JP6910043B2 - Hemagglutinin-binding peptide and preventive / therapeutic agents for influenza virus infections containing it - Google Patents

Description

The present invention relates to a hemagglutinin-binding peptide and a prophylactic / therapeutic agent for influenza virus infection containing the same.

Influenza A virus has a strong infectious ability and causes a seasonal epidemic every year in Japan and other parts of the world. On the other hand, there are concerns about the emergence of a new influenza virus as seen in 2009 and the acquisition of human infectivity for highly pathogenic avian influenza viruses such as H5N1 and H7N7, and a fatal pandemic is occurring worldwide. It is in a situation where it can occur at any time.

However, it is rapidly resistant to neuraminidase (NA) inhibitors such as osetamivir (trade name: Tamiflu®) and zanamivir (trade name: Relenza®), which are commonly used today. Is spreading, and there is an urgent need to establish a new drug for treating influenza.

On the particle surface of influenza A virus, hemagglutinin (hereinafter sometimes referred to as "HA"), which is involved in the binding of the virus to target cells, and NA, which is involved in the withdrawal of new virus particles from infected cells, 2 It is known that species of membrane-bound glycoproteins are present.

Furthermore, HA also plays a role in causing the fusion of the target cell membrane and the virus membrane after the virus binds to the target cell and releasing the viral gene into the cytoplasm, and plays an essential role in the virus growth cycle. It has been known. HA is also an antigen for many influenza vaccines.

Although the above suggests the importance of HA as a drug discovery target, at present, no therapeutic drug targeting HA has been put on the market.

HA has a trimer structure on the viral membrane, and one HA molecule recognizes and binds to one sialic acid molecule of glycoprotein or glycolipid existing on the host cell membrane. Therefore, a total of 3 molecules of sialic acid can be bound in the HA trimer. At this time, it is known that the binding affinity increases several thousand times in the 3: 3 binding as compared with the 1: 1 binding, and this phenomenon is called "cluster effect". For this reason, conventional techniques such as screening of small molecule compound libraries and phage display screening can basically be applied only to 1: 1 interactions, so that HA that functions by exerting a cluster effect is accepted. In principle, it is extremely difficult to identify an inhibitor by targeting a bacteriophage site. In fact, there have been no cases of obtaining small molecule compounds, and cases of obtaining HA-binding peptides by cyclic peptide library (Patent Document 1) or phage display screening (Patent Document 2) have been reported, but the cluster effect There have been no reports of isolation of inhibitory peptides directly targeting the receptor binding site of HA based on.

On the other hand, the present inventors have so far developed a polyvalent peptide library method as a technique for screening a molecule that inhibits a strong interaction based on the cluster effect (Patent Document 3). Since the polyvalent peptide library has a structure in which four random peptide libraries are bound to a tetravalent nuclear structure, it itself exerts a cluster effect. In addition, we have succeeded in developing an inhibitory molecule for various molecules that exert this cluster effect and function (for example, Patent Documents 4 to 6).

Furthermore, the present inventors have also established a technique for synthesizing a multivalent peptide on a sheet at a level of several hundreds by applying the polyvalent peptide library method (Patent Document 7).

Japanese Unexamined Patent Publication No. 2013-71904 Japanese Patent No. 5396111 Japanese Patent No. 4744443 Japanese Patent No. 5635779 Japanese Patent No. 5718574 Japanese Patent No. 5754008 Japanese Patent No. 5718574

By using the polyvalent peptide library method that has been established so far by targeting the receptor binding site of HA, the present invention can obtain a novel polyvalent peptide that binds to HA by the cluster effect. At the same time, the present invention has been completed based on the idea that a preventive / therapeutic agent for influenza virus infection can be established by using this polyvalent peptide as an active ingredient.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel hemagglutinin-binding peptide and a prophylactic / therapeutic agent for influenza virus infection containing the same.

In the hemagglutinin-binding peptide of the present invention, the peptide motif of SEQ ID NO: 1 directly or has a spacer on each of the four amino groups located at the end of the molecular nuclear structure formed by binding three lysines (Lys). It is characterized by being a tetravalent peptide bound via.

In this hemagglutinin-binding peptide, it is preferable that arginine (Arg) located on the N-terminal side of the peptide motif of SEQ ID NO: 1 is replaced with an unnatural isomer (D-Arg).

The prophylactic / therapeutic agent for influenza virus infection of the present invention is characterized by containing the above-mentioned hemagglutinin-binding peptide.

According to the hemagglutinin-binding peptide of the present invention, it can bind to hemagglutinin of influenza virus and inhibit cytotoxic activity due to infection with influenza virus. Further, according to the prophylactic / therapeutic agent for influenza virus infection of the present invention, influenza infection can be effectively prevented / treated.

FIG. A is a diagram showing the molecular weight and purity of wild-type HA (WT-HA) and mutant HA (L194A-HA) immobilized on Ni beads by electrophoresis. FIG. B is a diagram showing the measurement results of the binding activity of WT-HA and L194A-HA to fetish by the ELISA method. It is a figure which shows the chemical structure of the multivalent peptide library used in the screening of Example 2. FIG. A) is a diagram (table) showing the primary library used in the primary screening in Example 2 and the selectivity of amino acids at each position. B) is a diagram (table) showing the secondary library used in the secondary screening in Example 2 and the selectivity of amino acids at each position. HA inhibitor candidate substances (TKR-tet, RVH-tet) at each concentration were added to MDCK cells, and 30 minutes later, influenza A virus (A / PR / 8/34) was administered at a high titer (MOI = 10). It is a figure which shows the result of having measured the viability of a cell after infection and culturing for 24 hours by the WST method. It is a figure which shows the result of having blotting the sheet with ^{WT-HA which was radiation-labeled with 125 I, and measuring the bound radioactivity.} It is the figure which showed the appearance which the sheet was blotted with WT-HA and L194A-HA which were radiation-labeled with ^{125 I.} It is the figure which showed the appearance which the sheet was blotted with WT-HA and L194A-HA which were radiation-labeled with ^{125 I.} Inhibitors of each concentration (PVD-tet, PMH-tet, PVN-tet, PVF-tet, PVP-tet) were added to MDCK cells, and 30 minutes later, the A / PR / 8/34 influenza virus had a high titer (high titer). It is a figure which shows the result of having measured the viability of the cell after being infected with MOI = 10) and culturing for 24 hours by the WST method. It is a figure which shows the result of having added each inhibitor of 300 μg / ml to MDCK cell, and measured the viability of the cell after culturing for 48 hours by the WST method. It is a figure which shows the result (molecular weight) which treated PVF-tet and ^{(D) PVF-tet with trypsin.} It is a figure which shows the result of having measured the binding of PVF-tet with WT-HA or L194A-HA by the ELISA method. ^(D) It is a figure which shows the result of having measured the binding of PVF-tet with WT-HA or L194A-HA by the ELISA method. In A), each concentration of inhibitor (PVF-tet, ^(D) PVF-tet, RVH-tet) or fetishine was added to MDCK cells, and 30 minutes later, influenza virus was infected with a low titer (MOI = 0.001). It is a figure which shows the result of having measured the viability of the cell after culturing for 48 hours by the WST method. In B), each concentration of inhibitor (PVF-tet, ^(D) PVF-tet, RVH-tet) or fetishine was added to MDCK cells, and 30 minutes later, influenza virus was infected with a high titer (MOI = 10). It is a figure which shows the result of having measured the viability of the cell after culturing for 24 hours by the WST method. It is a figure which showed the result of having compared the inhibitory activity of PVF-tet under the condition of high titer infection (MOI = 10) with the monomer peptide which has the same sequence as the HA binding motif of PVF-tet. Each inhibitor of 300 μg / ml was added to MDCK cells, and after 30 minutes, A / PR / ^{8/34 influenza virus was infected with 6 × 10 4} pfu / ml, and the amount of new virus in the culture medium after 14 hours. Is quantified by the plaque assay method. 6-8 week old Balb / c mice (female) were nasally administered with A / PR / 8/34 influenza virus (2000 pfu) alone or in the ^{presence of each amount of (D)} PVF-tet. It is a figure which shows the result of having measured the change. It is a figure which shows the result of having compared the survival rate by nasal administration of influenza virus (2000 pfu) alone or in the ^{coexistence of each amount (D) PVF-tet to a mouse.} It is a figure which shows the result of having measured the amount of virus in the lung after 3 days and 5 days by nasal administration of influenza virus (2000 pfu) alone or in the coexistence of PVF-tet to a mouse by a plaque assay.

The hemagglutinin-binding peptide of the present invention contains the following molecular nuclear structure formed by binding three lysines (Lys).

The hemagglutinin-binding peptide of the present invention has the following peptide motifs on each of the four -NH groups located at the ends of the above molecular nuclear structure.
SEQ ID NO: 1: Arg-Arg-Pro-Val-Asn-His-Phe (RRPVNHF)
Is a tetravalent peptide bound directly or via a spacer.

Specifically, one embodiment of the hemagglutinin-binding peptide of the present invention is, for example, arranged in each of the four XXXX parts located at the ends of the molecular nuclear structure consisting of three lysines (Lys) in the following chemical formula. A tetravalent peptide incorporating the peptide motif of No. 1 is exemplified.

In the above chemical formula 2, the position where the peptide motif is incorporated is described as "XXXX" for convenience.

Hereinafter, a tetravalent peptide in which four peptide motifs of SEQ ID NO: 1 are incorporated in the XXXX portion located at the end of the molecular nucleus structure consisting of three lysines (Lys) may be described as "PVF-tet". ..

Further, in the above chemical formula 2, a form in which a spacer is bonded to each of the four amino groups located at the end of the molecular nucleus structure is illustrated, but each of the four amino groups is not mediated by a spacer. , The peptide motif of SEQ ID NO: 1 can also be directly linked. When binding the spacer, it is sufficient as long as it does not impair the binding property to hemagglutinin, and the specific molecule and length are not limited and can be appropriately designed. As the spacer, for example, a spacer having a chain length of about 4 to 10 carbon atoms having an amino acid at the terminal is preferable, and in particular, an amino hexanoic acid [NH _2- (CH ₂ ) _{5 represented by "U" in the above chemical formula 2 is preferable.} -COOH] (aminocaproic acid) can be preferably exemplified. Further, as the amino acid contained in the spacer, for example, alanine (A) can be exemplified.

Furthermore, the hemagglutinin-binding peptide of the present invention may have a modified molecule at each end of the peptide motif of SEQ ID NO: 1 incorporated in the XXXX portion of Chemical Formula 2. The peptide exemplified by the above chemical formula 2 has MA (Met-Ala) at the terminal, which exemplifies the peptide introduced at the time of screening in the examples described later, and the above chemical formula. MA of 2 is not always required in the hemagglutinin-binding peptide of the present invention.

_{Further, since NH 2} becomes a positive charge when NH 2 is exposed at the end of the peptide motif, from the viewpoint of charge regulation, a molecule having no charge as a modified molecule at each end of the peptide motif of SEQ ID NO: 1 and further Binding of hydrophobic molecules is also considered. When the therapeutic agent for influenza infection containing the hemagglutinin-binding peptide of the present invention is orally administered, the terminal NH ₂ can be protected by an acetyl group for the purpose of stabilization for suppressing degradation by protease in the gastrointestinal tract. .. As described above, the modified molecule at the end of the peptide motif can be appropriately selected.

The method for producing the hemagglutinin-binding peptide of the present invention is not particularly limited, and the hemagglutinin-binding peptide can be produced by a known method such as using a peptide synthesizer or the like. The peptide motif (peptide compound) of SEQ ID NO: 1 incorporated into the hemagglutinin-binding peptide of the present invention can be synthesized by sequentially adding amino acids to the tetravalent nuclear structure, and can be easily synthesized by the same method as monovalent peptide synthesis. Can be bulk synthesized.

In the hemagglutinin-binding peptide of the present invention, it is particularly preferable that arginine (Arg) located on the N-terminal side of the peptide motif of SEQ ID NO: 1 is replaced with an unnatural isomer (D-Arg) (hereinafter, hereafter. It ^{may be described as "(D)} PVF-tet"). Influenza virus infects the respiratory tract because HA-activated proteases such as trypsin-like proteases are localized in the respiratory tract. On the other hand, since the hemagglutinin-binding peptide (PVF-tet) of the present invention has the sequence of RRPVNHF (SEQ ID NO: 1) as an HA-binding motif, when used as a therapeutic agent, the trypsin-like protease of Arg at position 1 and Arg at position 2 There is a concern that the interval will be cut off and the inhibitory effect will be diminished. ^{As one embodiment of the hemagglutinin-binding peptide of the present invention, (D)} PVF-tet in which arginine (Arg) located on the N-terminal side of the peptide motif of SEQ ID NO: 1 is replaced with an unnatural isomer (D-Arg) is used. It has trypsin resistance and excellent stability.

As described above, the hemagglutinin-binding peptide of the present invention is a tetravalent peptide in which four peptide motifs of SEQ ID NO: 1 are bound to each end of the molecular nuclear structure formed by binding three lysines (Lys). Yes, it exerts a strong binding affinity for hemagglutinin due to the cluster effect.

Since the hemagglutinin-binding peptide of the present invention exhibits an anti-influenza virus effect by binding to hemagglutinin on the surface of influenza virus, the composition containing the hemagglutinin-binding peptide of the present invention is a prophylactic or therapeutic agent for influenza infection ( It is useful as a pharmaceutical composition).

In addition, the hemagglutinin-binding peptide of the present invention can be used as a kit for detecting influenza virus and diagnosing influenza, elucidating influenza virus infection caused by hemagglutinin, and various cell functions and biological phenomena associated therewith. It is also considered to be used as a tool for.

The prophylactic and / or therapeutic agent for influenza infection of the present invention contains the hemagglutinin-binding peptide of the present invention as described above.

When an influenza virus infects a cell, hemagglunitin contained in the influenza virus specifically binds to a receptor of a host cell, and the virus infects the cell using the receptor as a scaffold. Therefore, the hemagglutinin-binding peptide of the present invention can inhibit the binding of influenza virus to the host cell receptor, and can inhibit the cytotoxic activity of influenza virus.

The influenza virus targeted by the prophylactic or therapeutic agent for influenza infection of the present invention does not particularly limit the type and origin thereof, and other types such as type A, type B or type C or isolated human, pigs and horses, etc. It may be either a mammalian isolated type or a bird isolated type.

In the present specification, "infection" means a process in which a virus invades a living body through skin or mucous membrane, or a process in which a virus invades the cell by membrane fusion. Further, in the present specification, "virus infection" refers to a state in which a virus has invaded a living body regardless of the presence or absence of symptoms.

Further, in the present specification, "prevention or treatment of influenza infection" is used in the broadest sense thereof, for example, alleviation or prevention of exacerbation of one or more symptoms associated with influenza virus infection, infection. It causes suppression of the occurrence of later symptoms, prevention of virus infection to cells in vivo (delay or arrest), inhibition of virus growth in vivo (delay or arrest), reduction of the number of viruses in vivo, etc. Say that.

The administration form of the prophylactic or therapeutic agent for influenza infection of the present invention is not particularly limited, and may be oral administration or parenteral administration. Parenteral administration includes, for example, injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral cavity, eye, lung, vagina, and rectum). Etc. can be exemplified.

The prophylactic or therapeutic agent for influenza infection of the present invention may use the hemagglutinin-binding peptide as an active ingredient as it is, or may be formulated by adding a pharmaceutically acceptable carrier, excipient, additive or the like. .. Dosage forms include, for example, liquids (eg injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, Examples thereof include inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.

Formulations include, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjustments. It can be carried out by a conventional method by appropriately using an agent, a preservative, an antioxidant and the like.

Examples of ingredients used in formulation include purified water, saline solution, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose. , Hydroxypropyl cellulose, starch, corn starch, silicic anhydride, aluminum magnesium silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch , Pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, tragant, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohol, stearyl alcohol, stear Acids, human serum glucose and the like can be exemplified.

When the prophylactic or therapeutic agent for influenza infection of the present invention is administered to mammals (for example, humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), particularly humans, the dosage is determined by symptoms, patients. It depends on the age, sex, body weight, sensitivity difference, administration method, administration interval, type of active ingredient, type of preparation, and is not particularly limited, but for example, 10 mg to 200 mg, preferably 200 mg to 500 mg is 1 It can be administered in divided doses or in several divided doses. In the case of injection administration, 50 mg to 200 mg may be administered once or in several divided doses depending on the body weight of the patient.

The hemagglutinin-binding peptide and the prophylactic / therapeutic agent for influenza infection of the present invention are not limited to the above embodiments, and are appropriately designed as long as they do not impair the binding property to hemagglutinin and the cytotoxic activity inhibitory effect due to influenza infection. can do.
Further, the method for preventing / treating influenza infection of the present invention targets the above-mentioned preventive / therapeutic agents for influenza infection (for example, humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.). It is characterized by administration.

Hereinafter, the hemagglutinin-binding peptide and the prophylactic / therapeutic agent for influenza infection of the present invention will be described in detail together with Examples, but the hemagglutinin-binding peptide and the prophylactic / therapeutic agent for influenza infection of the present invention are limited to the following examples. It is not something that is done.

<Example 1> Large-scale preparation of wild-type HA (WT-HA) and mutant HA (L194A-HA) Multivalent peptide library screening requires a large amount of HA in a bead-fixed state. Therefore, a large amount of recombinant HA in which His-tag was introduced on the C-terminal side of the H1N1 subinfluenza virus-derived HA gene was prepared using a baculovirus expression system and immobilized on the beads using Ni-beads. In the preparation of HA mutants having mutations in HA receptor binding, L194A-HA, a mutant in which Leu194, which plays an essential role in binding to sialic acid, was replaced with Ala, was prepared in the same manner (Fig. 1A).

In the evaluation of the activities of the obtained WT-HA and L194A-HA, the specific binding to the sialic acid-rich protein fetishine was examined by the ELISA method (Fig. 1B). In the study, beads in which each HA was eluted were used. As a result, it was confirmed that WT-HA strongly binds to fetishin in a concentration-dependent manner, while L194A-HA completely eliminates the binding activity. Therefore, we decided to perform multivalent peptide library screening using this standard.

<Example 2> Identification of L194-specific HA-binding motif using the multivalent peptide library method The structure of the multivalent peptide library used for screening is shown in FIG. “M”, “A”, and “U” in FIG. 2 indicate Met, Ala, and aminocaproic acid, respectively. “XXXX” in FIG. 2 indicates the library part, and X (degenerate position) indicates that the synthesis was performed using a mixture of 19 kinds of amino acids other than Cys.

As the primary library, the library department screened using a XXXX (4 degenerate positions) multivalent peptide library. First, each HA-immobilized bead (250 μg in HA amount) and a multivalent peptide library (300 μg) were incubated in 200 μl PBS at 4 ° C. for 18 hours. After washing the beads, the peptides bound to each HA were eluted with 30% acetic acid, and after recovery, amino acid sequencing was performed sequentially from the N-terminal. For each degenerate position, the molar ratios of the 19 detected amino acids were calculated and corrected so that the sum was 19. Furthermore, for each amino acid, the value obtained when WT-HA was used was divided by the value obtained when L194A-HA was used to calculate the ratio, and the sum of the values for all amino acids was calculated again. Was corrected to be 19. Therefore, if there is no difference in the selectivity of each amino acid between WT-HA and L194A-HA, the value is all 1. As an index of selectivity, it is assumed that strong selectivity for WT-HA is observed when this value exceeds 1.2. As a result, it was clarified that Lys was strongly selected for all four degenerate positions, Val was selected for positions 1-3, and Gly was selected for position 4 (Fig. 3A).

Therefore, we created a secondary library in which Gly, Lys, and Val were introduced at the central position and three degenerate positions were introduced on both sides of the central position. Similar screening revealed that Gly-fixed libraries showed strong Gly selectivity at positions 6 and 7, but all other positions had low selectivity, while Lys and Val-fixed libraries showed strong selectivity. It was shown that all positoins yield amino acids showing strong selectivity of 1.2 or more (Fig. 3B). Therefore, for each of the Lys and Val-fixed libraries, a peptide motif in which the most strongly selected amino acids at each position are arranged,
SEQ ID NO: 2: His-His-Thr-Lys-Arg-Arg-Arg (HHTKRRR)
SEQ ID NO: 3: Arg-Arg-Arg-Val-Asn-His-His (RRRVNHH)
Was identified as a candidate HA-binding peptide motif.

Next, a tetravalent peptide compound (hereinafter referred to as "TKR-tet") in which the peptide motif of SEQ ID NO: 2 was incorporated into the XXXX portion of the polyvalent peptide library shown in FIG. 2 and the peptide of SEQ ID NO: 3 A tetravalent peptide compound (hereinafter referred to as "RVH-tet") in which the motif was incorporated into the XXXX portion of the polyvalent peptide library shown in FIG. 2 was synthesized as an HA inhibitor candidate.

For these compounds, each concentration of HA inhibitor candidate substances (TKR-tet, RVH-tet) was added to influenza A virus (A / PR / 8/34) MDCK cells, and 30 minutes later, A / PR / 8 / 34 Influenza virus was infected with a high titer (MOI = 10), and the survival rate of cells after culturing for 24 hours was measured by the WST method.

As a result, as shown in FIG. 4, it was clarified that both TKR-tet and RVH-tet inhibit the cytotoxic activity due to infection in a dose-dependent manner, and that the inhibitory effect is superior to RVH-tet. became. Therefore, based on the RRRVNHH motif of SEQ ID NO: 3, we attempted to obtain a motif with even better HA-binding activity by using a multivalent peptide sheet screening technique (Patent Document 7).

<Example 3> Identification of a high-affinity HA-binding motif using a polyvalent peptide sheet screening technique A spot peptide synthesizer manufactured by intavis AG was used for the synthesis of the polyvalent peptide on a cellulose sheet.

First, we attempted to optimize the valence of the multivalent peptide synthesized on the sheet, the distance from the sheet, and the synthesis density for HA. FMOC-βAla-OH and Boc-βAla-OH were used in different mixing ratios of 100: 0, 30:70, 10:90 in the first cycle with respect to the amino groups present on the sheet. The synthetic density of the peptides synthesized thereafter was changed (Boc-βAla-OH is resistant to deprotection, so that subsequent amino acid synthesis does not occur).

Subsequently, the spacer length was adjusted by changing the number of FMOC-aminocaproic acids to be added to 1, 2, or 3. Subsequently, one FMOC-Lys (FMOC) -OH was added, and an extension reaction was carried out so as to give the formed two amino groups an evenly subsequent sequence to obtain a divalent peptide, FMOC-. Lys (FMOC) -OH was reacted twice in succession, and the four amino groups formed were subjected to an extension reaction to synthesize tetravalent peptides. The sequence used after FMOC-Lys (FMOC) -OH addition was MA-RRRVNHH-A-, which contains the HA-binding motif RRRVNHH of SEQ ID NO: 3 previously determined.

The resulting sheet was ^{blotted with 125} I radiolabeled WT-HA and the bound radioactivity was measured with BAS 2500 (GE Healthcare). As a result, it was clarified that the peptide synthesized on the sheet had the strongest binding to HA under the conditions that the synthesis density was 100%, the spacer length was one aminocaproic acid, and the valence was tetravalence (Fig. 5). .. Therefore, in the subsequent experiments, it was decided to perform peptide synthesis on the cellulose sheet under these conditions.

The sequence of the HA binding motif synthesized on the sheet uses RRRVNHH of SEQ ID NO: 3 as the original sequence, and for all amino acids from the 1st Arg to the 7th His of this motif, the original amino acids and Cys are excluded in order. The ones substituted one by one with the other 18 kinds of amino acids were used. Therefore, a total of 7 (number of positions) x 18 (number of amino acids) = 126 types of tetravalent peptides were synthesized.

Similarly obtained sheets were ^{blotted with 125} I radiolabeled WT-HA and L194A-HA (Fig. 6) and bound radioactivity was measured with BAS 2500. Then, the binding ratio showing L194-specific binding, the binding force to WT-HA, and the product of both (WT / L194A * WT), which is an index showing that both are excellent, were used as indexes.

The measurement results are shown in Tables 1 and 2.

The top three motifs with the highest WT / L194A * WT values shown in Tables 1 and 2.
SEQ ID NO: 4: Arg-Arg-Pro-Val-Asn-His-His (RRPVNHH)
SEQ ID NO: 5: Arg-Arg-Arg-Asp-Asn-His-His (RRRDNHH)
SEQ ID NO: 6: Arg-Arg-Ser-Val-Asn-His-His (RRSVNHH)
Was determined as a candidate sequence for the next screening.

In addition, the top 15 motifs with a WT * WT / L194A value of 1.63 or higher are all substitutions at positions 2, 3, 4 or 7 of the original sequence, that is, these positions identify optimal inhibitory motifs. Was shown to be an important position.

Therefore, positions 2, 3, 4 or 7 of the three candidate motifs of RRPVNHH (SEQ ID NO: 4), RRRDNHH (SEQ ID NO: 5), and RRSVNHH (SEQ ID NO: 6) were shuffled with 19 other amino acids except Cys. A sequence-known library for the next screening was prepared on a sheet in a tetravalent state under the optimum conditions previously determined (secondary sheet library, FIG. 7).

Similarly obtained sheets were ^{blotted with 125} I-WT-HA and ²⁵ I-L194A-HA (Fig. 7) and bound radioactivity was measured with BAS 2500. Then, the product of the binding ratio showing L194-specific binding and the binding force to WT-HA (WT * WT / L194A) was used as an index.

The measurement results are shown in Tables 3 and 4.

Top 5 motifs with WT / L194A * WT values shown in Tables 3 and 4:
SEQ ID NO: 7: Arg-Arg-Pro-Val-Asn-His-Asp (RRPVNHD)
SEQ ID NO: 8: Arg-Arg-Pro-Met-Asn-His-His (RRPMNHH)
SEQ ID NO: 9: Arg-Arg-Pro-Val-Asn-His-Asn (RRPVNHN)
SEQ ID NO: 1: Arg-Arg-Pro-Val-Asn-His-Phe (RRPVNHF)
SEQ ID NO: 10: Arg-Arg-Pro-Val-Asn-His-Pro (RRPVNHP)
Was determined as the final HA binding sequence.

These motifs were incorporated into the XXXX section of the polyvalent peptide library shown in FIG. 2, respectively, and tetravalent peptide compounds, RR PV NH D -tet (hereinafter referred to as "PVD-tet"), and RR PM NH H. -tet (hereinafter referred to as "PMH-tet"), RR PV NH N -tet (hereinafter referred to as "PVN-tet"), RR PV NH F -tet (hereinafter referred to as "PVF-tet"), RR PV NH P -tet (hereinafter referred to as "PVP-tet") was synthesized as an influenza virus inhibitor candidate.

<Example 4> Evaluation of inhibitory ability against cytotoxic activity due to influenza virus infection
Inhibitors of each concentration (PVD-tet, PMH-tet, PVN-tet, PVF-tet, PVP-tet obtained in Example 3) were added to MDCK cells, and 30 minutes later, A / PR / 8/34. Influenza virus was infected with a high titer (MOI = 10), and the viability of cells after culturing for 24 hours was measured by the WST method.

As a result, it was clarified that PVF-tet showed the strongest inhibitory activity, and then PMH-tet, PVN-tet, and PVP-tet showed the same inhibitory activity (Fig. 8). On the other hand, PVD-tet showed only weak inhibitory activity (Fig. 8).

In addition, each inhibitor of 300 μg / ml (RVH-tet, TKR-tet, PVD-tet, PVP-tet, PVN-tet, PMH-tet, PVF-tet, ^(D) PVF-tet,) was added to MDCK cells. Fetuin was added, and the viability of the cells after culturing for 48 hours was measured by the WST method. It was confirmed that these compounds alone showed almost no cytotoxicity even after 48 hours (Fig. 9).

Based on the above results, PVF-tet was judged to be the most promising anti-influenza drug and was used in the following studies.

<Example 5> Improvement of biological stability of PVF-tet In order for influenza virus to acquire infectivity, the HA protein is cleaved by a protease and cleaved into the subunits of HA1 and HA2, and the fusion domain is formed. It needs to be exposed. Influenza virus infects the respiratory tract because HA-activated proteases such as trypsin-like proteases are localized in the respiratory tract. On the other hand, since PVF-tet has the sequence of RRPVNHF (SEQ ID NO: 1) as an HA binding motif, when used as a therapeutic agent, trypsin-like protease cleaves between Arg at position 1 and Arg at position 2, and has an inhibitory effect. There was concern that it would be diminished. In fact, after treating PVF-tet (molecular weight 5772) at 37 ° C. for 24 hours in the presence of 1 μg / ml trypsin, mass spectrometry with a mass spectrometer revealed that it was between Arg at position 1 and Arg at position 2. It was shown that a cleaved product (molecular weight 4138) was produced (Fig. 10).

^{Therefore, in order to impart trypsin resistance, (D)} PVF-tet in which Arg at position 1 was replaced with the unnatural isomer D-Arg was prepared, and the effect of trypsin treatment was examined in the same manner. ^{As a result, it was shown that (D)} PVF-tet remained stable without being cleaved even after trypsin treatment at 37 ° C. for 24 hours (Fig. 10).

The binding of PVF-tet and ^(D) PVF-tet to WT-HA or L194A-HA was examined by the ELISA method. As a result, both PVF-tet and ^(D) PVF-tet bind efficiently to WT-HA, while the binding activity to L194A-HA is attenuated, that is, they bind to HA in an L194-dependent manner. It was shown to be doing (FIGS. 11 and 12).

^{<Example 6> Inhibitory effect of PVF-tet and (D)} PVF-tet on cytotoxic activity due to influenza virus infection
Inhibitors of each concentration (PVF-tet, ^(D) PVF-tet, RVH-tet) or Fetwin were added to MDCK cells, and 30 minutes later, the A / PR / 8/34 influenza virus had a low titer (MOI = 0.001). ) Or high titer (MOI = 10), and the viability of the cells after culturing for 48 and 24 hours, respectively, was measured by the WST method. Fetwin is known to contain sialic acid at high density and efficiently bind to the receptor binding site of HA. 13A and 13B show the result of low titer infection (MOI = 0.001), and FIG. 13B) shows the result of high titer infection (MOI = 10).

As a result, although fetishin showed a high inhibitory effect against low-titer infection, it showed almost no inhibitory activity against high-titer infection (Fig. 13A). On the other hand, ^{it was clarified that PVF-tet and (D)} PVF-tet imparted with protease resistance show strong inhibitory activity against high titer infection (Fig. 13B).

Furthermore, it was shown that PVF-tet and RVH-tet show only weak inhibitory activity against low-titer infection, whereas ^(D) PVF-tet efficiently inhibits cytotoxic activity (Fig.). 13A). Under the condition of low titer infection, trypsin is added to the culture medium to promote the growth of the virus. Therefore, PVF-tet and RVH-tet are decomposed by trypsin in the culture, and the inhibitory effect is diminished. However, since ^(D) PVF-tet is resistant to trypsin, it is considered that sufficient inhibitory activity could be exhibited even under these conditions.

From the above, it was shown that ^(D) PVF-tet has the ability to efficiently inhibit the cytotoxic activity of the virus under both high-titer and low-titer infection conditions. Moreover, when the inhibitory activity of PVF-tet under high titer infection conditions was compared with the monomer peptide having the same sequence as the HA binding motif of PVF-tet, the monomer peptide had almost no inhibitory activity at any concentration. It was shown not to be seen (Fig. 14).

Furthermore, when the inhibitory effect on the actual virus production under the condition of low titer infection was examined, ^{it was confirmed that 300 μg / ml PVF-tet and (D)} PVF-tet significantly suppressed the virus production. (Fig. 15). From the above, PVF-tet and ^(D) PVF-tet bind to HA with high affinity by exerting a cluster effect and show antiviral activity. Such a motif was established by the present inventors. It was confirmed that it could not be identified except by the polyvalent peptide screening method.

<Example 7> ^{Inhibitory effect of (D)} PVF-tet on individual mouse toxicity due to influenza virus infection The inhibitory effect of a peptide HA inhibitor on individual mouse toxicity due to influenza virus infection was examined.

After trans-airway infection, influenza virus is cleaved by trypsin-like protease present in the respiratory tract, enabling efficient infection. ^{Therefore, here, (D)} PVF-tet, which has trypsin resistance, was used.

A / PR / 8/34 influenza virus alone or ^{nasally in the presence of each amount of (D)} PVF-tet was administered to 6-8 week old Balb / c mice, and body weight change (Fig. 16) and survival rate. (Fig. 17) were compared.

As a result, under the condition that all cases died from the virus alone, ^{10% of the mice in the 1.25 mg / kg (D)} PVF-tet administration group survived, and half survived at 2.5 mg / kg, and 12 surviving mice. It was shown that the body weight recovered to the same level as the non-infected group after the day.

Furthermore, in the 2.5 mg / kg ^(D) PVF-tet-administered group, the amount of virus in the lungs after 3 days and 5 days was measured by a plaque assay, and a clear decrease in virus production was observed in both cases. In particular, it became clear that it decreased to about 1/3 after 3 days (Fig. 18).

From the above results, ^{it is clear that (D)} PVF-tet can significantly suppress individual mouse toxicity due to influenza virus infection with just one administration, that is, it can be an excellent prophylactic / therapeutic drug for influenza virus infection. became.

Claims (3)

The peptide motif of SEQ ID NO: 1 is attached directly or via a spacer to each of the four amino groups located at the end of the molecular nuclear structure formed by binding three lysines (Lys). A hemagglutinin-binding peptide characterized by being a peptide. The hemagglutinin-binding peptide according to claim 1, wherein the arginine (Arg) located on the N-terminal side of the peptide motif of SEQ ID NO: 1 is replaced with an unnatural isomer (D-Arg). A prophylactic / therapeutic agent for influenza virus infection, which comprises the hemagglutinin-binding peptide according to claim 1 or 2.

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