ROMO1-derived antimicrobial peptides and variants thereof containing lysine substitutions

Abstract

The present invention relates to antimicrobial peptides derived from Romo1 protein, and specifically, a plurality of peptides are provided, each of which has an amino acid sequence of the α-helix 2 region of Romo1 protein, and its antimicrobial activity against Gram-positive, Gram-negative and multidrug-resistant bacteria is determined; and an antimicrobial composition comprising the peptide is provided. The Romo1-derived peptides and variants thereof of the present invention are superior to existing antibiotics and antibiotic peptides in terms of antibiotic ability against various types of bacteria and multidrug-resistant bacteria, and in particular, have antimicrobial activity against various bacteria in blood vessels, and therefore, are new antibiotics that can be provided as a substance for preventing or treating a wide range of bacterial infectious diseases. Therefore, the composition comprising the antimicrobial peptides of the present invention is expected to be used in a variety of fields for the purpose of preventing or treating infectious diseases, for example, as drugs, quasi-drugs, food and feed additives, and agricultural and cosmetic additives.

Description

Technical Field

[0001] The present invention relates to antimicrobial peptides derived from Romo1 protein, and in particular, provides a plurality of peptides having the amino acid sequence of the α-helix 2 region of Romo1 protein and an antibacterial composition comprising each peptide, since the peptides are determined to have antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and multidrug-resistant bacteria. Background Art

[0002] The biggest problem with antibiotics used as a treatment for bacterial infections is the acquisition of antibiotic resistance by bacteria. According to a UK government Welcome Trust report, it is expected that by 2050, the number of deaths worldwide due to superbugs that cannot be treated with existing antibiotics will exceed 10,000,000, and superbug infections will replace cancer as a cause of death.

[0003] In addition, the World Health Organization (WHO) selected carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Enterobacteriaceae sp. strains as bacteria in a key priority category among numerous multidrug-resistant bacteria, and announced that Gram-negative superbugs resistant to carbapenem-based antibiotics, known as last-resort antibiotics, should be addressed with the highest priority.

[0004] Meanwhile, bacteremia refers to a condition in which bacteria survive in the blood, while sepsis is caused by the direct or indirect effects of infection. That is, sepsis is a systemic inflammatory response syndrome caused by bacteria invading the human body infecting the blood, and is a disease in which the survival rate is less than 30% 6 hours after infection. The bacteria that cause sepsis are very diverse, including streptococcus, Staphylococcus aureus, Escherichia coli, pneumococcus, Pseudomonas aeruginosa, and fungi, and due to the diversity of these bacteria that cause sepsis, the treatment of sepsis is difficult.

[0005] To date, the best treatment for sepsis is mainly for the purpose of relieving symptoms, such as suppressing immune responses or inhibiting blood coagulation. For the fundamental treatment of sepsis, bacterial identification is required at the initial stage to identify the bacterial species that infect the patient. However, there is a problem that the time required for bacterial identification cannot be ensured due to the rapid systemic inflammatory response caused by sepsis as described above. In addition, even if the bacteria causing sepsis are successfully identified, when the bacteria causing sepsis are multidrug-resistant bacteria that are resistant to antibiotics, there is a problem that there is no drug suitable for its treatment. Therefore, it is necessary to develop antibiotics that have antibacterial activity against a wide range of bacteria without identifying the bacteria that cause sepsis, and it is particularly necessary to develop antibiotics that have antibacterial activity against multidrug-resistant bacteria.

[0006] As mentioned above, the biggest problem of human antibiotic use so far is that bacteria acquire resistance. In order to solve this problem, many studies have been conducted, and in particular, antimicrobial peptides (AMPs) have been actively developed to overcome antibiotic resistance acquired by bacteria. A variety of antimicrobial peptides derived from animal or human host defense proteins have been developed, but the problem is that bacteria in the human immune system are likely to acquire resistance, and they have a short half-life in the blood, are toxic to the human body, and show limited drug effects compared to existing antibiotics. Due to these problems, antimicrobial peptides have not yet been put into practical use. For example, magainin is an antimicrobial peptide developed in the United States in 1999, and after Phase 3 clinical trials, the FDA was requested to approve it, but the approval was rejected because its drug effect was not better than those traditional antibiotics. In addition, daptomycin in 2003 and oritavancin in 2014 were approved by the U.S. Food and Drug Administration, but both peptides only show antibacterial activity against Gram-positive bacteria, and they can only be applied to skin infectious diseases.

[0007] As mentioned above, due to its limited drug effect and application, antimicrobial peptides that can be used to treat bacterial infectious diseases such as bacteremia and sepsis have not yet been developed. The inventors found that the α-helix 2 structure of the Romo1 protein located in the inner membrane of the mitochondria exhibits antibacterial activity against Gram-positive, Gram-negative and multidrug-resistant bacteria, and therefore can be used to treat a wide range of bacterial infectious diseases, thereby completing the present invention.

[0008] [Prior art documents]

[0009] [Non-patent document]

[0010] (Non-patent document 1): WHO, Global Priority List of Antibiotic-ResistantBacteria to Guide Research, Discovery, and Development of New Antibiotics (2017.2.27)

[0011] (Non-patent document 2): Lee et al., J. Cell Biol., 217, 2059-2071, 2018

[0012] Public Content

[0013] Technical issues

[0014] One object of the present invention is to provide an antimicrobial peptide derived from Romo1 protein comprising lysine substitution, and a modified antimicrobial peptide produced by substitution or deletion of some amino acids of the peptide.

[0015] Another object of the present invention is to provide an antibiotic containing an antimicrobial peptide as an active ingredient, and a food and feed additive, a cosmetic composition, a biopesticide and an antibacterial quasi-drug composition each containing the antimicrobial peptide.

[0016] However, objects to be achieved by the present invention are not limited to the above objects, and other objects not mentioned herein will be clearly understood by those skilled in the art from the following description.

[0017] Technical Solution

[0018] One aspect provides an antimicrobial peptide comprising a sequence of 7 to 28 consecutive amino acids extending in the carboxyl (C-) terminal direction, the sequence starting from any amino acid selected from the 52nd to 79th amino acid region of the Romo1 protein sequence consisting of the amino acid sequence shown in SEQ ID NO:1.

[0019] One embodiment provides an antimicrobial peptide produced by substitution or deletion of at least one amino acid constituting the amino acid sequence of the antimicrobial peptide.

[0020] In another embodiment, the peptide may be a peptide comprising or consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 15.

[0021] In another embodiment, the peptide may be a peptide comprising or consisting of any one amino acid sequence selected from SEQ ID NOs: 16, 30, 40 and 45.

[0022] In another embodiment, the peptide consisting of the amino acid sequence of SEQ ID NO: 16 may consist of, for example, any one amino acid sequence selected from SEQ ID NOs: 17 to 29.

[0023] In another embodiment, the peptide consisting of the amino acid sequence of SEQ ID NO: 30 may consist of, for example, any one amino acid sequence selected from SEQ ID NOs: 31 to 39.

[0024] In another embodiment, the peptide consisting of the amino acid sequence of SEQ ID NO: 40 may consist of, for example, any one amino acid sequence selected from SEQ ID NOs: 41 to 44.

[0025] In another embodiment, the peptide consisting of the amino acid sequence of SEQ ID NO: 45 may consist of any one amino acid sequence selected from, for example, SEQ ID NO: 46 and 47.

[0026] In another embodiment, the peptide may preferably be a peptide comprising or consisting of any one amino acid sequence selected from SEQ ID NOs: 17 to 25, 27, 34, 37, 39, 42 and 44, more preferably a peptide comprising or consisting of any one amino acid sequence selected from SEQ ID NOs: 22, 23 and 24.

[0027] In another embodiment, the peptide may be an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 13, or a sequence comprising at least one amino acid substitution selected from the following (a) to (h) relative to the amino acid sequence of SEQ ID NO: 13. (a) K to R substitution at amino acid position 1, (b) T to K or R substitution at amino acid position 2, (c) Q to K or R substitution at amino acid position 5, (d) S to K, R or H substitution at amino acid position 6, (e) T to K or R substitution at amino acid position 9, (f) F to W substitution at amino acid position 10, (g) I to L substitution at amino acid position 16, and (h) I to G or L substitution at amino acid position 20.

[0028] In another embodiment, the antimicrobial peptide may be a peptide in which at least one methionine is replaced by norleucine or isoleucine.

[0029] In another embodiment, the antimicrobial peptide may be a peptide in which any one of the amino acids at positions 3, 4, 5, 7, 8, 9, 11, 17, 18 and 19 is deleted, or the amino acid at position 3 or 4 and the amino acid at position 7 or 8 are deleted.

[0030] In another embodiment, the antimicrobial peptide may further comprise an amino acid sequence linked to the C-terminus, wherein the amino acid sequence linked to the C-terminus may consist of one to three repeated sequences of R or K.

[0031] On the other hand, an antimicrobial peptide is provided, which comprises: the amino acid sequence of SEQ ID NO: 24; an amino acid sequence in which K at amino acid position 1 in the amino acid sequence of SEQ ID NO: 24 is substituted to T or T at amino acid position 12 is substituted to G; or an amino acid sequence in which T at amino acid position 2 in the amino acid sequence of SEQ ID NO: 24 is substituted.

[0032] According to one embodiment, the antimicrobial peptide may be a peptide in which at least one methionine is replaced by norleucine or isoleucine.

[0033] According to one embodiment, the antimicrobial peptide may further comprise any one modification selected from the following (a) to (s).

[0034] (a) deletion of any one of the amino acids at positions 1 to 12, 14 to 15, and 17 to 21; (b) deletion of the amino acid at position 14, and deletion of the amino acid at any one of positions 1 to 8, 12, 15, and 18; (c) deletion of the amino acid at position 14, deletion of the amino acid at position 18, and deletion of the amino acid at any one of positions 1 to 8, 12, and 15; (d) deletion of the amino acid at position 14, deletion of the amino acid at position 15, and deletion of the amino acid at position 1, 2, or 12; (e) deletion of the amino acid at position 1 or 2, deletion of the amino acid at position 3 or 4, and deletion of the amino acid at position 18; (f) deletion of the amino acid at position 3, 4, 14, and (g) a deletion of the amino acid at position 3 or 4, a deletion of the amino acid at position 14, a deletion of the amino acid at position 18, and a deletion of the amino acid at position 5, 6, 7, 8, 12 or 15; (h) a deletion of the amino acid at position 14, a deletion of the amino acid at position 15, a deletion of the amino acid at position 1 or 2, and a deletion of the amino acid at position 3, 4, 7 or 8; (i) a deletion of the amino acid at position 1, 2, 5 or 6, a deletion of the amino acid at position 7, 8 or 11, a deletion of the amino acid at position 14 or 15, and a deletion of the amino acid at position 18; (j) a deletion of the amino acid at position 5 or 6, a deletion of the amino acid at position 11, a deletion of the amino acid at position 15 (k) a deletion of an amino acid at position 1 or 2, a deletion of an amino acid at position 7 or 8, and a deletion of amino acids at positions 12, 14, and 15; (l) a deletion of an amino acid at position 1, 2, 5, or 6, a deletion of amino acids at positions 3 and 4, and a deletion of amino acids at positions 14 and 18; (m) a deletion of amino acids at positions 3, 4, 14, and 18, a deletion of an amino acid at position 5 or 6, and a deletion of amino acids at any one of positions 7 or 8, 12, and 15; (n) a deletion of an amino acid at position 1 or 2, a deletion of an amino acid at position 7 or 8, and a deletion of amino acids at positions 12, 14, and 15. (o) a deletion of an amino acid at position 3, 4, 14 and 18, a deletion of an amino acid at position 5 or 6, and a deletion of two amino acids selected from the amino acids at positions 7 or 8, 12 and 15; (p) a deletion of an amino acid at position 1 or 2, a deletion of an amino acid at position 5 or 6, a deletion of an amino acid at position 7 or 8, and a deletion of amino acids at positions 12, 14, 15 and 18; (q) a deletion of an amino acid at position 3, 4, 14 and 18, a deletion of an amino acid at position 5 or 6, and a deletion of three amino acids selected from the amino acids at positions 1 or 2, 7 or 8, 12 and 15;(r) a deletion of the amino acid at position 1 or 2, a deletion of the amino acid at position 3 or 4, a deletion of the amino acid at position 5 or 6, a deletion of the amino acid at position 7 or 8, and a deletion of the amino acids at positions 12, 14, 15, and 18; and (s) a deletion of the amino acid at position 1 or 2, a deletion of the amino acids at positions 3 and 4, a deletion of the amino acid at position 5 or 6, a deletion of the amino acid at position 7 or 8, and a deletion of the amino acids at positions 12, 14, 15, and 18.;

[0035] According to one embodiment, the antimicrobial peptide may be a peptide in which the amino acid pair at positions 10 and 14 or the amino acid pair at positions 14 and 18 is stapled together. Staples may be achieved by replacing the amino acid pair with pentenylalanine or octenylalanine and cross-linking the amino acids.

[0036] According to one embodiment, the antimicrobial peptide may further comprise an amino acid sequence linked to its C-terminus, wherein the amino acid sequence linked to the C-terminus may consist of one to three repeated sequences of R or K.

[0037] In another embodiment, the peptide may be one in which at least one amino acid in its sequence is modified.

[0038] In another embodiment, the modification may be pegylation, acetylation, carboxylation, lipidation, or amidation.

[0039] In another embodiment, the amino acids constituting the peptide may each independently be L- or D-amino acids, and they may be radioactively or fluorescently labeled amino acid analogs.

[0040] In another embodiment, the peptide may have antibacterial activity against one or more bacteria selected from Gram-positive bacteria, Gram-negative bacteria, and multidrug-resistant bacteria.

[0041] In another embodiment, the gram-positive bacteria can be a bacterium belonging to a genus selected from Staphylococcus sp., Bacillus sp., Enterococcus sp., Streptomyces sp. and Streptococcus sp. Preferably, the gram-positive bacteria can be one or more bacteria selected from Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, Streptomyces sindenensis, Enterococcus faecalis and Streptococcus pneumoniae.

[0042] In another embodiment, gram-negative bacteria can be a bacterium belonging to a genus selected from Escherichia coli (Escherichiasp.), Klebsiella sp., Acinetobacter sp., Pseudomonas sp. and Enterobacter. Preferably, gram-negative bacteria can be one or more bacterium selected from Escherichia coli, Klebsiella pneumoniae (Klebsiella pneumoniae), Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes (Enterobacter aerogenes).

[0043] In another embodiment, the multidrug-resistant bacteria can be the above-mentioned Gram-positive or Gram-negative bacteria, which are resistant to one or more antibiotics belonging to penicillins, carbapenems, cephalosporins, quinolones, macrolides, tetracyclines or glycopeptides. Preferably, the multidrug-resistant bacteria can be a bacterium belonging to a genus selected from methicillin-resistant Staphylococcus, multidrug-resistant Pseudomonas, vancomycin-resistant Enterococcus, multidrug-resistant Klebsiella, multidrug-resistant Acinetobacter and vancomycin-resistant Staphylococcus.

[0044] In another embodiment, the multidrug-resistant bacteria may be one or more bacteria selected from methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium, and vancomycin-resistant Staphylococcus aureus.

[0045] In another embodiment, the multidrug-resistant Pseudomonas aeruginosa may be resistant to one or more antibiotics selected from piperacillin, piperacillin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, and ciprofloxacin; the multidrug-resistant Acinetobacter baumannii may be resistant to one or more antibiotics selected from piperacillin, piperacillin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, and cefepime; and the multidrug-resistant Klebsiella pneumoniae may be resistant to one or more antibiotics selected from piperacillin-tazobactam, ceftazidime, cefepime, imipenem, gentamicin, and ciprofloxacin.

[0046] In another embodiment, the vancomycin-resistant Enterococcus faecium may be resistant to one or more antibiotics selected from rifampin, tetracycline, gentamicin, erythromycin, streptomycin and ampicillin, in addition to vancomycin resistance, and the vancomycin-resistant Staphylococcus aureus may be resistant to one or more antibiotics selected from oxacillin, benzylpenicillin, ampicillin and cefazolin, in addition to vancomycin resistance.

[0047] Another aspect provides an antibiotic containing an antimicrobial peptide as an active ingredient.

[0048] Yet another aspect provides a pharmaceutical composition for preventing or treating bacterial infectious diseases, comprising an antimicrobial peptide as an active ingredient.

[0049] Yet another aspect provides a method for preventing or treating a bacterial infectious disease, the method comprising the step of administering an antimicrobial peptide to a subject.

[0050] Another aspect provides use of an antimicrobial peptide for the manufacture of a medicament for preventing or treating a bacterial infectious disease.

[0051] In one embodiment, the bacterial infectious disease may be one or more diseases selected from skin infection, food poisoning, otitis media, cystitis, peritonitis, urinary tract infection, mastitis, pneumonia, endocarditis, conjunctivitis, arthritis, endometritis, bronchiolitis, bacteremia, sepsis and acne, and may preferably be pneumonia or sepsis.

[0052] Another aspect provides an antimicrobial quasi-drug composition, a cosmetic composition, a food additive, and a feed additive, each containing an antimicrobial peptide.

[0053] Beneficial Effects

[0054] The Romo1-derived peptides and variants thereof according to the present invention may have higher antibacterial activity against various types of bacteria compared to existing antibiotics and antibiotic peptides, and thus may simultaneously remove a variety of pathogenic bacteria from the blood of patients suffering from bacterial infectious diseases at an early stage. Specifically, the peptides of the present invention may exhibit high antibacterial activity against antibiotic-resistant bacteria, and thus may be provided as preventive or therapeutic agents for infectious diseases caused by multidrug-resistant bacteria, exceeding the application limitations of existing antibiotics. In addition, the Romo1-derived peptides and variants thereof according to the present invention are expected to be used for the purpose of preventing or treating a wide range of bacterial infectious diseases in a variety of applications including drugs, quasi-drugs, food and feed additives, pesticides, and cosmetic additives, because bacteria have a low probability of acquiring resistance to the Romo1-derived peptides and variants thereof, the Romo1-derived peptides and variants thereof have low toxicity, and particularly have higher antibacterial activity in the blood than existing antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Results comparing the antibacterial activities of KU-5878 and traditional antimicrobial peptides (LL-37, magainin 2, and daptomycin) against Gram-positive and Gram-negative bacteria are shown.

[0056] Figure 2 Results comparing the antibacterial activities of KU-5878 and traditional antimicrobial peptides (LL-37, magainin 2, and daptomycin) against multidrug-resistant bacteria are shown.

[0057] Figure 3 Shown are the results of comparing the antibacterial activities of KU-5878 and a traditional antibiotic (imipenem) against Gram-positive and Gram-negative bacteria.

[0058] Figure 4 Shown are the results of comparing the antibacterial activities of KU-5878 and a conventional antibiotic (imipenem) against multidrug-resistant bacteria.

[0059] Figure 5 Shown are the results comparing the cytotoxicity of KU-5878 and traditional antimicrobial peptides (magenin 2 and melittin) on human vascular endothelial cells (HUVEC).

[0060] Figure 6 Shown are the results of comparing the cytotoxicity of KU-5878 and KU-5878 variants (KU-5878-K4 and KU-5878-K4-D) with traditional antimicrobial peptides (LL-37, magainin 2, and melittin) on mouse erythrocytes by the degree of erythrocyte hemolysis.

[0061] Figure 7 The results of observing the weight of mice to determine the lack of toxicity of KU-5878 in vivo are shown. DETAILED DESCRIPTION

[0062] Reactive oxygen species modulator 1 (Romo1) protein is a membrane protein located in the inner membrane of mitochondria and is known to be involved in mitochondrial reactive oxygen species production, TNF-α signaling, cell senescence, cancer progression and invasion, and mitochondrial protein transport, etc. The structure of Romo1 protein is a protein containing two α-helices, of which α-helix 1 is mainly composed of hydrophobic amino acids and α-helix 2 has both hydrophilic and hydrophobic properties.

[0063] The inventors excluded the α-helix 1 region of the Romo1 protein from the study because the region does not dissolve well in water. The inventors have synthesized peptides of various lengths having the amino acid sequence of the α-helix 2 region (aa 52 to 79), evaluated their antibacterial activity, and found that variants of the peptides have antibacterial activity, thereby completing the present invention.

[0064] In a specific embodiment of the present invention, the inventors synthesized several peptides of different lengths, which comprised a continuous amino acid sequence starting from an amino acid selected from the 52nd to 79th amino acid region of the Romo1 protein, and the inventors evaluated the antibacterial activity of each peptide against Pseudomonas aeruginosa and Staphylococcus aureus using the minimum bactericidal concentration assay. As a result, it was determined that all peptides comprising a sequence of 18 consecutive amino acids (starting from any amino acid selected from the 52nd to 62nd amino acid region of the Romo1 protein) had antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In particular, it can be determined that the KU-5878 peptide comprising the 58th to 78th amino acid sequence of the Romo1 protein has excellent antibacterial activity (see Example 1).

[0065] Therefore, the present invention provides antimicrobial peptides derived from Romo1.

[0066] In the present specification, a "Romo1-derived peptide" is a peptide comprising a continuous amino acid sequence of the 52nd to 79th amino acid region of the Romo1 protein selected from the amino acid sequence shown in SEQ ID NO: 1, and a "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds. The Romo1-derived peptide can be obtained by fragmenting the Romo1 protein and can be produced according to a chemical synthesis method known in the art, particularly a solid phase synthesis technique or a liquid phase synthesis technique.

[0067] In addition, the length of the Romo1-derived peptide of the present invention is not limited, as long as the peptide contains a continuous amino acid sequence starting from any amino acid in the 52nd to 79th amino acid region of the Romo1 protein sequence selected from the amino acid sequence shown in SEQ ID NO: 1, but the length may preferably be 7 to 28 mers, more preferably 18 to 28 mers.

[0068] More specifically, the antimicrobial peptide may comprise a sequence of 18 consecutive amino acids starting from any amino acid selected from the 52nd to 62nd amino acid region of the Romo1 protein sequence. Preferably, the antimicrobial peptide may comprise any one amino acid sequence selected from SEQ ID NO: 2 to 15, more preferably selected from SEQ ID NO: 5, 8, 9, 13 and 14.

[0069] In the present specification, amino acids are listed in order from N-terminus to C-terminus, and the expression "continuous amino acid sequence" starting with an amino acid selected from a specific region of the Romo1 protein sequence refers to a continuous amino acid sequence starting with the selected amino acid and extending in the C-terminal direction. In addition, in the present specification, the expression "a sequence of 7 to 28 consecutive amino acids" starting with any amino acid selected from a specific region of the Romo1 protein refers to a sequence consisting of a total of 7 to 28 consecutive amino acids, including the starting amino acid selected from the specific region of the Romo1 protein, and including continuous amino acids starting from the starting amino acid and extending in the C-terminal direction.

[0070] In a specific embodiment of the present invention, the inventors selected the KU-5878 peptide comprising the amino acid sequence shown in SEQ ID NO: 13 as a representative to determine the application range of the Romo1-derived antimicrobial peptide, and evaluated the antibacterial activity of the peptide against the following: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, Streptomyces sendaiensis, Enterococcus faecalis, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes, methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium and vancomycin-resistant Staphylococcus aureus. As a result, it can be seen that the Romo1-derived antimicrobial peptide of the present invention exhibits a broad-spectrum antibacterial activity against all Gram-positive bacteria, Gram-negative bacteria and multidrug-resistant bacteria (see Example 2).

[0071] Furthermore, in order to identify peptides with better antibacterial activity, the present inventors classified the amino acids constituting KU-5878 into X 1 X 1 MMX 1 X 1 GGX 1 FGX 1FMAIGMGIR (SEQ ID NO: 16), KTX 2 X 2 QSGGTFGTFX 2 AX 2 GX 2 GX 2 R(SEQ ID NO: 30), KTMMQSGGTX 3 GTX 3 MAIGMGIR (SEQ ID NO: 40), and KTMMQSX 4 X 4 TFX 4 TFMX 4 IX 4 MX 4 IR (SEQ ID NO: 45), and synthesized amino acid X 1 To X 4 Each of the peptides was replaced and its antibacterial activity was evaluated.

[0072] In one embodiment of the present invention, the inventors examined whether the antibacterial activity of KU-5878 peptide would be changed when the hydrophilic amino acid residues in KU-5878 peptide were replaced by positively charged amino acids. In KU-5878 peptide, the positions of the hydrophilic amino acid residues are indicated by X in SEQ ID NO: 16. 1 Mark, and X 1 The amino acids at the positions are replaced independently. It has been determined that when the hydrophilic amino acid in the α-helix 2 region of the Romo1 protein is replaced by a positively charged amino acid (R, H or K) or another hydrophilic amino acid (T, S, Q or N), its antibacterial activity changes. As a result, it can be seen that the antibacterial activity of the peptide is maintained, or a better antibacterial activity is exhibited (see Example 4-1).

[0073] In another specific example of the present invention, the present inventors examined whether the antibacterial activity of the KU-5878 peptide would be changed when methionine (M) or isoleucine (I) in the KU-5878 peptide was replaced by a hydrophobic amino acid residue (M, L, I, V or Nle) having a structure similar thereto, and when methionine (M) or isoleucine (I) in the KU-5878 peptide was replaced by a hydrophobic amino acid residue (F, Y or W) having a structure different therefrom. In the KU-5878 peptide, methionine (M) and isoleucine (I) are replaced by X in SEQ ID NO: 30. 2 Mark, and X 2The amino acids at the positions are replaced independently. As a result, when methionine is replaced by norleucine (Nle), the antibacterial activity of the peptide is maintained, while when isoleucine is replaced by tryptophan (W), the antibacterial activity of the peptide is reduced. On the other hand, it was determined that when isoleucine is replaced by glycine, a peptide with improved antibacterial activity can be obtained (see Example 4-3).

[0074] In another specific example of the present invention, the inventors examined whether the antibacterial activity of KU-5878 peptide would be changed when the hydrophobic residue phenylalanine (F) in the KU-5878 peptide was replaced by a hydrophilic amino acid or a hydrophobic amino acid having a structure similar thereto (F, Y or W) or a hydrophobic amino acid having a structure different therefrom (M, L, I, V or Nle). In the KU-5878 peptide, phenylalanine is replaced by X in SEQ ID NO: 40. 3 Mark, and X 3 The amino acids at the positions were each independently replaced. As a result, it was determined that when phenylalanine was replaced with tryptophan (W), a peptide having improved antibacterial activity could be obtained (see Example 4-4).

[0075] In another specific example of the present invention, the inventors examined whether the antibacterial activity of KU-5878 would be changed when alanine (A) or glycine (G) in KU-5878 was replaced by glycine or alanine. In the KU-5878 peptide, alanine and glycine are replaced by X in SEQ ID NO: 45. 4 Mark, and X 4 The amino acids at the positions were each independently replaced. As a result, it was determined that when alanine was replaced by glycine, and when glycine was replaced by alanine, the peptide exhibited antibacterial activity, but compared to the KU-5878 peptide, it exhibited reduced antibacterial activity (see Examples 4-5).

[0076] In summary, the Romo1-derived peptide of the present invention comprises a sequence of 7 to 28 consecutive amino acids starting from any amino acid selected from the 52nd to 79th amino acid region of Romo1, and may comprise at least one amino acid substitution (within the range that it exhibits antibacterial activity).

[0077] In a specific example of the present invention, the inventors selected KU-5878-K4 as a representative to determine the application range of KU-5878 variants, and the inventors evaluated the antibacterial activity of KU-5878-K4 against a variety of bacteria. As a result, it can be seen that, similar to KU-5878, KU-5878-K4 also exhibits broad-spectrum antibacterial activity against all Gram-positive bacteria, Gram-negative bacteria and multidrug-resistant bacteria (see Example 4-2).

[0078] In addition, through some specific examples, the inventors can see that even though the peptides obtained by replacing some or all of the amino acids constituting the corresponding KU-5878 and KU-5878-K4 with D-amino acids exhibited antibacterial activity similar to that of KU-5878, and particularly in the presence of bovine serum, KU-5878 and KU-5878-K4 exhibited significantly reduced antibacterial activity, the reduction in the antibacterial activity of the variants obtained by replacing with D-amino acids was small (see Examples 4-6).

[0079] In summary, the amino acids constituting the Romo1-derived peptide of the present invention may each independently be an L- or D-amino acid, and each amino acid may be an amino acid analog, a radioactively labeled amino acid, or a fluorescently labeled amino acid.

[0080] Meanwhile, a portion of the amino acid sequence of the Romo1-derived peptide of the present invention can be selected and modified at the amino (N-) terminal or the carboxyl (C-) terminal to improve its activity. Through this modification, the peptide of the present invention can have an increased half-life when administered in vivo.

[0081] By way of a specific example, the present inventors determined that a KU-5878 variant amidated at the C-terminus had improved antibacterial activity (see Example 5).

[0082] Therefore, a protecting group, such as acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl or polyethylene glycol (PEG) can be attached to the amino terminus of the Romo1-derived peptide of the present invention, and the carboxyl terminus of the peptide can be substituted with a hydroxyl (-OH), amino (-NH 2 ), azido (-NHNH 2 ) and the like. In addition, fatty acids, oligosaccharide chains, any nanoparticles (gold particles, liposomes, heparin, hydrogels, etc.), amino acids, carrier proteins, etc. can be bound to the end of the peptide of the present invention or the R-group of the amino acid. The above-mentioned amino acid modifications play a role in improving the efficacy and stability of the peptide of the present invention. In this specification, the term "stability" refers not only to in vivo stability, but also to storage stability (including storage stability during room temperature storage, refrigerated storage or frozen storage).

[0083] Meanwhile, the Romo1-derived antimicrobial peptide of the present invention can be a peptide in which one or more amino acids constituting the peptide are missing. According to the foregoing, it can be determined that the peptide obtained by replacing, modifying or missing one or more amino acids constituting the Romo1-derived peptide of the present invention also exhibits antimicrobial activity. Therefore, the present invention can provide Romo1-derived peptide variants as antimicrobial peptides.

[0084] In the present invention, the term "variant" refers to a peptide in which one or more of the amino acids constituting the Romo1-derived peptide is replaced, modified and / or deleted (within the range in which it exhibits antibacterial activity). As long as the variant exhibits antibacterial activity, it is considered that the variant exhibits better antibacterial activity than the Romo1-derived peptide of the present invention, and is not particularly limited.

[0085] The antimicrobial peptide of the present invention may consist of the amino acid sequence of SEQ ID NO: 13, or a sequence comprising at least one amino acid substitution selected from the following (a) to (h) relative to the amino acid sequence of SEQ ID NO: 13.

[0086] (a) K to R substitution at amino acid position 1, (b) T to K or R substitution at amino acid position 2, (c) Q to K or R substitution at amino acid position 5, (d) S to K, R or H substitution at amino acid position 6, (e) T to K or R substitution at amino acid position 9, (f) F to W substitution at amino acid position 10, (g) I to L substitution at amino acid position 16, and (h) I to G or L substitution at amino acid position 20. According to Examples 8 and 9, the present inventors determined that when one or more amino acids in the amino acid sequence of KU-5878 were substituted by any one of the above (a) to (h), the antibacterial effect of the peptide was improved.

[0087] In another embodiment, the antimicrobial peptide can be a peptide in which at least one methionine is replaced by norleucine or isoleucine. Norleucine has the same structure as methionine except that sulfur (S) is replaced by carbon, and isoleucine has the same alkyl substituent as the alkyl substituent of norleucine in common in that it has an alkyl substituent with the same number of carbon atoms. The inventors determined that when 1 to 4 of the 4 methionines in the KU-5878 amino acid sequence were replaced by norleucine, the antimicrobial activity of the peptide was improved (see Table 13).

[0088] In another embodiment, the antimicrobial peptide may be a peptide in which any one of the amino acids at positions 3, 4, 5, 7, 8, 9, 11, 17, 18, and 19 is deleted, or an amino acid at position 3 or 4 and an amino acid at position 7 or 8 are deleted. According to Examples 6 and 7 of the present application, when any one of the amino acids at positions 3, 4, 5, 7, 8, 9, 11, 17, 18, and 19 in KU-5878 is deleted, the antibacterial activity of the peptide is improved, and the same effect can be expected for peptides in which some amino acids of KU-5878 are replaced.

[0089] In another embodiment, the antimicrobial peptide may further comprise an amino acid sequence linked to its C-terminus, wherein the amino acid sequence linked to the C-terminus may consist of one to three repeated sequences of R or K. According to Example 10, the present inventors determined that when an amino acid sequence consisting of RRR or KK was additionally added to the C-terminus of KU-5878, the antibacterial activity of the peptide was improved.

[0090] On the other hand, an antimicrobial peptide is provided, which comprises: the amino acid sequence of SEQ ID NO: 24; an amino acid sequence in which K at amino acid position 1 in the amino acid sequence of SEQ ID NO: 24 is substituted to T or T at amino acid position 12 is substituted to G; or an amino acid sequence in which T at amino acid position 2 in the amino acid sequence of SEQ ID NO: 24 is substituted.

[0091] According to one embodiment, the antimicrobial peptide may be a peptide in which at least one methionine is replaced by norleucine or isoleucine.

[0092] According to one embodiment, the antimicrobial peptide may further comprise any one modification selected from the following (a) to (s).

[0093] (a) deletion of any one of the amino acids at positions 1 to 12, 14 to 15, and 17 to 21; (b) deletion of the amino acid at position 14, and deletion of the amino acid at any one of positions 1 to 8, 12, 15, and 18; (c) deletion of the amino acid at position 14, deletion of the amino acid at position 18, and deletion of the amino acid at any one of positions 1 to 8, 12, and 15; (d) deletion of the amino acid at position 14, deletion of the amino acid at position 15, and deletion of the amino acid at position 1, 2, or 12; (e) deletion of the amino acid at position 1 or 2, deletion of the amino acid at position 3 or 4, and deletion of the amino acid at position 18; (f) deletion of the amino acid at position 3, 4, 14, and (g) a deletion of the amino acid at position 3 or 4, a deletion of the amino acid at position 14, a deletion of the amino acid at position 18, and a deletion of the amino acid at position 5, 6, 7, 8, 12 or 15; (h) a deletion of the amino acid at position 14, a deletion of the amino acid at position 15, a deletion of the amino acid at position 1 or 2, and a deletion of the amino acid at position 3, 4, 7 or 8; (i) a deletion of the amino acid at position 1, 2, 5 or 6, a deletion of the amino acid at position 7, 8 or 11, a deletion of the amino acid at position 14 or 15, and a deletion of the amino acid at position 18; (j) a deletion of the amino acid at position 5 or 6, a deletion of the amino acid at position 11, a deletion of the amino acid at position 15 (k) a deletion of an amino acid at position 1 or 2, a deletion of an amino acid at position 7 or 8, and a deletion of amino acids at positions 12, 14, and 15; (l) a deletion of an amino acid at position 1, 2, 5, or 6, a deletion of amino acids at positions 3 and 4, and a deletion of amino acids at positions 14 and 18; (m) a deletion of amino acids at positions 3, 4, 14, and 18, a deletion of an amino acid at position 5 or 6, and a deletion of amino acids at any one of positions 7 or 8, 12, and 15; (n) a deletion of an amino acid at position 1 or 2, a deletion of an amino acid at position 7 or 8, and a deletion of amino acids at positions 12, 14, and 15. (o) a deletion of an amino acid at position 3, 4, 14 and 18, a deletion of an amino acid at position 5 or 6, and a deletion of two amino acids selected from the amino acids at positions 7 or 8, 12 and 15; (p) a deletion of an amino acid at position 1 or 2, a deletion of an amino acid at position 5 or 6, a deletion of an amino acid at position 7 or 8, and a deletion of amino acids at positions 12, 14, 15 and 18; (q) a deletion of an amino acid at position 3, 4, 14 and 18, a deletion of an amino acid at position 5 or 6, and a deletion of three amino acids selected from the amino acids at positions 1 or 2, 7 or 8, 12 and 15;(r) a deletion of the amino acid at position 1 or 2, a deletion of the amino acid at position 3 or 4, a deletion of the amino acid at position 5 or 6, a deletion of the amino acid at position 7 or 8, and a deletion of the amino acids at positions 12, 14, 15, and 18; and (s) a deletion of the amino acid at position 1 or 2, a deletion of the amino acids at positions 3 and 4, a deletion of the amino acid at position 5 or 6, a deletion of the amino acid at position 7 or 8, and a deletion of the amino acids at positions 12, 14, 15, and 18.;

[0094] According to one embodiment, the antimicrobial peptide may be a peptide in which the amino acid pair at positions 10 and 14 or the amino acid pair at positions 14 and 18 are stapled together. The stapled peptide may be achieved by replacing the amino acid pair with pentenylalanine or octenylalanine and cross-linking the amino acids.

[0095] According to one embodiment, the antimicrobial peptide may further comprise an amino acid sequence linked to its C-terminus, wherein the amino acid sequence linked to the C-terminus may consist of one to three repeated sequences of R or K.

[0096] The Romo1-derived peptide and its variant according to the present invention have antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and multidrug-resistant bacteria. Therefore, the present invention can provide a pharmaceutical composition for preventing or treating bacterial infectious diseases, which comprises an antimicrobial peptide as an active ingredient.

[0097] Non-limiting examples of Gram-positive bacteria whose proliferation and / or growth is inhibited by the Romo1-derived peptide and variants thereof according to the present invention include Staphylococcus, Bacillus, Enterococcus, Streptomyces and Streptococcus. Preferably, the Gram-positive bacteria may be Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, Streptomyces sendaiensis, Enterococcus faecalis and / or Streptococcus pneumoniae.

[0098] In addition, non-limiting examples of Gram-negative bacteria whose proliferation and / or growth is inhibited by the Romo1-derived peptide and variants thereof according to the present invention include Escherichia, Klebsiella, Acinetobacter, Pseudomonas and Enterobacter. Preferably, the Gram-negative bacteria may be Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and / or Enterobacter aerogenes.

[0099] In addition, the Romo1-derived peptide and its variant according to the present invention have antibacterial activity against multidrug-resistant bacteria. Therefore, the multidrug-resistant bacteria whose proliferation and / or growth is inhibited by the Romo1-derived peptide and its variant can be the above-mentioned Gram-positive bacteria or Gram-negative bacteria, which are resistant to one or more antibiotics belonging to penicillins, carbapenems, cephalosporins, quinolones, macrolides, tetracyclines or glycopeptides. Preferably, the multidrug-resistant bacteria can be methicillin-resistant Staphylococcus, multidrug-resistant Pseudomonas, vancomycin-resistant Enterococcus, multidrug-resistant Klebsiella, multidrug-resistant Acinetobacter and / or vancomycin-resistant Staphylococcus.

[0100] The present invention can provide a pharmaceutical composition (i.e., an antibiotic) comprising a Romo1-derived peptide and / or a variant thereof as an active ingredient. The "bacterial infectious disease" to be prevented or treated by the pharmaceutical composition of the present invention may be a disease caused by Gram-positive bacteria, Gram-negative bacteria and / or multidrug-resistant bacterial infection. Preferably, the bacterial infectious disease may be a skin infection, food poisoning, otitis media, cystitis, peritonitis, urinary tract infection, mastitis, pneumonia, endocarditis, conjunctivitis, arthritis, endometritis, bacteremia, sepsis and / or acne caused by bacterial infection. More preferably, the bacterial infectious disease may be sepsis or pneumonia.

[0101] The present invention also provides a method for preventing or treating a bacterial infectious disease, comprising the step of administering a Romo1-derived peptide and / or a variant thereof to a subject.

[0102] In the present invention, the "subject" is not limited as long as it is a mammal. Preferably, the subject may be a human or a livestock.

[0103] In the present invention, the term "prevention" means any effect of delaying infection by Gram-positive bacteria, Gram-negative bacteria and / or multidrug-resistant bacteria or delaying the onset of a disease caused by infection by administering a pharmaceutical composition according to the present invention. The term "treatment" means any effect of alleviating or beneficially changing the symptoms of bacterial infection by administering a pharmaceutical composition according to the present invention.

[0104] In the present invention, the pharmaceutical composition may further include one or more known antibiotics in addition to the Romo1-derived peptide and / or its variants, and may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

[0105] In the present invention, "carrier" is also called a carrier and refers to a compound that promotes the incorporation of proteins or peptides into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that promotes the uptake of many organic compounds into cells or tissues of organisms.

[0106] In the present invention, "diluent" is defined as a compound diluted in water that will dissolve the target protein or peptide, as well as stabilize the biologically active form of the protein or peptide. In the art, salts dissolved in buffered solutions are used as diluents. A commonly used buffered solution is phosphate buffered saline because it simulates the salt conditions of human solutions. Since buffer salts can control the pH of a solution at low concentrations, buffered diluents rarely change the biological activity of the compound. Compounds including azelaic acid as used herein can be administered to human patients as is or as a pharmaceutical composition mixed with other ingredients or with a suitable carrier or excipient (such as in combination therapy).

[0107] In addition, the pharmaceutical composition comprising Romo1-derived peptide and / or its variant as an active ingredient for preventing or treating bacterial infectious diseases according to the present invention can be prepared and used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, external preparations such as aerosols and sterile injection solutions according to respective conventional methods. The antibacterial composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) according to the desired method. Its dosage varies depending on the patient's condition and body weight, the severity of the disease, the form of the drug, and the route and duration of administration, but can be appropriately selected by those skilled in the art. For example, the antibacterial composition can be administered in an amount of about 0.001 mg to 1000 mg in combination with a pharmaceutically acceptable carrier. The antibacterial composition of the present invention can be administered once or several times a day as needed, and can be used alone or in combination with surgery, hormone therapy, drug therapy, and methods using biological response modifiers.

[0108] In addition, the Romo1-derived peptide and / or its variant according to the present invention can provide a quasi-drug composition for the purpose of preventing or alleviating bacterial infectious diseases. The quasi-drug composition of the present invention can be used with other quasi-drugs or quasi-drug ingredients, and it can be used appropriately according to conventional methods. The quasi-drug composition can be used for, but is not limited to, disinfectant cleaners, bath foams, mouthwashes, wet wipes, detergent soaps, hand sanitizers, humidifier fillers, facial masks, ointments, or filter fillers.

[0109] In addition, the Romo1-derived peptide and / or its variant according to the present invention can be provided in the form of a cosmetic composition. The preparation of the cosmetic composition according to the present invention can be in the form of a skin lotion, a skin softener, a firming agent, a astringent, a lotion, a lotion lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, an essence, a nourishing essence, a peel-off mask, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, or a body cleanser. If necessary, the cosmetic composition of the present invention can be combined with other ingredients commonly used in cosmetics in addition to the above essential ingredients.

[0110] In addition, the present invention provides a food composition or feed composition containing a Romo1-derived peptide and / or its variant as an active ingredient. When the Romo1-derived peptide and / or its variant according to the present invention is used as an additive for food or feed, the Romo1-derived peptide and / or its variant can be added as is or used together with other foods, feeds or their components, and can be used appropriately according to conventional methods. The content of the active ingredient in the composition can be appropriately determined according to the intended use (preventive or therapeutic treatment of infectious diseases, or health treatment by inhibiting bacterial proliferation and growth). In general, in the production of feed, food or beverage, the amount of the Romo1-derived peptide and / or its variant added according to the present invention is 15wt% or less, preferably 10wt% or less, based on the total weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the lower limit of the above range, and since there is no problem in terms of safety, the amount of the active ingredient used may be greater than the upper limit of the above range. There is no particular restriction on the type of food and feed.

[0111] In the present invention, according to the IUPAC-IUB nomenclature, amino acids are abbreviated as follows.

[0112] Arginine (Arg, R), lysine (Lys, K), histidine (His, H), serine (Ser, S), threonine (Thr, T), glutamine (Gln, Q), asparagine (Asp, N), methionine (Met, M), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), alanine (Ala, A), glycine (Gly, G), proline (Pro, P), cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E) and norleucine (Nle).

[0113] Although the present invention can be modified differently and has multiple embodiments, some specific embodiments are shown in the drawings and described in detail in the specific embodiments. However, it should be understood that the present invention is not limited to the specific embodiments, and includes all changes, equivalents and alternatives included in the spirit and scope of the present invention. In the following description, when the relevant known technology may make the subject of the present invention obscure, its detailed description will be omitted.

[0114] [Example]

[0115] Example 1. Evaluation of the antibacterial activity of Romo1-derived peptides and identification of the best peptide

[0116] Romo1 protein contains two α-helices. The inventors synthesized an α-helix 2 region having both hydrophilicity and hydrophobicity, and the inventors used the minimum bactericidal concentration (MBC) assay to evaluate its antibacterial activity. The α-helix 2 region was named KU-5279 (amino acid region 52 to 79 of Romo1 protein). Then, in order to identify peptides that are beneficial to human application and have excellent antibacterial activity, the inventors synthesized several peptides containing a continuous amino acid sequence starting from any amino acid selected from the amino acid region 52 to 79 of Romo1 protein, and the antibacterial activity of the peptides was comparatively evaluated by the minimum bactericidal concentration assay (see Table 1 below for information on each peptide). The evaluation of the antibacterial activity of each peptide by the minimum bactericidal concentration assay was specifically carried out as follows.

[0117] First, Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 29213) were each cultured in 3% (w / v) TSB (Tryptic Soy Broth) liquid medium at 37°C and 200 rpm for 4 hours, and then further cultured for 3 hours under the same conditions to 5×10 6 Each further cultured strain was diluted to 2 × 10 CFU / ml with SP (sodium phosphate; 10 mM sodium phosphate, 1% TSB) buffer. 5 The strain solution was prepared with a final concentration of CFU / ml.

[0118] Then, different concentrations (0 μg / ml to 300 μg / ml) of each peptide were dispensed into each well of a 96-well microplate, and 100 μl (1 × 10 5 CFU / ml) of each prepared strain solution, mixed therewith, and allowed to react with the peptide in an incubator at 37°C for 1 hour.

[0119] In addition, 30 g / L of TS (tryptic soy) and 15 g / L of agar were dissolved in distilled water and sterilized, and then 25 ml of the solution was poured on a 100 mm circular plate and allowed to solidify at room temperature for one day or more to prepare a TS agar plate. 10 μl of each reaction solution of the peptide and strain was spread on a TS agar plate at a constant size and incubated in an incubator at 37 ° C for 18 hours, and then the colony formation on the plate was checked. The minimum bactericidal concentration was defined as the lowest peptide concentration at which no colonies were formed, and the experimental results are shown in Table 1 below.

[0120] [Table 1]

[0121]

[0122]

[0123] As shown in Table 1, pCM19 and KU-5279 peptides showed similar bactericidal activities, but pCM12 had low bactericidal activity, and thus its antibacterial activity could not be measured using the minimum bactericidal concentration assay. Meanwhile, it was determined that the KU-5878 peptide consisting of 21 amino acid residues had the best antibacterial activity, and it was determined that the antibacterial activity of KU-5878 was three times that of KU-5279.

[0124] Example 2. Evaluation of the antibacterial activity of KU-5878

[0125] 2-1. Evaluation of antibacterial activity against various bacteria

[0126] In order to identify the following bacterial types: the peptides comprising the 52nd to 79th amino acid region of the Romo1 protein or a portion of the region and determined to have antibacterial activity in Example 1 showed bactericidal activity against the bacterial types, KU-5878 was selected as a representative of the synthetic peptides shown in Table 1 above, and its antibacterial activity against the bacteria shown in Table 2 below was measured using the minimum bactericidal concentration assay. The minimum bactericidal concentration assay is the same as the method used in Example 1.

[0127] Specifically, each type of bacteria was cultured in 3% (w / v) TSB (Tryptic Soy Broth) liquid medium at 37°C and 200 rpm for 4 hours, and then further cultured under the same conditions for 3 hours to 5 × 10 6 CFU / ml concentration. Each further cultured strain was diluted to 2 × 10 5 The strain solution was prepared with a final concentration of CFU / ml.

[0128] Then, different concentrations (0 μg / ml to 300 μg / ml) of KU-5878 peptide were dispensed into each well of a 96-well microplate, and 100 μl (1×10 5 CFU / ml) of each prepared strain solution, mixed therewith, and allowed to react with the peptide in an incubator at 37°C for 1 hour.

[0129] In addition, 10 μl of each reaction solution of the peptide and strain was plated on a TS agar plate at a constant size and incubated in an incubator at 37°C for 18 hours, and then the colony formation on the plate was examined. The minimum bactericidal concentration was defined as the lowest peptide concentration at which no colonies were formed, and the experimental results are shown in Table 2 below.

[0130] Meanwhile, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Acinetobacter, and multidrug-resistant Klebsiella in Table 2 below are bacteria isolated from patients at Korea University Anam Hospital. It was determined that multidrug-resistant Pseudomonas aeruginosa was resistant to piperacillin, piperacillin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, and ciprofloxacin antibiotics, multidrug-resistant Acinetobacter was resistant to piperacillin, piperacillin-tazobactam, ceftazidime, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, and cefepime antibiotics, and multidrug-resistant Klebsiella was resistant to piperacillin-tazobactam, ceftazidime, cefepime, imipenem, gentamicin, and ciprofloxacin antibiotics. Then, these bacteria were used in the experiment.

[0131] [Table 2]

[0132]

[0133]

[0134] As shown in Table 2 above, it can be seen that KU-5878 exhibits broad-spectrum antibacterial activity against all Gram-positive bacteria, Gram-negative bacteria, and multidrug-resistant bacteria.

[0135] 2-2. Comparative evaluation of the antibacterial activity of KU-5878 and known antimicrobial peptides

[0136] In order to determine whether the KU-5878 peptide, which was determined to have antibacterial activity in vitro, effectively exhibits bactericidal activity against bacteria in vivo (especially in the blood vessels of animals), Staphylococcus aureus, which is a Gram-positive bacterium, Pseudomonas aeruginosa, which is a Gram-negative bacterium, methicillin-resistant Staphylococcus aureus, and multidrug-resistant Pseudomonas aeruginosa, which are multidrug-resistant bacteria, were selected as representatives, and the following experiments were then performed.

[0137] Specifically, Staphylococcus aureus, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and multidrug-resistant Pseudomonas aeruginosa were cultured in 3% (w / v) TSB liquid medium at 37°C and 200 rpm for 4 h, and then further cultured under the same conditions for 3 h to 5 × 10 9 CFU / ml concentration. Each further cultured strain was diluted to 1 × 10 5 The strain solution was prepared with a final concentration of CFU / μl.

[0138] Next, 100 μl of each strain solution was injected intravenously (IV) into the tail vein of each 10-week-old male mouse (C57BL / 6). One hour later, KU-5878 was injected intravenously at a concentration of 10 mg / kg. As a control, PBS was injected intravenously at a concentration of 10 mg / kg, and as a positive control, LL-37, magainin 2, or daptomycin was injected intravenously at a concentration of 10 mg / kg.

[0139] 40 minutes after the intravenous injection of each drug, about 1 cm was cut from the end of the tail of each mouse using surgical scissors, and 10 μl of blood was collected at the cut end and diluted in PBS. The diluted blood sample was spread on the previously prepared TSB agar plate and incubated in an incubator at 37°C for 18 hours, and the colonies formed were counted for comparison.

[0140] As a result, Figure 1 and 2 As shown in , it can be seen that the number of bacteria remaining in the blood vessels of mice is reduced by intravenous injection of the drug, and the reduction in the number of bacteria is most significant when KU-5878 is administered. Therefore, it can be seen that KU-5878 exhibits significantly better antibacterial activity than LL-37, magainin 2, and daptomycin, which are known to have antibacterial activity.

[0141] 2-3. Comparative evaluation of the antibacterial activity of KU-5878 and known antibiotics

[0142] According to Example 2-2, in order to evaluate the antibacterial activity of KU-5878 peptide in vivo, Staphylococcus aureus was selected as Gram-positive bacteria, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii were selected as Gram-negative bacteria, and multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, multidrug-resistant Klebsiella pneumoniae and multidrug-resistant Acinetobacter baumannii were selected as multidrug-resistant bacteria, and the following experiments were performed.

[0143] The experimental method is as described in Example 2-2 above. However, the final concentration of each strain administered to mice is shown in Table 3 below. For the positive group, each type of bacteria was injected into mice, and 30 mg / kg of imipenem (a carbapenem-based antibiotic) was injected intraperitoneally (IP) 24 hours later, and then the same amount of imipenem was intraperitoneally administered 4 times at 12-hour intervals. The survival rate of each control group and experimental group was measured at 24-hour intervals.

[0144] [Table 3]

[0145] bacteria Final concentration (CFU) Staphylococcus aureus <![CDATA[1×10 8 ]]> Pseudomonas aeruginosa <![CDATA[2×10 7 ]]> Klebsiella pneumoniae <![CDATA[8×10 8 ]]> Acinetobacter baumannii <![CDATA[4×10 8 ]]> Methicillin-resistant Staphylococcus aureus <![CDATA[3×10 8 ]]> Multidrug-resistant Pseudomonas aeruginosa <![CDATA[8×10 7 ]]> Multidrug-resistant Acinetobacter baumannii <![CDATA[5×10 8 ]]> Multidrug-resistant Klebsiella pneumoniae <![CDATA[8×10 8 ]]>

[0146] As a result, Figure 3As shown in , the survival rate of mice injected with Gram-positive or Gram-negative bacteria alone was 0 to 20%, the survival rate of mice injected with KU-5878 was 60%, and the survival rate of mice injected with imipenem was about 60 to 80%. In addition, as shown in Figure 4 As can be seen in the figure, mice injected with the multidrug-resistant bacteria alone showed a survival rate of 0 to 7%, mice injected with KU-5878 showed a survival rate of 60 to 67%, and mice injected with imipenem showed a survival rate of 7 to 27%.

[0147] Example 3. Toxicity evaluation of KU-5878

[0148] In order to determine whether the peptides comprising the amino acid region 52 to 79 of the Romo1 protein or a portion of the region and determined in Examples 1 and 2 to have bactericidal activity against Gram-positive, Gram-negative and multidrug-resistant bacteria are suitable for use in subjects, the toxicity of the peptides was measured. In the following experiments, KU-5878 was selected as a representative of the synthetic peptides shown in Table 1, and the toxicity of KU-5878 was evaluated.

[0149] 3-1. Evaluation of toxicity to human vascular endothelial cells (HUVEC)

[0150] Human vascular endothelial cells (HUVEC) were cultured in a 96-well microplate, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was dissolved in PBS at a concentration of 2 mg / ml and then sterile filtered through a 0.2 μm membrane filter. PBS was used as a control.

[0151] HUMEC culture medium was replaced with fresh culture medium, and KU-5878, melittin or magainin 2 were distributed at different concentrations (0 μg / ml to 300 μg / ml). Each well was then treated with 100 μl of MTT reagent and incubated at 37°C for 2 hours. The culture medium and peptide were then carefully removed to avoid separating the cells, and 100 μl of DMSO (dimethyl sulfoxide) was added to each well, and the absorbance at a wavelength of 570 nm was measured using a spectrophotometer. Relative cell viability was calculated as the percentage of viability of untreated control cells.

[0152] As a result, Figure 5 As shown in , it can be seen that KU-5878 showed very low toxicity to HUVEC.

[0153] 3-2. Evaluation of toxicity to red blood cells

[0154] Red blood cells were diluted with PBS, centrifuged 3 times at 900g for 10 minutes, and then washed. 100 μl of diluted red blood cell solution (10% v / v PBS) was distributed to each well of a 96-well microplate, and KU-5878, KU-5878-K4, KU-5878-K4-D, bee venom peptide, magainin 2 or daptomycin was added to each well, then filled to 200 μl with PBS, and then reacted by incubating at 37°C for 1 hour. Then, the supernatant of the reaction solution was separated, and the absorbance at a wavelength of 550 nm was measured using a spectrophotometer. As a control, PBS and 0.1% Triton X-100 (Triton X-100) solution were used.

[0155] Based on the absorbance measurement results, the extent of red blood cell destruction was calculated according to the following equation 1, and the calculation results were Figure 6 Shown in.

[0156] [Equation 1]

[0157] Intact RBC% = 100 (%) - [(absorbance of the red blood cell solution treated with peptide - absorbance of the red blood cell solution treated with PBS) / (absorbance of the red blood cell solution treated with Triton X-100 - absorbance of the red blood cell solution treated with PBS) × 100 (%)]

[0158] As a result, Figure 6 As shown in , it was determined that KU-5878 showed almost no hemolysis even at high concentrations. Similar to KU-5878, KU-5878-K4 and KU-5878-K4-D also showed almost no hemolysis of erythrocytes compared with magainin 2, daptomycin, and melittin, indicating that they have low toxicity to erythrocytes.

[0159] 3-3. Evaluation of in vivo toxicity

[0160] In order to evaluate whether the KU-5878 peptide, which was determined to be non-toxic in vitro, does not show toxicity to the subject in vivo, especially when injected into the blood vessels of animals (subjects), KU-5878 was intravenously injected into the tail vein of each 10-week-old male mouse (C57BL / 6) at a concentration of 100 mg / kg. At this time, the maximum volume of the injection dose was 150 μl. The behavior of the mice was observed within 1 hour after the injection, and then the change in the weight of the mice was measured at intervals of 24 hours for 12 days.

[0161] As a result, it was confirmed that all mice administered with KU-5878 survived, and KU-5878 had no effect on the weight of the mice. Figure 7 as shown in .

[0162] Example 4. Evaluation of antibacterial activity of peptides in which some of the amino acids constituting KU-5878 are replaced

[0163] 4-1. Evaluation of antibacterial activity of KU-5878 variants comprising the amino acid sequence of SEQ ID NO: 16

[0164] Next, in order to identify peptides with better antibacterial activity, the hydrophilic amino acid residues of KU-5878 were replaced with positively charged lysine (K) or arginine (R), and then the antibacterial activity of the variants was comparatively evaluated using the minimum bactericidal concentration assay. The experiment was performed in the same manner as in Example 1. Detailed information about KU-5878 variants and their MBC values ​​for each bacterium is shown in Table 4 below.

[0165] [Table 4]

[0166]

[0167]

[0168] As can be seen in Table 4, it can be seen that the peptides obtained by replacing the amino acid at position 59, 62, 63 or 66 of the Romo1 protein with lysine and the peptides obtained by replacing the amino acid at position 59 with arginine have better antibacterial activity than KU-5878. In addition, it can be determined that the KU-5878 variant obtained by replacing the amino acid at position 58 of the Romo1 protein with arginine also showed an antibacterial activity similar to that of KU-5878. From the above results, it can be seen that even when the hydrophilic amino acid in the α-helix 2 region of the Romo1 protein is replaced by a positively charged amino acid, particularly lysine (K) or arginine (R), the peptide can also exhibit similar antibacterial activity or better antibacterial activity.

[0169] From the above results, it can be seen that even when the hydrophilic amino acids in the α-helix 2 region of the Romo1 protein are replaced by positively charged amino acids, especially lysine (K) or arginine (R), the peptide can also exhibit similar or better antibacterial activity.

[0170] 4-2. Evaluation of the antibacterial activity of KU-5878-K4 against various bacteria

[0171] In order to identify the following bacterial types: the KU-5878 variants determined to have antibacterial activity in Example 4-1 showed bactericidal activity against the bacterial types, KU-5878-K4 was selected as a representative of the KU-5878 variants shown in Table 4 above, and the minimum bactericidal concentration assay was used to measure its antibacterial activity against the bacteria shown in Table 5 below. The minimum bactericidal concentration assay was performed in the same manner as in Example 1.

[0172] [Table 5]

[0173]

[0174] As can be seen in Table 5 above, it can be seen that KU-5878-K4 exhibits broad-spectrum antibacterial activity against all Gram-positive bacteria, Gram-negative bacteria, and multidrug-resistant bacteria.

[0175] 4-3. Evaluation of antibacterial activity of KU-5878 variants comprising the amino acid sequence of SEQ ID NO: 30

[0176] In Example 4-1, it was determined that even when the hydrophilic residues of KU-5878 were replaced with positively charged amino acids, its antibacterial activity was maintained or showed better antibacterial activity. Then, the hydrophobic methionine (M) or isoleucine (I) with a similar residue structure in KU-5878 was replaced with a hydrophilic amino acid or an amino acid with a different residue structure, and then the antibacterial activity of the variant was comparatively evaluated using the minimum bactericidal concentration assay. The experiment was performed in the same manner as in Example 1. Detailed information about KU-5878 variants and their MBC values ​​for each bacterium is shown in Table 6 below.

[0177] [Table 6]

[0178]

[0179] As can be seen in Table 6 above, when the hydrophobic methionine or isoleucine in KU-5878 with a similar residue structure is replaced by a hydrophilic amino acid, the antibacterial activity of the peptide is reduced. On the other hand, when methionine is replaced by norleucine (Nle) with a similar residue structure, the antibacterial activity of the peptide does not change significantly. In addition, it can be seen that when isoleucine is replaced by glycine (G), the antibacterial activity of the peptide is improved.

[0180] 4-4. Evaluation of antibacterial activity of KU-5878 variants comprising the amino acid sequence of SEQ ID NO: 40

[0181] Next, in order to identify KU-5878 variants having antibacterial activity comparable to or higher than that of KU-5878, the hydrophobic phenylalanine (F) in KU-5878 was replaced with a hydrophilic amino acid or an amino acid having a different residue structure, and then the antibacterial activity of the variant was comparatively evaluated using the minimum bactericidal concentration assay. The experiment was performed in the same manner as in Example 1. Detailed information about the KU-5878 variants and their MBC values ​​for each bacterium is shown in Table 7 below.

[0182] [Table 7]

[0183]

[0184] As a result, as can be seen in the above Table 7, it can be seen that when the hydrophobic phenylalanine at position 67 in KU-5878 is replaced with tryptophan having a similar residue structure, the antibacterial activity of the peptide is improved.

[0185] 4-5. Evaluation of antibacterial activity of KU-5878 variants comprising the amino acid sequence of SEQ ID NO: 45

[0186] After Example 4-4, alanine (A) or glycine (G) in KU-5878 was replaced with glycine or alanine, and then the antibacterial activity of the variant was comparatively evaluated using the minimum bactericidal concentration assay. The experiment was performed in the same manner as in Example 1. Detailed information about KU-5878 variants and their MBC values ​​for each bacterium is shown in Table 8 below.

[0187] [Table 8]

[0188]

[0189] As a result, as can be seen in the above Table 8, it can be seen that the peptides obtained by replacing alanine with glycine or glycine with alanine in KU-5878 showed antibacterial activity, but showed reduced antibacterial activity compared to the antibacterial activity of KU-5878.

[0190] 4-6. Wherein some or all of the amino acids constituting KU-5878 and KU-5878-K4 are replaced with D-amino acids Evaluation of the antimicrobial activity of peptides

[0191] After Examples 1-5, peptides (KU-5878-D(all) and KU-5878-K4-D) were synthesized by replacing the amino acids constituting KU-5878 and KU-5878-K4 with D-amino acids, and their antibacterial activities were comparatively evaluated. KU-5878-K4-D is a peptide obtained by replacing the lysine amino acid residues at positions 59, 62, 63, and 66 in KU-5878-K4 with D-amino acids, while KU-5878-D(all) is a peptide obtained by replacing all the amino acids of KU-5878 with D-amino acids.

[0192] The maximum bactericidal concentration of each peptide was measured in the same manner as in Example 1 in the presence or absence of bovine serum, and the results are shown in Table 9 below.

[0193] [Table 9]

[0194]

[0195] As can be seen in Table 9 above, it can be seen that in the antibacterial activity experiment conducted in the presence of bovine serum, KU-5878 and KU-5878-K4 have reduced antibacterial activity, but the reduction in the antibacterial activity of KU-5878-K4-D is very small compared to that of KU-5878-K4.

[0196] Example 5. Evaluation of the antibacterial activity of peptides in which some of the amino acids constituting KU-5878 are modified

[0197] In order to identify peptides with better antibacterial activity and higher stability than KU-5878, the C-terminus of KU-5878 was amidated, and then the antibacterial activity of the peptides was comparatively evaluated using the minimum bactericidal concentration assay. The experiment was performed in the same manner as in Example 1. Detailed information about KU-5878 variants and their MBC values ​​for each bacterium is shown in Table 10 below.

[0198] [Table 10]

[0199]

[0200] As shown in the above Table 10, it can be seen that the peptides obtained by modifying the termini of KU-5878 have improved antibacterial activity.

[0201] Example 6. Evaluation of the antibacterial activity of peptides obtained by deleting one amino acid from KU-5878

[0202] Variants were prepared by deleting one amino acid from the amino acid sequence of KU-5878 (SEQ ID NO: 13), and their antibacterial activity was evaluated. The evaluation results of the antibacterial activity are shown in Table 11 below.

[0203] As a result, regarding the amino acid sequence of KU05878 consisting of amino acids 58 to 78, deletion of an amino acid at position 60, 61, 62, 64, 65, 66, 68, 74, 75 or 76 resulted in an improvement in antibacterial activity. Considering that the same amino acids are consecutive, deletion of M at position 61 is the same as deletion of M at position 60, and deletion of G at position 65 is the same as deletion of G at position 64.

[0204] With respect to SEQ ID NO: 13, deletion of the amino acid at position 3, 4, 5, 7, 8, 9, 11, 17, 18 or 19 results in an increase in antibacterial activity.

[0205] [Table 11]

[0206]

[0207]

[0208] Example 7. Evaluation of antibacterial activity of peptides obtained by deleting two or more amino acids from KU-5878

[0209] Variants were prepared by deleting two or more amino acids from the amino acid sequence of KU-5878 (SEQ ID NO: 13), and their antibacterial activity was evaluated. The evaluation results of the antibacterial activity are shown in Table 12 below.

[0210] In most variants, the antibacterial activity was reduced, except for the variant in which the M at position 60 and the G at position 64 were deleted. Considering that the same amino acids are consecutive, the deletion of M at position 60 is the same as the deletion of M at position 61, and the deletion of G at position 64 is the same as the deletion of G at position 65.

[0211] Regarding SEQ ID NO: 13, deletion of the amino acid at position 3 or 4 and deletion of the amino acid at position 7 or 8 resulted in an improvement in antibacterial activity.

[0212] [Table 12]

[0213]

[0214] Example 8. Evaluation of the antibacterial activity of peptides obtained by replacing one amino acid in KU-5878

[0215] One amino acid in the amino acid sequence of KU-5878 (SEQ ID NO: 13) was replaced, and the antibacterial activity of the peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 13 below.

[0216] With respect to SEQ ID NO:13, a K to R substitution at amino acid position 1, a T to K or R substitution at amino acid position 2, a Q to K or R substitution at amino acid position 5, an S to K, R or H substitution at amino acid position 6, a T to K or R substitution at amino acid position 9, a F to W substitution at amino acid position 10, an I to L substitution at amino acid position 16, or an I to G or L substitution at amino acid position 20 results in improved antibacterial activity.

[0217] In addition, referring to KU-5878-4Nle in Table 13 below, it was determined that the antibacterial activity of the peptide was improved by replacing all four methionines in KU-5878 with norleucine. This is probably because the structures of methionine and norleucine are identical to each other except that sulfur is changed to carbon.

[0218] [Table 13]

[0219]

[0220]

[0221]

[0222] Example 9. Evaluation of the antibacterial activity of multi-substituted peptides based on amino acid substitution at position 59 in KU-5878

[0223] Substitutions of Q62K, S63K, and T66K were sequentially added to the amino acid sequence of U-5878-T59K, and the antibacterial activity of the peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 14 below.

[0224] The substitution positions of T59K, Q62K, S63K and T66K correspond to the amino acids at positions 2, 5, 6 and 9 with respect to SEQ ID NO:13.

[0225] [Table 14]

[0226]

[0227] Example 10. Evaluation of the antibacterial activity of KU-5878 containing an amino acid sequence added to the C-terminus

[0228] The amino acid repeat sequence was added to the C-terminus of KU-5878, and the antibacterial activity of the peptide was evaluated. According to Table 15 below, when the R (arginine) repeat sequence or the K (lysine) sequence was added to the C-terminus of KU-5878, the antibacterial activity of the peptide was improved.

[0229] [Table 15]

[0230]

[0231] Example 11. Evaluation of the antibacterial activity of KU-5878-K4 variants

[0232] Based on KU-5878-K4 having T59K, Q62K, S63K and T66K modifications in the KU-5878 sequence, the changes in the antibacterial activity of KU-5878-K4 after amino acid modification were examined. KU-5878-K4 may be referred to as K4 hereinafter.

[0233] The sequence of K4 is shown in SEQ ID NO: 24. In the following Table 16, K4-K58T refers to a peptide in which the amino acid at position 1 in SEQ ID NO: 24 is replaced by T; K4-4MI refers to a peptide in which four methionines present in the K4 sequence are all replaced by isoleucine; and K4-T69G refers to a peptide in which the amino acid at position 12 in SEQ ID NO: 24 is replaced by G. These variants all have higher antibacterial activity than KU-5878.

[0234] [Table 16]

[0235]

[0236] Example 12. Antibacterial activity of K4 peptide in which one amino acid is missing

[0237] One amino acid was deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 17 below.

[0238] Referring to Table 17 below, peptides in which any one of the amino acids at positions 1 to 12, the amino acids at positions 14 and 15, and the amino acids at positions 17 to 21 of SEQ ID NO: 24 is missing have higher antibacterial activity than 5878.

[0239] [Table 17]

[0240]

[0241]

[0242] Example 13. Antibacterial activity of K4 peptide in which two amino acids are missing

[0243] Two amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 18 below.

[0244] Referring to Table 18 below, peptides in which the amino acid at position 14 in SEQ ID NO: 24 is deleted and any one of the amino acids at positions 1 to 8, 12, 15 and 18 is further deleted have higher antibacterial activity than 5878.

[0245] [Table 18]

[0246]

[0247] Example 14. Antibacterial activity of K4 peptide in which three amino acids are missing

[0248] Three amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptides was evaluated. The evaluation results of the antibacterial activity are shown in Table 19 below, and these peptides had higher antibacterial activity than 5878.

[0249] [Table 19]

[0250]

[0251] Example 15. Antibacterial activity of K4 peptide in which four amino acids are missing

[0252] Four amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 20 below.

[0253] [Table 20]

[0254]

[0255] Example 16. Antibacterial activity of K4 peptide in which five amino acids are missing

[0256] Five amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 21 below.

[0257] [Table 21]

[0258]

[0259] Example 17. Antibacterial activity of K4 peptide in which six amino acids are missing

[0260] Six amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 22 below.

[0261] [Table 22]

[0262]

[0263] Example 18. Antibacterial activity of K4 peptide in which seven amino acids are missing

[0264] Seven amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 23 below.

[0265] [Table 23]

[0266]

[0267] Example 19. Antibacterial activity of K4 peptide in which eight amino acids are missing

[0268] Eight amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 24 below.

[0269] [Table 24]

[0270]

[0271] Example 20. Antibacterial activity of K4 peptide in which nine amino acids are missing

[0272] Nine amino acids were deleted from the amino acid sequence of K4, and the antibacterial activity of the resulting peptide was evaluated. The evaluation results of the antibacterial activity are shown in Table 25 below.

[0273] [Table 25]

[0274]

[0275] Example 21. Evaluation of the antibacterial activity of PEGylated K4

[0276] K4 in which four amino acids were deleted and K4 in which eight amino acids were deleted were PEGylated, and their antibacterial activities were measured.

[0277] Referring to Table 26 below, it was confirmed that the antibacterial activity was slightly decreased compared to the antibacterial activity before PEGylation, but it was significantly higher than that of 5878.

[0278] [Table 26]

[0279]

[0280] Example 22. Antibacterial activity of stapled peptides

[0281] Regarding the amino acid sequence of K4, the amino acid at position 2 was substituted with T (hereinafter may be referred to as K4-K59T), and the antibacterial activity of the resulting peptide was evaluated.

[0282] K4-K59T is a sequence having differences in the amino acid at position 2 and the amino acid at position 9 compared to K3 of SEQ ID NO: 23. In Table 27 below, "stapled" means a state in which an amino acid pair is replaced by pentenylalanine or octenylalanine and these amino acids are cross-linked. Stapled peptide-1 is a peptide in which the amino acid pair at positions 10 and 14 is replaced by pentenylalanine (labeled as S5) and the amino acids are cross-linked, and stapled peptide-2 is a peptide in which the amino acid pair at positions 14 and 18 is replaced by pentenylalanine and the amino acids are cross-linked.

[0283] The results of the antibacterial activity evaluation of K4-K59T and stapled K4-K59T are shown in Table 27 below.

[0284] [Table 27]

[0285]

[0286] Example 23. Evaluation of the antibacterial activity of K4 variants containing sequences added to the C-terminus

[0287] Amino acid R or K was added to the C-terminus of the K4 variant in which two amino acids were deleted or the C-terminus of the K4 variant in which four amino acids were deleted, and the antibacterial activity of the resulting variant was measured. Referring to Table 28 below, peptides to which a single R or K was added or a sequence consisting of repeated R or K was added had improved antibacterial activity.

[0288] [Table 28]

[0289]

[0290] Although the present invention has been described in detail with reference to some specific features, it is obvious to those skilled in the art that this detailed description is only of its preferred embodiment and does not limit the scope of the present invention. Therefore, the essential scope of the present invention will be defined by the appended claims and their equivalents. <110> Korea University Research and Buisness Foundation <120> ROMO1-derived antimicrobial peptides and their variants <130> PX200021PCT <150> KR 10-2019-0071635 <151> 2019-06-17 <160> 49 <170> KoPatentIn 3.0 <210> 1 <211> 79 <212> PRT <213> Artificial sequence <220> <223> Homo sapiens Romo1 <400> 1 Met Pro Val Ala Val Gly Pro Tyr Gly Gln Ser Gln Pro Ser Cys Phe 1 5 10 15 Asp Arg Val Lys Met Gly Phe Val Met Gly Cys Ala Val Gly Met Ala 20 25 30 Ala Gly Ala Leu Phe Gly Thr Phe Ser Cys Leu Arg Ile Gly Met Arg 35 40 45 Gly Arg Glu Leu Met Gly Gly Ile Gly Lys Thr Met Met Gln Ser Gly 50 55 60 Gly Thr Phe Gly Thr Phe Met Ala Ile Gly Met Gly Ile Arg Cys 65 70 75 <210> 2 <211> 27 <212> PRT <213> Artificial sequence <220> <223> KU-5278 <400> 2 Leu Met Gly Gly Ile Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe 1 5 10 15 Gly Thr Phe Met Ala Ile Gly Met Gly Ile Arg 20 25 <210> 3 <211> 28 <212> PRT <213> Artificial sequence <220> <223> KU-5279 <400> 3 Leu Met Gly Gly Ile Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe 1 5 10 15 Gly Thr Phe Met Ala Ile Gly Met Gly Ile Arg Cys 20 25 <210> 4 <211> 25 <212> PRT <213> Artificial sequence <220> <223> KU-5478 <400> 4 Gly Gly Ile Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr 1 5 10 15 Phe Met Ala Ile Gly Met Gly Ile Arg 20 25 <210> 5 <211> 26 <212> PRT <213> Artificial sequence <220> <223> KU-5479 <400> 5 Gly Gly Ile Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr 1 5 10 15 Phe Met Ala Ile Gly Met Gly Ile Arg Cys 20 25 <210> 6 <211> 23 <212> PRT <213> Artificial sequence <220> <223> KU-5678 <400> 6 Ile Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met 1 5 10 15 Ala Ile Gly Met Gly Ile Arg 20 <210> 7 <211> 24 <212> PRT <213> Artificial sequence <220> <223> KU-5679 <400> 7 Ile Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met 1 5 10 15 Ala Ile Gly Met Gly Ile Arg Cys 20 <210> 8 <211> 22 <212> PRT <213> Artificial sequence <220> <223> KU-5778 <400> 8 Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala 1 5 10 15 Ile Gly Met Gly Ile Arg 20 <210> 9 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> KU-5779 <400> 9 Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala 1 5 10 15 Ile Gly Met Gly Ile Arg Cys 20 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> KU-5875 <400> 10 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met <210> 11 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> KU-5876 <400> 11 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> KU-5877 <400> 12 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile 20 <210> 13 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> KU-5878 <400> 13 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 14 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> KU-5879 <400> 14 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg Cys 20 <210> 15 <211> 20 <212> PRT <213> Artificial sequence <220> <223> KU-6079 <400> 15 Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile Gly Met 1 5 10 15 Gly Ile Arg Cys 20 <210> 16 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Artificial peptides <220> <221> Variants <222> (1)..(2) <223> X = any of the following: R, K, H, S, T, Q or N <220> <221> Variants <222> (5)..(6) <223> X = any of the following: R, K, H, S, T, Q or N <220> <221> Variants <222> (9) <223> X = any of the following: R, K, H, S, T, Q or N <220> <221> Variants <222> (12) <223> X = any of the following: R, K, H, S, T, Q or N <400> 16 Xaa Xaa Met Met Xaa Xaa Gly Gly Xaa Phe Gly Xaa Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 17 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-T59K <400> 17 Lys Lys Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 18 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-Q62K <400> 18 Lys Thr Met Met Lys Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 19 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-S63K <400> 19 Lys Thr Met Met Gln Lys Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 20 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-T66K <400> 20 Lys Thr Met Met Gln Ser Gly Gly Lys Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 21 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> KU-5878-T69K <400> 21 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Lys Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 22 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> KU-5878-K2 <400> 22 Lys Lys Met Met Lys Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 23 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> KU-5878-K3 <400> 23 Lys Lys Met Met Lys Lys Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 24 <211> 21 <212> PRT <213> Artificial sequence <220> <223> KU-5878-K4 <400> 24 Lys Lys Met Met Lys Lys Gly Gly Lys Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 25 <211> 21 <212> PRT <213> Artificial sequence <220> <223> KU-5878-K58R <400> 25 Arg Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 26 <211> 21 <212> PRT <213> Artificial sequence <220> <223> KU-5878-K58A <400> 26 Ala Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 27 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-T59R <400> 27 Lys Arg Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 28 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-R78A <400> 28 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Ala 20 <210> 29 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-R78K <400> 29 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Lys 20 <210> 30 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Artificial peptides <220> <221> Variants <222> (3)..(4) <223> X = any of the following: Met, Leu, Ile, Val, or Nle <220> <221> Variants <222> (14) <223> X = any of the following: Met, Leu, Ile, Val, or Nle <220> <221> Variants <222> (16) <223> X = any of the following: Met, Leu, Ile, Val, or Nle <220> <221> Variants <222> (18) <223> X = any of the following: Met, Leu, Ile, Val, or Nle <220> <221> Variants <222> (20) <223> X = any of the following: Met, Leu, Ile, Val, Gly, or Nle <400> 30 Lys Thr Xaa Xaa Gln Ser Gly Gly Thr Phe Gly Thr Phe Xaa Ala Xaa 1 5 10 15 Gly Xaa Gly Xaa Arg 20 <210> 31 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-M60A <400> 31 Lys Thr Ala Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 32 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> KU-5878-I73A <400> 32 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ala 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 33 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> KU-5878-I73K <400> 33 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Lys 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 34 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> KU-5878-I73W <400> 34 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Trp 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 35 <211> 21 <212> PRT <213> Artificial sequence <220> <223> KU-5878-I77A <400> 35 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ala Arg 20 <210> 36 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-I77K <400> 36 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Lys Arg 20 <210> 37 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-I77T <400> 37 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Thr Arg 20 <210> 38 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-I77G <400> 38 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Gly Arg 20 <210> 39 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-4Nle <220> <221> Variants <222> (3)..(4) <223> X = Norleucine (Nle) <220> <221> Variants <222> (14) <223> X = Norleucine (Nle) <220> <221> Variants <222> (18) <223> X = Norleucine (Nle) <400> 39 Lys Thr Xaa Xaa Gln Ser Gly Gly Thr Phe Gly Thr Phe Xaa Ala Ile 1 5 10 15 Gly Xaa Gly Ile Arg 20 <210> 40 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Artificial peptides <220> <221> Variants <222> (10) <223> X = any of the following: F, Y, or W <220> <221> Variants <222> (13) <223> X = any of the following: F, Y, or W <400> 40 Lys Thr Met Met Gln Ser Gly Gly Thr Xaa Gly Thr Xaa Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 41 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-F67A <400> 41 Lys Thr Met Met Gln Ser Gly Gly Thr Ala Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 42 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-F67W <400> 42 Lys Thr Met Met Gln Ser Gly Gly Thr Trp Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 43 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-F70K <400> 43 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Lys Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 44 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-F70W <400> 44 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Trp Met Ala Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 45 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Artificial peptides <220> <221> Variants <222> (7)..(8) <223> X = any of the following: A or G <220> <221> Variants <222> (11) <223> X = any of the following: A or G <220> <221> Variants <222> (15) <223> X = any of the following: A or G <220> <221> Variants <222> (17) <223> X = any of the following: A or G <220> <221> Variants <222> (19) <223> X = any of the following: A or G <400> 45 Lys Thr Met Met Gln Ser Xaa Xaa Thr Phe Xaa Thr Phe Met Xaa Ile 1 5 10 15 I'm going to get it. 20 <210> 46 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-A72G <400> 46 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Gly Ile 1 5 10 15 Gly Met Gly Ile Arg 20 <210> 47 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> KU-5878-G76A <400> 47 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile 1 5 10 15 Gly Met Ala Ile Arg 20 <210> 48 <211> 19 <212> PRT <213> Artificial sequence <220> <223> pCM19 <400> 48 Cys Leu Arg Ile Gly Met Arg Gly Arg Glu Leu Met Gly Gly Ile Gly 1 5 10 15 Lys Thr Met <210> 49 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> pCM12 <400> 49 Lys Thr Met Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Met Gly 1 5 10 15 Ile Arg

Claims

1. An antimicrobial peptide consisting of: An amino acid sequence in which K at amino acid position 1 is replaced by T or T at amino acid position 12 is replaced by G based on SEQ ID NO:

24.

2. An antimicrobial peptide consisting of the amino acid sequence: An amino acid sequence in which all four methionines in SEQ ID NO: 24 are replaced by isoleucine.

3. An antimicrobial peptide consisting of the amino acid sequence: An amino acid sequence having one modification selected from (a) to (s) based on SEQ ID NO: 24: (a) deletion of any one of amino acids at positions 1 to 12, 14 to 15, and 17 to 21; (b) a deletion of the amino acid at position 14, and a deletion of the amino acid at any one of positions 1 to 8, 12, 15 and 18; (c) a deletion of the amino acid at position 14, a deletion of the amino acid at position 18, and a deletion of the amino acid at any one of positions 1 to 8, 12 and 15; (d) a deletion of the amino acid at position 14, a deletion of the amino acid at position 15, and a deletion of the amino acid at position 3, 4 or 12; (e) deletion of the amino acid at position 1 or 2, deletion of the amino acid at position 3 or 4, and deletion of the amino acid at position 18; (f) deletion of amino acids at positions 3, 4, 14 and 18; (g) deletion of the amino acid at position 3 or 4; deletion of the amino acid at position 14, deletion of the amino acid at position 18, and deletion of the amino acid at position 5, 6, 7, 8, 12 or 15; (h) a deletion of the amino acid at position 14, a deletion of the amino acid at position 15, a deletion of the amino acid at position 1 or 2, and a deletion of the amino acid at position 3, 4, 7 or 8; (i) a deletion of the amino acid at position 1 or 2, a deletion of the amino acid at position 7 or 8, a deletion of the amino acid at position 15, and a deletion of the amino acid at position 18; (j) deletion of the amino acid at position 5 or 6, deletion of the amino acid at position 11, deletion of the amino acid at position 15, deletion of the amino acid at position 18, and deletion of the amino acid at any of positions 1 or 2, 3 or 4, 7 or 8, 12 and 14; (k) deletion of amino acid at position 1 or 2, deletion of amino acid at position 7 or 8, and deletion of amino acid at position 12, 14 and 15; (l) deletion of amino acid at position 5 or 6, deletion of amino acid at position 3 and 4, and deletion of amino acid at position 14 and 18; (m) deletion of the amino acids at positions 3, 4, 14 and 18, deletion of the amino acid at position 5 or 6, and deletion of the amino acid at any of positions 7 or 8, 12 and 15; (n) deletion of amino acid at position 1 or 2, deletion of amino acid at position 7 or 8, and deletion of amino acid at position 12, 14, 15 and 18; (o) deletion of amino acids at positions 3, 4, 12, 14 and 18, deletion of amino acids at positions 5 or 6, and deletion of amino acids at positions 7 or 8; (p) deletion of amino acid at position 1 or 2, deletion of amino acid at position 5 or 6, deletion of amino acid at position 7 or 8, and deletion of amino acid at position 12, 14, 15 and 18; (q) deletion of amino acids at positions 3, 4, 14 and 18, deletion of amino acid at position 5 or 6, and deletion of three amino acids selected from amino acids at positions 1 or 2, 7 or 8, 12 and 15; (r) a deletion of the amino acid at position 1 or 2, a deletion of the amino acid at position 3 or 4, a deletion of the amino acid at position 5 or 6, a deletion of the amino acid at position 7 or 8, and a deletion of the amino acids at positions 12, 14, 15 and 18; and (s) deletion of amino acid at position 1 or 2, deletion of amino acid at position 3 and 4, deletion of amino acid at position 5 or 6, deletion of amino acid at position 7 or 8, and deletion of amino acid at position 12, 14, 15 and 18.

4. An antimicrobial peptide, wherein the antimicrobial peptide consists of the amino acid sequence: An amino acid sequence in which K at position 2 is replaced with T based on SEQ ID NO: 24, wherein the amino acid pair at positions 10 and 14 or the amino acid pair at positions 14 and 18 are stapled together, wherein the amino acid pair is replaced by pentenylalanine and cross-linked to achieve stapling.

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