an ointment.
  • a certain amount of the compound of the present invention is added to a fragrance, a colorant, a filler, a surfactant, a moisturizer, an emollient, a gelling agent, a carrier, a preservative, Manufactured by combining with one or more agents.
  • a certain amount of the compound of the present invention is mixed with a high-melting low-melting solid such as higher esters such as palmitic acid myristyl ester, polyethylene glycol, cocoa butter, and mixtures thereof. It can be performed using a suppository or the like.
  • the administration can be performed, for example, by using a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection such as a solution or suspension.
  • a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection such as a solution or suspension.
  • a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection such as a solution or suspension.
  • a non-toxic liquid carrier suitable for injection purposes such as an aqueous or oily medium, then the suspension or solution. It is manufactured by sterilizing.
  • a peptide having a cell membrane permeation function for example, Pegelin, Penetratin, etc.
  • a cell membrane permeation function for example, Pegelin, Penetratin, etc.
  • Doxorubi cm— peptide conjugates overcome multidrug resistance.
  • Anti-Cancer Drugs 2001, 12, Dcrossi D. et al The third helix of the antennapedia homeodomain translocates th rough biological membranes, J. Biol. Chem. 1994, 269, 10444-10450.
  • a method for introducing the peptide of the present invention into cells (1) one or more copies of a gene encoding a peptide sequence may be incorporated into a viral vector (eg, adenovirus). (2) a method of directly introducing the target peptide by intravenous injection, (3) a target peptide or a DNA encoding the target peptide or DNA encoding it physically.
  • the method for example, particle gun
  • the particle gun is an extremely powerful gene transfer method that introduces nucleic acid attached to gold or tandastene microparticles (microcarriers) into target cells by the pressure of helium gas. This method is much easier to operate than the microinjection method, and can be used in a wide range of applications regardless of the cell type. This is expected to be applied to the introduction of peptides.
  • the present invention relates to a method for screening a drug for treating or preventing HCV infection.
  • a test compound is contacted with the peptide described in the following ( a ) or (b).
  • the state of the peptide used in the first embodiment is not particularly limited, and may be, for example, a purified state, a state expressed in a cell, a state expressed in a cell extract, or the like.
  • Examples of cells expressing the peptide include cells expressing a foreign peptide.
  • a cell expressing the exogenous peptide can be prepared, for example, by introducing a vector containing DNA encoding the peptide into the cell. Introduction of a vector into a cell can be performed by a general method such as calcium phosphate precipitation, electric pulse perforation, ribophetamine, or microinjection.
  • the biological species from which a cell into which such an exogenous peptide is introduced is derived is not limited to mammals, and any biological species that has established a technique for expressing a foreign protein in the cell may be used.
  • the cell extract in which the peptide is expressed is included in, for example, an in vitro transcription / translation system.
  • An example is a cell extract obtained by adding a vector containing DNA encoding a peptide.
  • the in vitro transcription / translation system it is possible to use a commercially available in vitro transcription / translation kit without particular limitation.
  • test compound in the method of the present invention is not particularly limited, for example, natural compounds, organic compounds, inorganic compounds, single compounds such as proteins and peptides, and compound libraries. Gene library expression products, cell extracts, cell culture supernatants, fermentation microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts, etc. Can be mentioned.
  • the above test compound can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label.
  • the “plurality of test compounds” is not particularly limited. For example, in addition to the above test compounds, a mixture of a plurality of these test compounds is also included.
  • “contact” is performed depending on the state of the peptide.
  • the peptide if it is in a purified state, it can be carried out by adding a test compound to the purified sample. Moreover, if it is in a state expressed in a cell or in a cell extract, it can be carried out by adding a test compound to the cell culture solution or the cell extract, respectively.
  • the cell in the present invention is not particularly limited, but a cell derived from a mammal including yeast or human is preferable.
  • the test compound is a protein
  • a vector containing DNA encoding the protein is introduced into a cell in which the peptide is expressed! /, Or the vector is expressed in the peptide. It can also be carried out by adding to the cell extract. Further, for example, a two-hybrid method using yeast or animal cells can be used.
  • the binding between the peptide and the test compound is detected.
  • the means for detecting or measuring the binding between proteins can be performed, for example, by using a label attached to the protein.
  • the label type include fluorescent labels and radiolabels.
  • it can also be measured by a known method such as an enzyme two-hybrid method or a measurement method using BIACORE.
  • a test compound bound to the peptide is then selected. Some of the selected test compounds treat HCV infection. Or drugs to prevent. Alternatively, the selected test compound may be used as a test compound for the following screen.
  • test compound is added to sphingomyelin simultaneously with the peptide described in the following (a) or (b).
  • the binding ability of the peptide described in (a) or (b) above and the sphingomyelin is measured next.
  • the binding ability can be measured by the method described above.
  • the test compound is then selected as a pharmaceutical agent for treating or preventing HCV infection when the binding ability is reduced as compared with the case where no test compound is added.
  • the present invention also relates to a method for evaluating the efficacy of a drug for treating or preventing HCV infection.
  • a test compound is brought into contact with the peptide described in the following ( a ) or (b).
  • the binding between the peptide and the test compound is then detected. Detection of the binding between the peptide and the test compound can be performed by the method described above.
  • the efficacy of the therapeutic or prophylactic effect of HCV infection of the test compound bound to the above peptide is evaluated.
  • the test compound can detect HCV infection.
  • the efficacy of the therapeutic or preventive effect can be evaluated. For example, when the test compound shows a significant binding ability to the peptide, it can be evaluated that the test compound shows a significant therapeutic or preventive effect on HCV infection.
  • a test compound is added to sphingomyelin simultaneously with the peptide described in the following (a) or (b).
  • the binding ability of the peptide described in (a) or (b) above and sphingomyelin is measured, and the test compound has an effect of treating or preventing HCV infection.
  • the binding ability can be measured by the method described above.
  • the test compound By detecting the binding ability of the peptide and sphingomyelin (eg, equilibrium binding constant, binding rate constant, dissociation rate constant, etc.), the test compound has the ability to inhibit the binding of the peptide to sphingomyelin. Can be detected.
  • the efficacy of the test compound for treating or preventing HCV infection can be evaluated.
  • test compound when the test compound is added and the binding ability of the peptide and sphingomyelin is significantly reduced compared to the case where the test compound is not added, the test compound is significantly more susceptible to HCV infection. It can be evaluated that it shows an effective therapeutic or preventive effect.
  • the above-described evaluation method of the present invention is not limited to a screening method for finding a new drug for treating or preventing HCV infection, but an evaluation of the efficacy of the drug for the development of a drug candidate compound. It is also useful as an evaluation method when evaluating the efficacy for quality control required in the manufacture and supply of pharmaceuticals.
  • the present invention relates to a kit for use in the screening method or evaluation method described above.
  • a kit may include those used in the detection process and measurement process of the screening method or evaluation method described above.
  • reagents and instruments required for measuring the binding ability of the peptide and sphingomyelin.
  • Other steaming Retention water, salt, buffer solution, protein stabilizer, preservative and the like may be contained. All prior art documents cited in the present specification are incorporated herein by reference.
  • HIV-lgpl20-derived V3 loop (Fig. 1A, SEQ ID NO: 4) and prion-derived PrP protein have sphingolipid binding domains.
  • structures similar to these were searched using the CE program (cl. Sdsc.edu/ce.html).
  • FIG. 1 in HCV-NS5B sequence E230-G263 (FIG. 1A, SEQ ID NO: 3), a similar structure having a helix 'turn' helix motif (FIG. 1) was observed.
  • the full length base sequence of HCV-NS5B is shown in SEQ ID NO: 8 and the amino acid sequence is shown in SEQ ID NO: 9.
  • Figure 1B shows the structure of HCV-NS5B.
  • the vertical line is the putative sphingolipid binding region identified by HCV-NS5B.
  • the diagonal line is the sphingolipid of the HIV-1 V3 loop. Binding area).
  • NS5B-SBD peptide and SM were examined using Biacore S51.
  • SM was dissolved in chloroform and vacuum-dried, and then PBS buffer was added and suspended to 10 mM. The suspension was repeatedly frozen and thawed, passed through a membrane with a pore size of 50 nm, and ribosomes were prepared and immobilized on the sensor chip L1.
  • NS5B DIRVEESIYQCCDL APEARQAIKSLTERLY (SEQ ID NO: 1), synthesized by Sigma genosys) and PrP (KQHT VTTTTKGENFTETDVKMMER (SEQ ID NO: 2), synthesized by Sigma genosys) were used as synthetic peptides.
  • the amount of specific binding was determined by measuring the amount of binding using a sensorgram without a peptide as a blank and subtracting the sensorgram force at each concentration.
  • NS5B-SBD peptide was found to bind to sphingomyelin in a concentration-dependent manner (Fig. 2A, sensorgram of NS5B-SBD peptide). Similar binding was observed in prion protein (PrP) (Fig. 2B, RU value at 80 s sensorgram in Figure 2A).
  • HCV revlikon inhibitory activity of NS5b sphingo binding domain peptide (SEQ ID NO: 1) and its derivative peptides (SEQ ID NO: 10, 11).
  • DMEM was added in O.lmL and further cultured. 20 and 40 hours after peptide addition, replicon with Steady-Glo Luciferase Assay System (Promega, Cat.No. E2520) Activity was measured. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 12 was used as a negative peptide.
  • NS5B sphingo-binding domain peptide and its derivatives showed 20-30% anti-HCV replication inhibition at a final concentration of 0.1 mg / mL (FIG. 3). On the other hand, no inhibition was observed with the negative peptide.
  • Myriocin or a compound represented by the following formula (II) was given to the replicon cell Huh-3-l in the range of ⁇ to 100 ⁇ and cultured at 37 ° C in the presence of 5% CO.
  • the Northern analysis used the following. That is, NorthernMax transfer buffer (Ambion # 8672), transfer film BrightStar-Plus (Ambion # 10100), filter paper (Sigma P-6664), ULTRAhy b (Ambion # 8670).
  • the probe was labeled biotinylated with BiotinStar Psoralen-Biotin kit (Ambion # 9860 G3).
  • High Stringency buffer Amibion # 8674
  • BrightStar BioD etect kit Wash buffer, Ambion # 8650G; Blocking buffer, Ambion # 8651G; Streptavi din-Alkaline Phosphatase, Ambion # 2374G; Assay buffer, Ambion # 8652G; CDP— Starr, (Ambion # 8653G)
  • RNA was run on a 1% agarose gel, and after electrophoresis, RN A was stained and photographed. After decolorization, it was transferred to a transfer film for 2 hours using NorthernMax transfer buffer. In a wet state, RNA was immobilized on the transfer membrane with a UV crosslinker. Using a hybrid rotor, after pre-rotating for 42 minutes at ULTRAhyb for 30 minutes, the pre-treatment solution was discarded, and the neomycin resistance gene and 10 ml of ULTRAhyb solution were added and shaken overnight at 42 degrees.
  • the electrophoresed protein was transferred to a mini-trans blot cell (BIO-RAD # l 70-393 0) was transferred to a membrane (PROTRAN BA85, Nitrocellulose transfer membrane (Shleicher & Sc huell # 10401196)) Western analysis was performed using an anti-NS3 Usagi antibody derived from HCV protein, and an anti-actin Usagi antibody was used as an internal standard.
  • myriocin and the compound represented by formula (II) were found to have an effect of reducing HCV protein expression by 50% at a concentration of 1-lOnM (FIGS. 7 and 8).
  • HCV replicon inhibitory activity of fumonisin B1 which specifically inhibits dihydrosphingosine synthase, which produces dihydrosphingosine, a dihydroceramide, was measured.
  • Thin B1 was diluted three-fold in order, added to a final concentration of 1.37 M to 1000 M, and further cultured for 3 days. Two assembly plates were prepared, one for the white plate and the other for the clear plate. After completion of the culture, the white plate was used for Steady-Glo Luciferase Assay System (Promega cat. No. E2520). That is, 100 ⁇ l of reagent was added per well, mixed 3-4 times with a pipette, allowed to stand for 5 minutes, and then luminescence was measured with 1450 MicroBeta TRILUX (WALLAC).
  • WALLAC MicroBeta TRILUX
  • the IC50 (50% inhibitory concentration) of the drug was calculated by subtracting all values from the value with no added cells as the background, and the value with no drug added as 0% inhibition.
  • Cell's Count Kit 8 (DOJIN Laboratories, cat.No.341-07771) was put in 10 ⁇ l per well, mixed 3-4 times with a pipette, and left at about OD450nm power of about 1.0 after about 30 minutes. It measured when it became.
  • the IC50 (50% inhibitory concentration) of the drug was calculated by subtracting all values from the value with no added cells as the background and the value with no drug added as 0% inhibition.
  • fumosin B1 exhibits HCV replicon inhibitory activity at a concentration of 10-1000 M (FIG. 9).
  • siRNA targeting LCB1 one subunit of SPT heterodimer.
  • siRNAs two specific The siRNAs (si246, si633) were designed based on the LCBlcDNA sequence (GenBank Accession No. Y08685) and synthesized by Qiagen.
  • SEQ ID NO: 15 As a control siRNA (SEQ ID NO: 15), a sequence that does not affect the expression of LCB1 was used.
  • the synthesized siRNA sequence is shown in SEQ ID NO: 13 (si24 6) and SEQ ID NO: 14.
  • Cell lysis buffer 50 mM Tris-HC1 (pH 7.5), 0.5% Triton, 3 mM EDTA, 1 50 mM NaCl, 12 mM glycerophosphate, 50 mM NaF, 1 mM Na ⁇ O ⁇ , 0.5 mM PMSF, Suspended in 0.5 mM aporotinin) and left on ice for 10 minutes. The supernatant was collected by centrifugation at 15,000 X g for 10 minutes.
  • the cells treated with si246 and si633 that suppressed the expression of LCB1 significantly inhibited HCV replicon activity compared to the cells treated with control siRNA.
  • This inhibitory effect was strongly observed with si246, which strongly suppressed the expression of LCB1.
  • the cytotoxicity of siRNA treatment was examined. Almost no effect was observed.
  • the HCV replicon assay and cytotoxicity test were conducted on the compounds represented by formula (I) or their derivatives.
  • HCV-RNA with a firefly-derived luciferase gene introduced as a reporter gene.
  • IRES Internal Ribosome Entry Site
  • a luciferase gene was introduced immediately below the neomycin resistance gene.
  • the RNA was synthesized in vitro, then introduced into Huh7 cells by the electopore method, and isolated as a G418 metaclone.
  • Firefly 'Luciferase HCV replicon cells (Huh-3-l) were suspended in Dulbecco's MEM (Gibco cat. No. 10569-010) containing 5% urine fetal serum (Hyclone cat. No. SH30071.03). Seed the well plate with 5000 cells Zwell, 5% CO
  • Cell counting kit-8 (Dojindo catalog No. CK04) was used for the measurement of cytotoxicity. That is, 10 1 Cell counting kit-8 was added to a clear plate and incubated at 37 degrees for 30-60 minutes. Absorbance was measured with a 96-well plate reader at a wavelength of 450 and a control wavelength of 630 nm. All values were subtracted from the value with no cell added, and the CC (50% cell inhibitory concentration) of the drug was calculated with the value without drug added as 0% inhibition.
  • HCV replicon cells were treated with the compound represented by formula (II) at the concentration shown in FIG. 12, and the replicon replication inhibitory activity and cell survival inhibitory activity were measured.
  • Steady— JLO luciferase assay system (Promega, cat. No. E251 0), cell viability inhibitory activity) by cell count counting kit— 8 (Dojin Laboratories, cat.
  • IC50 2 nM
  • the cytotoxicity of the compound represented by the formula (II) was not observed (IC50> 50 nM).
  • HCV replicon cells were treated with 100 nM of the compound represented by formula (II) for 96 hours, and then the cells were fixed with 3.7% formaldehyde. After blocking with 3% BSA and incubating with NS3 antibody (provided by F. Hoffman Laroche), the washed cells were incubated with Texas-Red labeled Usagi IgG (Molecular probe) and analyzed with a fluorescence microscope (Fig. 13). As a result, as shown in Fig. 13, the HCV-NS3 protein disappeared due to the addition of the compound represented by the force equation (II) existing around the nucleus (the part shown in white is the NS3 protein). The part shown in gray shows the nucleus stained with Hoechst 33342 (Sigma, cat. No. B2261)).
  • Replicon cells were treated with 100 nM of the compound represented by formula (II) for the times shown in FIG. 14 (48 hours and 96 hours).
  • Western plot analysis was performed in the same manner as in Reference Example 5.
  • the non-structural proteins NS3, NS5A and NS5B of HCV were detected with each antibody in a time-dependent manner, and a decrease in the expression level of the viral protein was observed (Fig. 14).
  • LCBl and LCB2 cDNAs were obtained from human liver cDNA library (Clontech, cat. No. 639307) by RT-PCR, and His-tagged pBudCE4.1 vector (Invitogen, cat. No. V532-20) ).
  • the gene was introduced into HEK293 cells (ATCC, cat. No. CRL-1573). After 72 hours, the cells were lysed, and the protein was purified with N ⁇ NTA agarose (Qiagen, cat. No. 1018244).
  • SPT activity is reaction buffer [200 mM HEPES buffer (pH 8.0), 5 mM EDTA, 10 mM DTT, 0.05 mM pyridoxal 5 -phosphate, 0.2 mM palmitoy CoA, 0.1 mM L-serine, and 1 mCi [3 H] Serine (Amer sham, cat. no. TRK308)] was added and the reaction was allowed to proceed at 37 ° C for 15 minutes. After extraction with black mouth form: methanol (1: 2, v / v), the organic layer was re-extracted twice with water, and then the radioactivity of the organic layer was measured with a liquid scintillation counter. As a result, as shown in Figure 15, It was revealed that the compound represented by the formula (II) has an SPT inhibitory activity with an IC50 of about 10 nM.
  • HCV replicon cells were treated with the compound represented by formula (II) at the concentration shown in FIG. 16 for 48 hours, and then labeled with [14C] serine for 3 hours. After extraction with black mouth form: methanol (1: 2, v / v), de novo synthesized ceramide (Fig. 16A) and sphingomyelin (Fig. 16B) were separated by thin layer chromatography. As a result, as shown in FIG. 16, the compound represented by the formula (II) inhibited de novo synthesis of intracellular ceramide and sphingomyelin in a concentration-dependent manner.
  • HCV replicon cells were separated from the known SPT inhibitor myriocin (Sigma, cat. No. M1177), ceramide synthesis inhibitor fumosin Bl (Sigma, cat. No. F1147), and ceramide transport inhibitor H PA-12 [ After treatment with Kobayashi et al., Org.lett. (2002), the levulincon activity and viable cell count were measured 72 hours later. As a result, each compound was found to have an effect of suppressing HCV replication at a concentration where no cytotoxicity was observed (FIG. 18).
  • ImM compound (II) represented by the formula (II) was added to HCV replicon cells for 72 hours, and then the cell extract was treated with 1% NP-40 for 1 hour. Raft proteins (solubilizing agent resistance) and non-raft proteins were separated by sucrose density gradient fractionation, diluted with PBS, concentrated and quantified by ELISA analysis. As a result, the compound represented by the formula (II) was significantly dissociated from the raft in NS5B (FIG. 20).
  • the knowledge of the present invention greatly contributes to the development of anti-HCV agents targeting novel binding of sphingolipids and HCV proteins.

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Abstract

L’invention concerne une tentative pour déterminer le site de liaison protéique du HCV sur un sphingolipide et pour examiner la capacité de liaison entre une protéine HCV et le sphingolipide. En conséquence, il s’avère qu'une sphingomyéline (composant d'un radeau lipidique) peut fortement se lier à un site spécifique de la HCV-NS5B (une des protéines HCV) et qu’un virus HCV peut se reproduire grâce à cette liaison. Il s’avère aussi que la réplication de virus HCV peut être empêchée en inhibant la liaison entre une sphingomyéline et une protéine HCV.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8957199B2 (en) 2008-11-26 2015-02-17 Chugai Seiyaku Kabushiki Kaisha Oligoribonucleotide or peptide nucleic acid capable of inhibiting activity of hepatitis C virus
US8981123B2 (en) 2011-12-12 2015-03-17 Microbial Chemistry Research Foundation Compound and asymmetric synthesis reaction

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NEHETE P. ET AL.: "A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains", ANTIVIRAL RESEARCH, vol. 56, 2002, pages 233 - 251, XP003010462 *
SAKAMOTO H. ET AL.: "Host sphingolipid biosynthesis as a target for hepatitis C virus therapy", NATURE CHEMICAL BIOLOGY, vol. 1, no. 6, November 2005 (2005-11-01), pages 333 - 337, XP003010463 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8957199B2 (en) 2008-11-26 2015-02-17 Chugai Seiyaku Kabushiki Kaisha Oligoribonucleotide or peptide nucleic acid capable of inhibiting activity of hepatitis C virus
US8981123B2 (en) 2011-12-12 2015-03-17 Microbial Chemistry Research Foundation Compound and asymmetric synthesis reaction
US9187498B2 (en) 2011-12-12 2015-11-17 Microbial Chemistry Research Foundation Compound and asymmetric synthesis reaction

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