WO2007043640A1

WO2007043640A1 - Substance for use in treatment or prevention of hcv infection

Info

Publication number: WO2007043640A1
Application number: PCT/JP2006/320440
Authority: WO
Inventors: Masayuki Sudo; Hiroshi Sakamoto
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2005-10-14
Filing date: 2006-10-13
Publication date: 2007-04-19
Also published as: JPWO2007043640A1

Abstract

An attempt has been made to specify the HCV protein-binding site on a sphingolipid and to examine the binding capability between an HCV protein and the sphingolipid. As a result, it is found that a sphingomyelin (a constituent of a raft) can strongly bind to HCV-NS5B (one of HCV proteins) at a specific site and that HCV virus can be replicated through this binding. It is also found that the replication of HCV virus can be prevented by inhibiting the binding between a sphingomyelin and an HCV protein.

Description

Specification

Drugs for treating or preventing HCV infection

Technical field

[0001] 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.

Background art

[0002] It is estimated that there are 100-200 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. It is said that hepatitis C is responsible for about 90% of liver cancer. In Japan, more than 30,000 patients die from liver cancer associated with HCV infection every year.

[0003] 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.

[0004] 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. However, it is also known that many patients with liver cancer recurrence due to inflammation that continues in non-cancerous areas even after surgery.

[0005] Therefore, establishment of an effective treatment method for hepatitis C is desired. Among them, apart from coping therapy that suppresses inflammation with anti-inflammatory agents, HCV is reduced or eradicated in the affected liver. There is a strong demand for development of drugs that can be used.

[0006] Currently, interferon treatment is known as the only effective treatment for HCV elimination. However, about 1Z3 of all patients are effective for interferon. In particular, 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.

[0007] In recent years, ribavirin: 1—β-D-ribofuranosyl 1 一 — 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. In addition, 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.

[0008] Since the HCV gene has been cloned, molecular biological analysis has progressed rapidly due to the mechanism and function of the viral gene and the function of the protein of each virus. However, mechanisms such as persistent infection and pathogenicity have not been fully elucidated, and a reliable HCV infection experiment system using cultured cells has been established and has been very powerful. Therefore, in the past, when evaluating anti-HCV drugs, it was necessary to use an alternative virus-assy method using other closely related viruses.

[0009] In recent years, it has become possible to observe HCV replication in vitro using non-structural regions of HCV, so that anti-HCV drugs can be easily evaluated by the levicon assembly assay. (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.

[0010] Information on prior art documents related to the invention of the present application is shown below.

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

Disclosure of the invention

Problems to be solved by the invention

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).

[0012] 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) 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.

[0013] 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.

Means for solving the problem

[0014] In order to solve the above problems, the inventors of the present application have developed HCV protein and sphingolipid

The binding site of (sphingomyelin) was identified and the binding ability was examined.

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 with a helix-turn-helix motif was observed in the NS5B sequence E230-G263 (Fig. 1).

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[0015] Next, using Biacore S51, the binding ability of NS5B sphingo binding domain (NS5B-SBD) and sphingomyelin (SM) was confirmed. NS5B-SBD peptide bound to sphingomyelin in a concentration-dependent manner. It became clear that doing this (Figure 2A). In prion protein (PrP) However, similar binding was observed (Figure 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 their binding. It became power. Moreover, it became clear that inhibition of HCV virus replication 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 containing a compound that inhibits the binding of sphingomyelin and HCV protein as an active ingredient, Thus, the present invention has been completed.

[0017] 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] 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).

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

(b) SEQ ID NO: 1 peptide in which one or more amino acids are substituted, deleted, added, and Z or inserted in the amino acid sequence described in any one of 4, 10, or 11

[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).

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

(b) SEQ ID NO: 1 in the amino acid sequence described in any one of 4, 10 or 11, in which one or more amino acids are substituted, deleted, added, and amino acid sequence is not Z or inserted. Peptide

[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.

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[5] The drug according to [1] to [4], wherein the HCV protein 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 according to the following (a) or (b)

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

(B) a step of detecting the binding between the peptide according to (A) and a test compound

(C) a step of selecting a test compound that binds to the peptide according to (A)

[8] A method for screening a drug for treating or preventing HCV infection, comprising the following steps (A) to (C):

(A) A step of adding the test compound to the sphingomyelin simultaneously with the peptide described in (a) or (b) below

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

(b) 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

(B) a step of measuring the binding ability of the peptide according to (a) or (b) above and sphingomyelin

(C) a step of selecting a test compound having a reduced binding ability compared to the case without adding a test compound

[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) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

(C) A step of evaluating the efficacy of the therapeutic or prophylactic effect of HCV infection of a test compound that binds to the peptide of (A)

[12] A method for evaluating the efficacy of a drug for treating or preventing HCV infection, comprising the following steps (A) and (B):

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

(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.

[13] A kit for use in the evaluation method according to any one of [11] and [12].

[14] The peptide according to the following (a) or (b).

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

Brief Description of Drawings

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 (Π).

[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 (Π) 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. BEST MODE FOR CARRYING OUT THE INVENTION

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. [0020] In the present invention, 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.

[0021] In the present invention! As HCV proteins, HCV-NS2, HCV-NS3, HCV-NS4A, HCV-NS4B, HCV-NS5A, HCV-NS5B can be mentioned, more preferably HCV-NS5B. it can.

[0022] 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) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[0023] SEQ ID NO: 1 to 4, 10 or 11, for example, 1 to 10, preferably 1 to 5 amino acid residues in the amino acid sequence substitution, deletion, addition, and Z or insertion 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. For example, 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, Υ), 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, Η), 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). An amino acid sequence modified by deletion or addition of one or more amino acid residues to a certain amino acid sequence, and substitution by Z or another amino acid. It is already known that a polypeptide having a sequence maintains its biological activity.

[0024] 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.

[0025] 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. .

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

The oligonucleotide of the present invention can be used by incorporating it into an appropriate vector in a form capable of expressing the peptide.

[0026] 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 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.

[0027] 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.

(a) SEQ ID NO: 1 to 4, 10, or 11 Chido

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.

[0028] An antibody recognizing the peptide used in the present invention can be obtained as a polyclonal or monoclonal antibody using a known means. As the antibody recognizing the peptide used in the present invention, 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.

[0029] 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.

[0030] Specifically, an antibody that recognizes the peptide may be prepared as follows!

After inserting a base sequence encoding the peptide into a known expression vector system and transforming an appropriate host cell, 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. In addition, a fusion protein of the peptide and another protein may be used as a sensitizing antigen.

[0031] In the present specification, the term "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. The term “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.

[0032] The compound of the present invention can be used in medicine. The salt is not particularly limited as long as it is pharmacologically acceptable. For example, 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. .

[0033] 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. In this case, it can be formulated using known adjuvants that can be usually used. In the case of forming into a tablet form, 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. 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.

[0034] Furthermore, 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. For example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and tar; And binders such as tragacanth powder, gelatin and ethanol; and disintegrants such as laminaran agar. In the case of forming into a suppository, 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. When prepared as injections, 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. 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 solubilizing agent, buffering agent, soothing agent may be used. An agent or the like may be added. In addition, it may contain colorants, preservatives, fragrances, flavors, sweeteners, and other medicines as necessary.

[0035] 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. Of course, the pharmaceutical composition is administered in a dosage form suitable for these administration methods.

[0036] 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. However, for humans, 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. When administering a large amount, it is desirable to divide it into several times a day.

[0037] 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.

[0038] 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.

[0039] The topical administration (eg, transdermal 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. For these, 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.

[0040] In the above-mentioned rectal administration, 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.

[0041] 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 consist of suspending or dissolving 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, then the suspension or solution. It is manufactured by sterilizing.

As a method for transporting a protein such as the peptide or antibody of the present invention into a cell, for example, a peptide having a cell membrane permeation function (for example, Pegelin, Penetratin, etc.) is attached. (Martine Mazel etal, 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.), It is possible to transport the protein of the present invention into cells.

[0042] In addition, as 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) introduced into the organization can be mentioned. 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.

[0043] The present invention relates to a method for screening a drug for treating or preventing HCV infection.

In the first embodiment of the screening method of the present invention, first, _a test compound is contacted with the peptide described in the following ( _a ) or (b).

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[0044] 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.

[0045] 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. As the in vitro transcription / translation system, it is possible to use a commercially available in vitro transcription / translation kit without particular limitation.

[0046] The "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.

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 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. 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 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.

[0048] In the first embodiment, next, 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 label type include fluorescent labels and radiolabels. Moreover, 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. 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.

[0049] In 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 the following (a) or (b).

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[0050] In the second embodiment, 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. In this method, 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.

[0051] The present invention also relates to a method for evaluating the efficacy of a drug for treating or preventing HCV infection.

In the 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) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

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. Next, the efficacy of the therapeutic or prophylactic effect of HCV infection of the test compound bound to the above 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 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.

[0052] In a second embodiment 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) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[0053] In the second aspect, in the following, 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. Evaluate efficacy. The binding ability can be measured by the method described above. 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. 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 more susceptible to HCV infection. It can be evaluated that it shows an effective therapeutic or preventive effect.

[0054] 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. 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. 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.

Example

[0055] 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-lgpl20-derived V3 loop (Fig. 1A, SEQ ID NO: 4) and prion-derived PrP protein have sphingolipid binding domains. In order to search for the presence of a sphingolipid binding region in HCV proteins, 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 closely matched that of the V3 loop (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).

[0057] [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. 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. 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 observed in prion protein (PrP) (Fig. 2B, RU value at 80 s sensorgram in Figure 2A).

[0058] From the above results, Sphingomyelin, which is a component of raft, and HCV protein HCV-NS5B are strongly bound at a specific site, and the HCV virus is replicated by their binding. Clearly became a force. These findings suggest that replication of HCV virus can be suppressed by inhibiting the binding of sphingomyelin and HCV protein.

[Example 3] Measurement of HCV replicon inhibitory activity of NS5B sphingo binding domain peptide and its derivatives

Next, using HCV revlikon cells, measure the HCV revlikon inhibitory activity of NS5b sphingo binding domain peptide (SEQ ID NO: 1) and its derivative peptides (SEQ ID NO: 10, 11).

[Table 1] Types of peptides N-terminal → C-terminal sequence SEQ ID NO:

Pep-1 derivative 1 D 1 RVEES 1 YQCCALAPAAAQA 1 KSLTERLY 10

Pep-2 derivative 2 D 1 RVEES IAQCCDAAPEARQAAKSLTERLY 11

Pep-3 NS5b protein D 1 RVEES 1 YQCCDLAPEARQA 1 KSLTERLY 1

Pep-4 negative peptide YLRETLSK 1 AQRAEPALDCCQY 1 SEEVR 1 D 12

Place HCV replicon cells in a 96-well plate at 10,000 cells per well (O.lmL), 5% Fetal

Dulbecco 's Modified including Bovine Serum (J¾ BS) (Hv lone, Cat.No. SH30071)

37 in Eagle Medium (DMEM) (Invitrogen, Cat. No. 10569-010). C, 5% CO

2 Cultured for 20 hours. The medium in the 96-well plate was discarded, and O.lmL of DMEM without FBS was added. BioPORTER (Gene Therapy System, Cat. No. BP502401) was dissolved in black mouth form and filmed by air drying. This was redissolved with a peptide (2 mg / mL) dissolved in Phosphate Buffered Saline (Sigma, Cat. No. D8537). After 5 minutes, each peptide of O.OlmL was added to a 96-well plate and cultured at 37 ° C. in the presence of 5% CO for 3 to 4 hours. Includes 10% FBS

2

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.

As a result, it became clear that 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.

[0060] Examples described in WO2006 / 16657 are given below as reference examples.

[Reference Example 1] Measurement of HCV RNA replication inhibitory activity of compounds represented by formula (II) derived from microorganisms such as myriocin or Aureobasidium by Northern blot analysis

Myriocin or a compound represented by the following formula (II) was given to the replicon cell Huh-3-l in the range of ΙρΜ to 100 μΜ and cultured at 37 ° C in the presence of 5% CO.

2

Formula (Π):

[0062] 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 in accordance with the Northern Max Kit method 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), 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)

[0063] 1 μg of total 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.

[0064] The ULTRAhyb solution was discarded, 15 ml of High Stringency buffer kept at 42 ° C was added, and the mixture was shaken at 42 ° C for 30 minutes. The same operation was repeated once more. The transfer film was washed with a wash buffer that was soaked. The transfer membrane was shaken with Blocking buffer for 30 minutes. The solution was discarded, and 2 ml of Streptavidin-Alkaline Phosphatase in 10 ml Blocking buffer was added and shaken for 30 minutes at room temperature. 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 concentrate the band. The amount of RNA was compared. As a result, the effect of reducing replicon RNA by 50% at a concentration of 1-lOnM was recognized for myriocin and the compound represented by formula (II) (FIGS. 5 and 6).

[0065] [Reference Example 2] Measurement of inhibition of HCV protein synthesis by myriocin or a compound represented by formula (II) by Western plot analysis

Western analysis was performed by the following method. Myriocin or a compound represented by the formula (II) is given to the replicon cell Huh-3-l in the range of ΙρΜ to 100 / z M, 37 ° C in the presence of 5% CO.

2

Incubated in 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 glycerophosp hate, 50 mM NaF, 1 mM Na VO, 0.5 mM PMSF, 0.5 mM aporotinin) Pipette

3 4

Cells were disrupted by ting and the supernatant was collected by high speed centrifugation. Protein quantification was performed with Dye Reagent (Nacalai tesque # 074-27) (USH γ globulin standard solution, BIO-RAD # 500-0005). 5 g of the obtained protein was electrophoresed with Tris-Glycine-SDS buffer (BIO-RAD # 161-0772) on 911% gradient gel (Daiichi Kagaku, # 3 17552). Molecular weight size ~~ force '~~ i Rain. ow molecular weight markers (AmershamBioscience # RPN7 56) were used. 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. As a result, 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).

[0066] [Reference Example 3] Measurement of HCV replicon inhibitory activity of fumosin B1

In the middle stage of sphingolipid biosynthesis, HCV replicon inhibitory activity of fumonisin B1, which specifically inhibits dihydrosphingosine synthase, which produces dihydrosphingosine, a dihydroceramide, was measured.

Suspension of firefly luciferase HCV replicon cells (Huh-3-l) in Dulbecco's MEM (G¾co cat. No. 10569) containing 5% urine fetal serum (Hyclone cat. No. SH30071.03) The cells were seeded with 5000 cells ZWell and cultured overnight at 37% with 5% CO. About 20 hours later

2

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 μl 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). 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. On the other hand, in the cell toxicity test, Cell's Count Kit 8 (DOJIN Laboratories, cat.No.341-07771) was put in 10 μl 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.

As a result, it was found that fumosin B1 exhibits HCV replicon inhibitory activity at a concentration of 10-1000 M (FIG. 9).

[0067] [Reference Example 4] siRNA synthesis of serine palmitoyltransferase

In order to confirm whether inhibition of SPT inhibited HCV replicon activity, we synthesized siRNA targeting LCB1 (one subunit of SPT heterodimer). Two specific The siRNAs (si246, si633) were designed based on the LCBlcDNA 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 sequence is shown in SEQ ID NO: 13 (si24 6) and SEQ ID NO: 14.

[Reference Example 5] Inhibition of protein expression of LCB1 by siRNA using Western plot analysis 1.2 × 10 ⁵ Huh-3-l cells were seeded in a 6-well plate and cultured 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, and then add 4 / z L of RNAiFect transfection reagent (Qiagen cat. No. 3). 01605) was added and the mixture was gently stirred and allowed to stand at room temperature for 10 minutes. The siRNA was culled to a final concentration of 35 nM and cultured for 4 days.

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. After protein quantification, the amount of protein in each sample was adjusted to 10 g, and 1/4 volume of 5 X SDS sample buffer (125 mM Tris-HC1 (pH 6.5), 25% Glycerol, 5% SDS, 0.25) % BPB, 5% 2-Mercaptoethanol) Treat at 98 ° C for 5 minutes. After electrophoresis on polyacrylamide gel PAG mini 9/11 (Daiichi Kagaku, cat. No. 317552), the gel was blotted onto -trocellulose membrane PROTRAN BA85 (Schleicher & Schuell cat. No. 10404496) (70V, 3 hours) To do. Block the membrane with Blocking Buffer (10% skim milk, 0.1% Tween 20 / PBS). Incubate with anti-LCB1 antibody (Transduction cat. No. L89820) diluted 1000-fold and anti-actin (20-33) antibody (Sigma cat. No. A5060) diluted 250-fold 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 (Cel 1 Signaling cat. No. A7074) is reacted for 1 hour at room temperature. The membrane was washed with 0.1% Tween 20 / PBS, reacted with ECL for 1 minute, and the 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 inhibition of expression. I was cut off.

[0069] [Reference Example 6] Effect of LCB1 knockdown on HCV levicon control activity

Based on the results of Reference Example 5, the effects of cell toxicity and HCV revlikon activity under conditions where Huh-3-l cells decrease LCB1 expression were measured by the following methods. That is, 3500 cells per well were seeded in a 96-well plate and cultured at 37 ° C and 5%. Add 1.75 L of 2 M 3 «^ to 23.3 μL of ECR buffer (buffered in RNAiFect Kit), mix vigorously, and add 0.31 μL of RNAiFect transfection reagent gently. And allowed to stand at room temperature for 10 minutes. siRNA was added to a final concentration of 35 nM and cultured for 4 days. To determine the effects of cytotoxicity, add 10 μL of Cell counting kit-8 (DOJI N Laboratories cat. No. 341-07761) to the medium and mix 3-4 times with a pipette, and leave at 37 ° C for 1 hour. Thereafter, the absorbance was measured at 450 nm using a microplate reader Emax (Molecular devices). HCV levulincon activity is replaced with fresh medium after completion of culture, Steady-Glo Luciferase Assay System (Promega cat.no.E2520) is used, 100 μl of reagent is added per well, and mixed by pipetting 3-4 times. After standing for 5 minutes, luminescence was measured with 1450 MicroBeta TRILUX (WAL LAC). The IC50 (50% inhibitory concentration) of the drug was calculated by subtracting all values from the value of the non-cell-added caro as the background, and the value with no drug added as 0% inhibition.

As a result, as shown in FIG. 11, 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. Under the same conditions, the cytotoxicity of siRNA treatment was examined. Almost no effect was observed.

[0070] [Reference Example 7] HCV rebricon assembly and cytotoxicity test

The HCV replicon assay and cytotoxicity test were conducted on the compounds represented by formula (I) or their derivatives.

First, in order to quantify the number of HCV-RNA copies, we constructed HCV-RNA with a firefly-derived luciferase gene introduced as a reporter gene. According to the method of Krieger et al. (J. Virol. 75: 4614), the HCV 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

2

Incubated overnight at 37 ° C. After about 20 hours, 10 1 of diluted test compound was added per well, and further cultured for 3 days. Two assembly plates were prepared, one for the white plate and the other for the plate. After completion of the culture, the white plate was used for Steady-Glo Luciferas e 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 (WAL LAC). Subtract all the values from the value of un-added caroline as a background, and set the IC (50% inhibitory concentration) of the drug to 0% as the value without the test compound added.

50

) Was calculated.

[0071] 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.

50

The results of the above HCV replicon assay and cytotoxicity test are shown in Tables 2 and 3.

[0072] [Table 2]

Lebricon Atsey Cytotoxic Compound No. I C50 [M] CC50 Ifl M]

(1) 0. 002> 5

(2) 0. 01 0> 5

(3) <0. 001> 5

(4) 0. 001> 5

(5) 0. 002> 5

(6) 0. 007> 5

(7) 0. 004> 1

(8) 0. 01 4> 1

(9) 0. 01 7> 1

(1 0) 0. 01 1> 1

(1 1) 0. 009> 1

(1 2) 0. 01 7> 1

(1 3) 0. 01 0> 1

(1 4) 0. 009> 1

(1 5) 0. 006> 1

(1 6) 0. 008> 1

(1 7) 0. 01 2> 1

(1 8) 0. 068> 1

(1 9) 0. 01 2> 1

(20) 0. 055> 1

(21) 0. 080> 1

(22) 0. 500> 1

(23) 0. 21 0> 1

(24) 0. 024> 1

(25) 0. 020> 1

(26) 0. 001> 1

(27) 0. 002> 1

Lebricon Atsey Cytotoxicity

Compound number CC50 [jU M]

(28) 0. 001> 1

(29) 0. 003> 1

(30) 0. 001> 1

(31) 0. 005> 1

(32) 0.800> 5

(33) 0. 250> 1

(34) 0. 003> 1

(35) 0. 004> 1

(36) 0. 004> 1

(37) 0. 01 7> 1

(38) o 0. 024> 1

(39) 0. 002> 1

(40) 0. 002> 5

(41) 0. 1 28> 1

(42) 0. 076> 1

(43) 0. 1 03> 1

(44) 0. 082> 1

(45) 0. 007> 1

(46) 0. 002> 5

(47) 0. 005> 5

(48) 0. 020> 5

(49) 0. 245> 50

(50) 0. 262> 5

(51) 0. 072> 5

(52) 0. 1> 50

(53) 0. 020 22

(54) 0. 020> 50

[0074] [Reference Example 8] Inhibition of HCV rebricon by 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. Steady— (JLO luciferase assay system (Promega, cat. No. E251 0), cell viability inhibitory activity) by cell count counting kit— 8 (Dojin Laboratories, cat. As a result of measurement using no. 341-07761), as shown in FIG. 12, HCV levulincon inhibitory activity was observed in a concentration-dependent manner by treatment with the compound represented by formula (II) (IC50 = 2 nM). In addition, the cytotoxicity of the compound represented by the formula (II) was not observed (IC50> 50 nM).

[0075] [Reference Example 9] Inhibition of HCV-NS3 protein expression by a compound represented by formula (II) by immunostaining Harm confirmation

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)).

[Reference Example 10] Inhibition of NS3, NS5A and NS5B protein expression of compound represented by formula (II) by Western blot analysis

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. As a result, 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).

[0077] [Reference Example 11] SPT inhibitory activity of the compound represented by formula (II)

in vitr. In order to measure the SPT inhibitory activity in, human recombinant SPT (heterodimer LCB1 and LCB2) proteins were prepared. 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 卜 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.

[0078] [Reference Example 12] Inhibition of de novo synthesis of ceramide and sphingomyelin by a 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 [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.

[0079] [Reference Example 13] Recovery of replication inhibition of compound represented by formula (II) by C2-ceramide

To elucidate whether the HCV revlikon inhibitory activity of the compound represented by the formula (II) depends on the sphingolipid biosynthesis pathway, a cellular permeable ceramide: C2-ceramide (S igma, cat. No A7191) was added to the HCV replicon cells simultaneously with the compound represented by formula (II) and cultured for 96 hours. Using the cell extract, Western blot analysis was performed in the same manner as in Example 5. As a result (FIG. 17), it was revealed that inhibition of HCV replication by the compound represented by the formula (II) is suppressed depending on the concentration of C2 ceramide.

[0080] [Reference Example 14] Inhibition of HCV replication by low molecular weight compounds related to raft biosynthesis

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).

[0081] [Reference Example 15] Effect of raft protein by the compound represented by formula (II) (1)

After adding the compound represented by formula (II) of ImM to HCV replicon cells for 72 hours, the cell extract was solubilized with 1% Nonidet P-40 (Nacalai tesque, cat. No. 252-23). Treated for 1 hour. Fractions 1-9 were separated by sucrose density gradient fractionation, and Western plot 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. While doing NS5A and the host raft-binding protein strength beolin-2 were not affected by the compound represented by formula (II). The expression of strength veolin-2 was confirmed with the strength veolin-2 antibody (BD Transduction, cat. No. 610684).

[0082] [Reference Example 16] Influence of raft protein by compound represented by formula (II) (2)

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).

Industrial applicability

[0083] So far, the mechanism and function of the HCV gene has been elucidated, and the development of HCV therapeutics targeting non-structural proteins of HCV has progressed. However, the non-structural proteins themselves mutated during the replication process. Therefore, it was difficult to develop an effective therapeutic agent.

[0084] According to the present invention, it was found that strong binding was performed at a specific site of the sphingolipid protein, and the mechanism of viral replication in the host cell became clear. 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.

[0085] Conventional anti-HCV agents have unclear target cascades and have been worried about side effects. However, the agents of the present invention have a binding action of targeting sphingolipids and HCV proteins, and the binding sites are also more It is clear that side effects can be easily eliminated and the effects of drugs can be easily adjusted.

Claims

The scope of the claims

[2] The drug according to claim 1, which is a compound having the ability to inhibit the binding of sphingomyelin and HCV protein, which is the peptide according to the following (a) or (b).

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[3] The drug according to claim 1, which is an oligonucleotide encoding the peptide according to (a) or (b) below, which is a compound ability to inhibit the binding of sphingomyelin and HCV protein.

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[4] The drug according to claim 1, which is an antibody that recognizes the peptide according to (a) or (b) below, which has a compound ability to inhibit the binding of sphingomyelin and HCV protein.

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[5] The drug according to any one of claims 1 to 4, wherein the HCV protein is HCV-NS5B!

[6] The drug according to any one of claims 1 to 5, wherein the HCV infection is hepatitis C, cirrhosis, liver fibrosis, or liver cancer.

[7] An agent for treating or preventing HCV infection comprising the following steps (A) to (C): Screening method.

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[8] A screening method for a drug for treating or preventing HCV infection, comprising the following steps (A) to (C):

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[9] The HCV infection is hepatitis C, cirrhosis, liver fibrosis, or liver cancer, or 7

The screening method according to 8.

[10] A kit for use in the screening method according to any one of claims 7 to 9.

(A) A step of bringing a test compound into contact with the peptide according to the following (a) or (b) (a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability

[13] A kit for use in the evaluation method according to any one of claims 11 and 12.

[14] The peptide according to the following (a) or (b).

(a) SEQ ID NO: 1 to 4, 10 or 11 peptide having amino acid sequence ability