JPWO2007043640A1 - Drugs for treating or preventing HCV infection - Google Patents

Abstract

The inventors of the present invention have identified the binding site of HCV protein and sphingolipid and examined the binding ability. As a result, it was clarified that sphingomyelin, which is a component of raft, and HCV-NS5B, which is an HCV protein, bind strongly at specific sites, and that HCV virus is replicated by these bonds. . It was also revealed that replication of HCV virus can be suppressed by inhibiting the binding of sphingomyelin and HCV protein.

Description

  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. Moreover, it is related with the screening method of the drug for treating or preventing HCV infection, and the kit for using these.

  It is estimated that there are 1 to 200 million people infected with HCV worldwide and more than 2 million people in Japan. About 50% of these patients transition to chronic hepatitis, of which about 20% develop cirrhosis and liver cancer more than 30 years after infection. About 90% of liver cancer is said to be caused by hepatitis C. In Japan, more than 30,000 patients die from liver cancer associated with HCV infection every year.

  HCV was discovered in 1989 as a major causative virus for non-A non-B hepatitis after blood transfusion. HCV is an RNA virus having an envelope, and its genome consists of single-stranded (+) RNA, and is classified into the genus Hepacivirus of the Flaviviridae family.

  HCV avoids the immune system of the host due to an unclear cause, so persistent infection is often established even when infected with an adult with an advanced immune system, which progresses to chronic hepatitis, cirrhosis, and liver cancer. It is also known that even after excision, there are many patients whose liver cancer recurs due to inflammation that continues in the non-cancerous part.

  Therefore, establishment of an effective treatment method for hepatitis C is desired. In particular, apart from coping therapy that suppresses inflammation with anti-inflammatory agents, development of drugs that reduce or eradicate HCV in the affected liver is strong. It is desired.

  Currently, interferon therapy is known as the only effective treatment for HCV exclusion. However, about 1/3 of all patients have effective interferon. In particular, the response rate of interferon against HCV genotype 1b is very low. Therefore, development of an anti-HCV drug that can replace or be used in combination with interferon is strongly desired.

  In recent years, ribavirin (1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) has been marketed as a therapeutic agent for hepatitis C in combination with interferon. It is still low and further novel hepatitis C therapeutics are desired. In addition, attempts have been made to eliminate viruses by enhancing the immunity of patients, such as interferon agonists and interleukin-12 agonists, but no effective drug has been found yet.

  Since the HCV gene was cloned, molecular biological analysis of viral gene mechanisms and functions, protein functions of each virus, etc. has rapidly progressed, but virus replication, persistent infection, and pathogenicity in host cells. Such a mechanism has not been fully elucidated, and a reliable HCV infection experiment system using cultured cells has not been established. Therefore, in the past, alternative virus assays using other closely related viruses had to be used to evaluate anti-HCV drugs.

  However, in recent years, it has become possible to observe HCV replication in vitro using a non-structural region portion of HCV, so that anti-HCV drugs can be easily evaluated by a replicon assay method (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. Thus, this system can be referred to as a cell-based assay system useful for identifying compounds that inhibit HCV replication.

Prior art document information related to the invention of the present application is shown below.
International Publication WO98 / 56755 Pamphlet International Publication WO04 / 71503 Pamphlet International Publication WO05 / 05372 Pamphlet Buoy Lohmann et al., Science, 1999, 285, 110-113

  The inventors of the present application are disclosed in International Publication No. WO98 / 56755 (Patent Document 1), and a series of compounds derived from microorganisms such as the genus Aureobasidium have high HCV in the above replicon assay method. It was found to have a replication inhibitory activity (Patent Document 2). In addition, the present inventors have found that the compound has minimal in vitro cytotoxicity and is extremely useful as a preventive or therapeutic agent for HCV infection, and further constructed a method for synthesizing the compound and derivative ( Patent Document 3). In addition, the present inventors 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, is a constituent of rafts on cell membranes, and viruses such as influenza and HIV are replicated via rafts (Takeda M. et al. (2003) PNAS, 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)).
From these facts, 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 the object thereof is 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. Is to provide. 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.

In order to solve the above-mentioned problems, the present inventors have investigated the binding site and binding ability of HCV protein and sphingolipid (sphingomyelin).
First, in order to search for a sphingolipid-binding region in the HCV protein, a similar structure was searched by the CE program. As a result, a similar structure having a helix-turn-helix motif was observed in NS5B sequence E230-G263 (Fig. 1).

Next, Biacore S51 was used to confirm the binding ability of the NS5B sphingo-binding domain (NS5B-SBD) and sphingomyelin (SM). The NS5B-SBD peptide could bind to sphingomyelin in a concentration-dependent manner. It became clear (Fig. 2A). Similar binding was also observed in prion protein (PrP) (FIG. 2B).
Furthermore, when HCV replicon inhibitory activity of NS5B sphingo-binding domain peptides and derivatives thereof was measured, it was revealed that these peptides exhibited significant anti-HCV replication inhibitory activity (FIG. 3).
From the above results, it is clear that sphingomyelin, which is a component of raft, and HCV-NS5B, which is an HCV protein, bind strongly at a specific site, and that the HCV virus is replicated by these bonds. It was. It was also revealed that replication of HCV virus can be suppressed by inhibiting the binding of sphingomyelin and HCV protein.

  That is, the present inventors have succeeded in developing 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. It came to be completed.

More specifically, the present invention provides the following [1] to [14].
[1] 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] The drug according to [1], wherein the compound that inhibits the binding of sphingomyelin and HCV protein is the peptide according to the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 A peptide comprising an amino acid sequence in which an amino acid is substituted, deleted, added and / or inserted [3] A compound that inhibits the binding of sphingomyelin and HCV protein encodes the peptide described in (a) or (b) below: The drug according to [1], which is an oligonucleotide.
(A) a peptide comprising the amino acid sequence described in any one of SEQ ID NOs: 1 to 4, 10 or 11 (b) one or more of the amino acid sequences described in any one of SEQ ID NOs: 1 to 4, 10 or 11 A peptide comprising an amino acid sequence substituted, deleted, added and / or inserted [4] A compound that inhibits the binding of sphingomyelin and HCV protein recognizes the peptide described in (a) or (b) below The drug according to [1], wherein
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 The peptide according to any one of [1] to [4], wherein the peptide [5] HCV protein consisting of an amino acid sequence substituted, deleted, added and / or inserted is HCV-NS5B.
[6] The drug according to any one of [1] to [5], wherein the HCV infection is hepatitis C, cirrhosis, liver fibrosis, or liver cancer.
[7] A screening method for a drug for treating or preventing HCV infection, comprising the following steps (A) to (C):
(A) A step of bringing a test compound into contact with the peptide described in (a) or (b) below: (a) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 (b A peptide (B) (A) comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 to 4, 10 or 11 Step (C) for detecting the binding of the described peptide to the test compound (C) The step of selecting the test compound that binds to the peptide described in (A) [8] The following steps (A) to (C) are included: A screening method for drugs for treating or preventing HCV infection.
(A) A step of adding a test compound to sphingomyelin simultaneously with the peptide described in (a) or (b) below: (a) the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 A peptide (b) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 ) Step of measuring the binding ability of the peptide described in (a) or (b) above and sphingomyelin (C) Select a test compound having a reduced binding ability compared to the case where no test compound is added [9] The screening method according to [7] or [8], wherein the HCV infection is hepatitis C, cirrhosis, liver fibrosis, or liver cancer.
[10] A kit for use in the screening method according to any one of [7] to [9].
[11] A method for evaluating the efficacy of a drug for treating or preventing HCV infection, comprising the following steps (A) to (C):
(A) A step of bringing a test compound into contact with the peptide described in (a) or (b) below: (a) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 (b A peptide (B) (A) comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 to 4, 10 or 11 Step (C) for detecting the binding of the described peptide to the test compound (C) The step [12] for evaluating the efficacy of the therapeutic or prophylactic effect of HCV infection of the test compound binding to the peptide described in (A) A method for evaluating the efficacy of a drug for treating or preventing HCV infection, comprising the steps of (A) and (B).
(A) A step of adding a test compound to sphingomyelin simultaneously with the peptide described in (a) or (b) below: (a) the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 A peptide (b) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 ) Step [13] [11] or [12] for evaluating the efficacy of the therapeutic or prophylactic effect of HCV infection by measuring the binding ability of the peptide described in (a) or (b) above and sphingomyelin A kit for use in the evaluation method according to any one of the above.
[14] The peptide according to the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted

A is a figure which shows the amino acid sequence of sequence E230-G263 and V3 loop of HCV-NS5B. B is a figure which shows the structure of HCV-NS5B. The vertical line is the putative sphingolipid binding region identified by HCV-NS5B. The diagonal line indicates the sphingolipid binding region of the HIV-1 V3 loop. C is a diagram showing a helix-turn-helix motif in HCV-NS5B. It is a figure which shows the result of the binding examination of HCV-NS5B sphingo binding region peptide (NS5B-SBD, sequence number: 1) and sphingomyelin (SM) using Biacore. A is a diagram showing a sensorgram of NS5B-SBD 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. Indicates the RU value at sensorgram 80s. It is a figure which shows the result of the HCV replicon inhibitory activity measurement of NS5B sphingo binding domain peptide and its derivative (s). It is a figure which shows the sphingolipid synthetic pathway (synthetic pathway from palmitoyl CoA to sphingomyelin). It is a photograph which shows the HCV RNA replication inhibitory activity of myriocin by Northern blot analysis. The horizontal axis represents the concentration of myriocin. It is a photograph which shows the HCV RNA replication inhibitory activity of the compound represented by Formula (II) by Northern blot analysis. The horizontal axis represents the concentration of the compound represented by formula (II). It is a photograph which shows the HCV protein synthesis inhibitory activity of myriocin by Western blot analysis. The horizontal axis represents the concentration of myriocin. It is a photograph which shows the HCV protein synthesis inhibitory activity of the compound represented by Formula (II) by Western blot analysis. The horizontal axis represents the concentration of the compound represented by formula (II). It is a figure which shows the HCV replicon inhibitory activity of fumonisin B1. It is a photograph which shows protein expression inhibition of serine palmitoyltransferase (LCB1) by siRNA. It is a figure which shows the influence of HCV replicon inhibitory activity and cytotoxicity by siRNA. It is a figure which shows inhibition of the HCV replicon by the compound represented by Formula (II), and toxicity to a host cell. 2 is a photograph showing inhibition of expression of HCV-NS3 protein by a compound represented by formula (II). After immunostaining, it was observed with a fluorescence microscope. White indicates NS3 protein and gray indicates nuclei stained with Hoechst 33342. 2 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 blot analysis. It is a figure which shows SPT inhibitory activity by the compound represented by Formula (II). 2 is a photograph showing inhibition of de novo synthesis of ceramide and sphingomyelin by a compound represented by formula (II). It is a photograph which shows suppression of HCV replication inhibition of the compound represented by Formula (II) by C2-ceramide. It is a figure which shows HCV replication inhibition by the raft biosynthesis related low molecular weight compound. It is a photograph which shows the influence on the raft protein by the compound represented by Formula (II). It is a figure which shows the influence on raft protein by the compound represented by Formula (II).

  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.

  In the present invention, the compound that inhibits the binding between sphingomyelin and HCV protein may be any compound that can directly or indirectly inhibit the binding reaction between sphingomyelin and HCV protein. Moreover, it is a compound which produces | generates or increases these inhibitors, Comprising: The compound which indirectly inhibits the binding reaction of sphingomyelin and HCV protein may be sufficient.

  In the present invention, examples of the HCV protein include HCV-NS2, HCV-NS3, HCV-NS4A, HCV-NS4B, HCV-NS5A, and HCV-NS5B, 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).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted

  In the amino acid sequence described in any one of SEQ ID NOs: 1 to 4, 10 or 11, for example, 1 to 10, preferably 1 to 5 amino acid residues are substituted, deleted, added, and / or inserted. A peptide that includes an amino acid sequence and is functionally equivalent to the peptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 4, 10, or 11 is also included in these peptides. 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. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, 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 group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids having amino acids (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic-containing side chains The amino acids (H, F, Y, W) that can be used can be listed (the parentheses indicate the single letter of the amino acid). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by one or more amino acid residues to a certain amino acid sequence maintains its biological activity. Yes.

The peptide consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 includes SEQ ID NOs: 5 to 5. Peptides consisting of the amino acids described in any of 7 can be mentioned.
These peptides are considered to specifically bind to the HCV protein binding site in sphingomyelin, and thus reduce the binding activity between sphingomyelin and HCV protein. As a result, it is considered that replication of HCV virus via rafts is suppressed.

Preferable examples of the compound that inhibits the binding of sphingomyelin and HCV protein of the present invention include oligonucleotides encoding the peptides described in the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 A peptide comprising an amino acid sequence in which an amino acid is substituted, deleted, added and / or inserted. The oligonucleotide of the present invention can be used by incorporating it into an appropriate vector in a form capable of expressing the peptide.

  In the present specification, 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 is included in a form that can be expressed. That is, for example, it means that the coding DNA is arranged under the control of a promoter suitable for the animal species 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 the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 A peptide comprising an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted. 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. Can be mentioned.

  The antibody that recognizes the peptide used in the present invention can be obtained as a polyclonal or monoclonal antibody using a known means. As an antibody that recognizes the peptide used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. This antibody inhibits the binding of HCV protein and sphingomyelin by binding to HCV protein. As a result, it is considered that replication of HCV virus via rafts is suppressed.

  An antibody-producing hybridoma that recognizes the peptide can be basically produced using a known technique as follows. That is, using the peptide as a sensitizing antigen, this is immunized according to a normal immunization method, the resulting immune cells are fused with a known parent cell by a normal cell fusion method, and a monoclonal antibody is obtained by a normal screening method. It is possible to prepare by screening for an antibody-producing cell.

Specifically, an antibody that recognizes the peptide may be prepared as follows.
A base sequence encoding the peptide is inserted into a known expression vector system to transform an appropriate host cell, and then the desired peptide is purified from the host cell or culture supernatant by a known method. This purified peptide may be used as a sensitizing antigen. Moreover, you may use the fusion protein of this peptide and another protein as a sensitizing antigen.

  In the present specification, the term “treatment” means that HCV is extinguished or reduced, further spread of HCV is suppressed, and symptoms due to HCV infection are reduced by administering the drug of the present invention to a subject. means. Moreover, the description of "prevention" means that it is administered to a subject before HCV is infected to prevent infection of HCV or suppress proliferation. Symptoms caused by HCV infection preferably include hepatitis C, cirrhosis, liver fibrosis, liver cancer and the like.

The compounds of the present invention can be used in medicine. The salt is not particularly limited as long as it is pharmacologically acceptable, and examples thereof include salts with mineral acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid; acetic acid, tartaric acid, lactic acid, citric acid. Salts with organic acids such as acid, fumaric acid, maleic acid, succinic acid, methane sulfonic acid, ethane sulfonic acid, benzene sulfonic acid, toluene sulfonic acid, naphthalene sulfonic acid and 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 pharmaceutical preparation is not particularly limited and is appropriately selected within a wide range, and is, for example, 0.1 to 99.5% by weight, preferably 0.5 to 90% by weight.

  The compounds of the present invention are usually used in the pharmaceutical formulation technical field as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. Can be formulated using known adjuvants. In molding into tablets, conventionally known carriers can be widely used as carriers, such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and the like. Binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; dry starch, sodium alginate, agar powder, laminaran powder , Disintegrating agents such as sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, starch and lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter and hydrogenated oil; quaternary Ann Absorption promoters such as nium salts and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; purified talc, stearate, boric acid powder, polyethylene glycol, etc. Examples of such abundant agents can be given.

  Furthermore, the tablets can be made into tablets with ordinary coatings, if necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets. In molding into the form of pills, those conventionally known in this field can be widely used as carriers, for example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, talc; Examples include binders such as tragacanth powder, gelatin and ethanol; disintegrants such as laminaran agar. In molding into the form of suppository, conventionally known carriers can be widely used as carriers, such as polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. it can. When prepared as injections, the solutions and suspensions are preferably sterilized and isotonic with blood, and when formed into these solutions, emulsions and suspensions, this is used as a diluent. Any of those commonly used in the field can be used, and examples thereof include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of sodium chloride, glucose, or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a normal solubilizer, buffer, soothing agent, etc. May be added. Furthermore, a coloring agent, a preservative, a fragrance | flavor, a flavor agent, a sweetening agent, etc. and other pharmaceuticals can be contained as needed.

  The pharmaceutical composition is preferably administered in dosage unit form, and is administered orally, intra-tissue (subcutaneous, intramuscular, intravenous, etc.), topical (transdermal etc.) or rectally. can do. Of course, the pharmaceutical composition is administered in a dosage form suitable for these administration methods.

  When the compound of the present invention is administered as a medicine, 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. For humans, the amount of the active ingredient of the present invention for adults is in the range of 0.1 to 2000 mg per day. In some cases, a dose lower than the above range may be sufficient, and conversely, a dose exceeding the above range may be required. When administering a large amount, it is desirable to divide the dose into several times a day.

  The oral administration can be performed in solid, powder or liquid dosage units, for example, powders, powders, tablets, dragees, capsules, drops, sublingual agents, other dosage forms, and the like. .

  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 suspend or dissolve a certain amount of a compound of the invention in a non-toxic liquid carrier suitable for injection purposes, such as an aqueous or oily medium, and then sterilize the suspension or solution. It is manufactured by.

  The above-mentioned topical administration (transdermal administration etc.) can be performed by using forms of external preparations such as liquids, creams, powders, pastes, gels, ointments and the like. These compounds can be used for a certain amount of the compound of the present invention, such as a fragrance, a coloring agent, a filler, a surfactant, a moisturizer, an emollient, a gelling agent, a carrier, a preservative, It is manufactured by combining with one or more of agents.

  In the above-mentioned rectal administration, a certain amount of the compound of the present invention is mixed with a suppository mixed with a low-melting-point solid comprising, for example, higher esters such as myristyl palmitate, polyethylene glycol, cocoa butter, and mixtures thereof. Can be used.

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. These are those in which a certain amount of a compound of the invention is suspended or dissolved in a non-toxic liquid carrier adapted to the purpose of injection, for example, an aqueous or oily medium, and the suspension or solution then sterilized. It is manufactured by doing.
As a technique for transporting a protein such as the peptide or antibody of the present invention into a cell in order to act in the cell, for example, adding a peptide having a cell membrane permeation function (for example, Pegelin or Penetratin) (Martine Mazel) etal., Doxorubicin-peptide conjugates overcome multidrug resistance.Anti-Cancer Drugs 2001, 12, Dcrossi D. et al., The third helix of the antennapedia homeodomain translocates through biological membranes, J. Biol. Chem. 1994, 269, 10444- 10450.), the protein of the present invention can be transported into cells.

  In addition, the method of introducing the peptide of the present invention into cells includes (1) linking one or more copies of a gene encoding a peptide sequence to a viral vector (for example, adenovirus) and infecting the virus. (2) a method of directly introducing the target peptide by intravenous injection, (3) a method of physically introducing the target peptide or DNA encoding it into a cell or tissue (for example, a particle gun), etc. I can list them. The particle gun is a very powerful gene transfer method that introduces nucleic acid attached to gold or tungsten fine particles (microcarriers) into target cells by the pressure of helium gas. This method is much easier to operate than the microinjection method, and a wide range of applications can be expected regardless of the cell type. Utilizing this fact, it 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.
As a first aspect of the screening method of the present invention, first, a test compound is brought into contact with the peptide described in the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted

  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 the vector into the cells can be carried out by a general method such as calcium phosphate precipitation, electric pulse perforation, lipophetamine, microinjection and the like. The biological species from which the cell into which such an exogenous peptide is introduced is derived is not limited to mammals, and any biological species may be used as long as a technique for expressing a foreign protein in the cell is established.

  Examples of the cell extract in which the peptide is expressed include those obtained by adding a vector containing DNA encoding the peptide to the cell extract contained in the in vitro transcription / translation system. The in vitro transcription / translation system is not particularly limited, and a commercially available in vitro transcription / translation kit can be used.

  There is no restriction | limiting in particular as "test compound" in the method of this invention, For example, a single compound, such as a natural compound, an organic compound, an inorganic compound, protein, a peptide, and an expression product of a compound library and a gene library And cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, animal cell extracts, and the like. The test compound can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label. Moreover, there is no restriction | limiting in particular as "a some test compound", For example, in addition to the said test compound, the mixture which mixed multiple types of these test compounds is also contained.

  In the present invention, “contact” is performed depending on the state of the peptide. For example, if the peptide is in a purified state, it can be carried out by adding a test compound to the purified preparation. Moreover, if it is the state expressed in the cell or the state expressed in the cell extract, it can be carried out by adding a test compound to the cell culture solution or the cell extract, respectively. Although it does not restrict | limit especially as a cell in this invention, The cell derived from mammals including yeast or a human is preferable. When the test compound is a protein, for example, a vector containing DNA encoding the protein is introduced into a cell in which the peptide is expressed, or the vector is added to a cell extract in which the peptide is expressed. It is also possible to do this. For example, a two-hybrid method using yeast or animal cells can be used.

  Next, in the first embodiment, 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. Examples of the type of label include a fluorescent label and a radiolabel. Further, it can also be measured by a known method such as an enzyme two-hybrid method or a measurement method using BIACORE. In this method, a test compound bound to the peptide is then selected. Among the selected test compounds are agents for treating or preventing HCV infection. In addition, the selected test compound may be used as a test compound for the following screening.

As a second embodiment of the screening method of the present invention, first, a test compound is added to sphingomyelin simultaneously with the peptide described in (a) or (b) below.
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted

Next, in the second embodiment, the binding ability of the peptide described in (a) or (b) above and sphingomyelin is measured. The binding ability can be measured by the method described above.
In the present method, the test compound is then selected as a drug for treating or preventing HCV infection when the binding ability is reduced as compared to 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.
As a first aspect of the method for evaluating a drug of the present invention, first, a test compound is brought into contact with the peptide described in the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptide consisting of an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted In the first embodiment, 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. Subsequently, the efficacy of the therapeutic or prophylactic effect of HCV infection of the test compound bound to the peptide is evaluated. By detecting the binding ability of the test compound to the peptide (for example, equilibrium binding constant, binding rate constant, dissociation rate constant, etc.), the efficacy of the test compound for treating or preventing HCV infection is evaluated. I can do it. 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.

As a second aspect of the method for evaluating a drug of the present invention, first, a test compound is added to sphingomyelin simultaneously with the peptide described in the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted

  In the second embodiment, the binding ability of the peptide described in (a) or (b) above and sphingomyelin is then measured, and the efficacy of the test compound for treating or preventing HCV infection is evaluated. The binding ability can be measured by the method described above. Detecting the binding inhibitory ability of the test compound to the peptide and sphingomyelin by detecting the binding ability of the peptide and sphingomyelin (eg, equilibrium binding constant, binding rate constant, dissociation rate constant, etc.) Can do. Furthermore, based on these values, the efficacy of the test compound for treating or preventing HCV infection can be evaluated. For example, 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 different from 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 also evaluation of efficacy and drug production for the development of drug candidate compounds. It is also useful as an evaluation method when evaluating the efficacy for quality control required at the time of supply.
The present invention relates to a kit for use in the screening method or evaluation method described above. Such a kit may include those used in the detection process and measurement process of the screening method or evaluation method described above. For example, there can be mentioned reagents and instruments required for measuring the binding ability of the peptide and sphingomyelin. In addition, distilled water, salt, buffer solution, protein stabilizer, preservative and the like may be contained.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Search for sphingolipid-binding motif of HCV-NS5B
HIV-1 gp120-derived V3 loop (FIG. 1A, SEQ ID NO: 4) and prion-derived PrP protein have a sphingolipid-binding domain. In order to search for a sphingolipid binding region in HCV protein, structures similar to these were searched using the CE program (cl.sdsc.edu/ce.html). As a result, as shown in 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. The size of this structural domain almost coincided with the V3 loop (Fig. 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 binding of the HIV-1 V3 loop. region).

[Example 2] Binding of putative NS5B sphingo-binding domain peptide (NS5B-SBD) to sphingomyelin (SM)
The binding ability of NS5B-SBD peptide and SM was examined using Biacore S51. After SM was dissolved in chloroform and vacuum-dried, 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, liposomes were prepared, and immobilized on the sensor chip L1. NS5B (DIRVEESIYQCCDLAPEARQAIKSLTERLY (SEQ ID NO: 1), synthesized by Sigma genosys) and PrP (KQHTVTTTTKGENFTETDVKMMER (SEQ ID NO: 2), synthesized by Sigma genosys) were used as synthetic peptides. As the specific binding amount, a sensorgram to which no peptide was added was used as a blank, and the amount subtracted from the sensorgram at each concentration was used for the measurement of the binding amount. As a result, NS5B-SBD peptide was found to bind to sphingomyelin in a concentration-dependent manner (FIG. 2A, sensorgram of NS5B-SBD peptide). Similar binding was also observed in the prion protein (PrP) (RU value at 80 s in the sensorgram of FIG. 2B and FIG. 2A).

  From the above results, it is clear that sphingomyelin, which is a component of raft, and HCV-NS5B, which is an HCV protein, bind strongly at a specific site, and that the HCV virus is replicated by these bonds. It was. These findings suggest that replication of HCV virus can be suppressed by inhibiting the binding of sphingomyelin and HCV protein.

HCVレプリコン細胞を96穴プレートの１穴当たり10,000細胞（0.1mL）入れ、５％Fetal Bovine Serum（以下FBS）(HyClone, Cat. No. SH30071)を含むDulbecco's Modified Eagle Medium(以下DMEM) (Invitrogen, Cat. No. 10569-010)中で37℃、５％CO_２存在下20時間培養した。96穴プレートの培地を捨て、FBSを含まないDMEMを0.1ｍL加えた。BioPORTER (Gene Therapy System, Cat. No.BP502401)をクロロホルムで溶解し、風乾にてフィルム化した。これをPhosphate Buffered Saline（Sigma, Cat. No. D8537）に溶解したペプチド（2mg/mL）で再溶解した。5分後、96穴プレートに0.01mLの上記各ペプチドを添加し、37℃、５％CO_２存在下で3〜4時間培養した。10%FBSを含むDMEMを0.1ｍL加え、更に培養を行なった。ペプチド添加から20時間および40時間後に、Steady-Glo Luciferase Assay System (Promega, Cat. No. E2520)でレプリコン活性を測定した。ネガティブペプチドとして配列番号：１２に記載のアミノ酸配列からなるペプチドを使用した。
その結果、NS5Bスフィンゴ結合領域ペプチドおよびその誘導体は終濃度0.1mg/ｍLにおいて、20〜30％の抗HCV複製阻害を示すことが明らかとなった（図３）。一方、ネガティブペプチドでは阻害が観測されなかった。[Example 3] Measurement of HCV replicon inhibitory activity of NS5B sphingo binding domain peptide and derivatives thereof Next, NS5b sphingo binding domain peptide (SEQ ID NO: 1) and its derivative peptide (SEQ ID NO: 10, 11) HCV replicon inhibitory activity was measured.

HCV replicon cells are put in 10,000 cells (0.1 mL) per well of a 96-well plate. Dulbecco's Modified Eagle Medium (hereinafter DMEM) containing 5% Fetal Bovine Serum (hereinafter FBS) (HyClone, Cat. No. SH30071) (Invitrogen, Cat. No. 10569-010) was cultured at 37 ° C. in the presence of 5% CO _{2 for} 20 hours. The medium in the 96-well plate was discarded, and 0.1 mL of DMEM without FBS was added. BioPORTER (Gene Therapy System, Cat. No. BP502401) was dissolved in chloroform and formed into a film by air drying. This was redissolved with a peptide (2 mg / mL) dissolved in Phosphate Buffered Saline (Sigma, Cat. No. D8537). After 5 minutes, 0.01 mL of each peptide was added to a 96-well plate and cultured at 37 ° C. in the presence of 5% CO ₂ for 3 to 4 hours. 0.1 mL of DMEM containing 10% FBS was added and further cultured. Replicon activity was measured with Steady-Glo Luciferase Assay System (Promega, Cat. No. E2520) 20 and 40 hours after peptide addition. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 12 was used as a negative peptide.
As a result, it was revealed that NS5B sphingo-binding domain peptide and derivatives thereof exhibited 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.

Examples described in WO2006 / 16657 are given below as reference examples.
[Reference Example 1] Measurement of HCV RNA replication inhibitory activity of a compound represented by the formula (II) derived from microorganisms such as Myriocin or Aureobasidium by Northern blot analysis Myriocin or the following formula (II) The compounds represented were given to the replicon cells Huh-3-1 in the range of 1 pM to 100 μM and cultured at 37 degrees in the presence of 5% CO ₂ .
Formula (II):

After 72 hours, cells were collected and total RNA was extracted, and then Northern analysis was performed using a neomycin resistance gene as a probe according to the method of Northern Max Kit of Ambion.
The Northern analysis used the following. That is, NorthernMax transfer buffer (Ambion # 8672), transfer film BrightStar-Plus (Ambion # 10100), filter paper (Sigma P-6664), ULTRAhyb (Ambion # 8670). The probe was labeled with biotinylated with BiotinStar Psoralen-Biotin kit (Ambion # 9860G3). High Stringency buffer (Amibion # 8674); BrightStar BioDetect kit (Wash buffer, Ambion # 8650G; Blocking buffer, Ambion # 8651G; Streptavidin-Alkaline Phosphatase, Ambion # 2374G; Assay buffer, Ambion # 8652G; CDP-Star, Ambion # 8653G )

  1 μg of total RNA was electrophoresed on a 1% agarose gel, and after electrophoresis, RNA was stained with ethidium bromide and photographed. After decolorization, it was transferred to a transfer membrane for 2 hours using a NorthernMax transfer buffer. In a wet state, RNA was immobilized on the transfer membrane with a UV crosslinker. Using a hybrid rotor, after pre-rotation treatment with ULTRAhyb at 42 degrees for 30 minutes, the pretreatment liquid was discarded, biotinylated neomycin resistance gene and 10 ml of ULTRAhyb liquid were added, and the mixture was shaken at 42 degrees overnight.

  The ULTRAhyb solution was discarded, 15 ml of High Stringency buffer kept at 42 degrees was added, and the mixture was shaken at 42 degrees for 30 minutes. The same operation was repeated once more. The transfer film was washed with a wash buffer soaked. The transfer membrane was shaken with Blocking buffer for 30 minutes. The solution was discarded and 2 μl of Streptavidin-Alkaline Phosphatase was added to 10 ml of Blocking buffer and shaken at room temperature for 30 minutes. Shake for 10 minutes with Blocking buffer soaking the transfer membrane. Wash with Wash buffer for 5 minutes, wash the transfer film with Wash buffer, cover the transfer film with CDP-Star, remove excess liquid after 5 minutes, expose to X-ray film after 1 hour, and darken the band. RNA amount was compared. As a result, the effect of reducing replicon RNA by 50% at a concentration of 1-10 nM was observed in myriocin and the compound represented by formula (II) (FIGS. 5 and 6).

[Reference Example 2] Measurement of inhibition of HCV protein synthesis by myriocin or compound represented by formula (II) by Western blot analysis Western analysis was performed by the following method. Myriocin or a compound represented by formula (II) was given to replicon cells Huh-3-1 in the range of 1 pM to 100 μM and cultured at 37 ° C. in the presence of 5% CO ₂ . After 72 hours, the medium was discarded, PBS (Phosphate buffered saline) was added, the cells were removed by pipetting, and the cells were collected by centrifugation. Phosphate solution (50 mM Tris-HCl (pH 7.5), 0.5% Triton, 3 mM EDTA, 150 mM NaCl, 12 mM glycerophosphate, 50 mM NaF, 1 mM Na ₃ VO ₄ , 0.5 mM PMSF, 0.5 mM aporotinin) The cells were disrupted by pipetting and the supernatant was recovered by high speed centrifugation. Protein quantification was performed with Dye Reagent (Nacalai tesque # 074-27) (bovine γ globulin standard solution, BIO-RAD # 500-0005). 5 μg of the obtained protein was electrophoresed on a 9-11% gradient gel (Daiichi Kagaku, # 317552) with Tris-Glycine-SDS buffer (BIO-RAD # 161-0772). Rainbow molecular weight markers (AmershamBioscience # RPN756) were used as molecular weight size markers. The electrophoresed protein was transferred to a membrane (PROTRAN BA85, Nitrocellulose transfer membrane (Shleicher & Schuell # 10401196)) using a mini-trans blot cell (BIO-RAD # 170-3930), Western analysis using an anti-NS3 rabbit antibody derived from HCV protein The anti-actin rabbit antibody was used as an internal standard, and as a result, the compound represented by myriocin and formula (II) had an effect of reducing HCV protein expression by 50% at a concentration of 1-10 nM. (FIGS. 7 and 8).

[Reference Example 3] Measurement of HCV replicon inhibitory activity of fumonisin B1 HCV replicon of fumonisin B1 that specifically inhibits dihydrosphingosine-N-acyltransferase that produces dihydroceramide from dihydrosphingosine during the process of sphingolipid biosynthesis. Inhibitory activity was measured.
Firefly luciferase HCV replicon cells (Huh-3-1) suspended in Dulbecco MEM (Gibco cat. No. 10569) containing 5% fetal calf serum (Hyclone cat. No. SH30071.03) and 5000 cells in a 96-well plate / Welled with Well and cultured overnight at 37 ° C. with 5% CO ₂ . After about 20 hours, fumonisin B1 was diluted three-fold sequentially, added to a final concentration of 1.37 μM to 1000 μM, and further cultured for 3 days. Two assay 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 μl of reagent was added per well, mixed 3-4 times with a pipette, allowed to stand for 5 minutes, and luminescence was measured with 1450 MicroBeta TRILUX (WALLAC). The IC50 (50% inhibitory concentration) of the drug was calculated by subtracting the value without addition of cells from all values as the background, and the value without drug addition as 0% inhibition. On the other hand, in the cytotoxicity test, Cell Count Kit-8 (DOJIN Laboratories, cat. No.341-07761) was put in 10 μl per well, mixed with a pipette 3-4 times, and after standing for about 30 minutes, OD450nm became about 1.0. Measured at the time. The IC50 (50% inhibitory concentration) of the drug was calculated by subtracting the value without addition of cells from all values as the background, and the value without drug addition as 0% inhibition.
As a result, it was found that fumonisin B1 exhibits HCV replicon inhibitory activity at a concentration of 10-1000 μM (FIG. 9).

[Reference Example 4] siRNA synthesis of serine palmitoyltransferase
In order to confirm whether inhibition of SPT inhibits HCV replicon activity, siRNA targeting LCB1 (one subunit of SPT heterodimer) was synthesized. Two specific siRNAs (si246, si633) were designed based on the LCB1 cDNA sequence (GenBank Accession No. Y08685) and synthesized by Qiagen. As a control siRNA (SEQ ID NO: 15), a sequence that does not affect the expression of LCB1 was used. The synthesized siRNA sequences are shown in SEQ ID NO: 13 (si246) and SEQ ID NO: 14.
[Reference Example 5] Inhibition of protein expression of LCB1 by siRNA using Western blot analysis
1.2 × 10 ⁵ Huh-3-1 cells were seeded in a 6-well plate and cultured overnight at 37 ° C. and 5%. Add 2.3 μL of 20 μM siRNA to 100 μL of ECR buffer (Buffer included in RNAiFect Kit), mix vigorously, add 4 μL of RNAiFect transfection reagent (Qiagen cat.No. 301605), mix gently, and mix at room temperature. Left for 10 minutes. siRNA was added to a final concentration of 35 nM and cultured for 4 days.

Cell lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5% Triton, 3 mM EDTA, 150 mM NaCl, 12 mM glycerophosphate, 50 mM NaF, 1 mM Na ₃ VO ₄ , 0.5 mM PMSF, 0.5 suspended in mM aporotinin) and left on ice for 10 minutes. The supernatant was collected by centrifugation at 15,000 × g for 10 minutes. After protein quantification, the amount of protein in each sample was adjusted to 10 μg, and 1/4 volume of 5 × SDS sample buffer (125 mM Tris-HCl (pH 6.5), 25% Glycerol, 5% SDS, 0.25% BPB, 5% 2-Mercaptoethanol), then treat at 98 ° C for 5 minutes. After electrophoresis on polyacrylamide gel PAG mini 9/11 (Daiichi Kagaku, cat. No. 317552), the gel is blotted (70 V, 3 hours) on a nitrocellulose membrane PROTRAN BA85 (Schleicher & Schuell cat. No. 10404496). Block the membrane with Blocking Buffer (10% skim milk, 0.1% Tween 20 / PBS). The reaction is carried out with an anti-LCB1 antibody (Transduction cat. No. L89820) diluted 1000 times and an anti-actin (20-33) antibody (Sigma cat. No. A5060) diluted 250 times at room temperature for 2 hours. After washing the membrane with 0.1% Tween 20 / PBS, the secondary antibodies were diluted 1000-fold with anti-mouse IgG-HRP (Cell Signaling cat. No. A7076) and anti-rabbit IgG-HRP (Cell Signaling cat. No. A7074). ) For 1 hour at room temperature. The membrane was washed with 0.1% Tween 20 / PBS, reacted with ECL for 1 minute, and a signal was detected by autoradiography.
As a result, as shown in FIG. 10, a decrease in the protein expression level of LCB1 was observed for both si246 and si633 as compared to the control siRNA. In particular, si246 strongly inhibited expression.

[Reference Example 6] HCV replicon inhibitory activity effect by LCB1 knockdown Based on the results of Reference Example 5, the effects of cytotoxicity and HCV replicon activity under the condition that Huh-3-1 cells decrease LCB1 expression are as follows: It measured by the method of. That is, 3500 cells per well were seeded in a 96-well plate and cultured overnight at 37 ° C. and 5%. Add 1.75 μL of 2 μM siRNA to 23.3 μL of ECR buffer (buffer included in RNAiFect Kit), stir vigorously, add 0.31 μL of RNAiFect transfection reagent, and gently stir at room temperature for 10 Left for a minute. siRNA was added to a final concentration of 35 nM and cultured for 4 days. To determine the effect of cytotoxicity, add 10 μL of Cell counting kit-8 (DOJIN Laboratories cat. No. 341-07761) to the medium, mix 3-4 times with a pipette, leave at 37 ° C. for 1 hour, then microplate reader Emax The absorbance was measured at 450 nm using (Molecular devices). For the HCV replicon activity, replace the culture medium with fresh one after the culture, add 100 μl of reagent per well using Steady-Glo Luciferase Assay System (Promega cat. No. E2520), mix 3-4 times with pipette, and mix for 5 minutes After standing, luminescence was measured with 1450 MicroBeta TRILUX (WALLAC). The IC50 (50% inhibitory concentration) of the drug was calculated by subtracting the value with no cell added from all values as the background, and setting the value with no drug added to 0% inhibition.
As a result, as shown in FIG. 11, HCV replicon activity was significantly inhibited in the cells treated with si246 and si633 in which the expression of LCB1 was suppressed compared to the cells treated with control siRNA. This inhibitory effect was strongly observed with si246 that strongly suppressed the expression of LCB1. Moreover, when the cytotoxicity by siRNA treatment was investigated under the same conditions, almost no effect was observed.

[Reference Example 7] HCV Replicon Assay and Cytotoxicity Test The compound represented by formula (I) or a derivative thereof was subjected to HCV replicon assay and cytotoxicity test.
First, in order to quantify the copy number of HCV-RNA, a HCV-RNA having a firefly-derived luciferase gene introduced as a reporter gene was constructed. According to the method of Krieger et al. (J. Virol. 75: 4614), the luciferase gene was introduced in the form of fusing with the neomycin resistance gene directly under the IRES (Internal Ribosome Entry Site) of the HCV gene. After synthesizing the RNA in vitro, it was introduced into Huh7 cells by electroporation and isolated as a G418 resistant clone. Firefly luciferase HCV replicon cells (Huh-3-1) are suspended in Dulbecco's MEM (Gibco cat. No. 10569-010) containing 5% fetal calf serum (Hyclone cat. No. SH30071.03) and 96-well plate Were seeded at 5000 cells / well and cultured overnight at 37 ° C. with 5% CO ₂ . After about 20 hours, 10 μl of diluted test compound was added per well and further cultured for 3 days. Two assay plates were prepared, one was a white plate and the other was a 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 μl of reagent was added per well, mixed 3-4 times with a pipette, allowed to stand for 5 minutes, and luminescence was measured with 1450 MicroBeta TRILUX (WALLAC). The IC ₅₀ (50% inhibitory concentration) of the drug was calculated by subtracting the value with no cell added from all values as the background, and setting the value with no test compound added to 0% inhibition.

In addition, Cell counting kit-8 (Dojindo catalog No. CK04) was used for the measurement of cytotoxicity. That is, 10 μl of Cell counting kit-8 was added to the 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 nm and a control wavelength of 630 nm. The value with no cells added was subtracted from all the values as the background, and the CC ₅₀ (50% cell inhibitory concentration) of the drug was calculated with the value without drug added as 0% inhibition.
The results of the above HCV replicon assay and cytotoxicity test are shown in Tables 2 and 3.

[Reference Example 8] Inhibition of HCV replicon by a compound represented by formula (II) and toxicity to host cells
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. The luciferase activity by replicon was measured using Steady-GLO luciferase assay system (Promega, cat.no.E2510), and the cell survival inhibitory activity was measured using Cell counting counting kit-8 (Dojin Laboratories, cat.no. 341-07761). As a result, as shown in FIG. 12, HCV replicon inhibitory activity was recognized in a concentration-dependent manner by treatment with the compound represented by formula (II) (IC50 = 2 nM). Further, cytotoxicity of the compound represented by the formula (II) was not recognized (IC50> 50 nM).

[Reference Example 9] Confirmation of inhibition of HCV-NS3 protein expression of the compound represented by formula (II) by immunostaining
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 rabbit IgG (Molecular probe) and analyzed with a fluorescence microscope (FIG. 13). . As a result, as shown in FIG. 13, the HCV-NS3 protein was present around the nucleus, but disappeared by the addition of the compound represented by the formula (II) (the part shown in white is NS3 protein, gray The part indicated by is a nucleus stained with Hoechst 33342 (Sigma, cat. No. B2261).

[Reference Example 10] Inhibition of NS3, NS5A and NS5B protein expression of compound represented by formula (II) by Western blot analysis Replicon cells were shown in FIG. 14 with 100 nM of compound represented by formula (II). Processed for hours (48 and 96 hours). Western blot analysis was performed in the same manner as in Reference Example 5. As a result, the non-structural proteins NS3, NS5A and NS5B of HCV were detected with each antibody in a time-dependent manner. As a result, a decrease in the expression level of the viral protein was observed (FIG. 14).

[Reference Example 11] SPT inhibitory activity of a compound represented by formula (II)
In order to measure SPT inhibitory activity in vitro, human recombinant SPT (heterodimer LCB1 and LCB2) proteins were prepared. From the human liver cDNA library (Clontech, cat. No. 639307), the cDNAs of LCB1 and LCB2 were obtained by RT-PCR and transferred to HisB-tagged pBudCE4.1 vector (Invitogen, cat. No. V532-20). Incorporated. The gene was introduced into HEK293 cells (ATCC, cat. No. CRL-1573), 72 hours later, the cells were lysed, and the protein was purified with Ni-NTA agarose (Qiagen, cat. No. 1018244). SPT activity was measured in reaction buffer [200 mM HEPES buffer (pH 8.0), 5 mM EDTA, 10 mM DTT, 0.05 mM pyridoxal 5-phosphate, 0.2 mM palmitoyl-CoA, 0.1 mM L-serine, and 1 mCi [3H] serine Purified SPT was added to (Amersham, cat. No. TRK308)] and reacted at 37 ° C. for 15 minutes. After extraction with chloroform: 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 FIG. 15, it was revealed that the compound represented by the formula (II) has an SPT inhibitory activity with an IC50 of about 10 nM.

[Reference Example 12] Inhibition of de novo synthesis of ceramide and sphingomyelin by the compound represented by formula (II)
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 [14 C] serine for 3 hours. After extraction with chloroform: 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.

[Reference Example 13] Recovery of replication inhibition of compound represented by formula (II) by C2-ceramide Whether or not the HCV replicon inhibitory activity by the compound represented by formula (II) depends on the sphingolipid biosynthesis pathway In order to clarify the above, a cellular osmotic ceramide: C2-ceramide (Sigma, cat. No. A7191) was added to the HCV replicon cells simultaneously with the compound represented by the formula (II) and cultured for 96 hours. Western blot analysis was performed by the same method as in Example 5 using the cell extract. As a result (FIG. 17), it was revealed that inhibition of HCV replication by the compound represented by formula (II) is suppressed depending on the concentration of C2 ceramide.

[Reference Example 14] Inhibition of HCV replication by raft biosynthesis-related low molecular weight compounds
HCV replicon cells were isolated from the known SPT inhibitor myriocin (Sigma, cat. No. M1177), the ceramide synthesis inhibitor fumonisin B1 (Sigma, cat. No. F1147), and the ceramide transport inhibitor HPA-12 [Kobayashi et al. Org. The replicon activity and the number of viable cells were measured 72 hours later after treatment with lett. (2002). As a result, any compound was found to suppress HCV replication at a concentration at which no cytotoxicity was observed (FIG. 18).

[Reference Example 15] Influence of raft protein by compound represented by formula (II) (1)
After 1 mM of the compound represented by the formula (II) is added to HCV replicon cells for 72 hours, the cell extract is added with 1% Nonidet P-40 (Nacalai tesque, cat. No. 252-23). Time processed. Fraction 1-9 was separated by sucrose density gradient fractionation, and Western blot analysis was performed in the same manner as in Reference Example 5. As a result, as shown in FIG. 19, the compound represented by the formula (II) decreased the expression level of NS5B in the solubilizer-resistant fraction. However, NS5A and the host raft-binding protein caveolin-2 were not affected by the compound represented by formula (II). The expression of caveolin-2 was confirmed by caveolin-2 antibody (BD Transduction, cat. No. 610684).

[Reference Example 16] Influence of raft protein by compound represented by formula (II) (2)
After 1 mM of the compound represented by the formula (II) was added to HCV replicon cells for 72 hours, the cell extract was treated with 1% NP-40 for 1 hour. Raft protein (solubilizing agent resistance) and non-raft protein were separated by sucrose density gradient fractionation, diluted with PBS, concentrated, and then quantified by ELISA analysis. As a result, the compound represented by the formula (II) was significantly dissociated from the raft in NS5B (FIG. 20).

  So far, the mechanism and function of the HCV gene has been elucidated, and the development of HCV therapeutic agents targeting non-structural proteins of HCV has been underway, but the possibility of non-structural proteins themselves to mutate during the replication process Therefore, it has been difficult to develop an effective therapeutic agent.

According to the present invention, it has been found that sphingolipids bind strongly at specific sites of the HCV protein, and the mechanism of viral replication in the host cell has been clarified. Compounds that inhibit these bindings are considered to be extremely useful therapeutic or preventive agents for HCV infection.
The knowledge of the present invention greatly contributes to the development of anti-HCV agents targeting novel binding of sphingolipids and HCV proteins.

  Conventional anti-HCV agents have unclear target cascades and are worried about side effects, but the agents of the present invention have a binding action of sphingolipids and HCV proteins, and the binding site is also clearer Therefore, it is considered that side effects can be easily eliminated and the effect of the drug can be easily adjusted.

Claims (14)

  An agent for treating or preventing HCV infection, comprising a compound that inhibits the binding of sphingomyelin and HCV protein as an active ingredient. The drug according to claim 1, wherein the compound that inhibits the binding of sphingomyelin and HCV protein is the peptide according to the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted The agent according to claim 1, wherein the compound that inhibits the binding of sphingomyelin and HCV protein is an oligonucleotide encoding the peptide according to the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted The drug according to claim 1, wherein the compound that inhibits the binding between sphingomyelin and HCV protein is an antibody that recognizes the peptide according to the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted   The drug according to any one of claims 1 to 4, wherein the HCV protein is HCV-NS5B.   The drug according to any one of claims 1 to 5, wherein the HCV infection is hepatitis C, cirrhosis, liver fibrosis, or liver cancer. A method for screening a drug for treating or preventing HCV infection, comprising the following steps (A) to (C):
(A) A step of bringing a test compound into contact with the peptide described in (a) or (b) below: (a) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 (b A peptide (B) (A) comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 to 4, 10 or 11 A step of detecting the binding between the peptide described and a test compound (C) A step of selecting a test compound binding to the peptide described in (A) A method for screening a drug for treating or preventing HCV infection, comprising the following steps (A) to (C):
(A) A step of adding a test compound to sphingomyelin simultaneously with the peptide described in (a) or (b) below: (a) the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 A peptide (b) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 ) Step of measuring the binding ability of the peptide described in (a) or (b) above and sphingomyelin (C) Select a test compound having a reduced binding ability compared to the case where no test compound is added Process   The screening method according to claim 7 or 8, wherein the HCV infection is hepatitis C, cirrhosis, liver fibrosis, or liver cancer.   A kit for use in the screening method according to any one of claims 7 to 9. A method for evaluating the efficacy of a drug for treating or preventing HCV infection, comprising the following steps (A) to (C):
(A) A step of bringing a test compound into contact with the peptide described in (a) or (b) below: (a) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 (b A peptide (B) (A) comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 to 4, 10 or 11 The step of detecting the binding between the peptide described and the test compound (C) The step of evaluating the efficacy of the therapeutic or prophylactic effect of the test compound binding to the peptide described in (A) on the HCV infection A method for evaluating the efficacy of a drug for treating or preventing HCV infection, comprising the following steps (A) and (B):
(A) A step of adding a test compound to sphingomyelin simultaneously with the peptide described in (a) or (b) below: (a) the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 A peptide (b) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10 or 11 ) A step of evaluating the efficacy of the therapeutic or prophylactic effect of HCV infection by measuring the binding ability of the peptide described in (a) or (b) above and sphingomyelin   A kit for use in the evaluation method according to claim 11. The peptide according to the following (a) or (b).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, 10, or 11 (b) one or more of the amino acid sequences of SEQ ID NOs: 1 to 4, 10 or 11 Peptides consisting of amino acid sequences in which amino acids are substituted, deleted, added and / or inserted

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