JP2013071904A - Peptide having anti-influenza virus activity - Google Patents

Abstract

[PROBLEMS] To provide a preventive or therapeutic agent for influenza having a novel mechanism of action targeting a molecule different from existing drugs.
A peptide having a specific amino acid sequence and having a length of about 12 amino acids, which can bind to hemagglutinin of influenza virus. Also, a screening method for selecting an anti-influenza drug from candidate peptides, a step of expressing a peptide library in a cell-free translation system using a nucleic acid containing a specific base sequence as a template, and contacting the library with hemagglutinin Incubating and selecting a hemagglutinin-binding peptide.
[Selection figure] None

Description

  The present invention relates to peptides having anti-influenza virus activity.

  The outbreak and spread of new influenza viruses have historically caused great damage to mankind, and even today, highly pathogenic avian influenza is expected to pose a major threat when mutated from viruses to humans. ing.

Influenza viruses are enveloped RNA viruses and are classified into A, B, and C types according to the antigenicity of nucleoprotein (NP) and matrix protein (M). The epidemic in humans is mainly type A and type B.
Even if the types such as A, B, and C are the same, they are further classified into a plurality of subtypes based on the antigenicity difference between hemagglutinin (HA) and neuraminidase (NA), which are molecules on the envelope surface, and H1N1, H5N1, etc. It is expressed.

As existing anti-influenza drugs, zanamivir (Relenza (registered trademark)) (for example, Patent Document 1) and oseltamivir (Tamiflu (registered trademark)) (for example, Patent Document 2) are widely used.
Zanamivir and oseltamivir act by suppressing the activity of neuraminidase, which is required when influenza virus infects other cells from infected cells. Furthermore, there are amantadine and rimantadine that target M2 protein as anti-influenza drugs having other action mechanisms, which inhibit virus unshelling after cell infection.
These drugs that inhibit the function of neuraminidase or M2 protein, like other drugs that target enzymes and ion channels, are derivatized from substrates, molecular design based on three-dimensional structures, or discovery of functions from existing compounds It has been developed through such processes. That is, it belongs to an analog of a molecule present in a living body or a chemically prepared small molecule.

However, M2 protein inhibitors are only effective against influenza A, and neuraminidase inhibitors do not succeed against influenza C without neuraminidase.
Antiviral drugs generally have limited efficacy due to the acquisition of viral drug resistance due to mutations. Antiviral viruses such as zanamivir, oseltamivir, amantadine, etc., which have been used for a long time, have emerged. ing. In addition to the reduction of drug affinity due to mutation of the target molecule, the acquisition of drug resistance by the virus is also due to the indirect mechanism of restoring the growth potential by mutation of a different molecule, and complete acquisition of resistance over the long term It is difficult to create drugs that can be avoided.

US Pat. No. 5,360,817 US Pat. No. 5,763,483

  An object of the present invention is to provide an anti-influenza drug having a novel mechanism of action that targets a molecule different from existing drugs.

As a result of diligent studies to solve the above-mentioned problems, the present inventors have selected many types of compounds that bind to hemagglutinin that are present on the surface of influenza viruses and play an important role in cell infection. We thought it would lead to the development of a new drug that shows an inhibitory action against influenza virus.
As a means for efficiently obtaining many kinds of compounds that bind to hemagglutinin, screening was performed using a library of peptides prepared in a cell-free translation system as a population. This screening succeeded in obtaining dozens of peptides having high affinity for hemagglutinin.
The obtained peptide was synthesized, the inhibitory activity was confirmed by an influenza virus inhibition test, and the present invention was completed.
That is, the present invention
[1] Any of the following peptides:
(I) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1-24;
(Ii) a peptide comprising a partial sequence of the peptide described in (i) above, which is capable of binding hemagglutinin; and (iii) in the amino acid sequence of the peptide described in (i) or (ii) above, Or a peptide in which several amino acids are deleted, added or substituted, and capable of binding hemagglutinin;
[2] The peptide according to [1] above, which is cyclized;
[3] The peptide according to [2] above, which contains chloroacetylamino acid and cysteine, and is cyclized by a thioether bond between chloroacetylamino acid and cysteine;
[4] The peptide according to [3] above, having a chloroacetyl amino acid within 3 amino acids from the N-terminus and a cysteine within 3 amino acids from the C-terminus;
[5] A peptide consisting of the amino acid sequence represented by SEQ ID NO: 25-48, which is cyclized by a thioether bond between N-terminal chloroacetyl-tryptophan and the second cysteine from the C-terminal;
[6] A pharmaceutical composition for preventing or treating influenza comprising the peptide according to any one of [1] to [5] above;
[7] Use of the peptide derivative according to any one of [1] to [5] above for producing a preventive or therapeutic agent for influenza;
[8] A screening method for selecting a preventive or therapeutic agent for influenza from candidate peptides,
Expressing a peptide library in a cell-free translation system using a nucleic acid containing the following sequence as a template;
ATG (NNK) n (SEQ ID NO: 49)
[Wherein n represents an integer of 4 to 12, N corresponds to A, T, G or C, and K corresponds to T or G. ];
Contacting the peptide library with hemagglutinin and incubating to select a peptide that binds to hemagglutinin;
A method comprising:
[9] A template containing TGC at the 3 ′ end of (NNK) n is used as the template.
In the cell-free translation system, a chloroacetylated amino acid is used as an aminoacyl tRNA corresponding to the codon ATG,
The method according to [8] above, comprising a step of cyclizing the peptide prior to the step of contacting and incubating the peptide library with hemagglutinin;
[10] An influenza virus detection agent comprising the peptide according to any one of [1] to [5] above; and [11] an influenza virus detection kit comprising the influenza detection agent according to [10] above ,
About.

Since the peptide of the present invention exhibits an inhibitory activity against influenza viruses by binding to hemagglutinin, it also has an antiviral effect against influenza viruses resistant to neuraminidase inhibitors and M2 protein inhibitors.
In addition, it is considered that the probability that a drug-resistant mutant appears can be further reduced by combining the peptide of the present invention with a drug targeting neuraminidase, M2 protein or the like.

FIG. 1 is a graph showing the change in the recovery rate in a selection experiment for a special cyclic peptide. FIG. 2 is a photograph of a plate showing the results of a growth inhibition test with the peptide of the present invention against influenza virus H5N1-Vac3. FIG. 3 is a photograph of a plate showing the results of a growth inhibition test with the peptide of the present invention against influenza virus H5N1-Vac3. FIG. 4 is a graph showing the number of plaques in each well created based on the results of FIGS. FIG. 5 is a graph showing the number of plaques in each well created based on the results of FIGS. FIG. 6 is a graph showing the diameter of the plaque of each well prepared based on the results of FIG. 2 and FIG. 3, and the vertical axis indicates mm. FIG. 7 is a photograph of a plate showing the results of a growth inhibition test with the peptide of the present invention against influenza virus H1N1. FIG. 8 is a photograph of a plate showing the results of a growth inhibition test conducted by previously mixing a peptide with influenza virus and contacting the mixture with cells in order to confirm the neutralizing activity of the peptide against influenza virus. FIG. 9 shows treatment conditions for peptides and virus solutions. FIG. 10 is a photograph of a plate showing the results of examining the neutralizing activity of the peptide of the present invention against influenza virus H5N1-Vac3. FIG. 11 is a photograph of a plate showing the results of examining the neutralizing activity of Tamiflu and Relenza against influenza virus H5N1-Vac3.

(peptide)
One embodiment of the peptide according to the present invention is any of the following (i) to (iii).
(I) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1-24.
(Ii) A peptide comprising a partial sequence of the peptide described in (i) above, which is capable of binding to hemagglutinin.
(Iii) A peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence of the peptide described in (i) or (ii), and can bind to hemagglutinin.

In the present specification, a peptide refers to a peptide in which two or more amino acids are linked by peptide bonds, and for example, a peptide in which 8-20 amino acids are peptide-bonded.
In this specification, “amino acid” is used in its broadest sense, and includes artificial amino acid variants, derivatives, and analogs in addition to natural amino acids. In the present specification, amino acids are natural proteinaceous L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; natural non-protein amino acids such as norleucine, β-alanine, ornithine; And chemically synthesized compounds having properties known in the art. Examples of unnatural amino acids include α-methyl amino acids (such as α-methylalanine), D-amino acids, histidine-like amino acids (2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine and α -Methyl-histidine, etc.), amino acids with extra methylene in the side chain ("homo" amino acids) and amino acids in which the carboxylic acid functional amino acids in the side chain are replaced with sulfonic acid groups (such as cysteic acid).

In the present specification, amino acids other than the 20 natural amino acids used for translation synthesis on ribosomes in living cells, that is, amino acids corresponding to the following (1) to (3) are referred to as “special amino acids”. There is.
(1) Amino acids corresponding to amino acid residues on polypeptides that have been modified after translation synthesis (for example, phosphorylated tyrosine, acetylated lysine, farnesylated cysteine)
(2) Naturally occurring amino acids that are not used for translational synthesis on ribosomes (3) Non-naturally occurring artificial amino acids (non-natural amino acids)

The amino acids represented by SEQ ID NOs: 1-24 are shown in the following table.

  The peptide of (i) may be a polypeptide consisting only of the amino acid sequence represented by SEQ ID NO: 1-24, or may be one in which one or more amino acids are bonded to at least one end thereof. The total length of the peptide of (i) can be, for example, 15 amino acids or less, 20 amino acids or less, or 30 amino acids or less.

  The peptide of (ii) may be any peptide as long as it is a peptide containing a partial sequence of the peptide described in (i) and can bind to hemagglutinin. For example, the peptide of (i) (I) one or more amino acids bonded to at least one end thereof; (i) a peptide comprising eight consecutive amino acids and one or more amino acids bonded to at least one end thereof; (i) The peptide comprises 10 consecutive amino acids, and one or more amino acids bonded to at least one end thereof. The total length of the peptide of (ii) can be, for example, 15 amino acids or less, 20 amino acids or less, or 30 amino acids or less.

  The peptide of (iii) is a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence of the peptide described in (i) or (ii) and can bind to hemagglutinin As long as it is any peptide. Whether or not it can bind to hemagglutinin can be confirmed by those skilled in the art according to a known method.

  In the present specification, when “a peptide in which one or several amino acids are deleted, added, or substituted” is referred to, the number of amino acids to be deleted is as long as the peptide maintains its ability to bind hemagglutinin. Although it does not specifically limit, For example, it can be set to 1-5 pieces, 1-3 pieces, or 1-2 pieces. Or it is good also as less than 10% of the whole length, or less than 5%. The location of deletion, addition, or substitution may be at the end of the peptide, in the middle, at one location, or at two or more locations.

The peptide of the present invention includes various derivatives thereof as long as the problem of the present invention is solved. Such derivatives include, for example, those in which the C-terminus is an amide or ester, or those fused with a membrane-penetrating peptide (CPP) so as to be introduced into cells when administered. Can be mentioned. CPP is a generic term for peptides having affinity for cell membranes and ability to migrate into cells, and is a domain consisting of 11 amino acids of a trans-activator of transcription protein (TAT protein) expressed by HIV-1 virus. Protein-transduction domain (PTD), Drosophila Antennapedia, VP22 derived from herpes virus, oligoarginine and the like are known. In general, many CPPs have a high content of basic amino acids such as arginine, lysine and histidine.
Examples of the peptide derivative of the present invention include those with modifications such as phosphorylation, methylation, acetylation, adenylylation, ADP ribosylation, and glycosylation, and fusion peptides with other peptides and proteins. It is done. These derivatives can be prepared by those skilled in the art by a known method or a method analogous thereto.

  Moreover, the peptide of this invention also includes the salt, as long as it solves the subject of this invention. As salts of peptides, salts with physiologically acceptable bases and acids are used. For example, addition salts of inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), organic salts Addition salts of acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic bases (ammonium hydroxide or alkali or Alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), amino acid addition salts, and the like.

In this specification, hemagglutinin refers to an antigenic glycoprotein present on the surface of many bacteria and viruses including influenza virus, and is represented as “HA”. Hemagglutinin is involved in the process of virus attachment to host cells. Specifically, when hemagglutinin on the surface of the virus binds to sialic acid on the surface of the target host cell, the virus is encapsulated in the cell membrane and taken up into the cell in the form of endosomes containing the virus. Subsequently, the endosomal membrane and the viral membrane fuse, the viral genome is inserted into the cell, and proliferation begins.
Hemagglutinin has at least 16 subtypes and is called H1 to H16. The influenza subtype name H indicates hemagglutinin.

  The peptide of the present invention can be produced by a known peptide production method such as a liquid phase method, a solid phase method, a chemical synthesis method such as a hybrid method combining a liquid phase method and a solid phase method;

In the solid-phase method, for example, the hydroxyl group of a resin having a hydroxyl group is esterified with the carboxy group of the first amino acid (usually the C-terminal amino acid of the target peptide) in which the α-amino group is protected with a protecting group. . As the esterification catalyst, a known dehydrating condensing agent such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), or the like can be used.
Next, the α-amino group protecting group of the first amino acid is removed, and a second amino acid in which all functional groups other than the carboxy group of the main chain are protected is added to activate the carboxy group. , Combining the first and second amino acids. Further, the α-amino group of the second amino acid is deprotected, a third amino acid in which all functional groups other than the carboxy group of the main chain are protected is added, the carboxy group is activated, and the second and Bind a third amino acid. This is repeated, and when a peptide having a desired length is synthesized, all functional groups are deprotected.

Resins for solid phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck) Is mentioned. These resins can be used after being washed with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).
Protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, allyloxycarbonyl (Alloc). ) Group and the like. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine.
For protecting the α-carboxy group, methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester and the like can be used.
As other functional groups of amino acids, the hydroxy group of serine or threonine can be protected with a benzyl group or a tert-butyl group, and the hydroxy group of tyrosine is protected with a 2-bromobenzyloxycarbonyl dilute or tert-butyl group. The amino group of the lysine side chain and the carboxy group of glutamic acid or aspartic acid can be protected in the same manner as the α-amino group and α-carboxy group.

  The activation of the carboxy group can be performed using a condensing agent. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy) tris. (Dimethylamino) phosphonium hexafluorophosphate (BOP), 1- [bis (dimethylamino) methyl] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU) and the like.

  The peptide chain can be cleaved from the resin by treatment with an acid such as TFA or hydrogen fluoride (HF).

Production of a peptide by a genetic recombination method (translation synthesis system) can be performed using a nucleic acid encoding the peptide of the present invention. The nucleic acid encoding the peptide of the present invention may be DNA or RNA.
The nucleic acid encoding the peptide of the present invention can be prepared by a known method or a method analogous thereto. For example, it can be synthesized by an automatic synthesizer. In order to insert the obtained DNA into a vector, a restriction enzyme recognition site may be added, or a base sequence encoding an amino acid sequence for cutting out the resulting peptide chain with an enzyme or the like may be incorporated.
As described above, when the peptide of the present invention is fused with a membrane-permeable peptide or the like, the nucleic acid includes a nucleic acid encoding the membrane-permeable peptide.
In order to suppress degradation by a host-derived protease, a chimeric protein expression method in which the target peptide is expressed as a chimeric peptide with another peptide can also be used. In this case, as the nucleic acid, a nucleic acid encoding a target peptide and a peptide that binds to the peptide is used.

  Subsequently, an expression vector is prepared using a nucleic acid encoding the peptide of the present invention. The nucleic acid can be inserted downstream of the promoter of the expression vector, as it is, after digestion with a restriction enzyme, or by adding a linker. Examples of vectors include Escherichia coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids ( pSH19, pSH15, YEp, YRp, YIp, YAC, etc., bacteriophage (e phage, M13 phage, etc.), virus (retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus , Baculovirus, etc.) and cosmids.

The promoter can be appropriately selected depending on the type of host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is E. coli, trp promoter, T7 promoter, lac promoter, etc. can be used.
Expression vector encodes DNA replication origin (ori), selection marker (antibiotic resistance, auxotrophy, etc.), enhancer, splicing signal, poly A addition signal, tag (FLAG, HA, GST, GFP, etc.) It is also possible to incorporate nucleic acid or the like.

  Next, an appropriate host cell is transformed with the expression vector. The host can be appropriately selected in relation to the vector. For example, Escherichia coli, Bacillus subtilis, Bacillus sp.), Yeast, insects or insect cells, animal cells and the like are used. As animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells can be used. Transformation can be performed according to a known method such as a lipofection method, a calcium phosphate method, an electroporation method, a microinjection method, or a particle gun method, depending on the type of host. The desired peptide is expressed by culturing the transformant according to a conventional method.

For peptide purification from transformant cultures, cultured cells are collected, suspended in an appropriate buffer, disrupted by sonication, freeze-thawing, etc., and roughly extracted by centrifugation or filtration. Obtain a liquid. When the peptide is secreted into the culture solution, the supernatant is collected.
Purification from a crude extract or culture supernatant is a known method or a method equivalent thereto (for example, salting out, dialysis method, ultrafiltration method, gel filtration method, SDS-PAGE method, ion exchange chromatography, affinity chromatography, Reversed phase high performance liquid chromatography).
The obtained peptide may be converted from a free form to a salt or from a salt to a free form by a known method or a method analogous thereto.

The translation synthesis system may be a cell-free translation system. Cell-free translation systems include, for example, ribosomal proteins, aminoacyl tRNA synthetases (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF) elongation factor (EF), termination factor (RF), and ribosome Includes regeneration factor (RRF), as well as other factors required for translation. In order to increase the expression efficiency, an Escherichia coli extract or a wheat germ extract may be added. In addition, a rabbit erythrocyte extract or an insect cell extract may be added.
By supplying energy continuously to a system containing these using dialysis, a protein of several hundred μg to several mg / mL can be produced. In order to perform transcription from gene DNA together, a system containing RNA polymerase may be used. As cell-free translation systems that are commercially available, E. coli-derived systems such as RTS-100 (registered trademark) from Roche Diagnostics, PURESYSTEM (registered trademark) from PGI, etc., are systems using wheat germ extracts. For example, those from Zoigene or Cell Free Science can be used.
According to the cell-free translation system, the expression product can be obtained in a highly purified form without purification.

In a cell-free translation system, an artificial aminoacyl tRNA in which a desired amino acid or hydroxy acid is linked (acylated) to a tRNA may be used instead of the aminoacyl tRNA synthesized by a natural aminoacyl tRNA synthetase. Such aminoacyl-tRNA can be synthesized using an artificial ribozyme.
Such ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami, D. Kourouklis, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 1077-1084; H. Murakami, A. Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3, 357-359 "The flexizyme system: a highly flexible tRNA aminoacylation tool for the synthesis of nonnatural peptides "; N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & Medicinal Chemistry Letters 19, 3892-3894" A flexizyme that selectively charges amino acids activated by a water-friendly leaving group "; and WO2007 / 066627). Flexizyme is also known by the name of the original flexizyme (Fx) and dinitrobenzyl flexizyme modified from this (dFx), enhanced flexizyme (eFx), amino flexizyme (aFx) and the like.

The desired codon can be translated in association with the desired amino acid or hydroxy acid by using the tRNA linked to the desired amino acid or hydroxy acid produced by the flexizyme. Special amino acids may be used as the desired amino acids.
Examples of special amino acids are shown in the table below, but are not limited thereto. In the table, DBE and CME are ester types when a special amino acid is bound to tRNA by flexizyme, DBE means 3,5-dinitrobenzyl ester, and CME means cyanomethyl ester.

The peptide of the present invention may be cyclized as one embodiment, and the cyclized peptide is also included in the peptide of the present invention. In this specification, cyclization means that two amino acids separated by one or more amino acids are bound directly or indirectly through a linker or the like in one peptide to form a cyclic structure in the molecule. To do.
Cyclization includes disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, C—S—C bond, C—N—C bond, C ═N—C bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, and the like, but is not limited thereto.
By cyclizing the peptide, the structure of the peptide may be stabilized and the affinity for the target may be increased.

For example, for cyclization, a chloroacetylated amino acid may be used as a special amino acid, and a complex of the special amino acid and tRNA may be generated by flexizyme and used in a translation synthesis system. Such a complex can be prepared by reacting a chloroacetylated amino acid with tRNA in the presence of flexizyme.
Examples of chloroacetylated amino acids include N-Chloroacetyl-L-alanine, N-Chloroacetyl-L-phenylalanine, N-Chloroacetyl-L-tyrosine, N-Chloroacetyl-L-tryptophan among the special amino acids shown in the above table. , N-Chloroacetyl-D-alanine, N-Chloroacetyl-D-phenylalanine, N-Chloroacetyl-D-tyrosine, N-Chloroacetyl-D-tryptophan, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L -tryptophane can be used.
Since these are used as starting amino acids, they become the N-terminal amino acids of the peptides. Therefore, when these special amino acids are used, a thioether bond is generated between the N-terminal amino acid and cysteine in the same peptide, and a cyclic structure is formed.
If Nγ- (2-chloroacetyl) -α, γ-diaminobutylic acid or Nγ- (2-chloroacetyl) -α, γ-diaminopropanoic acid is used as the chloroacetylated amino acid, it is introduced into any part of the peptide chain. As a result, a thioether bond is generated between the amino acid at an arbitrary position and cysteine in the same peptide, and a cyclic structure is formed.
Cyclization methods using special amino acids include, for example, Kawakami, T. et al., Nature Chemical Biology 5, 888-890 (2009); Yamagishi, Y. et al., ChemBioChem 10, 1469-1472 (2009); Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934 (2008); Goto, Y. et al., ACS Chemical Biology 3, 120-129 (2008); Kawakami T. et al, Chemistry & Biology 15, 32-42 (2008) can be performed, the disclosure of which is incorporated herein by reference in its entirety.

Specifically, for example, chloroacetyl-L / D-tryptophan can be arranged at the N-terminus of the amino acid sequence represented by SEQ ID NO: 1-24, and cysteine can be arranged at the C-terminal side of the amino acid sequence. .
The position of the chloroacetylated amino acid and cysteine may be anywhere in the peptide as long as the cyclization does not reduce the affinity for hemagglutinin. For example, the chloroacetylated amino acid may be arranged within 3 amino acids from the N-terminal, and the cysteine may be arranged within 3 amino acids from the C-terminal.

The thioether bond formed between the chloroacetylated amino acid and cysteine is not susceptible to degradation even under reducing conditions in vivo, so it is possible to increase the blood half-life of the peptide and maintain the bioactive effect. .
As a method for introducing a chloroacetylated amino acid into a peptide, a peptide is synthesized by an arbitrary method without using a special amino acid, and any amino acid residue of the peptide is chloroacetylated to form a peptide with cysteine in the peptide. It can also be cyclized by forming a thioether bond therebetween.

(Pharmaceutical composition)
As shown in Examples described later, the peptide of the present invention exhibits an anti-influenza virus effect by binding to hemagglutinin on the surface of influenza virus. Therefore, the composition containing the peptide of the present invention is useful as a preventive or therapeutic agent for influenza.
In this specification, influenza refers to an acute infection caused by an influenza virus. Infection causes cold-like symptoms with high fever and muscle pain in humans. It may be accompanied by gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea. Complications include pneumonia and influenza encephalopathy.
In the present specification, the term “infection” is used as a term indicating either a process in which a virus enters a living body through skin or mucous membrane, or a process in which a virus enters a cell by membrane fusion. In addition, viral infection in this specification refers to a state in which a virus has entered a living body regardless of the presence or absence of symptoms.

As used herein, the treatment or prevention of influenza is used in its broadest sense, for example, alleviation or prevention of one or more symptoms associated with influenza virus infection, suppression of the occurrence of symptoms after infection. Inhibiting (delaying or stopping) infection of cells with a virus in vivo, preventing growth (delaying or stopping) of virus in vivo, reducing the number of viruses in the living body, and the like. When showing at least one of these effects, it is judged useful for the treatment or prevention of influenza.
Moreover, since the peptide of this invention has the neutralization activity with respect to hemagglutinin as shown in the Example mentioned later, it is understood that the same effect as an influenza vaccine can be acquired.

In this specification, the dosage form of the pharmaceutical composition is not particularly limited, and may be orally or parenterally administered. Parenteral administration includes, for example, intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, transmucosal administration (nasal, oral cavity, ocular, pulmonary, vaginal, transrectal) administration. Etc. are raised.
The pharmaceutical composition can be variously modified in view of the property that the polypeptide is easily metabolized and excreted. For example, polyethylene glycol (PEG) or a sugar chain can be added to a polypeptide to increase the residence time in blood and reduce antigenicity. In addition, biodegradable polymer compounds such as polylactic acid / glycol (PLGA), porous hydroxyapatite, liposomes, surface modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. are sustained release bases It may be used as a polypeptide. In the case of transdermal administration, a weak current can be passed through the skin surface to permeate the stratum corneum (iontophoresis method).

The above-mentioned pharmaceutical composition may be used as it is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives and the like. Examples of the dosage form include liquids (for example, injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, Examples include inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.
Formulation includes, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjustments It can be carried out by a conventional method using an agent, preservative, antioxidant and the like as appropriate.
Examples of ingredients used for formulation include purified water, saline, 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, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch , Pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglyse Emissions, glycerine, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohol, stearyl alcohol, stearic acid, human serum albumin, and the like without limitation.
In view of the fact that the peptide is difficult to be absorbed through the mucosa, the pharmaceutical composition can contain an absorption enhancer that improves the absorption of the poorly absorbable drug. Examples of such absorption promoters include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; Fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, nitric oxide donors, etc. Can be used.

Pills or tablets can also be coated with sugar coatings, gastric and enteric substances.
The injection can contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols and the like. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives and the like can be added.

  When the pharmaceutical composition 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 the symptoms, patient age, sex, It varies depending on body weight, sensitivity difference, administration method, administration interval, active ingredient type, formulation type, and is not particularly limited.For example, 30 μg to 100 g, 100 μg to 500 mg, 100 μg to 100 mg are administered once or divided into several times can do. In the case of injection administration, 1 μg / kg to 3000 μg / kg, 3 μg / kg to 1000 μg / kg may be administered once or divided into several times depending on the weight of the patient.

(Screening method)
The present invention also includes a screening method for selecting preventive or therapeutic agents for influenza from candidate peptides.
In one aspect of the screening method of the present invention,
A step of expressing a peptide library in a cell-free translation system using a nucleic acid comprising the following sequence as a template, and ATG (NNK) n (SEQ ID NO: 49)
[Wherein n represents an integer of 4 to 12, N corresponds to A, T, G or C, and K corresponds to T or G. ];
Incubating the peptide library with hemagglutinin and selecting a peptide that binds to hemagglutinin.
The nucleic acid containing ATG (NNK) n may have, for example, a TGC encoding cysteine at the 3 ′ end or a base sequence encoding glycine serine linker (for example, GGCAGCGGCAGCGGCAGGC; SEQ ID NO: 50).
The step of expressing a peptide library in a cell-free translation system can be performed according to the peptide production method described above.

In addition, in one aspect of the screening method of the present invention, when a peptide library is expressed in a cell-free translation system, a starting amino acid of a special amino acid may be used as an aminoacyl tRNA corresponding to ATG.
If a chloroacetylated amino acid is used among the starting amino acids, a thioether bond is formed with cysteine, and a cyclic peptide library can be obtained.

(Influenza virus detection drug and detection kit)
The present invention also includes an influenza virus detection agent comprising the peptide of the present invention. The peptide of the present invention specifically binds to hemagglutinin on the surface of influenza virus. Therefore, for example, instead of an anti-influenza antibody in an immunoassay such as an ELISA method, an influenza virus in a sample can be detected using the peptide of the present invention.
When used as a detection agent, the peptide of the present invention may be detectably labeled. Peptide labels include, for example, enzymes such as peroxidase and alkaline phosphatase, radioactive materials such as ¹²⁵ I, ¹³¹ I, ³⁵ S, and ³ H, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near red An antibody labeled with a fluorescent substance such as an external fluorescent material, or a luminescent substance such as luciferase, luciferin, or aequorin is used. In addition, antibodies labeled with nanoparticles such as colloidal gold and quantum dots can also be detected.
In an immunoassay, the peptide of the present invention can be detected by labeling with biotin and binding avidin or streptavidin labeled with an enzyme or the like.
Among immunoassays, an ELISA method using an enzyme label is preferable because it can easily and quickly measure an antigen. For example, an antibody that specifically recognizes a portion other than hemagglutinin of influenza virus is immobilized on a solid phase carrier, a sample is added and reacted, and then the labeled peptide of the present invention is added and reacted. After washing, reaction with an enzyme substrate, color development, and measurement of absorbance can detect influenza virus. After reacting the antibody immobilized on the solid phase carrier with the sample, an unlabeled peptide of the present invention may be added, and an antibody against the peptide of the present invention may be enzyme-labeled and further added.
As the enzyme substrate, when the enzyme is peroxidase, 3,3′-diaminobenzidine (DAB), 3,3′5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used. In this case, p-nitropheny phosphate (NPP) or the like can be used.

  In the present specification, the “solid phase carrier” is not particularly limited as long as it can immobilize the antibody, and is made of a microtiter plate made of glass, metal or resin, substrate, beads, nitrocellulose membrane, nylon membrane, PVDF. Examples include a membrane, and the target substance can be immobilized on these solid phase carriers according to a known method.

  The test kit according to the present invention includes reagents and instruments necessary for the above detection (including but not limited to the peptide, antibody, solid phase carrier, buffer solution, enzyme reaction stop solution, microplate reader, etc. of the present invention). including.

The following examples are merely illustrative and are intended to describe the present invention in detail together with the above-described embodiments, and are not intended to limit the present invention.

[Selection of special cyclic peptides that bind to hemagglutinin]
<Screening 1>
A protein obtained by recombinantly expressing hemagglutinin of H5N1 influenza virus (1203/04) (PROSPEC) in a baculo system, dissolved in DMSO, and adjusted to pH 8.0-8.2 with an aqueous sodium phosphate solution, about 3 equivalents of biotin activity It was made to react with the ester body (Sulfo-NHS-LC-Biotin (PIERCE)). This reaction solution was mixed with a suspension of streptavidin-immobilized magnetic beads (Dynabeads M-280 Streptavidin (Invitrogen)) without purification and used as target beads for selection experiments except for non-biotinylated hemagglutinin.
ATG (NNK) nTGCGGGCAGCGGCAGCGGCAGC (SEQ ID NO: 51) was used as a template molecule for the special cyclic peptide library. In the formula, n represents an integer of 4 to 12, N corresponds to A, T, G or C, and K corresponds to T or G.
This DNA molecule has a transcription initiation sequence and translation promoting sequence (GGGTTAACTTTAAGAAGGAGATATACAT: SEQ ID NO: 53) on the 5 ′ side, and a tag sequence (GGCAGGCGCAGCGGCAGC; SEQ ID NO: 50) for matching with the glycine serine linker sequence on the 3 ′ side. Have each. This was transcribed into mRNA, and a nucleic acid derivative used for matching was covalently bound (see International Publication No. 2011/049157 pamphlet). 240 pmol of this complex was used for initial translation.
In addition to 19 kinds of natural amino acids except methionine, a complex in which chloroacetyl-D / L-tryptophan was bound to tRNA corresponding to the start codon was added to the translation system at a concentration corresponding to about 50 μM. This complex was prepared by reacting chloroacetyl-D / L-tryptophan cyanomethyl ester with tRNA in the presence of flexizyme (H. Murakami et al., Nat. Methods (2006) 3: 357-359; Y. Goto et al., ACS Chem. Biol. (2008) 3: 120-129; Y. Goto et al., RNA (2008) 14: 1390-1398).
48 μl of 100 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution was added to about 240 μL of the translation solution and allowed to stand for 30 minutes, and then mixed with streptavidin-fixed magnetic beads and mixed at 4 ° C. for 1 hour. The supernatant was collected and combined with biotinylated hemagglutinin-bound target beads, mixed for 1 hour at 4 ° C., and washed 3 times with TBST (50 mM trishydroxymethylaminomethane-hydrochloric acid buffer solution, 120 mM sodium chloride, 0.05% Tween 20). did. A reverse transcription reaction mixture was added to the beads and reacted at 42 ° C. for 1 hour. The supernatant was diluted with a PCR reaction solution, and a part thereof was quantified by real-time PCR. The number of cycles was determined according to the quantitative result, and the remaining solution was subjected to PCR reaction.
The obtained PCR reaction product was transferred to a template, and a second selection experiment was performed on the 1/12 scale. The second and subsequent reverse transcription reactions were performed after translation and reacted with target beads in the form of double strands. The PCR reaction solution was added to the target beads and heated at 95 ° C. to recover the template nucleic acid. This selection experiment was repeated up to the fifth time when the amount of nucleic acid recovered was 10 times or more higher when hemagglutinin was immobilized. The change in the recovery rate after each selection experiment is shown in FIG.
The final recovered DNA was ligated to a plasmid (pGEM-T Easy (Promega)), cloned, and analyzed for 64 sequences. As a result, 52 types of nucleic acids containing 2 to 5 duplications were supported. Was.

<Screening 2>
Next, three types were selected from these 52 types of nucleic acids, and template molecules containing the following coding regions were designed based on these three types of nucleic acid sequences based on empirical rules.
ATGVVKNNKHWTVVKNNKVDAVDATWTNNKHWTVVKTGGTGTGGGTCGGGGTCGGGTTCG (SEQ ID NO: 52)
In this sequence, V represents C, A or G, H represents T, C or A, W represents T or A, and D represents T, A or G.
The nucleic acid library was transcribed in the presence of tRNA ester-linked with chloroacetyl-D-tryptophan at the 3 ′ position, and about 50 pmol of the library combined with the nucleic acid derivative was translated with 50 μl of a cell-free translation system. After adding 10 μl of EDTA (100 mM) aqueous solution and allowing to stand at 37 ° C. for 30 minutes, cDNA was formed by reverse transcription reaction.
Using this complex as an initial translation library, affinity selection for hemagglutinin was repeated in the same manner as in screening 1. However, the interaction with hemagglutinin was carried out at room temperature instead of 4 ° C. The selection experiment was repeated up to 6 times, and finally recovered DNA was amplified by PCR and cloned. The 17 sequences analyzed were all different but converged throughout and a high degree of homology was observed.

[Chemical synthesis of special cyclic peptides that bind to hemagglutinin]
For the purpose of confirming whether the peptide considered to correspond to the nucleic acid obtained as a result of selection actually has a binding activity to hemagglutinin or an inhibitory activity against the growth of influenza virus, it was obtained in the above screenings 1 and 2. The 24 peptides shown in the table below selected from the 69 peptides were chemically synthesized.
Peptide synthesis was performed using an automatic synthesizer SyroI (Biotage Japan) according to a general solid phase synthesis method using a 9-fluorenylmethoxycarbonyl group (Fmoc group) as a protective group for the α-amino group.
3 μmol equivalent of Rink Amide AM resin (Novabiochem) was added to the chip for synthesis and protected with 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and Fmoc group Amino acids (both Novabiochem) were reacted sequentially.
After linking N-terminal tryptophan to remove Fmoc, 6 equivalents of chloroacetyl N-hydroxysuccinate dissolved in 1-methyl-2-pyrrolidinone (NMP; Nacalai Tesuque) was added and left to stand for 1 hour.
The obtained peptide resin was washed three times with N, N-dimethylformamide (DMF; Nacalai Tesuque) and methylene chloride, dried, and a mixture of trifluoroacetic acid-water-triisopropylsilane (90: 5 by volume): 5) was added and stirred at room temperature for 2 hours.
The crude peptide cleaved by ether precipitation was recovered, washed twice with diethyl ether and dried, and then added with an 80% aqueous DMSO solution containing 125 mM trishydroxymethylaminomethane to a final concentration of 10 mM. For 1 hour.
The cyclic peptide solution was acidified and filtered, and the target product was purified by reverse phase high performance liquid chromatography using a C4 column (Cosmosil AR300 5C ₄ (10 × 250 mm, Nacalai Tesuque)). Water-acetonitrile containing fluoroacetic acid was used, and the resulting peptide was identified by MALDI-TOF-MS using Autoflex II (Bruker Daltonics).
The following 24 types of peptides were used in the influenza virus growth inhibition test.

[Evaluation of peptide inhibitory activity against influenza virus growth]
1. Inhibition against influenza virus H5N1-Vac3 Inhibition test against influenza virus H5N1-Vac3 was carried out according to the following procedure.
1) Three days before the assay, MDCK cells were seeded at 2 × 10 ⁵ cells / well in 6-well plates.
2) On the day before influenza virus infection, the cells were pretreated by replacing with a cell culture medium containing 0.01 μM, 0.1 μM and 1 μM of each peptide.
3) 0.1 ml of influenza virus H5N1 (1 × 10 ³ PFU / ml) virus solution diluted with MEM medium containing 1% BSA was added to each well and spread throughout.
4) Adsorbed at 37 or 34 ℃ for 1 hour. The plate was shaken every 15 minutes.
5) The virus solution was removed and washed once with BSA (-) / MEM.
6) Peptides 0.01 μM, 0.1 μM, 1 μM, 10 μg / ml acetyltrypsin, 0.8% agarose and 1% BSA / MEM 2 ml were added to each well.
7) After agarose coagulation, the plate was turned upside down and placed in a CO ₂ incubator.
8) Cultured at 37 or 34 ° C for 2-3 days.
9) 10% formalin was added. Fixed at room temperature for 1 hour.
10) Formalin and agarose medium were removed and washed with water.
11) Stained with 1% crystal violet for 1 hour.
12) The number of plaques was determined after washing and drying.

FIGS. 2 and 3 show photographs of the plate after culturing. Compared to the negative control (NC), No. 11, 12, 18 and 24 (FIG. 2), and 19 and No. In FIG. 23 (FIG. 3), there was a clear difference in plaque size and number. The amount of virus used was confirmed by performing back-titration, but it was a predetermined amount of virus.
Figures 4-6 show a comparison of plaque number and plaque diameter. As shown in FIGS. 4 and 5, as a whole, the number of plaques tends to decrease compared to the negative control. In 11 and 12, it decreased to less than half (FIG. 4). No. No. 14, the number of plaques is less than half. No. 19 has a plaque number of zero at 0.1 μM or more. 23 had a plaque number of 1/10 at 1 μM.
In addition, as shown in FIG. 6, the plaque diameter tended to decrease in a dose-dependent manner for any of the peptides.
No. In 1 μM of No. 18, both the number and the diameter of the plaques were decreased in the same manner as Zanamivir (trade name Relenza). At 1 μM of 24, the plaque diameter was reduced by approximately half. No. In 14, 19, and 23, the plaque diameter was also less than half compared to the negative control (data not shown).

2. Similarly to the inhibition against the influenza virus H1N1 strain, an inhibition test with a peptide against the influenza virus H1N1 was conducted.
An inhibition test against influenza virus H1N1 strain A / Tokyo / 2619/2009 was performed according to the following procedure.
1) Three days before the assay, MDCK cells were seeded at 2 × 10 ⁵ cells / well in 6-well plates.
2) On the day before influenza virus infection, the cells were pretreated by replacing with a cell culture medium containing 0.01 μM, 0.1 μM and 1 μM of each peptide.
3) 0.1 ml of influenza virus H5N1 (1 × 10 ³ PFU / ml) virus solution diluted with MEM medium containing 1% BSA was added to each well and spread throughout.
4) Adsorption for 1 hour at 37 or 34 ℃. The plate was shaken every 15 minutes.
5) The virus solution was removed and washed once with BSA (-) / MEM.
6) Peptides 0.01 μM, 0.1 μM, 1 μM, 10 μg / ml acetyltrypsin, 0.8% agarose and 1% BSA / MEM 2 ml were added to each well.
7) After coagulation of the agarose, turn the plate upside down and place it in the CO ₂ incubator.
8) Cultured at 37 or 34 ° C for 2-3 days.
9) 10% formalin was added. Fixed at room temperature for 1 hour.
10) Formalin and agarose medium were removed and washed with water.
11) Stained with 1% crystal violet for 1 hour.
12) The number of plaques was determined after washing and drying.
The results are shown in FIG. A peptide having an inhibitory effect on growth was also observed against H1N1, for which Relenza had no effect. In particular, no. 23 and 24 showed a high growth inhibitory effect.

3. Neutralizing activity against influenza virus strain H5N1-Vac3 In addition, in order to confirm the neutralizing activity of the peptide against influenza virus, a test was performed in which the peptide and influenza virus were first mixed, and then the mixture was brought into contact with cells. Specific test methods are shown below.
1) Three days before the assay, MDCK cells were seeded at 2 × 10 ⁵ cells / well in 6-well plates.
2) On the day before influenza virus infection, the cells were pretreated by replacing with a cell culture medium containing 0.01 μM, 0.1 μM and 1 μM of each peptide.
3) 110 μl of 55 PFU virus solution (H5N1 Vac-3) was mixed with 110 μl of cell culture medium containing 0.01 μM, 0.1 μM and 1 μM of each peptide, and incubated at 37 ° C. for 1 hour in a CO ₂ incubator.
4) 0.2 ml of virus solution incubated with the peptide was added to each well and spread throughout.
5) Adsorption for 1 hour at 37 or 34 ℃. The plate was shaken every 15 minutes.
6) The virus solution was removed and washed once with BSA (-) / MEM.
7) Peptide 0.01 μM, 0.1 μM, 1 μM, 10 μg / ml acetyltrypsin, 2% 1% BSA / MEM with 0.8% agarose was added to each well. Alternatively, an agarose medium containing 0.01 μM, 0.1 μM, and 1 μM of oseltamivir (trade name Tamiflu) or zanamivir (trade name Relenza) was overlaid.
8) After agarose coagulation, the plate was turned upside down and placed in a CO ₂ incubator.
9) Cultured at 37 or 34 ° C for 2-3 days.
10) 10% formalin was added. Fixed at room temperature for 1 hour.
11) Formalin and agarose medium were removed and washed with water.
12) Stained with 1% crystal violet for 1 hour.
13) The number of plaques was determined after washing and drying.
The results are shown in FIG. FIG. 9 shows the treatment conditions for the test peptide and virus solution. The number in the upper right of each well represents the number of plaques. Peptide No. 11, 14, and 18 were only two conditions in FIG. 9, that is, neutralization reaction, and the number of plaques was reduced to less than half compared with the ineffective condition. This result shows that peptide No. The 11, 14, 18 peptide suggests the possibility of having neutralizing activity against influenza virus

Furthermore, no. In order to confirm the neutralizing activity of the 23,24 peptide, the peptide and influenza virus were first mixed, and then a test was conducted in which the mixture was contacted with cells. Specific test methods are shown below.
1) Three days before the assay, MDCK cells were seeded at 2 × 10 ⁵ cells / well in 6-well plates.
2) 110 μl of 55 PFU virus solution (H5N1 Vac-3) was mixed with 110 μl of cell culture medium containing 0.01 μM, 0.1 μM and 1 μM of each peptide, and incubated at 37 ° C. for 1 hour in a CO ₂ incubator.
3) 0.2 ml of virus solution incubated with the peptide was added to each well and spread throughout.
4) Adsorption for 1 hour at 37 or 34 ℃. The plate was shaken every 15 minutes.
5) The virus solution was removed and washed once with BSA (-) / MEM.
6) 2 ml of 1% BSA / MEM with 10 μg / ml acetyltrypsin and 0.8% agarose was added to each well. As a positive control of the test, an agarose medium containing 0.01 μM, 0.1 μM, and 1 μM of oseltamivir (trade name Tamiflu) or zanamivir (trade name Relenza) was overlaid.
7) After agarose coagulation, the plate was turned upside down and placed in a CO ₂ incubator.
8) Cultured at 37 or 34 ° C for 2-3 days.
9) 10% formalin was added. Fixed at room temperature for 1 hour.
10) Formalin and agarose medium were removed and washed with water.
11) Stained with 1% crystal violet for 1 hour.
12) The number of plaques was determined after washing and drying.
FIG. 10 shows the results of the neutralization test, and FIG. 11 shows the results of the positive control. The amount of virus used was confirmed by performing back-titration, but it was a predetermined amount of virus. The number in the upper right of each well represents the number of plaques. In all peptides, the number of plaques was reduced to less than half compared with the ineffective condition. This result suggests the possibility that the peptide of the present invention has neutralizing activity against influenza virus.

SEQ ID NOs: 1 to 24 are amino acid sequences of peptides having anti-influenza virus activity.
SEQ ID NOs: 25-48 are the amino acid sequences of cyclic peptides having anti-influenza virus activity.
SEQ ID NO: 49 is an example of a template nucleic acid sequence used for expressing a peptide library in a cell-free translation system.
SEQ ID NO: 50 is the base sequence of the nucleic acid encoding glycine-serine linker.
SEQ ID NO: 51 is an example of a template nucleic acid sequence used for expressing a peptide library in a cell-free translation system.
SEQ ID NO: 52 is an example of a template nucleic acid sequence used for expressing a peptide library in a cell-free translation system.
SEQ ID NO: 53 is a translation promoting sequence added to the 5 ′ side of a template nucleic acid sequence used when a peptide library is expressed in a cell-free translation system.

Claims (11)

Any of the following peptides:
(I) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1-24;
(Ii) a peptide comprising a partial sequence of the peptide described in (i) above, which is capable of binding hemagglutinin; and (iii) in the amino acid sequence of the peptide described in (i) or (ii) above, Alternatively, a peptide in which several amino acids are deleted, added or substituted and which can bind to hemagglutinin.   The peptide according to claim 1, which is cyclized.   The peptide according to claim 2, comprising chloroacetylamino acid and cysteine, and cyclized by a thioether bond between chloroacetylamino acid and cysteine.   The peptide according to claim 3, which has a chloroacetyl amino acid within 3 amino acids from the N-terminus and a cysteine within 3 amino acids from the C-terminus.   A peptide consisting of the amino acid sequence represented by SEQ ID NO: 25-48, which is cyclized by a thioether bond between the N-terminal chloroacetyl-tryptophan and the second cysteine from the C-terminal.   A pharmaceutical composition for preventing or treating influenza comprising the peptide according to any one of claims 1 to 5.   Use of the derivative | guide_body of the peptide of any one of Claim 1 to 5 for manufacturing the preventive or therapeutic agent of influenza. A screening method for selecting preventive or therapeutic agents for influenza from candidate peptides,
Expressing a peptide library in a cell-free translation system using a nucleic acid containing the following sequence as a template;
ATG (NNK) n (SEQ ID NO: 49)
[Wherein n represents an integer of 4 to 12, N corresponds to A, T, G or C, and K corresponds to T or G. ];
Contacting the peptide library with hemagglutinin and incubating to select a peptide that binds to hemagglutinin;
Including methods. As the template, one containing TGC at the 3 ′ end of (NNK) n,
In the cell-free translation system, a chloroacetylated amino acid is used as an aminoacyl tRNA corresponding to the codon ATG,
9. The method according to claim 8, comprising a step of cyclizing the peptide prior to the step of contacting and incubating the peptide library with hemagglutinin.   The influenza virus detection agent containing the peptide of any one of Claim 1 to 5.   An influenza virus detection kit comprising the influenza detection agent according to claim 10.