Novel peptide and antiviral use thereof

Abstract

A peptide provided in the present application has antiviral activity, and thus is useful for the prevention and / or treatment of viral infections or virus-induced diseases.

Description

Novel peptides and their antiviral uses

[0001] The present application relates to a novel peptide and its antiviral use.

[0002]

[0003] The general mechanisms of action of antiviral peptides are known to include inhibition of viral membrane protein function, inhibition of viral RNA amplification within the nucleus of infected cells, and inhibition of infection through damage to the viral envelope. In the case of enveloped viruses, the envelope consists of a lipid bilayer structure, so inactivation of the envelope means loss of viral infectivity.

[0004] Among the various mechanisms of action, a characteristic of peptides that exhibit antiviral efficacy by attacking the viral outer membrane is their amphipathic nature. An amphipathic peptide is a peptide that possesses both hydrophilicity (polarity) and hydrophobicity (nonpolarity) properties within a single helix. Amphipathic peptides have hydrophilic and hydrophobic amino acids coexisting within a single helix, and they acquire amphipathicity through a biased distribution within the alpha helix structure. This characteristic allows them to easily act on lipid bilayers, which have a hydrophobic exterior and a hydrophilic interior, and various amphipathic peptides are known to have antiviral efficacy.

[0005]

[0006] The purpose of the present application is to provide a peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 100.

[0007] Another object of the present application is to provide a polynucleotide encoding the above peptide.

[0008] Another object of the present application is to provide a recombinant vector comprising the polynucleotide.

[0009] Another object of the present application is to provide a microorganism comprising at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, and a recombinant vector comprising the polynucleotide.

[0010] Another object of the present application is to provide a method for producing the peptide, comprising a step of culturing the microorganism in a medium.

[0011] Another object of the present application is to provide a composition for producing the peptide, comprising at least one selected from the group consisting of the microorganism and a medium in which the microorganism is cultured.

[0012] Another object of the present application is to provide an antiviral composition comprising the peptide.

[0013] Another object of the present application is to provide a pharmaceutical composition for preventing or treating viral infection and / or diseases caused by viruses, comprising the peptide.

[0014] Another object of the present application is to provide a feed composition, food composition and / or cosmetic composition for preventing or improving viral infection and / or diseases caused by viruses, comprising the peptide.

[0015] Another object of the present application is to provide a feed additive comprising the above peptide.

[0016] Another object of the present application is to provide a disinfectant comprising the peptide.

[0017] Another object of the present application is to provide a detergent comprising the peptide.

[0018]

[0019] This is specifically explained as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below. Furthermore, those skilled in the art will recognize or ascertain numerous equivalents to the specific embodiments of this application described in this application using only routine experimentation. Furthermore, such equivalents are intended to be encompassed by this application.

[0020]

[0021] Definition of terms

[0022] The definitions of terms used in this specification are as follows.

[0023] approximately

[0024] The term "about" as used herein means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0025] Amino acid sequence notation

[0026] Unless otherwise stated, when describing amino acid sequences in this specification, amino acid single-letter notation or three-letter notation is used, and is written in the N-terminal to C-terminal direction. For example, when written as RNVP, it means a peptide in which arginine, asparagine, valine, and proline are sequentially connected from the N-terminal to the C-terminal. Another example, when written as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected from the N-terminal to the C-terminal. In the case of amino acids that cannot be expressed in the single-letter notation, other letters are used and additional explanations are provided.

[0027] The notation for each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Ty, Y); and Valine (Val, V).

[0028] Nucleic acid sequence notation

[0029] The symbols A, T, C, G, and U used herein are to be interpreted as meanings understood by a person skilled in the art. They may be appropriately interpreted as bases, nucleosides, or nucleotides in DNA or RNA, depending on the context and technology. For example, when referring to a base, they may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) themselves, respectively; when referring to a nucleoside, they may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively; and when referring to a nucleotide in a sequence, they should be interpreted to mean a nucleotide containing each of the above nucleosides.

[0030] In this specification, the symbol N may be appropriately interpreted as a base, a nucleoside, or a nucleotide in DNA or RNA, depending on the context and technology. For example, when referring to a base, it may be interpreted as any one of adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), respectively; when referring to a nucleoside, it may be interpreted as any one of adenosine (A), thymidine (T), cytidine (C), guanosine (G), and uridine (U), respectively; and when referring to a nucleotide in a sequence, it should be interpreted as meaning a nucleotide containing each of the above nucleosides.

[0031] antiviral peptides

[0032] HCV is an enveloped, single-stranded, positive-sense RNA virus. The single-stranded HCV RNA genome is approximately 9,500 nucleotides long and contains a single open reading frame (ORF) that encodes a single large protein of approximately 3,000 amino acids. In infected cells, this protein is cleaved at multiple sites by cellular and viral proteases, generating structural and non-structural proteins.

[0033] In HCV, the production of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is influenced by two viral proteases. The first is a metalloprotease located in NS2 that cleaves the NS2-NS3 junction in cis. The second is a serine protease contained within the N-terminal region of NS3 (hereafter referred to as NS3 protease) that mediates all subsequent cleavage of NS3 in cis at the NS3-NS4A cleavage site and in trans at the remaining NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites. The NS4A protein appears to perform several functions, including acting as a cofactor for the NS3 protease and aiding in membrane localization of NS3 and other viral replicase components.

[0034] Meanwhile, among the nonstructural proteins of HCV is NS5A. Several studies have examined the amino acid sequence of NS5A and confirmed that it possesses an amphipathic alpha-helix structure at the NH2-terminus. This amphipathic alpha-helix is ​​known to be necessary for NS5A's membrane binding and to anchor the protein by inserting it within the membrane plane. Notably, disruption of the amphipathic alpha-helix of NS5A is known to impair its membrane binding ability and halt HCV RNA replication.

[0035] Cheng, Guofeng, et al. identified an antiviral peptide from the NS5A protein of HCV (Cheng, Guofeng, et al. "A virocidal amphipathic α-helical peptide that inhibits hepatitis C virus infection in vitro." Proceedings of the National Academy of Sciences 105.8 (2008): 3088-3093.). Specifically, Cheng, Guofeng, et al. confirmed that an amphipathic α-helical peptide (C5A) derived from the membrane anchor domain of the NS5A protein of hepatitis C virus (HCV) had an antiviral effect against HCV in vitro. C5A has a sequence identical to amino acids 3-20 near the N-terminus of the NS5A protein. Because C5A is derived from the membrane anchor domain of NS5A, which is predicted to be an in-plane amphipathic helix, it can penetrate the envelope and destabilize HCV virions. Furthermore, Cheng, Guofeng, et al. compared the antiviral activity of C5A analogs derived from various HCV genotypes. Specifically, they compared the antiviral activity of C5A analogs derived from HCV 1b, 2a, 3a, 4a, 5a, and 6a, and confirmed that C5A derived from HCV 1a and C5A analogs derived from HCV 3a (e.g., a peptide having the amino acid sequence of SEQ ID NO: 101) exhibited higher antiviral activity (Table 1 of Cheng, Guofeng, et al.).

[0036] In this specification, the phrase "a polynucleotide (which may be used interchangeably with "gene") or a peptide (which may be used interchangeably with "polypeptide" or "protein") "comprises a specific nucleic acid sequence or amino acid sequence" or "consists of a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or peptide essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a "substantially equivalent sequence" in which a mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence within the scope of maintaining the original function and / or desired function of the polynucleotide or peptide.

[0037] In one example, a polynucleotide or peptide "comprises a particular nucleic acid sequence or amino acid sequence" or "consists of or is represented by a particular nucleic acid sequence or amino acid sequence" means that the polynucleotide or peptide (i) essentially comprises the particular nucleic acid sequence or amino acid sequence, or (ii) is at least 60%, 65%, 70%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, It may mean that it consists of or essentially contains a nucleic acid sequence or amino acid sequence having a homology or identity of 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more or 99.9% or more and maintains the original function and / or the desired function. In the present specification, the original function may be an antiviral activity (in the case of an amino acid sequence) and / or a function of encoding a protein having an antiviral activity (in the case of a nucleic acid sequence).

[0038] As used herein, the term "identity" refers to the degree of identity with a given nucleic acid sequence or amino acid sequence, and can be expressed as a percentage (%). For nucleic acid sequences, homology can be determined, for example, using the algorithm BLAST by the literature (see: Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see: Methods Enzymol., 183, 63, 1990). Based on the algorithm BLAST, programs called BLASTN and BLASTX have been developed (see: http: / www.ncbi.nlm.nih.gov).

[0039] The protein or peptide provided herein may be isolated and / or purified from nature, or may be recombinantly or chemically synthesized. If the amino acid sequence of the protein or peptide provided herein includes a methionine (Met, M) or Met-Ala-Ser (MAS) sequence as the first amino acid residue from the N-terminus, the protein or peptide may be recombinantly produced, and the methionine at the first amino acid residue from the N-terminus may be encoded by an initiation codon. Therefore, if the amino acid sequence of a protein or peptide provided herein includes a methionine at the N-terminus by recombinant production, it can be interpreted that the protein or peptide includes an amino acid sequence starting from the second amino acid residue excluding the methionine at the first position of the N-terminus by recombinant production, or an amino acid sequence starting from the amino acid residue following the MAS sequence (e.g., the fourth amino acid residue), if the protein or peptide is obtained by another method (e.g., chemical synthesis or isolation from nature).

[0040] The peptides provided herein may not be of natural origin and may be recombinantly or chemically synthesized. When the peptides are produced recombinantly, they may be in a form in which conventional signal peptides, cleavage sites, tags, etc. are combined for purification. Accordingly, in a non-limiting example, the peptides provided herein may be in a form that additionally includes one or more selected from the group consisting of signal peptides, cleavage sites, tags (e.g., His tag, GST (glutathione-s-transferase) tag, MBP (maltose binding protein) tag, etc.) that can be conventionally used in the recombinant production process of proteins, or may be in a purified form in which these are removed.

[0041]

[0042] peptides

[0043] One aspect provides a peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 100.

[0044] In one example, the peptide may comprise any one amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 100.

[0045] In one example, the peptide may comprise any one amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 46, 52, 79, and 81.

[0046] Peptide length

[0047] The number of amino acid residues constituting the peptide of the present disclosure is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36. In other words, the antiviral peptide is a 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27mer, 28mer, 29mer, 30mer, 31mer, 32mer, 33mer, 34mer, 35mer, or 36mer peptide.

[0048] For example, the number of amino acid residues constituting the peptide may be between the two values ​​selected in the immediately preceding sentence. For example, the number of amino acid residues constituting the peptide may be 16 to 27.

[0049] Preferably, the number of amino acid residues constituting the peptide may be 18. In other words, the peptide may be composed of 18 amino acid residues. In another expression, the peptide may be an 18-mer peptide.

[0050] Amino acid residues in peptides

[0051] The peptide of the present disclosure may include hydrophilic amino acid residues and hydrophobic amino acid residues. Here, the hydrophilic amino acid residues may include arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), lysine (Lys, K), serine (Ser, S), and / or threonine (Thr, T). The hydrophobic amino acid residues may include alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), proline (Pro, P), valine (Val, V), phenylalanine (Phe, F), tyrosine (Tyr, Y), and / or tryptophan (Trp, W). In some embodiments, the peptide may include hydrophilic amino acid residues including K, R, S, and D, and hydrophobic amino acid residues including C, W, V, L, F, and I.

[0052] In some embodiments, the number of hydrophilic amino acid residues and hydrophobic amino acid residues contained in the peptide may be the same. In other embodiments, the number of hydrophilic amino acid residues contained in the peptide may be 1, 2, 3, 4, or 5 more than the number of hydrophobic amino acid residues contained in the peptide. In still other embodiments, the number of hydrophobic amino acid residues contained in the peptide may be 1, 2, 3, 4, or 5 more than the number of hydrophilic amino acid residues contained in the peptide. In preferred embodiments, the number of hydrophobic amino acid residues contained in the peptide may be 4 more than the number of hydrophilic amino acid residues contained in the peptide.

[0053] In some embodiments, when the peptide is an 18-mer peptide, the peptide may comprise seven hydrophilic amino acid residues and eleven hydrophobic amino acid residues. In some other embodiments, when the peptide is an 18-mer peptide, the peptide may comprise eight hydrophilic amino acid residues and ten hydrophobic amino acid residues. In some other embodiments, when the peptide is an 18-mer peptide, the peptide may comprise nine hydrophilic amino acid residues and nine hydrophobic amino acid residues. In some other embodiments, when the peptide is an 18-mer peptide, the peptide may comprise ten hydrophilic amino acid residues and eight hydrophobic amino acid residues. In some other embodiments, when the peptide is an 18mer peptide, the peptide may comprise 11 hydrophilic amino acid residues and 7 hydrophobic amino acid residues.

[0054] Peptide structure - alpha helix structure

[0055] The peptide of the present disclosure can form an alpha helix (α-helix) structure when reacting with a lipid membrane. In this case, the peptide reacting with a lipid membrane may mean that the peptide comes into contact with a lipid membrane (specifically, a phospholipid bilayer).

[0056] When the peptide has an alpha helix structure, the positions of amino acid residues can be diagrammed in the direction of looking down at the alpha helix structure from top to bottom, as shown in Fig. 1. The amino acid sequence of the peptide illustrated in Fig. 1 is N-terminal-DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101)-C-terminal. When the peptide has an alpha helix structure that rotates around an imaginary first axis, when looking at the peptide from one direction parallel to the imaginary first axis, each amino acid can be seen as illustrated in Fig. 1. At this time, if one imaginary plane including the imaginary first axis (the imaginary plane is indicated by a dashed dotted line in Fig. 1) is well selected, the peptide can be divided into a hydrophilic region and a hydrophobic region by the imaginary plane. At this time, each of the two regions divided by the imaginary plane includes nine amino acid residues.

[0057] In the present specification, the hydrophilic region may mean a region containing a greater number of hydrophilic amino acid residues among the two regions divided by the virtual plane, and the hydrophobic region may mean a region other than the hydrophilic region among the two divided regions.

[0058] In some embodiments, the hydrophilic region may comprise only hydrophilic amino acid residues and no hydrophobic amino acid residues. That is, the hydrophilic region may comprise nine hydrophilic amino acid residues.

[0059] In some other embodiments, the hydrophilic region may comprise eight hydrophilic amino acid residues and one hydrophobic amino acid residue.

[0060] In some other embodiments, the hydrophilic region may comprise seven hydrophilic amino acid residues and two hydrophobic amino acid residues.

[0061] In some other embodiments, the hydrophilic region may comprise six hydrophilic amino acid residues and three hydrophobic amino acid residues.

[0062] In some embodiments, the hydrophobic region may comprise only hydrophobic amino acid residues and no hydrophilic amino acid residues. That is, the hydrophobic region may comprise nine hydrophobic amino acid residues.

[0063] In some other embodiments, the hydrophobic region may comprise eight hydrophobic amino acid residues and one hydrophilic amino acid residue.

[0064] In some other embodiments, the hydrophobic region may comprise seven hydrophobic amino acid residues and two hydrophilic amino acid residues.

[0065] In some other embodiments, the hydrophobic region may comprise six hydrophobic amino acid residues and three hydrophilic amino acid residues.

[0066] In one embodiment, each hydrophilic amino acid or hydrophilic amino acid residue can be any one selected from arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), lysine (Lys, K), serine (Ser, S), and threonine (Thr, T).

[0067] In one embodiment, each hydrophobic amino acid or hydrophobic amino acid residue can be any one selected from alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), proline (Pro, P), valine (Val, V), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W).

[0068] Characteristics of Peptides - Amphipathy

[0069] The peptide of the present disclosure may be an amphipathic peptide. Specifically, the peptide does not have amphipathic characteristics when it has a linear structure, but has amphipathic characteristics when it has an alpha-helical structure. This is because, due to the characteristics of the amino acid sequence of the peptide, when it has a linear structure, there is no region where hydrophilic or hydrophobic amino acid residues are concentrated within a single structure, but when it has an alpha-helical structure, there is a region where hydrophilic or hydrophobic amino acid residues are concentrated within a single structure. In other words, the peptide has amphipathic characteristics when it reacts with a lipid membrane or forms an alpha-helical structure for other reasons.

[0070] The above amphipathic characteristic means that when the target peptide has a three-dimensional structure such as an alpha helix structure, one side of the target peptide within the three-dimensional structure exhibits hydrophilic (polar, hydrophilicity) properties, and the other side exhibits hydrophobic (non-polar, hydrophobicity) properties. In other words, the fact that the target peptide has amphipathic properties means that the target peptide has both hydrophobic and hydrophilic properties.

[0071] The amphipathic characteristic of the above peptide can be explained through the distribution of hydrophilic and hydrophobic amino acid residues within the three-dimensional helical structure of the peptide. Specifically, since the alpha helix structure is divided into two regions by an imaginary plane, in one region, hydrophilic amino acid residues are distributed in greater numbers than hydrophobic amino acid residues, and in the other region, hydrophobic amino acid residues are distributed in greater numbers than hydrophilic amino acid residues, the peptide having the alpha helix structure has both hydrophilic and hydrophobic properties within a single structure.

[0072] The above amphipathic characteristic is presumed to be one of the reasons for the efficacy of the peptide in acting on the phospholipid bilayer.

[0073] For example, the amphipathic characteristic of the peptide can be measured using the EMBOSS (European Molecular Biology Open Software Suite) program. Specifically, when the sequence of the peptide is input into the EMBOSS program, the hydrophobic moment value can be any one of 0.568 to 0.633. That is, the peptide can have a hydrophobic moment value (hydrophobic moment value calculated from the EMBOSS program) any one of 0.568 to 0.633.

[0074] Examples of peptide design methods

[0075] The inventors of the present application designed a novel peptide sequence by changing the positions of amino acid residues of a peptide having an amino acid sequence of DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101) derived from HCV genotype 3a. At this time, in order to maintain the structure and characteristics of the peptide having an amino acid sequence of SEQ ID NO: 101, the peptides disclosed by the present disclosure were designed using the peptide having an amino acid sequence of SEQ ID NO: 101 in the following manner.

[0076] The method of changing the positions of the amino acid residues refers to a method of changing the positions of amino acid residues included in the hydrophilic region and changing the positions of amino acid residues included in the hydrophobic region when the peptide represented by the amino acid sequence of SEQ ID NO: 101 has an alpha helix structure. At this time, since the method is a method without adding new amino acid residues, the types of amino acids included in the amino acid sequence of the newly designed peptide and the number of each type are the same as the types of amino acids included in DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101) and the number of each type.

[0077] For example, when a peptide having an amino acid sequence of DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101) has an alpha helix structure, the specific positions of the amino acid residues included in the hydrophilic region and the amino acid residues included in the hydrophobic region are as follows: amino acid residues corresponding to positions 1, 4, 5, 8, 11, 12, 15, 16, and 18 in SEQ ID NO: 101 are amino acid residues included in the hydrophobic region, and amino acid residues corresponding to positions 2, 3, 6, 7, 9, 10, 13, 14, and 17 in SEQ ID NO: 101 are amino acid residues included in the hydrophilic region. That is, when the positions of amino acid residues are changed according to the above design method, amino acid residues D, R, I, D, C, S, S, D, and K corresponding to positions 1, 4, 5, 8, 11, 12, 15, 16, and 18 in SEQ ID NO: 101 are randomly positioned at positions 1, 4, 5, 8, 11, 12, 15, 16, and 18 in the amino acid sequence (from the N-terminal to the C-terminal direction) of the newly designed 18-mer peptide. In addition, amino acid residues W, L, I, W, W, V, V, V, and F corresponding to positions 2, 3, 6, 7, 9, 10, 13, 14, and 17 in SEQ ID NO: 101 are randomly located at positions 2, 3, 6, 7, 9, 10, 13, 14, and 17 in the amino acid sequence (from the N-terminus to the C-terminus) of the newly designed 18-mer peptide. In the present disclosure, this design method is called the scrambling method.

[0078] In the end, the number of mutated peptides that can be obtained by scrambling the hydrophilic region for the peptide having the amino acid sequence of SEQ ID NO: 101 is 30,240, which is the value obtained by dividing 9*8*7*6*5*4*3*2*1 by {(9C5) / (3*2*1) * (2*1)} = {(9*8*7*6*5 / 5*4*3*2*1) / (6*2)} = 12 to remove duplicate counts due to the presence of three identical amino acids D and two identical amino acids S, and the number of mutated peptides that can be obtained by scrambling the hydrophobic region for the peptide having the amino acid sequence of SEQ ID NO: 101 is 9*8*7*6*5*4*3*2*1, which is the value obtained by dividing 9*8*7*6*5*4*3*2*1 by the value obtained by removing duplicate counts due to the presence of three identical amino acids W and three identical amino acids V. {(9C6) / (3*2*1) * (3*2*1)} = {(9*8*7*6*5*4 / 6*5*4*3*2*1) / (6*6)} = 10,080 can be derived, which is the value divided by 36, so the total number of mutated peptides that can be derived by the scrambling method described above is 30,240*10,080=304,819,200.

[0079] A newly designed peptide using the scramble method targeting a peptide having an amino acid sequence of DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101) may be a peptide including any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 100.

[0080] The peptides of the present disclosure are selected from among a plurality of peptides designed by a scramble method using a peptide having an amino acid sequence of DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101). At this time, the criteria for the selection were whether the hydrophobic moment value calculated by the EMBOSS program (European Molecular Biology Open Software Suite) based on the sequences of the designed plurality of peptides is within a range of 0.568 to 0.633; and / or whether the Hydrophobic moment (μH) value calculated by the HeliQuest program (IPMC & CNRS, Institute Pharmacology Molιculaire Et Cellulaire, Centre national de la recherche scientifique) is within a range of 0.54 to 0.80.

[0081] Peptide activity

[0082] The above peptide may have antiviral activity.

[0083] The above antiviral activity may be an activity that causes damage and / or destruction of the outer membrane (or phospholipid bilayer) of the virus, as described below. Alternatively, the above antiviral activity may be an activity that causes a reduction or inactivation of the virus's ability to infect cells.

[0084] The above peptide can cause damage to the outer membrane (or phospholipid bilayer) of the virus.

[0085] The above peptide can cause destruction of the outer membrane (or phospholipid bilayer) of the virus. Specifically, the peptide can cause destruction of the phospholipid bilayer of the virus by causing damage to the phospholipid bilayer of the virus.

[0086] The peptide may cause a reduction or inactivation of the virus's ability to infect cells. Specifically, the peptide may cause a reduction or inactivation of the virus's ability to infect cells by causing damage (or destruction) of the phospholipid bilayer.

[0087] The above peptide can cause a reduction or inactivation of the virus's ability to multiply (or replicate). Specifically, the peptide can cause a reduction or inactivation of the virus's ability to multiply (or replicate) by causing damage (or destruction) to the phospholipid bilayer.

[0088] The viral outer membrane damaging (or destroying) effect of peptides

[0089] The peptide of the present disclosure can cause damage to the outer membrane (or phospholipid bilayer) of a virus. Furthermore, the peptide can cause destruction of the outer membrane (or phospholipid bilayer) of the virus by causing damage to the outer membrane (or phospholipid bilayer).

[0090] Here, the damage (or destruction) of the outer membrane (or phospholipid bilayer) of the virus by the peptide may be caused by the insertion of the peptide into the phospholipid bilayer of the virus. It is understood by those skilled in the art studying the peptide disclosed herein that the mechanism by which the phospholipid bilayer of the virus is damaged (or destroyed) due to the insertion of the peptide into the phospholipid bilayer of the virus is related to the structural characteristics of the virus according to its size. This is explained as follows.

[0091]

[0092] Compared to cells, viruses are very small, surrounded by an outer membrane. Due to this size difference, the phospholipid bilayer of the viral outer membrane possesses structural features distinct from those of the cell outer membrane. To specifically explain the structural features of the phospholipid bilayer of the viral outer membrane, for convenience of calculation, we assume that viruses and cells are perfectly spherical in shape. We compare these differences using the formula for calculating the surface area of ​​a sphere.

[0093] First, the phospholipids that make up the outer membrane's phospholipid bilayer can be divided into an inner layer and an outer layer. The inner layer refers to the layer of phospholipids in the phospholipid bilayer whose phosphate groups face the cell interior. The outer layer refers to the layer of phospholipids in the phospholipid bilayer whose phosphate groups face the cell exterior.

[0094] Let the distance between one phosphate group part of the outer layer and the farthest phosphate group part be the diameter (= 2R), and let the length of the fatty acid part of the phospholipid be a (assuming that the phosphate group part of the phospholipid has no volume or length),

[0095] (Surface area composed of phosphate groups in the outer layer) = 4 × π × R 3

[0096] (Surface area composed of phosphate groups in the inner layer) = 4 × π × (R - 2a) 3

[0097] (Surface area ratio) = (Surface area composed of phosphate groups in the inner layer) / (Surface area composed of phosphate groups in the outer layer) = (R - 2a) 3 / R 3 = (1-(2a / R)) 3 This is it.

[0098] At this time, the length of the fatty acid (a) is generally a fixed value, and the value of R varies greatly depending on the size of the virus or cell. Considering this, as the value of R increases, the (surface area ratio) approaches 1, and as the value of R decreases, the (surface area ratio) decreases. That is, since a virus with a small R value has a larger difference in the surface area ratio between the inner layer and the outer layer than a cell with a large R value, the virus has a structure in which the spacing between the phosphate groups of the outer layer is further apart than that of the cell (see Figure 16).

[0099] That is, the phospholipid bilayer of a virus may have a structure that creates gaps that are absent in cells or are larger than those of cells. Considering these structural differences, it is expected that the peptide of the present disclosure will be more easily inserted into the phospholipid bilayer of a virus than into a cell. However, while there are experimental results demonstrating that peptides inserted into the phospholipid bilayer of a virus can cause damage or destruction of the phospholipid bilayer, the precise mechanism for this is unknown.

[0100] Therefore, the peptides of the present disclosure are expected to only cause damage or destruction of the phospholipid bilayer of the virus, without significantly affecting the cell. This is because, as previously explained, viruses have a smaller overall size (or diameter) and thus have a more curvature-enhanced outer membrane compared to cells.

[0101] According to this mechanism, the peptides of the present disclosure can cause damage or destruction of the phospholipid bilayer not only of specific viruses, but also of viruses having an outer membrane of a phospholipid bilayer, generally.

[0102] It can be confirmed through the experimental methods and results disclosed in Experimental Examples 5 and 6 that the peptide of the present disclosure can cause damage or destruction of the phospholipid bilayer of the virus.

[0103] Furthermore, the results of the experiment in Experimental Example 5.2, in which the liposomes were of different sizes, show that the peptides of the present disclosure have a high level of destruction (or damage) of the phospholipid bilayer of liposomes with small diameters. These results demonstrate that the peptides of the present disclosure can cause damage (or destruction) of the phospholipid bilayer of viruses, but not of cells, due to the difference in size between cells and viruses.

[0104] In one example, the peptide has a diameter of 5 to 1000 nm, 5 to 900 nm, 5 to 800 nm, 5 to 700 nm, 5 to 600 nm, 5 to 500 nm, 5 to 450 nm, 5 to 400 nm, 5 to 350 nm, 5 to 300 nm, 5 to 250 nm, 5 to 200 nm, 5 to 150 nm, 5 to 100 nm, 10 to 1000 nm, 10 to 900 nm, 10 to 800 nm, 10 to 700 nm, 10 to 600 nm, 10 to 500 nm, 10 to 450 nm, 10 to 400 nm, 10 to 350 nm, 10 to 300 nm, 10 250 nm, 10 to 200 nm, 10 to 150 nm, 10 to 100 nm, 15 to 1000 nm, 15 to 900 nm, 15 to 800 nm, 15 to 700 nm, 15 to 600 nm, 15 to 500 nm, 15 to 450 nm, 15 to 400 nm, 15 to 350 nm, 15 to 300 nm, 15 to 250 nm, 15 to 200 nm, 15 to 150 nm, 15 to 100 nm, 20 to 1000 nm, 20 to 900 nm, 20 to 800 nm, 20 to 700 nm, 20 to 600 nm, 20 to 500 nm, 20 to 450 nm, 20 to 400 nm, 20 to 350 nm, 20 to 300 nm, 20 to 250 nm, 20 to 200 nm, 20 to 150 nm, 20 to 100 nm, 30 to 1000 nm, 30 to 900 nm, 30 to 800 nm, 30 to 700 nm, 30 to 600 nm, 30 to 500 nm, 30 to 450 nm, 30 to 400 nm, 30 to 350 nm, 30 to 300 nm, 30 to 250 nm, 30 to 200 nm, 30 to 150 nm, 30 to 100 nm, 40 to 1000 nm, 40 to 900 nm, 40 to 800 nm, 40 to 700 nm, 40 to 600 nm, 40 to 500 nm,It can be effective in damaging or destroying a phospholipid bilayer having a thickness of 40 to 450 nm, 40 to 400 nm, 40 to 350 nm, 40 to 300 nm, 40 to 250 nm, 40 to 200 nm, 40 to 150 nm, 40 to 100 nm, 50 to 1000 nm, 50 to 900 nm, 50 to 800 nm, 50 to 700 nm, 50 to 600 nm, 50 to 500 nm, 50 to 450 nm, 50 to 400 nm, 50 to 350 nm, 50 to 300 nm, 50 to 250 nm, 50 to 200 nm, 50 to 150 nm, 50 to 100 nm.

[0105] Target virus

[0106] In the present disclosure, the virus that can cause damage to the phospholipid bilayer, destruction of the phospholipid bilayer, etc. by the peptide is a virus having an outer coat (or membrane) of the phospholipid bilayer.

[0107] The above virus may be a virus belonging to a family such as Paramyxoviridae, Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, or Herpesviridae.

[0108] In one example, the virus may be a pathogenic virus.

[0109] In one example, the pathogenic virus may be at least one selected from the group consisting of Paramyxoviridae, Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae.

[0110] For example, the above viruses include Sin Nombre Hantavirus belonging to the family Verniaviridae; Coronaviruses involved in various acute respiratory syndromes belonging to the family Coronaviridae; Ebola virus and Marburg virus belonging to the family Filoviridae; West Nile virus, Yellow Fever virus, Dengue Fever virus, and Hepatitis C virus belonging to the family Flaviviridae; Hepatitis B virus belonging to the family Hepadnaviridae; Herpes Simplex 1 virus and Herpes Simplex 2 virus belonging to the family Herpesviridae; Influenza virus belonging to the family Orthomyxoviridae; The virus may be at least one selected from the group consisting of Smallpox virus, Vaccinia virus, Molluscum contagiosum virus, and Monkeypox virus belonging to the Poxviridae family; Rabies virus belonging to the Rhabdoviridae family; Human Immunodeficiency virus (HIV) belonging to the Retroviridae family; Chikungunya virus belonging to the Togaviridae family; Pseudorabies virus and HHV virus belonging to the Herpesviridae family; and influenza virus belonging to the Orthomyxoviridae family, but is not limited thereto.

[0111] In one example, the virus may be a virus of the genus matapneumovirus, subfamily pneumovirinae, belonging to the family paramyxoviridae.

[0112] In one example, the virus may be Respiratory Syncytial Virus (RSV).

[0113] The above respiratory syncytial virus may be, but is not limited to, hMPV, RSV A, RSV B, etc.

[0114] In one example, the virus may be a dengue virus.

[0115] In one example, the virus may be a severe acute respiratory syndrome-related coronavirus (SARSr-CoV, SARS-CoV).

[0116] The above SARS-related coronavirus may be SARS-CoV and / or SARS-CoV-2.

[0117] Virus neutralizing ability of peptides

[0118] In one example, the antiviral activity of the peptide can be measured by a plaque reduction neutralization test (PRNT). Virus-infected cells are destroyed as the virus proliferates, forming plaques. However, the peptide exhibits antiviral activity and can inhibit such plaque formation. The degree of plaque formation can be expressed as neutralizing activity (the degree of plaque formation calculated relative to an untreated control).

[0119] In one example, the peptide may have a neutralizing capacity (or a neutralizing capacity as measured by a plaque reduction neutralization test) of at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0120] In one example, the peptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 21, 22, 27, 28, 33, 35, 36, 37, 39, 42, 46, 47, 51, 52, 53, 54, 60, 73, 75, 77, 79, 81, 82, 83, 86, 95, 96, and 98.

[0121] In one example, the peptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 16, 19, 22, 27, 35, 39, 46, 47, 51, 52, 53, 77, 79, 81, 83, 86, 95, 96, and 98.

[0122] In one example, the peptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19, 22, 27, 39, 46, 52, 79, 81, and 98.

[0123]

[0124] In one example, the peptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19, 39, 46, 52, 79, 81, and 98.

[0125] In one example, the peptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19, 46, 52, 79, and 81.

[0126]

[0127] Method for producing polynucleotides, recombinant vectors, microorganisms and peptides

[0128] Another aspect provides a polynucleotide encoding the peptide.

[0129] Another aspect provides a recombinant vector comprising the polynucleotide. The recombinant vector can be used as an expression vector.

[0130] Another aspect provides a microorganism (or cell, etc.) comprising at least one member selected from the group consisting of the peptide, a polynucleotide encoding the peptide, and a recombinant vector comprising the polynucleotide.

[0131] Another aspect provides a method for producing the peptide, comprising a step of expressing the peptide in a microorganism. The step of expressing the peptide in the microorganism may be performed by culturing a recombinant cell containing the polynucleotide or a recombinant vector containing the polynucleotide. The method may further comprise a step of isolating and / or purifying the expressed peptide after the expression step.

[0132] Another aspect provides a composition for producing the peptide, comprising at least one selected from the group consisting of the above microorganism and a medium in which the above microorganism is cultured.

[0133] The introduction of the polynucleotide or vector into a microorganism can be performed by a person skilled in the art by appropriately selecting a known transformation method. As used herein, the term "transformation" refers to introducing a vector containing a polynucleotide encoding the peptide into a microorganism so that the peptide encoded by the polynucleotide can be expressed in the microorganism. The transformed polynucleotide can be located either within the chromosome of the microorganism or outside the chromosome, as long as it can be expressed in the microorganism. There is no limitation on the form in which the polynucleotide is introduced, as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette may typically include expression control elements such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, which are operably linked to the polynucleotide. The above expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence necessary for expression in the microorganism. The term "operably linked" as used herein refers to a polynucleotide that is functionally linked to an expression control element (e.g., a promoter) so that transcriptional regulation (e.g., transcription initiation) of the polynucleotide can be achieved. Operable linking can be achieved using genetic recombination techniques known in the art.

[0134] The method for transforming the polynucleotide into a microorganism can be performed by any method for introducing a nucleic acid into a microorganism, and can be performed by appropriately selecting a transformation technique known in the art depending on the microorganism. Examples of the known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation, polyethylene glycol-mediated uptake, DEAE-dextran, cationic liposome, lipofection, and lithium acetate-DMSO.

[0135] As used herein, the term "vector" refers to a DNA construct containing a polynucleotide sequence operably linked to suitable regulatory sequences so as to enable expression of a target protein in a suitable host. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into a suitable microorganism, the vector may be expressed independently of the genome (genetics) of the microorganism, or may be integrated into the genome of the microorganism.

[0136] The vector usable in this specification is not particularly limited as long as it can replicate in a microorganism, and can be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc. in a natural or recombinant state. For example, as the vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as a phage vector or a cosmid vector, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as a plasmid vector. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be exemplified, but are not limited thereto.

[0137] The vector may further comprise a selection marker to confirm whether the vector has been inserted into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the polynucleotide has been inserted. The selection marker may be selected from genes that confer a selectable phenotype, such as drug resistance, nutritional requirement, cytotoxic agent resistance, or surface protein expression. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypic characteristics, thereby enabling the selection of transformed cells.

[0138]

[0139] Uses of Peptides

[0140] According to one aspect of the present disclosure, the peptide of the present disclosure can be used to cause the destruction or damage of the lipid bilayer of a virus. The destruction or damage of the lipid bilayer reduces the infectivity of the virus, and thus, the effect of preventing or treating viral infection (or related diseases) can be expected. In other words, the peptide can be used as an effective therapeutic agent for viral infection (or related diseases) by inducing the destruction or damage of the lipid bilayer. Here, the virus is a virus having an outer coat (or membrane) of a phospholipid bilayer. Therefore, the peptide can be usefully used for antiviral and / or the prevention, improvement, and / or treatment of viral infection or diseases caused by the virus.

[0141] In the following applications, any one peptide containing any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 100 may be used alone or in combination of two or more peptides.

[0142]

[0143] Antiviral composition

[0144] Another aspect provides an antiviral composition comprising at least one member selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0145] Another aspect provides a method for inhibiting or killing a virus, comprising administering or treating to a subject in need of virus inhibition or killing at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0146] Another aspect provides a use for inhibiting or killing a virus, comprising at least one member selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0147] In this specification, the term "antiviral composition" may refer to a composition having antiviral activity. The antiviral activity may be as described above, and may refer to inhibiting the growth of a virus, and / or killing the virus. The term "antiviral activity" may be used interchangeably with antibiotic activity, and the term "antiviral composition" encompasses all forms of preparations having growth inhibition and / or killing abilities against viruses, and may be used interchangeably with antibiotic compositions, antiviral agents, antibiotics, etc., unless otherwise specified.

[0148]

[0149] Pharmaceutical composition

[0150] Another aspect provides a pharmaceutical composition for preventing and / or treating a viral infection or a disease caused by a virus, comprising at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0151] Another aspect provides a method for preventing and / or treating a viral infection or a disease caused by a virus, comprising administering to a subject in need of prevention and / or treatment of a viral infection or a disease caused by a virus at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector. The method for preventing and / or treating may further comprise, prior to the administering step, a step of identifying a subject in need of prevention or treatment of a viral infection or a disease caused by a virus.

[0152] Another aspect provides a use for preventing and / or treating a viral infection or a disease caused by a virus, comprising at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0153] Another aspect provides a use for producing a composition for preventing and / or treating a viral infection or a disease caused by a virus, comprising at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0154] In the present application, "prevention" means any act of inhibiting or delaying the onset of a disease (illness) by administering a composition according to an example, "treatment" means any act of improving or beneficially changing the symptoms of a suspected or affected individual by administering a composition according to an example, and "improvement" may mean any act of at least reducing a parameter related to a state in which a disease is treated, for example, the degree of a symptom, by administering a composition according to an example. The disease may mean a viral infection or a disease caused by a virus.

[0155] The virus is as described above. The virus infection and / or disease caused by the virus infection may be at least one selected from dengue fever, coronavirus disease-19 (COVID-19), symptoms such as sputum, cough, sore throat, headache, hemoptysis, dyspnea, upper respiratory tract infection, lower respiratory tract infection, respiratory tract infection, pneumonia, and cytokine storm syndrome.

[0156] The above respiratory infections may include, but are not limited to, respiratory infections caused by influenza virus, respiratory syncytial virus (RSV), and respiratory infections caused by human metapneumovirus (hMPV).

[0157] In one example, the viral infection and / or disease caused by the viral infection may be at least one selected from the group consisting of acute respiratory disease, cold, flu, cough, sneezing, runny nose, muscle pain, pharyngitis, nasal congestion, laryngitis, sore throat, hoarseness, headache, sinus pain, rhinitis, pharyngitis, bronchitis, asthma, fever, dyspnea, general weakness, chills, nausea, vomiting, diarrhea, gastrointestinal symptoms such as abdominal pain, muscle pain, joint pain, and myocarditis.

[0158] In one example, the viral infection and / or disease caused by the viral infection may be dengue fever.

[0159] In one example, the viral infection and / or disease caused by the viral infection may be coronavirus disease-19 (COVID-19).

[0160] As used herein, the pharmaceutical effective amount refers to the content or dosage of an effective ingredient that can achieve a desired effect. The effective ingredient may be at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector. The content or dosage of the effective ingredient in the pharmaceutical composition may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate, and response sensitivity. For example, when the active ingredient is a peptide, a single dose may be in the range of, but is not limited to, 0.001 to 1000 mg / kg, 0.01 to 100 mg / kg, 0.01 to 50 mg / kg, 0.01 to 20 mg / kg, 0.01 to 10 mg / kg, 0.01 to 5 mg / kg, 0.1 to 100 mg / kg, 0.1 to 50 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 1 to 100 mg / kg, 1 to 50 mg / kg, 1 to 20 mg / kg, 1 to 10 mg / kg, or 1 to 5 mg / kg.

[0161] In one example, the pharmaceutical composition may be administered to the subject once a day or in divided doses of two or more times a day, and in one example, may be administered at intervals of 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 1 month, and / or 3 months.

[0162] The above dosage and / or administration cycle do not limit the scope of the present application in any way.

[0163] In another example, the content of the active ingredient in the pharmaceutical composition is, based on the total weight of the pharmaceutical composition, 0.01 wt% to 99.9 wt%, 0.01 wt% to 90 wt%, 0.01 wt% to 80 wt%, 0.01 wt% to 70 wt%, 0.01 wt% to 60 wt%, 0.01 wt% to 50 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 1 wt% to 99.9 wt%, 1 wt% to 90 wt%, 1 wt% to 80 wt%, 1 wt% to 70 wt%, 1 wt% to 60 wt%, 1 wt% to 50 wt%, 1 wt% to 40 wt%, 1 wt% to 30 wt%, 5 wt% to 99.9 wt%, 5 wt% to It may be, but is not limited to, 90 wt%, 5 wt% to 80 wt%, 5 wt% to 70 wt%, 5 wt% to 60 wt%, 5 wt% to 50 wt%, 5 wt% to 40 wt%, 5 wt% to 30 wt%, 10 wt% to 99.9 wt%, 10 wt% to 90 wt%, 10 wt% to 80 wt%, 10 wt% to 70 wt%, 10 wt% to 60 wt%, 10 wt% to 50 wt%, 10 wt% to 40 wt%, or 10 wt% to 30 wt%.

[0164] In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or adjuvant in addition to the active ingredient. The pharmaceutically acceptable carrier is one commonly used in the formulation of drugs containing proteins, nucleic acids, or cells, and may refer to a carrier that does not stimulate a living organism and does not inhibit the biological activity and / or properties of the active ingredient. In one example, the carrier may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. The above pharmaceutical composition may further include at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions.

[0165] In one example, the pharmaceutically acceptable carrier is one commonly used in formulations, and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other conventional additives such as antioxidants and buffers as needed. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, each ingredient can be preferably formulated using the method disclosed in Remington's Pharmaceutical Sciences (19th edition, 1995). In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable carrier, such as a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; a sweetener; a flavoring agent; a syrup; a liquid carrier such as a fatty oil; a sterile aqueous solution; an injectable ester such as propylene glycol; polyethylene glycol; ethyl oleate; a suspending agent; an emulsion; a freeze-dried preparation; an external preparation; a stabilizer; a buffering agent; an animal oil; a vegetable oil; a wax; paraffin; a starch; tragacanth; a cellulose derivative; a polyethylene glycol; a silicone; a bentonite; a silica; talc; a zinc oxide, or a suitable combination thereof.

[0166] The subject of administration of the pharmaceutical composition provided herein may be a mammal including a human, a dog, a cat, a horse, a cow, a pig, a goat, a rabbit, a mouse, a rat, etc., or a cell, tissue, or culture thereof isolated therefrom. In one example, the subject may be a subject (a mammal such as a human) that requires prevention, improvement, and / or treatment of a viral infection and / or a disease caused by a viral infection as described above, or a cell, tissue, or culture thereof isolated therefrom having a viral infection and / or a disease caused by a viral infection.

[0167] The pharmaceutical composition may be administered orally or parenterally, or by contact with cells, tissues, or body fluids. Specifically, in the case of parenteral administration, it may be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, since proteins or peptides are digested, the oral composition must be formulated to coat the active agent or protect it from degradation in the stomach. When administered intranasally, the pharmaceutical composition may be diluted and administered via nasal spray through a nebulizer or nebulizing system so that it is absorbed into the nasal cavity. Examples of nasal sprays or respiratory formulations for the nasal spray include aerosols.

[0168] In addition, the pharmaceutical composition may be formulated in the form of a solution, injection, suspension, syrup, emulsion, application agent, patch, extract, powder, granule, tablet, capsule, aerosol, etc. in an oil or aqueous medium, and may additionally include a dispersing agent or stabilizer for formulation.

[0169] In one embodiment, the pharmaceutical composition may be formulated as a troche, lozenge, tablet, aqueous suspension, oily suspension, prepared powder, granules, emulsion, hard capsule, soft capsule, syrup, or elixir. In another embodiment, the pharmaceutical composition may be formulated as an injection, a suppository, a powder for respiratory inhalation, an aerosol for spraying, an ointment, a powder for application, an oil, or a cream. In yet another embodiment, the pharmaceutical composition may be formulated as an injection. Specifically, a therapeutically effective amount of an antiviral peptide may be mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which may be formulated for unit dose in ampoules or vials. In another embodiment, the pharmaceutical composition may be formulated as an aerosol by mixing a propellant or the like with additives to prepare a water-dispersed concentrate or a wet powder, and then formulated as an aerosol. In another embodiment, when the pharmaceutical composition is formulated for transdermal use, an ointment, cream, powder for application, oil, external skin preparation, etc. can be prepared by adding animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. as a carrier to a therapeutically effective amount of an antiviral peptide.

[0170]

[0171] Feed compositions, food compositions and cosmetic compositions

[0172] Another aspect provides a feed composition for preventing and / or improving a viral infection or a disease caused by a virus, comprising at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0173] In this specification, “feed” may mean any natural or artificial diet, meal, etc., or a component of said meal, intended for or suitable for eating, ingesting, or digesting by an animal.

[0174] In this specification, the feed composition may be used as feed itself or in the form of a feed additive added to feed.

[0175] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant-based feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal-based feed such as proteins, inorganic substances, oils, minerals, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0176] The feed composition may further include excipients, diluents, and / or additives. In addition to the above components, the feed composition may include ingredients effective for promoting animal growth, nutrients, nutritional supplements, ingredients for increasing preservation stability, coating ingredients, amino acids, vitamins, enzymes, non-protein nitrogen compounds, silicates, buffers, extractants, probiotics for disease prevention, etc.; enzymes such as amylase and lipase; vitamins such as L-ascorbic acid, choline chloride, and inositol; minerals such as potassium chloride, iron citrate, magnesium oxide, and phosphates; amino acids such as lysine, alanine, and methionine; organic acids such as fumaric acid, butyric acid, and lactic acid or salts thereof; antioxidants such as vitamin C and vitamin E, antifungals such as calcium propionate, and emulsifiers such as lecithin and glycerin fatty acid esters; and / or pigments. Even if not described above, the feed composition may additionally contain other nutritional ingredients within the range that would be expected by a person skilled in the art.

[0177] The subject (animal) that can be fed the feed containing the composition according to an example is not particularly limited, but may be mammals, fish, crustaceans and / or shellfish, and for example, may be pigs, cows, horses, goats, deer, sheep, chickens, ducks, geese, turkeys, dogs, cats, rabbits, and fish.

[0178] When the above feed composition is used in the form of a feed additive, it can be manufactured in the form of a commercially available feed with the feed additive added thereto. The feed in which the feed additive can be used, according to an example, is preferably in powder or pellet form, or in liquid form, but is not limited thereto. Furthermore, the amount of the feed additive of the present application added to the feed does not require any particular limitations.

[0179] The feed can be manufactured by separately manufacturing the composition of the present application in the form of a feed additive and mixing it into the feed, or by directly adding it during the manufacture of the feed. The feed additive of the present application included in the feed can be in a liquid or dry state, for example, can be in the form of a dried powder. The feed additive can be included in an amount of 0.005 to 10 wt%, 0.05 to 10 wt%, 0.1 to 10 wt%, 0.005 to 5 wt%, 0.05 to 5 wt%, 0.1 to 5 wt%, 0.005 to 2 wt%, 0.05 to 2 wt%, or 0.1 to 2 wt% of the total feed weight, but is not limited thereto. In addition, the feed can additionally include conventional additives that can increase the preservative properties of the feed in addition to the feed additive.

[0180] The feed to which the above feed additive can be added may be selected from the group consisting of commercially available feed, grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, grain by-products, proteins, inorganic substances, mineral substances, single-cell proteins, animal plankton, leftover food, etc., but is not limited thereto.

[0181]

[0182] Another aspect provides a food composition for preventing and / or improving a viral infection or a disease caused by a virus, comprising at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0183] The above food composition may be a food itself, a food additive added to a food, or a health functional food.

[0184] A food composition according to an example may mean dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the conventional sense.

[0185] The above functional food is the same term as food for special health use (FoSHU), and refers to a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. Here, "function" means regulating nutrients for the structure and function of the human body or obtaining a useful effect for health purposes such as physiological function. The food of the present application can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the food can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present application can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term administration of drugs since it uses food as a raw material, and is highly portable, so the food of the present application can be consumed as a supplement to enhance the effect of preventing or improving viral infection or diseases caused by viruses.

[0186] The term "health food" refers to foods that have a more active health maintenance or promotion effect than regular foods, while "health supplement food" refers to foods intended for health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement food" may be used interchangeably.

[0187] Specifically, the health functional food is a food product manufactured by adding a composition according to the example to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing it in the form of encapsulation, powder, suspension, etc., and means that when consumed, it brings about a specific health effect, but unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material.

[0188] A food composition according to an example can be used very usefully because it can be consumed on a daily basis and is expected to be highly effective in preventing or improving viral infections or diseases caused by viruses.

[0189] The above food composition may additionally include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0190] In addition, the food composition may include additional ingredients commonly used in food compositions to improve odor, taste, sight, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.

[0191] In addition, the food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), sterilizers (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar colorants, etc.), color developers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glucose fillers), film-forming agents, gum-forming agents, foam suppressants, solvents, and improvers. The above additives can be selected according to the type of food and used in an appropriate amount.

[0192] The composition according to an example can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on its purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the food composition of the present application can be added to the food or beverage in an amount of 50 parts by weight or less, specifically 20 parts by weight or less. However, when consumed for a long period of time for health and hygiene purposes, the content below the above range can be included, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the above range.

[0193] As an example of the food composition, the above-mentioned food composition may be used as a health beverage composition, and in this case, various flavoring agents or natural carbohydrates may be contained as additional ingredients, as in a conventional beverage. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener may be a natural sweetener such as thaumatin and stevia extract; or a synthetic sweetener such as saccharin and aspartame. The proportion of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g, per 100 mL of the health beverage composition of the present application.

[0194] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, or carbonating agents. In addition, it may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present application.

[0195]

[0196] Another aspect provides a cosmetic composition for preventing and / or improving a viral infection or a disease caused by a virus, comprising at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0197] A cosmetic composition according to an example may be a cosmetic composition or a skin external application composition, and such cosmetic composition or skin external application composition may exhibit antiviral activity and thus exhibit an effect of preventing and / or improving a viral infection or a disease caused by a virus.

[0198] There is no particular limitation on the formulation of the external skin composition or cosmetic composition provided herein, and may be formulated as, for example, a solution, a suspension (anhydrous and aqueous), an emulsion, a paste, a gel, a cream (oil-in-water, water-in-oil, multiphase, etc.), a powder, an ointment, a patch, a toner, an essence, a gel, a lotion, a solution, an anhydrous product (oil and glycol), a mask, a pack, a powder, or a capsule with a film such as gelatin (soft capsule, hard capsule), or a spray.

[0199] The above cosmetic composition may be in any one formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a powder, an ointment, a patch, a toner, an essence, a gel, a lotion, a mask, a pack, a powder, a capsule, and a spray.

[0200] In the present application, the skin is a concept that includes not only the face but also the scalp and the entire body, and the external skin composition that can be applied to the scalp includes shampoo, rinse, treatment, hair growth agent, etc., and can be manufactured in various forms for use as a body cleanser that can be applied to the entire body.

[0201] The above composition may include a solvent commonly used in the manufacture of a skin external preparation and / or cosmetic composition, and the usable solvent may be at least one selected from the group consisting of purified water, rock water, hot spring water, glacial water, seawater, deep ocean water, plant extract water, etc., but is not limited thereto.

[0202]

[0203] Feed additives, food additives and drinking water additives

[0204] Another aspect provides a feed additive comprising at least one member (such as a peptide) selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0205] Another aspect provides a feed comprising the above feed additive.

[0206] The above feed can be manufactured by separately manufacturing the above peptides, etc. in the form of feed additives and mixing them into the feed, or by directly adding them during feed manufacturing.

[0207] The feed additive may be in a liquid or dry state, for example, in the form of a dry powder. The feed additive may be included in an amount of 0.005 to 10 wt%, 0.05 to 10 wt%, 0.1 to 10 wt%, 0.005 to 5 wt%, 0.05 to 5 wt%, 0.1 to 5 wt%, 0.005 to 2 wt%, 0.05 to 2 wt%, or 0.1 to 2 wt% of the total feed weight, but is not limited thereto. In addition, the feed may additionally include conventional additives that can increase the preservative properties of the feed in addition to the feed additive.

[0208] In the present specification, the feed to which the feed additive can be added may be selected from the group consisting of commercially available feed, grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, grain by-products, proteins, inorganic substances, oils, minerals, single-cell proteins, animal plankton, leftover food, etc., but is not limited thereto.

[0209] Another aspect provides a food additive and / or drinking water additive comprising at least one member selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0210] By mixing the above peptides and the like into feed, food, and / or drinking water and supplying them, the number of viruses in the feed, food, and / or drinking water can be reduced. The viruses are as described above.

[0211]

[0212] Disinfectants and cleaners

[0213] Another aspect provides a disinfectant comprising at least one member selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0214] Another aspect provides a disinfection method comprising the step of applying to a subject requiring disinfection at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0215] Another aspect provides a detergent comprising at least one member selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0216] Another aspect provides a washing method comprising the step of applying to a subject requiring washing at least one selected from the group consisting of the peptide, a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a microorganism comprising the polynucleotide or the recombinant vector.

[0217] The above peptides, etc. can be used for the purpose of disinfecting and / or cleaning objects that have been or may be exposed to viruses (body parts of humans or non-human animals (e.g., skin, etc.), buildings such as poultry farms and livestock sheds, rooms, tables, chairs, utensils, dishes, automobiles, etc.). The viruses are as described above.

[0218]

[0219] The peptide provided in this application has antiviral activity and can therefore be useful for preventing and / or treating viral infections or diseases caused by viruses.

[0220]

[0221] Figure 1 shows the positions of the expected amino acid residues when a peptide with the amino acid sequence DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101) has an alpha helix structure. In SEQ ID NO: 16, the N-terminal is indicated at the first position, aspartic acid (Asp, D), and the C-terminal is indicated at the last position, lysine (Lys K). At this time, starting from the N-terminus, D, a line is connected in order from the next amino acid residue after D, W, to the next amino acid residue, L, and the last position where the line is connected is the C-terminus, K. Figure 1 shows that a hydrophilic region and a hydrophobic region are distinguished within a single structure of the peptide, and that each region contains nine amino acid residues. At this time, the types of amino acid residues included in the hydrophilic region are tryptophan (Trp, W), valine (Val, V), leucine (Leu, L), phenylalanine (Phe, F), and isoleucine (Ile, I), which are all hydrophilic amino acid residues. The types of amino acid residues included in the hydrophobic region are composed of hydrophobic amino acid residues lysine (Lys, K), arginine (Arg, R), serine (Serine, S), and aspartic acid (Asp, D), and hydrophilic amino acid residues cysteine ​​(Cysteine, C) and isoleucine (Ile, I), and 7 of the 9 amino acid residues included in the hydrophobic region are hydrophobic amino acid residues. Among the amino acid residues shown in Figure 1, those having a checkered pattern are hydrophilic amino acid residues, and those having other patterns are hydrophobic amino acid residues, so the characteristics of the amino acid residues distributed in each region can be more easily confirmed.

[0222] Figure 2 shows the results of confirming the Ct value for dengue virus RNA according to treatment with the control peptide, 3a-3 / 20 peptide (SEQ ID NO: 101).

[0223] Figure 3 shows the results of confirming the Ct value for dengue virus RNA according to treatment with DS-1 peptide (SEQ ID NO: 01).

[0224] Figure 4 shows the results of confirming the Ct value for dengue virus RNA according to treatment with DS-4 peptide (SEQ ID NO: 04).

[0225] Figure 5 shows the results of confirming the Ct value for dengue virus RNA according to treatment with DS-5 peptide (SEQ ID NO: 05).

[0226] The inhibition rate on the vertical axis of FIGS. 2 to 5 is the average Ct value of wells infected with dengue virus without peptide treatment, converted to an infection rate of 100%, and the average Ct value of wells infected with dengue virus treated with each peptide at each concentration is converted to an inhibition rate.

[0227] The Log(concentration) on the horizontal axis of FIGS. 2 to 5 means Log(concentration of peptide in the peptide solution treated in each well).

[0228] Figure 6 shows the results of confirming liposome leakage efficacy when the concentration of DS-1 to DS-8 peptides is 250 nM.

[0229] Figure 7 shows the results of confirming liposome leakage efficacy when the concentration of DS-1 to DS-8 peptides is 125 nM.

[0230] Figure 8 shows the results of confirming liposome leakage efficacy when the concentration of DS-9 to DS-15 peptides is 250 nM.

[0231] Figure 9 shows the results of confirming liposome leakage efficacy when the concentration of DS-9 to DS-15 peptides is 125 nM.

[0232] The graphs in Figures 6 to 9 represent the results of measuring fluorescence levels over time in each well, with the horizontal axis representing time and the vertical axis representing fluorescence levels. The data in each graph can be analyzed to indicate that the peptides treated in wells with high fluorescence levels exhibited superior viral lipid membrane damage (or destruction) efficacy.

[0233] Fig. 10 shows the results of confirming the liposome rupture efficacy of DS-1 to DS-15 peptides. The graph of Fig. 10 shows the results of measuring the change in fluorescence value according to the type of peptide treated in each well, and the horizontal axis indicates the type of peptide treated in each well. The change in fluorescence value on the vertical axis is calculated as ((fluorescence value before peptide treatment - fluorescence value after peptide treatment) / fluorescence value before peptide treatment), and the calculated value when treated with Triton X-100 is converted to 100 (% Rupture), and means the value compared and analyzed by calculating the calculated value of each peptide.

[0234] Figure 11 shows the changes in fluorescence signals observed immediately, 2 minutes, 4 minutes, 6 minutes, 8 minutes, and 10 minutes after treatment with 3a-3 / 20 peptide in each well with attached liposomes of different sizes of 50 nm, 150 nm, and 350 nm.

[0235] Figure 12 shows the changes in fluorescence signals observed immediately, 2 minutes, 4 minutes, 6 minutes, 8 minutes, and 10 minutes after treatment with DS-5 peptide in each well with attached liposomes of different sizes of 50 nm, 150 nm, and 350 nm.

[0236] Figure 13 is a graph showing the fluorescence signals measured at 20-second intervals after treating each well with the 3a-3 / 20 peptide, where the attached liposomes had different sizes of 50 nm, 150 nm, and 350 nm. The vertical axis of the graph represents the normalized fluorescence signal, which is the fluorescence value immediately after treating the peptide (0 seconds) converted to 1.

[0237] Figure 14 is a graph showing the fluorescence signals measured at 20-second intervals after treating each well with DS-5 peptide, with attached liposomes of different sizes (50 nm, 150 nm, and 350 nm). Here, the vertical axis of the graph represents the normalized fluorescence signal by converting the fluorescence value immediately after peptide treatment (0 seconds) to 1.

[0238] Figure 15 shows the results of measuring the amount of dengue virus RNA remaining undigested by RNase after treating dengue virus with peptides. The treated peptides were 3a-3 / 20 peptide and DS-5 peptide, and the concentrations of each peptide were varied to 250, 500, and 1000. The vertical axis of the graph represents the average Ct value of the negative control group that was not treated with the peptides, converted to 100% and normalized to a normalized value.

[0239] Figure 16 illustrates an example of a structure in which a gap may be created in the phospholipid bilayer of a virus due to the small size of the virus.

[0240] Figures 17a and 17b show the results of an antiviral activity experiment against dengue virus obtained by performing a plaque reduction neutralization test on 85 peptides (DS-16 to DS-100).

[0241] Figures 18a to 18e show the plaque reduction neutralization test results and IC50 values ​​of five peptides (DS-19, 46, 52, 79, 81) that exhibited excellent antiviral activity.

[0242] Figure 19a is a graph summarizing the results of liposome rupture efficacy evaluation experiments of the above five peptides, and Figures 19b to 19h show the results of experiments of each of the five peptides and the control group.

[0243] Figures 20a to 20c show the results of antiviral activity experiments against the SARS-CoV-2 virus of three peptides (DS-19, 52, 79) that exhibited excellent antiviral activity.

[0244]

[0245] The present invention will be described in more detail below with reference to the following examples. However, these examples are provided solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0246]

[0247] Experimental Example 1. Peptide design according to one embodiment of the present application.

[0248] A peptide was designed by randomly scrambling nine amino acids located in the hydrophilic region and nine amino acids located in the hydrophobic region of a peptide having the amino acid sequence DWLRIIWDWVCSVVSDFK (SEQ ID NO: 101, referred to herein as “3a-3 / 20”) derived from HCV genotype 3a, respectively, for each hydrophilic region and each hydrophobic region. The structure of the peptide having the amino acid sequence of SEQ ID NO: 101 is disclosed in Fig. 1. Fig. 1 discloses the wheel projection when the peptide forms an alpha helical structure.

[0249] Specifically, peptides were designed using the EMBOSS program (European Molecular Biology Open Software Suite). The hydrophobic moment value of the EMBOSS program was set to 0.568 to 0.633, and the amino acids within the peptides were scrambled. At this time, the amino acids in the hydrophilic region were positioned relative to other amino acids in the hydrophilic region, and the amino acids in the hydrophobic region were positioned relative to other amino acids in the hydrophobic region. Through this, scrambled peptides (DS-1 to DS-15) with hydrophobic moment values ​​of 0.568 to 0.633 (hydrophobic moment values ​​calculated from the EMBOSS program) were designed.

[0250] In addition, among the sequences generated by random scramble, 85 peptides were randomly selected from among those with a hydrophobicity value of 0.841 and a hydrophobic moment (μH) value of 0.54 to 0.80 in the HeliQuest program (IPMC & CNRS, Institute Pharmacology Molιculaire Et Cellulaire, Centre national de la recherche scientifique) and were named DS-16 to DS-100, respectively. The amino acid sequences and hydrophobic moment values ​​of the 100 selected peptides (DS-1 to DS-100) are summarized in Table 1 below.

[0251] PeptideSequenceHydrophobic momentSEQ ID NODS-1DIFCDWWRWVISVLDSVK0.5731DS-2DVVSIWFKWIDCLWDSVR0.5732DS-3RLVKSIWDWFCIVVDSWD0.5953DS-4DVWCSWVSWVIKLFRDID0.5744DS-5DWVDCFWSWLIKVIDRVS0.5785DS-6SVVCDWWDVIIKWFDRLS0.5886DS-7DWVSKFWRILICVWDSVD0.5817DS-8DVVDDWWSVLCKWFRIIS0.6338DS-9RVFCIVWDWVKSWIDDLS0.5799DS-10SWVDSFWDWLIRVIKCVD0.60610DS-11DVVDIVLRWICSWWSDFK0.57111DS-12RVIDCWVDWWSIVFKSLD0.58412DS-13SVFCSLWRWVIDWVDDIK0.56813DS-14KLVDSIWSWFICVVRDWD0.57714DS-15DVFCIWIDWVKSLWRDVS0.57315DS-16KVWCDWVDWISRLFDSVI0.71516DS-17KWVISVWDFLDDIVRCWS0.65617DS-18RVWDIWFDVVCKWISDLS0.67418DS-19CWWDDVVKVIDRLWSIFS0.65819DS-20RFVCKWIIWVDDWVDSLS0.55620DS-21RIVDIWWDFWSSLVDKVC0.66621DS-22DFWRSLVKIVSDVWIDWC0.66422DS-23RVWKSILDWVDDVFSCWI0.71523DS-24DWVIRIWSLVDDVWKSFC0.64324DS-25KVWCDWISWVDRVLIDFS0.57225DS-26SWVRSFVDVLIDWICDWK0.60026DS-27DIFKSWVDVVIDWWSRLC0.72427DS-28DLWRKFVDWWSDVVISIC0.66728DS-29SVLKIIVSWFDDVWDCWR0.70829DS-30SIWDCWVKLFIRVWSDVD0.62630DS-31DVWCKWVRVLSDFIIDWS0.66631DS-32CVWRDFWKIVDDVLISWS0.55432DS-33KVVSDLWDWVIDIWSRFC0.69933DS-34RVWSCVWIIWSKFVDDLD0.56534DS-35DWVDKWLDFVIRIVSCWS0.68935DS-36RFWKDVVCWVIDLWSDIS0.58736DS-37CWWSKVVDVWDIFLRSID0.59837DS-38DVVKDFWSVLICWIDSWR0.61438DS-39DLVDIWWRFVKSWVCDIS0.59939DS-40RWWKDWFDVICIVLSDVS0.62440DS-41SVWKDWLCIFISVVDDWR0.58141DS-42SWVIDLIRFWSKVVDCWD0.64742DS-43SWWRIVWDLIDDVFCSVK0.55443DS-44DVWDRVICFWSKVWSDLI0.68244DS-45IVVDCWISVLDRWFSDWK0.59045DS-46KWVDRVWSWLIDIFSDVC0.64546DS-47SWWIDVIDWVRSFLKDVC0.59347DS-48RVWDCWFDLVSDIVISWK0.62048DS-49KLFDSIVRWWSDVVICWD0.66549DS-50DVLRSVFDWIKDVWCIWS0.63550DS-51CWVKDWLSVVSDFWDIIR0.64151DS-52CWVSRVLDWIDIFVKSWD0.58952DS-53DWWDSVVRWFICVISKLD0.57953DS-54SILDSWWDVVIDFVRCWK0.68454DS-55KWFDCWLSVVIDWVDRIS0.65555DS-56KLVSRWVDFVICWWSDID0.59456DS-57RFVSDIWDWVCKVWIDLS0.59857DS-58DWWKIWVSFVSCLVRDID0.66758DS-59DVWDCWVIFISKLVDRWS0.62659DS-60RWIDIWVDLVKSWVDCFS0.79460DS-61SLFSCVWRWIDKVVDDWI0.64961DS-62KVWIDIWSLVRDFVCSWD0.56162DS-63DVVSDILRWWISWFCKVD0.57663DS-64DVWCRFWSVVSIWLKDID0.56864DS-65SVWIKVLDWFRDVICSWD0.55165DS-66DWVRSVWSIWIDFLKDVC0.67066DS-67SWFIKIVSLWDDWVDRVC0.63767DS-68SLWDDWVCVFISWVDKIR0.59168DS-69SFLKCWVIIWRDVVSDWD0.60169DS-70SIVDIVWKWWDCLVRSFD0.65670DS-71IVFKCIVDWWDRWLSDVS0.60671DS-72CWIKIVFSLVSDWVRDWD0.60672DS-73DFWDDWVCILKIVWRSVS0.58373DS-74IWWDCIFKVWSSLVDDVR0.60474DS-75ILVSDFVRIWDDVWCSWK0.54875DS-76KWIDDWVIVWCRFVSSLD0.62076DS-77KWWSDILDVFSIWVDCVR0.67677DS-78CLWIRVWKIWSSVVDDFD0.54778DS-79CWLDIIVDFVDSVWSKWR0.58879DS-80KVWRCVIDVWIDLWSSFD0.66980DS-81SLVDDWFRVVIKWWSCID0.66781DS-82KVISSWWIFVCDWVDRLD0.56782DS-83DWVSDIWCVLIDWVRKFS0.60683DS-84SFWKCWLDIVDIVWDRVS0.59884DS-85KWIRDWVDVWISVLDSFC0.75385DS-86IWWDSVFRWVCKVIDDLS0.56886DS-87SVVDDFIIVWCSWWRKLD0.57987DS-88RLWDDFIDVWKIVVSCWS0.69288DS-89DWIRKWVCWLSIVVDDFS0.57289DS-90SWVDCVWDIWRIFVDKLS0.61090DS-91DLVCSWFKVWSRVWDIID0.68391DS-92SLVIDIWKFVDSWWRDVC0.61892DS-93RWFKDIWDVLSDVWSCVI0.71393DS-94DFIDKWVCVLRDWWSSVI0.65294DS-95DIWIDVWKFLDSVVSRWC0.66295DS-96DVWDDIWSVFRKLWCIVS0.62696DS-97DLWKIVIRWFSDVVDSWC0.69897DS-98DWFIDIVKVLRDWWSSVC0.67998DS-99CVVSRLWDIWIKFVSDWD0.58899DS-100DWVRIVIDWLSDFWSCVK0.6701003a-3 / 20DWLRIIWDWVCSVVSDFK0.582101.

[0252] Experimental Example 2. Peptide Production

[0253] The above 100 designed peptides were synthesized by Anygen Co., Ltd. Each peptide was synthesized using a standard SPPS (Solid-Phase Peptide Synthesis) peptide synthesis method. The Fmoc protecting group was removed by reaction with 20% piperidine in DMF for 10 minutes (twice), and coupling was performed using Fmoc amino acid (6 equivalents), HOBT (6 equivalents), HBTU (6 equivalents), and DIPEA (12 equivalents). At each step, the resin was washed with DMF and methanol (repeated 2 to 3 times). The synthesized crude peptides were reacted for 2 hours using a mixture of TFA / EDT / Thioanisole / TIS / DW (90 / 2.5 / 2.5 / 2.5 / 2.5 Volume) to remove the protecting groups of the resin and amino acid. Ether was added to the resulting mixed solution, centrifuged, and the peptides were recovered, followed by lyophilization. Crude peptides were dissolved in deionized water and purified by reverse-phase HPLC using a C18 reverse-phase column (Water-Acetonitrile linear gradient containing 0.1% (v / v) Trifluoroacetic acid; Acetonitrile concentration 10–75% (v / v)). The purified fractions were then lyophilized.

[0254]

[0255] Experimental Example 3. Confirmation of the antiviral efficacy of peptides (1)

[0256] The antiviral efficacy (or lipid membrane disruption) of the peptide manufactured in Experimental Example 2 was examined. To this end, the virus and peptide were co-cultured, the culture medium was applied to cells, and the RNA levels of the virus co-cultured with the cells were measured. A lower measured RNA level indicates greater antiviral efficacy of the peptide.

[0257] The specific experimental methods and results are as follows.

[0258] Vero cells (KCLB; 10081) were seeded at 1.5 × 10 4 per well in a 96-well plate. After the seeded cells were cultured for one day, each of the peptides (DS-1 to DS-15 peptides (experimental group) and 3a-3 / 20 (control group) of Experimental Example 2) at a concentration of 200 μM dissolved in DMSO was serially diluted two-fold in MEM medium (Welgene; LM007-54) to prepare peptide solutions with a maximum concentration of 1000 nM and a minimum concentration of 7.8 nM (concentration: 1000 nM; 500 nM; 250 nM; 125 nM; 62.5 nM; 31.3 nM; 15.6 nM; 7.8 nM). In addition, a virus solution diluted to 0.03 MOI when treated with dengue virus (NCCP; 43248) was prepared. The diluted peptide solution and the diluted virus solution were mixed at a ratio of 1:1 (v / v), and then incubated at 37°C for 1 hour to prepare a mixture of virus and peptide. 200 μL of the prepared virus and peptide mixture was treated to the inoculated cells, and incubated at 37°C and 5% CO2 for 1 hour. After the mixture was removed, 200 μL of MEM medium containing 10% FBS was additionally treated, and then incubated at 37°C and 5% CO2 for 2 days.

[0259] Specifically, viral RNA was extracted from cell culture fluid using the QIAamp Viral RNA Kit (Qiagen; Cat no. 52906), and each Ct value was confirmed by RT-qPCR. RT-qPCR was performed using One Step PrimeScript III RT-qPCR Mix (TaqMan) (Takara; Cat no. #RR600A) with forward primer 5'-GAAAGACCAGAGATCCTGCTGTCT-3' (SEQ ID NO: 102), reverse primer 5'-ACCATTCCATTTTCTGGCGTT-3' (SEQ ID NO: 103), and probe 5'-FAM-AGCATCATTCCAGGCAC-3BHQ1-3' (AGCATCATTCCAGGCAC (SEQ ID NO: 104)) at 42°C for 30 min, 95°C for 5 min, 95°C for 30 s, and 60°C for 60 s, repeated 45 times.

[0260] The percent inhibition (% inhibition) for each well was calculated based on the formula for a standard curve generated using the average Ct values ​​of dengue virus-infected wells within the same plate, converted to a 100% infection rate. Each value was obtained through duplicate experiments, and IC 50 values ​​were derived using GraphPad Prism 5 software. A lower IC 50 value indicates a greater antiviral effect.

[0261]

[0262] The results of the analysis on the antiviral effect or virus proliferation inhibition effect of the peptide according to one embodiment of the present application are disclosed in Tables 2 to 4 and FIGS. 2 to 5 below. Specifically, the Ct value of viral RNA according to each peptide treatment is disclosed in Table 2. The virus inhibition rate of each peptide is disclosed in Table 3. The IC 50 of each peptide is disclosed in Table 4. The virus inhibition rate (% inhibition) and IC 50 -value of each peptide are disclosed in FIGS. 2 to 5 (corresponding to the results in Tables 3 and 4). FIG. 2 discloses the results for the control peptide 3a-3 / 20. FIG. 3 discloses the results for the peptide (DS-1) having the amino acid sequence of SEQ ID NO: 1. FIG. 4 discloses the results for the peptide (DS-4) having the amino acid sequence of SEQ ID NO: 4. Figure 5 discloses the results for a peptide (DS-5) having the amino acid sequence of SEQ ID NO: 5.

[0263] The results of Experimental Example 3 demonstrate that the number of dengue viruses infecting cells is reduced by DS-1 to DS-15. These results demonstrate that DS-1 to DS-15 have an antiviral effect.

[0264] PeptideCt Value125 nM62.5 nMDS-136.2436.24DS-221.8622.08DS-321.8621.51DS-435.9926.00DS-537.7837.83DS-626.6823.52DS-724.4322.99DS-822.5122.44DS-921.7 021.50DS-1027.3626.30DS-1124.9723.66DS-1221.8222.54DS-1325.5422.13DS-1422.5021.79DS-1520.9521.463a-3 / 20(control)26.4523.88

[0265] control peptide (3a)DS-1DS-4DS-5Conc.(nM)Ct Value% InhibitionCt Value% InhibitionCt Value% InhibitionCt Value% Inhibition50036.485100.0035.87100.0036.865100.0038.64100.0025026.61598.7534.43100.0024.8494. 5936.285100.0012523.1377.7222.7669.7423.2679.9945100.0062.522.1449.4722.7469.1121.97542.0734 .71100.0031.321.82534.4222.2955.1821.6826.0626.0497.9915.622.1449.4722.2152.3121.29-2.0924.87594.747.821.6926.6721.6322.6221.80533.3322.9874.783.921.5920.3521.4510.2021.417.5622.4360.08

[0266] PeptideIC 50 (nM)control peptide (3a)31.95DS-123.88DS-463.79DS-53.62

[0267] Experimental Example 4. Confirmation of liposome leakage efficacy of peptides.

[0268] The inventors of the present application attempted to confirm the antiviral efficacy (or lipid membrane destruction efficacy) of the peptide prepared in Experimental Example 2 by using a liposome solution in which a fluorescent substance is encapsulated (specifically, by utilizing the fact that the measured fluorescence level is low when the fluorescent substance is encapsulated in the liposome, and high when the fluorescent substance leaks out of the liposome).

[0269] As a specific experimental method to confirm the efficacy, after a liposome solution encapsulating a fluorescent substance was treated with a solution containing the peptides manufactured in Experimental Example 2 (peptides DS-1 to DS-15 (experimental group) and 3a-3 / 20 (control group) of Experimental Example 2), a change in the fluorescence level was observed. At this time, data indicating an increase in the fluorescence level means that the fluorescent substance leaked out of the liposome, and such leakage means that the liposome (specifically, the lipid membrane) was damaged or destroyed.

[0270]

[0271] Experimental Example 4.1 Preparation of liposomes encapsulating 6-carboxyfluorescein

[0272] 2.5 μmol of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) was filmed for 16 hours to completely remove the organic solvent through a rotary evaporator, and the fluorescent substance 6-carboxyfluorescein (6CF) was dissolved in a buffer solution (10 mM Tris, 150 mM NaCl, pH 7.5) to prepare a 100 mM concentration. The filmed POPC was dissolved in 1 mL of a 100 mM 6CF solution to prepare a 2.5 mM POPC solution, and then freezing and thawing were repeated 7 times using liquid nitrogen and a constant temperature water bath at 60°C.

[0273] The POPC solution was converted into liposomes using a small extruder. A 1 μm filter membrane was mounted inside the extruder, through which the POPC solution passed 15 times. A 30 nm filter membrane was then mounted on the extruder, through which the POPC solution passed 15 times. This resulted in liposomes of uniform size (55 nm). Fluorescent molecules not encapsulated in the liposomes were removed using a PD-10 desalting column.

[0274]

[0275] Experimental Example 4.2 Liposome Leakage Assay

[0276] Each of the peptides DS-01~DS-15 (experimental group) and 3a-3 / 20 (control group) dissolved in DMSO at a concentration of 200 μM was diluted in a buffer solution to a concentration of 500 nM and 250 nM, respectively, and the liposome solution encapsulated with a fluorescent material was diluted in a buffer solution to a POPC concentration of 20 μM. The diluted peptides, 0.06% Triton X-100 as a positive control, and the buffer solution as a negative control were dispensed into three wells of a 96-well black plate (50 μL each) in triplicate. Here, the positive control 0.06% Triton X-100 is a solution containing 0.06% Triton X-100, a surfactant well known to destroy lipid membranes.

[0277] After that, 50 μL of diluted liposome solution was dispensed into each well, and immediately a plate reader was used to measure the fluorescence value (RFU) at an excitation wavelength of 492 nm and an emission wavelength of 517 nm at 1-minute intervals for 20 minutes.

[0278] After 20 minutes of fluorescence measurement, 50 μL of 0.06% Triton X-100 was added to all wells and the fluorescence was re-measured. The reason for re-measuring the fluorescence after 20 minutes of 0.06% Triton X-100 addition to all wells was to confirm 1) that the increase in fluorescence level over time in each well was due to the destruction of liposomes and 2) that the total amount of fluorescent material contained in each well did not differ significantly.

[0279]

[0280] The measured fluorescence levels were plotted in graphs and shown in Figures 6 to 9.

[0281] The results shown in FIGS. 6 to 9 show that the fluorescent material encapsulated inside the liposome leaked to the outside of the liposome by DS-1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, and 15. These results show that DS-1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, and 15 can cause damage (or destruction) of the lipid bilayer of the virus, and among them, DS-1, 4, 5, 7, 9, 11, and 13 cause a higher level of damage (or destruction) of the lipid bilayer of the virus than 3a-3 / 20, which is a peptide before being scrambled.

[0282]

[0283] Experimental Example 5. Confirmation of the liposome rupture efficacy of peptides.

[0284] The present inventors attempted to confirm the antiviral efficacy (or lipid membrane destruction efficacy) of the peptides prepared in Experimental Example 2 (DS-1 to DS-15 peptides (experimental group) and 3a-3 / 20 (control group) of Experimental Example 2) using liposomes in which a fluorescent material was labeled on the lipid membrane.

[0285] As a specific experimental method to confirm the efficacy, the inventors treated a plate to which liposomes labeled with a fluorescent substance were attached to the lipid membrane with a solution containing the peptide prepared in Experimental Example 2, and observed the change in the fluorescence level.

[0286] Here, the data indicating that the fluorescence level decreased over time indicates that the lipid membrane of the liposome was destroyed and removed during the washing process. In other words, the results of this experiment can confirm whether the peptides manufactured in Experimental Example 2 can induce liposome rupture.

[0287]

[0288] Experimental Example 5.1 Evaluation of the Liposome Rupture Efficacy of Peptides

[0289] Experimental Example 5.1.1 Preparation of Rhodamine Fluorescently Labeled Liposomes

[0290] 2.23 μmol of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 4.8 nmol of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000) Biotin), and 168 nmol of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(18:1 Liss Rhod PE) were mixed and evaporated on a rotary evaporator for 16 h to completely remove the organic solvent and form a film. Biotin was used for binding to streptavidin on the plate, and rhodamine was used as a fluorescent material.

[0291] The film-formed phospholipid solution was dissolved in 1 mL of buffer solution (10 mM Tris, 150 mM NaCl, pH 7.5) to prepare a phospholipid solution with a concentration of 2.5 mM. The solution was then frozen and thawed seven times using liquid nitrogen and a 60°C water bath. The phospholipid solution was passed 15 times through a small extruder equipped with a 30 nm filter membrane to produce liposomes of uniform size (55 nm).

[0292]

[0293] Experimental Example 5.1.2 Liposome Rupture Assay

[0294] Streptavidin-coated 96-well black plates were treated with 100 μL of 50 μM liposomes per well, and the plates were incubated at room temperature for 2 hours to allow the liposomes to attach to the plates. Unattached liposomes were removed and washed three times with 200 μL of buffer, and the fluorescence values ​​(RFU) at wavelengths of excitation; 575 nm, emission; 595 nm were measured using a plate reader. Then, each of the 200 μM DS-01~DS-15 and 3a-3 / 20 peptides dissolved in DMSO was diluted in buffer to make a concentration of 2 μM. 100 μL each of 1% Triton-X 100 as a positive control, buffer as a negative control, and diluted peptides were treated in triplicate to the liposome-attached wells, and the plates were incubated at room temperature for 1 hour. After the reaction was completed, the peptide was removed and washed three times with 200 μl of buffer solution. The fluorescence value (RFU) at wavelengths of excitation; 575 nm, emission; 595 nm was measured using a plate reader.

[0295]

[0296] The results were calculated as ((fluorescence value before peptide treatment - fluorescence value after peptide treatment) / fluorescence value before peptide treatment) and compared and analyzed by converting the calculated value when treated with Triton X-100 to 100 (%Rupture). The results are shown in Table 5 and Fig. 10.

[0297] The experimental results in Table 5 and Figure 10 demonstrate that the lipid membranes of fluorescently labeled liposomes were disrupted by the DS-1 to DS-15 and 3a-3 / 20 peptides, resulting in a decrease in fluorescence values. These results demonstrate that the DS-1 to DS-15 and 3a-3 / 20 peptides can cause damage (or destruction) of the lipid bilayer of the virus.

[0298] Peptide (peptide)%RuptureDS-141.21DS-224.32DS-362.59DS-462.08DS-581.48DS-653.68DS-748.17DS-865.81DS-97.02DS-1059.43DS-1183.13DS-1217.59DS-1360.86DS-1426.10DS-1540.643a-3 / 2062.85NC(buffer)3.71PC(Triton-X)100.00

[0299] Experimental Example 5.2 Evaluation of Liposome Rupture Efficacy of Peptides (TIRF)

[0300] Experimental Example 5.2.1 Preparation of Rhodamine Fluorescently Labeled Liposomes

[0301] 2.49 μmol of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 2.51 nmol of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000](DSPE-PEG(2000) Biotin), and 17.58 nmol of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(18:1 Liss Rhod PE) were mixed and the organic solvent was completely removed through a rotary evaporator for 16 hours to form a film. The film-formed phospholipid solution was dissolved in 1 mL of buffer solution (10 mM Tris, 150 mM NaCl, pH 7.5) to prepare a phospholipid solution with a concentration of 2.5 mM, and then frozen and thawed 7 times using liquid nitrogen and a constant temperature water bath at 60°C.

[0302] The phospholipid solution was produced into liposomes of three sizes using a small extruder. The phospholipid solution was passed 15 times through an extruder equipped with a 30 nm filter membrane to produce liposomes of uniform size (55 nm), 11 times through an extruder equipped with a 200 nm filter membrane to produce liposomes of uniform size (150 nm), and 11 times through an extruder equipped with an 800 nm filter membrane to produce liposomes of uniform size (350 nm).

[0303]

[0304] Experimental Example 5.2.2 TIRF Microscopy Measurements

[0305] Biotin-coated coverslips were attached to channel-type Sticky slides (IBIDI, Cat. 80608), and then 100 μl of 10 μg / mL Neutravidin diluted in buffer was treated and reacted for 10 minutes. Unreacted Neutravidin was removed and washed five times, and then the prepared liposomes were diluted to a phospholipid concentration of 0.03 μM. 100 μl of the diluted liposomes were treated and reacted for 10 minutes to attach the liposomes to the coverslip, after which the unattached liposomes were removed and washed five times.

[0306] After that, each of the 200 μM concentrations of DS-05 and 3a-3 / 20 peptides dissolved in DMSO were diluted in a buffer solution to produce diluted peptides with a concentration of 100 nM. Using a peristaltic pump, the diluted peptides were flowed at a rate of 150 μL / min into the liposome-attached wells, and the fluorescence signal changes of a single liposome were measured in the TRITC channel at 20-second intervals for 25 minutes using a TIRF microscope (Total Internal Reflection Fluorescence Microscope). The measurements were repeated three times, and the fluorescence signal changes of 200 to 300 liposomes were measured for each measurement, and the fluorescence value at 0 second was converted to 1, calculated, and graphed.

[0307]

[0308] The observed fluorescence signal changes and graphs are shown in Figures 11 to 14. Figures 11 and 13 show the results for the 3a-3 / 20 peptide, and Figures 12 and 14 show the results for the DS-05 peptide.

[0309] The experimental results in Figures 11 to 14 demonstrate that the lipid membranes of fluorescently labeled liposomes were destroyed by the DS-05 and 3a-3 / 20 peptides, resulting in a decrease in fluorescence values. These results demonstrate that the DS-1 to DS-15 and 3a-3 / 20 peptides possess antiviral efficacy (or lipid membrane-destroying efficacy).

[0310] Referring to the experimental results of FIGS. 11 to 14, the phospholipid bilayer disruption effect of the antiviral peptide of the present disclosure was greatest for the liposome particle having the smallest diameter (largest curvature), and the phospholipid bilayer disruption effect of the antiviral peptide of the present disclosure was smallest for the liposome particle having the largest diameter (smallest curvature). Consequently, the experimental results show that the phospholipid bilayer disruption effect of the antiviral peptide of the present disclosure is dependent on the curvature of the lipid membrane. The greater the curvature of the target particle having the phospholipid bilayer (the smaller the diameter of the target particle), the greater the phospholipid bilayer disruption effect of the antiviral peptide of the present disclosure. These results are believed to be a phenomenon that occurs because the greater the curvature of the phospholipid bilayer of the particle, the greater the stress applied to the lipid membrane. When the antiviral peptide of the present disclosure is inserted into the phospholipid bilayer structure in a situation where greater stress is applied, the structure of the lipid membrane becomes more easily destroyed.

[0311] These results suggest that the antiviral peptides of the present disclosure are very effective in damaging (or destroying) the phospholipid bilayer of viruses (referring to the size or diameter of viruses, which is typically about 20 to 200 nm), but will not have a significant effect on the phospholipid bilayer of other cells (referring to the size or diameter of animal cells, which is typically about 10 to 30 μm).

[0312]

[0313] Experimental Example 6. RNase digestion assay of peptides

[0314] The present inventors attempted to confirm the antiviral efficacy (or lipid membrane destruction efficacy) of the DS-05 and 3a-3 / 20 peptides prepared in Experimental Example 2 by using a nuclease capable of entering the virus through a pore in the lipid membrane of the virus and degrading RNA.

[0315] To confirm efficacy, the inventors treated dengue virus with the peptide and then incubated it with Micrococcal Nuclease (NEB, Cat. M0247S). The level of viral RNA was then measured. A low level of viral RNA indicates that the lipid membrane of the virus has been destroyed, resulting in a significant amount of RNA being degraded by RNase entering the virus.

[0316] The specific experimental methods and results are as follows.

[0317] 1x10 7 Dengue virus stored at a concentration of 4x10 pfu / ml 5pfu / ml in buffer solution (50 mM Tris-HCl, 5 mM CaCl2). Then, peptide solutions were prepared by serially diluting 2-fold in buffer solution each of 3a-3 / 20 and DS-05 peptides at a concentration of 400 μM dissolved in DMSO to obtain peptide solutions at concentrations of 2 μM, 1 μM, and 0.5 μM. The diluted peptides, 2% Triton X-100 as a positive control, and buffer solution as a negative control were dispensed into three microtubes (triplicate) at each concentration, 50 μL each, and the diluted virus solutions were mixed at a ratio of 1:1 (v / v), and then incubated at 37°C for 1 hour. After incubation, 14 μg of BSA and 960 gel units of Micrococcal Nuclease (NEB, Cat. M0247S) were added to each microtube to make a final volume of 140 μL and incubated at 37°C for 1 hour. At this time, Micrococcal Nuclease (NEB, Cat. M0247S) was used to degrade the viral RNA. That is, if the lipid membrane of the virus is destroyed, the viral RNA will be degraded by the Micrococcal Nuclease (NEB, Cat. M0247S) that has entered the virus. On the other hand, if the lipid membrane of the virus is not destroyed and Micrococcal Nuclease (NEB, Cat. M0247S) does not enter the virus, the viral RNA will not be degraded.

[0318] Viral RNA from the cultured mixture was extracted using the QIAamp Viral RNA Kit (Qiagen; Cat no. 52906), and each Ct value was confirmed by RT-qPCR. RT-qPCR was performed using One Step PrimeScript III RT-qPCR Mix (TaqMan) (Takara; Cat no. #RR600A) with forward primer 5'-GAAAGACCAGAGATCCTGCTGTCT-3' (SEQ ID NO: 102), reverse primer 5'-ACCATTCCATTTTCTGGCGTT-3' (SEQ ID NO: 103), and probe 5'-FAM-AGCATCATTCCAGGCAC-3BHQ1-3' (AGCATCATTCCAGGCAC (SEQ ID NO: 104)) at 42°C for 30 min, 95°C for 5 min, 95°C for 30 s, and 60°C for 60 s, repeated 45 times.

[0319] The % Amplifiable genome was calculated based on the formula for the standard curve, which was generated by converting the average Ct values ​​of the negative controls to 100%. Each value was obtained through three replicate experiments.

[0320]

[0321] The experimental results are shown in Table 6 and Fig. 15.

[0322] The experimental results show that the lipid membrane of the virus was destroyed by the DS-05 and 3a-3 / 20 peptides, and the RNA of the virus was degraded by the RNase that entered the virus.

[0323] That is, it shows that DS-05 and 3a-3 / 20 peptides have antiviral efficacy (or lipid membrane disruption efficacy).

[0324] 3a-3 / 20DS-05Conc. (nM)Ct Value% Amplifiable genomeCt Value% Amplifiable genome100026.9111.7428.792.3450025.5936.1226.6215.0525024.5985.2425.5437.70

[0325] Experimental Example 7. Plaque reduction neutralization test (PRNT, Plaque assay)

[0326] 1x10 per well in a 6-well plate 6 Vero cells were inoculated. After culturing the inoculated cells for one day, 85 peptides (Experimental Example 2, DS-16 to DS-100) and 3a-3 / 20 peptide (control) dissolved in DMSO at a concentration of 4 mM were diluted to 250 nM in MEM medium to prepare solutions.

[0327] The prepared peptide sample and DENV2 virus (NCCP; 43248) were mixed 1:1 (v / v) and incubated at 37°C for 1 hour to prepare a mixture. After removing the medium from the wells inoculated with cells, 500 μL of MEM medium was added, and 100 μL of the mixture was treated per well and incubated for a total of 1 hour at 37°C and 5% CO2 with shaking at 15-minute intervals. After incubation, the treated mixture was removed, washed once with DPBS, and then solidified with DMEM / F12 medium supplemented with 0.9% agarose. The solidified plates were incubated at 37°C and 5% CO2 for 7 days, fixed with 3.7% paraformaldehyde, stained with 0.3% crystal violet solution, and the neutralizing ability (% Inhibition) was calculated by observing the number of formed plaques. The neutralizing activity % was calculated based on the number of plaques formed in the control (virus only). A higher neutralizing activity % (% Inhibition) value indicates a higher antiviral efficacy.

[0328]

[0329] The results are shown in Table 7 below and Figures 17a and 17b.

[0330] 펩타이드# of Plaque% Inhibition3a-3 / 2018.985.21DS-01644.358.72DS-01763.745.62DS-01896.323.49DS-0193.488.76DS-020132.7-1.13DS-02187.029.81DS-0226.384.47DS-023133.0-1.36DS-024104.717.84DS-025130.00.67DS-026135.0-4.86DS-02711.081.14DS-02870.339.99DS-029120.75.09DS-030129.3-0.92DS-031120.75.09DS-032126.01.38DS-03399.020.11DS-034124.72.31DS-03529.768.19DS-03666.742.53DS-03786.328.89DS-038109.712.71DS-0394.785.52DS-040117.07.62DS-041129.3-0.26DS-04282.336.18DS-043104.718.86DS-044128.00.78DS-045118.08.53DS-0467.394.32DS-04751.060.47DS-048122.35.17DS-049127.01.55DS-050109.015.50DS-05126.079.58DS-0520.399.74DS-05335.372.25DS-05483.734.29DS-055127.7-0.26DS-056121.04.97DS-057117.77.59DS-058118.76.81DS-059122.33.93DS-06065.348.69DS-061122.73.66DS-062124.02.62DS-063123.33.14DS-064119.36.28DS-065124.07.46DS-066139.0-3.73DS-067134.00.00DS-068122.08.96DS-069112.016.42DS-070123.08.21DS-071133.00.75DS-072143.0-6.72DS-07388.034.33DS-074126.05.97DS-07584.037.31DS-076128.04.48DS-07767.050.00DS-078118.011.94DS-0790.0100.00DS-080147.0-9.70DS-0815.396.02DS-082104.022.39DS-08343.066.92DS-084 129.00.77DS-085132.0-1.54DS-08632.375.13DS-087125.03.85DS-088132.0-1.54DS-089128.01. 54DS-090133.0-2.31DS-091130.00.00DS-092127.02.31DS-093126.03.08DS-094128.01.54DS-095 42.367.44DS-09632.774.87DS-097128.51.15DS-09815.787.95DS-099129.00.77DS-100129.50.38.

[0331] As can be confirmed in Table 7 above, various peptides exhibited antiviral activity. The DS-16, DS-17, DS-18, DS-19, DS-21, DS-22, DS-27, DS-28, DS-33, DS-35, DS-36, DS-37, DS-39, DS-42, DS-46, DS-47, DS-51, DS-52, DS-53, DS-54, DS-60, DS-73, DS-75, DS-77, DS-79, DS-81, DS-82, DS-83, DS-86, DS-95, DS-96, and DS-98 peptides exhibited excellent antiviral activity, with a neutralizing ability of 20% or more. The DS-19, DS-22, DS-27, DS-39, DS-46, DS-51, DS-52, DS-53, DS-79, DS-81, DS-86, DS-96 and DS-98 peptides exhibited antiviral activity equivalent to that of the 3a-3 / 20 peptide, while the DS-19, DS-39, DS-46, DS-52, DS-79, DS-81 and DS-98 peptides exhibited antiviral activity superior to that of the 3a-3 / 20 peptide.

[0332] Among these, the five peptides selected were diluted to produce peptide solutions with a highest concentration of 250 nM and a lowest concentration of 1.95 nM to calculate IC50 (concentration: 250 nM; 125 nM; 62.5 nM; 31.25 nM; 15.63 nM; 7.81 nM; 3.91 nM; 1.95 nM), and the number of plaques was observed and the neutralizing activity % was calculated to calculate IC50. The results are shown in Tables 9 to 13 below (corresponding to Figures 18a to 18e, respectively).

[0333] Peptide IC50 (nM)DS-0194.79DS-04615.74DS-05222.03DS-07915.53DS-0818.66

[0334] DS-19Conc. (nM)# of Plaque% Inhibition125.000.00100.0062.500.00100.0031.250.00100.0015.631. 0099.177.8118.0085.123.9184.0030.581.95120.000.830.98118.332.200 (NC)121.000.00

[0335] DS-46Conc. (nM)# of Plaque% Inhibition125.000.00100.0062.500.3399.7431.2517.3386.5315.6364.3 350.007.81106.0017.623.91116.679.331.95125.332.590.98123.334.150 (NC)128.670.00

[0336] DS-52Conc. (nM)# of Plaque% Inhibition125.000.00100.0062.506.6795.0031.2526.6780.0015.6399.3 325.507.81121.339.003.91132.670.501.95137.00-2.750.98129.672.750 (NC)133.330.00

[0337] DS-79Conc. (nM)# of Plaque% Inhibition125.000.00100.0062.500.00100.0031.255.3395.6215.6361.0 049.867.81115.005.483.91119.671.641.95127.67-4.930.98123.00-1.100 (NC)121.670.00

[0338] DS-81Conc. (nM)# of Plaque% Inhibition125.000.00100.0062.500.3399.7431.252.6797.9115.6312.0 090.587.8175.6740.583.91108.6714.661.95117.677.590.98123.003.400 (NC)127.330.00

[0339] Experimental Example 8. Evaluation of Liposome Rupture Efficacy of Peptides

[0340] Experimental Example 8.1. Liposome Production

[0341] 2.5 μmol of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) was filmed for 16 h to completely remove the organic solvent using a rotary evaporator. The filmed POPC was dissolved in 1 mL of buffer (10 mM Tris, 150 mM NaCl, pH 7.5) to prepare a 2.5 mM POPC solution, which was then frozen and thawed seven times using liquid nitrogen and a 60°C water bath. The POPC solution was then passed 15 times through a small extruder equipped with a 30 nm filter membrane to produce a liposome solution of uniform size (55 nm).

[0342]

[0343] Experimental Example 8.2. Confirmation of peptide liposome rupture using dynamic light scattering (DLS) measurements.

[0344] DLS measurements were performed at 25°C using a nanoSAQLA (Otsuka Electronics, Osaka, Japan) equipped with a 660 nm laser and a photodiode detector. 4 mM 3a-3 / 20 peptide (control) and five selected peptides (DS-019, DS-046, DS-052, DS-079, and DS-081) dissolved in DMSO were diluted in buffer to a concentration of 20 μM, and the liposome solution was diluted in buffer to a POPC concentration of 100 μM. 700 μL of the liposome solution was dispensed into 15 mL tubes, and 700 μL each of the diluted peptide and the negative control (NC) buffer were added, respectively, and the mixture was incubated at RT for 20 min. The reaction solution was then transferred to a RATIOLAB disposable cuvette with a path length of 1 cm, and the size was determined by averaging 25 consecutive measurements. Data analysis to calculate the mean hydrodynamic diameter (Dh) and polydispersity was performed using the software provided with the device.

[0345] The results are shown in Figures 19a to 19h. Figure 19a shows the combined results of the experiments on five peptides (DS-019, DS-046, DS-052, DS-079, DS-081), the 3a-3 / 20 peptide, and the negative control, and Figures 19b to 19h show the respective experimental results.

[0346] As can be seen in Fig. 19a, compared to the group that was not treated with anything (NC, Negative Control), the group treated with five peptides (DS-019, DS-046, DS-052, DS-079, DS-081) showed an increase in size, which means that the phospholipid bilayer of the liposome was damaged by the peptides.

[0347]

[0348] Experimental Example 9. Evaluation of the Neutralizing Potential of Peptides Against SARS-CoV2

[0349] 1.5x10 per well in a 96-well plate 4 Vero cells were inoculated. After culturing the inoculated cells for one day, three selected peptides (DS-019, DS-052, DS-079) at a concentration of 4 mM dissolved in DMSO were diluted 1 / 100 in DPBS and serially diluted two-fold to prepare solutions with a highest concentration of 40 μM and a lowest concentration of 0.08 μM (concentration: 40 μM; 20 μM; 10 μM; 5 μM; 2.5 μM; 1.25 μM; 0.63 μM; 0.31 μM; 0.16 μM; 0.08 μM). The prepared sample was mixed 1:1 (v / v) with 200 TCID50 of SARS-CoV-2 virus (NCCP; 43326) and incubated at 37°C for 1 hour to prepare a mixture. The mixture was treated with 40 μL of each concentration to four wells seeded with cells and cultured at 37°C, 5% CO2 for 4 days. After incubation, the cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. The neutralizing activity % was calculated by observing CPE (cytopathic effect). The neutralizing activity % was normalized to the positive control (mock) and negative control (0.5% DMSO) and the 50% inhibitory dose (ID50) was calculated.

[0350]

[0351] The results are shown in Table 14 below and Figures 20a to 20c.

[0352] % of neutralization (Neutralization ability)ID50(μM)Concentration(μM)Sample201052.51.30.60.30.20.10.04DS-019-10 0100750000002.422DS-052100100505050000003.751DS-07910010075250000003.574

[0353] Figures 20a to 20c show the inhibition rate according to the treatment concentration of three peptides (DS-019, DS-052, DS-079).

[0354] As can be seen in Table 14 above and FIGS. 20a to 20c, the peptide provided in the present application exhibited excellent antiviral effects against the SARS-CoV-2 virus.

[0355]

[0356] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO:

100.

2. A peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 21, 22, 27, 28, 33, 35, 36, 37, 39, 42, 46, 47, 51, 52, 53, 54, 60, 73, 75, 77, 79, 81, 82, 83, 86, 95, 96, and 98 in the first paragraph.

3. A peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 39, 46, 52, 79, 81, and 98 in the first paragraph.

4. A peptide having a neutralizing ability of 20% or more as measured by a plaque reduction neutralization test in the first paragraph.

5. A peptide according to claim 1, characterized in that the peptide is amphipathic.

6. A peptide according to claim 1, characterized in that the peptide has an alpha helical structure.

7. A peptide according to claim 1, characterized in that the peptide has antiviral activity.

8. A peptide according to claim 7, characterized in that the peptide can cause damage and / or destruction of the lipid bilayer of the virus.

9. A peptide according to claim 8, characterized in that the virus is a virus belonging to at least one family selected from the group consisting of Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, and Herpesviridae.

10. A polynucleotide encoding a peptide according to any one of claims 1 to 9.

11. A recombinant vector comprising the polynucleotide of Article 10.

12. A microorganism comprising the polynucleotide of Article 10 or a recombinant vector comprising the polynucleotide.

13. A peptide according to any one of claims 1 to 9; A polynucleotide encoding the above peptide; A recombinant vector comprising the above polynucleotide; and An antiviral composition comprising at least one selected from the group consisting of microorganisms, comprising the polynucleotide or a recombinant vector comprising the polynucleotide.

14. A feed additive comprising the antiviral composition of Article 13.

15. A disinfectant comprising the antiviral composition of Article 13.

16. A detergent comprising the antiviral composition of Article 13.

17. A peptide according to any one of claims 1 to 9; A polynucleotide encoding the above peptide; A recombinant vector comprising the above polynucleotide; and A pharmaceutical composition for preventing or treating a viral infection or a disease caused by a viral infection, comprising at least one selected from the group consisting of microorganisms, including the polynucleotide or a recombinant vector containing the polynucleotide.

18. A pharmaceutical composition for preventing or treating a viral infection or a disease caused by a viral infection, wherein the virus is a virus belonging to one or more families selected from the group consisting of Paramyxoviridae, Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, and Herpesviridae.

19. A pharmaceutical composition for preventing or treating a viral infection or a disease caused by a viral infection, wherein the viral infection or a disease caused by a viral infection is at least one selected from dengue fever, coronavirus disease-19 (COVID-19), phlegm, cough, sore throat, headache, hemoptysis, dyspnea, upper respiratory tract infection, lower respiratory tract infection, respiratory infection, pneumonia, and cytokine storm syndrome.

20. A peptide according to any one of claims 1 to 9; A polynucleotide encoding the above peptide; A recombinant vector comprising the above polynucleotide; and A feed composition for preventing or improving a viral infection or a disease caused by a viral infection, comprising at least one selected from the group consisting of microorganisms, including the polynucleotide or a recombinant vector containing the polynucleotide.

21. A peptide according to any one of claims 1 to 9; A polynucleotide encoding the above peptide; A recombinant vector comprising the above polynucleotide; and A food composition for preventing or improving a viral infection or a disease caused by a viral infection, comprising at least one selected from the group consisting of microorganisms, including the polynucleotide or a recombinant vector containing the polynucleotide.

22. A peptide according to any one of claims 1 to 9; A polynucleotide encoding the above peptide; A recombinant vector comprising the above polynucleotide; and A cosmetic composition for preventing or improving a viral infection or a disease caused by a viral infection, comprising at least one selected from the group consisting of microorganisms, including the polynucleotide or a recombinant vector containing the polynucleotide.

Images