KR101875057B1 - Antimicrobial peptides with high synergistic effect with antibiotics against multidrug resistant gram-negative bacteria and their uses - Google Patents

Abstract

The present invention relates to an antimicrobial peptide having antimicrobial activity against Gram negative multidrug resistant bacteria. More particularly, the present invention relates to an antimicrobial peptide having antimicrobial activity against Gram negative multidrug resistant bacteria, A food additive, a feed additive, a composition for an antimicrobial, and a composition for quasi-antimicrobial use. The antibiotic peptide of the present invention not only exhibits significant antimicrobial activity against Gram-negative bacteria but also exhibits a strong antimicrobial activity only against Gram-positive bacteria and an antibiotic which does not have antimicrobial activity against gram-negative bacteria or has a low antimicrobial activity and the antibiotic peptide of the present invention , It is possible to exhibit an excellent antimicrobial effect against gram-negative bacteria and their antibiotic-resistant bacteria as well as Gram-positive bacteria.

Description

Antimicrobial peptides with high synergistic effect with antibiotics against multidrug resistant gram-negative bacteria and their uses,

The present invention relates to an antimicrobial peptide having antimicrobial activity against Gram negative multidrug resistant bacteria. More particularly, the present invention relates to an antimicrobial peptide having antimicrobial activity against Gram negative multidrug resistant bacteria, A food additive, a feed additive, a composition for an antimicrobial, and a composition for quasi-antimicrobial use.

Bacterial infection is one of the most common and deadly causes of human disease. Numerous antibiotics have been developed since penicillin and have been used to combat bacterial infiltration into the body. Recently, however, strains resistant to these antibiotics have emerged and are regarded as a big problem. Bacterial species such as Enterococcus faecalis , Mycobacterium tuberculosis and Pseudomonas aeruginosa , which can pose a life threat, are resistant to all known antibiotics (Stuart B. Levy, Scientific American , (1988): 46-53).

Tolerance to antibiotics is a distinct phenomenon from resistance to antibiotics, which was first discovered in Pneumococcus sp. In the 1970s and provided important clues to the mechanism of action of penicillin (Tomasz et al., Nature , 227, (1970): 138-140). Conventional chemical antibiotics, such as penicillin, Cephalosporin, etc., exhibit antibiotic action by inhibiting the synthesis of cell walls or proteins of microorganisms. However, tolerant species stop growing in the presence of conventional concentrations of antibiotics, but do not die as a result. Resistance occurs when antibiotics inhibit the cell wall synthetase because autolytic enzymes such as autolysin do not activate. This suggests that penicillin activates the endogenous hydrolytic enzyme By killing bacteria, bacteria also inhibit their activity, resulting in the survival of antibiotics. Therefore, it is urgent to develop antibiotics having a novel action mechanism capable of eliminating these resistant strains. Antibiotic peptides showing different antibiotic mechanisms from conventional chemical antibiotics are attracting attention as a new concept of next generation antibiotics (Zasloff M. Curr Opin Immunol 4 (1992): 3-7; Boman, HG, Cell , 65, 205 (1991); Boman, HG J Intern Med. 254.3 (2003): 197-215; Hancock, RE, & Scott, MG, Proc. Natl. Acad. Sci. USA 97 (2000): 8856-8861, Zasloff, M., Nature 415 (2002): 389-395).

Recently, Gram-negative bacteria such as P. aeruginosa and A. baumannii have been known to be resistant to antimicrobial resistance because they belong to the most frequently acquired species and are endogenous. In P. aeruginosa and A. baumannii , multidrug resistance means a strain resistant to all three types of drugs, including aminoglycoside, fluoroquinolone, and carbapenem. Although carbapenem was almost the only effective antimicrobial agent, it has been shown that there has been a great deal of limitation in antimicrobial treatment due to an increase in the number of strains resistant to carbapenem for 10 years. Pseudomonas aeruginosa is a Gram-negative bacterium that is a common infectious pathogen and has low susceptibility to antibiotics. Therefore, it can easily acquire antimicrobial resistance naturally. It is also known that respiratory infections such as respiratory tract infection, burn infection, cyst It is a cause of pneumonia and urinary tract infections in patients with sexually transmitted fibrosis. It is present in infected areas such as artificial ring apparatus, aspiration catheter, and contaminated water, and is directly or indirectly transferred to the patient, Is known to have high mortality due to difficulty in treatment.

Acinetobacter baumannii is a Gram-negative aerobic strain that is a major cause of hospital infection in many hospitals. Recently, it has been reported that aminoglycosides, cephalosporins, fluoroquinolones, beta- lactamase inhibitors, and carbapenem resistant multidrug-resistant Acinetobacter baumannii (MRAB). In 2010, a total of 46 people were infected with Acinetobacter germs at the University of Tokyo Hospital, and 10 of them died, raising awareness about the rapidly increasing antibiotic resistant MRAB in the world in recent 10 years, spurring the development of antibiotics have. Acinetobacter itself is also present in water, soil or human skin, and healthy people do not develop infection even when infected. However, when a person with a weakened immune system is infected, it can die from pneumonia or sepsis. Since the 1990s, it has started to increase in the United States and Europe. From 2000, almost no antibiotics were heard. In general, a multi-drug resistant Acinetobacter baumannii (MDRAB) refers to a strain resistant to all three types of drugs, including aminoglycosides, fluoroquinolones, and carbapenems. In this case, Colistin ) Remain the only susceptible, and Acinetobacter bacteria may be susceptible to Tigecycline. Acetobacter, the major causative agent of medical infection, was the only effective antimicrobial agent because of its resistance to multidrug resistance. However, the number of strains resistant to carbapenem increased for 10 years. Recently, the resistance rate of P. aeruginosa was about 20%, but the acenetobacterium rapidly increased, exceeding 50% in most large hospitals, and the increase of carbapenem resistance rate led to the increase of acenetobacter, In the related infection rate survey, P. aeruginosa is overtaken, followed by MRSA, Enterococcus sp. Therefore, the development of therapeutic agents is urgent because of the high incidence of fatal bacterial infections in Korea and the high mortality rate.

In a broad sense, the antigen-antibody reaction, termed the immune response, has played a major role in biological defense in invertebrates, including humans. However, insects and amphibians protect themselves by enhancing innate immune mechanisms that are different from acquired immune mechanisms such as antigen-antibody reactions. Thus, innate immunity plays an important defense against early infections in insects, amphibians, and vertebrates including humans. The most important means of innate immune mechanisms is to obtain antimicrobial peptides with sterilization and inactivation actions against various microorganisms. Antibacterial peptides and proteins having an antimicrobial action mainly on peptides are highly likely to be used as antimicrobial agents caused by diseases such as livestock including human beings and infection of various pathogens. Recently, antibiotic resistance has become a serious problem, and new antibiotic peptides are attracting interest in new drug candidates. Antibiotic peptides have antimicrobial activity against bacterial membranes, so they appear to retain their activity against strains resistant to existing antibiotics. Therefore, a variety of naturally occurring antibiotic peptides are emerging as important biomarkers for the search for a new concept of antibiotics to replace existing antibiotics, which are threatened by the emergence of resistant strains.

The antimicrobial proteins and peptides of the insects discovered so far have been isolated from about 200 insect species since the first report of secrophin by Boman's team in Secopia moth, , And a peptide containing a large amount of a glycine and a glycine amino acid.

Seckropin is a basic protein composed of 31-39 amino acids. A new antimicrobial peptide (papillosin) from the larvae of the tiger butterfly, which the team has recently patented, has a N-terminal amphipathic helix and a hydrophobic helix at the C- Helix-hinge-α-helix structure including the α-helix-hinge-α-helix structure and has excellent antimicrobial activity and anti-inflammatory activity against various pathogens (Non-Patent Document 1). Particularly, it has been known that a region having a helix structure at the N-terminus plays an important role in the antimicrobial activity, thereby confirming the excellent antimicrobial activity and anti-inflammatory activity against resistant strains (Patent Document 1, Non-Patent Document 2).

MA-MA is an amphipathic amphipathic peptide that has antimicrobial activity against a variety of bacteria. We have developed a CA-MA peptide which is prepared by conjugating the N-terminal peptide of schepyrin with the N-terminal peptide of magainin The three-dimensional structure was identified and it was confirmed that the antimicrobial and anticancer activities were excellent (Non-Patent Document 3). In addition, recently, PapMA-1, which was constructed by connecting 8 residues of N-terminal region of Papiliocin and 9 of N-terminal residues of Magainin, was designed and synthesized, and excellent Gram-negative bacteria and Gram- (Non-Patent Document 4). However, the synergistic effect of PapMA-1 by mixing with existing antibiotics has not been elucidated.

Accordingly, the present inventors have made efforts to produce a novel synthetic peptide having antimicrobial activity from peptides reported to have antibacterial activity. As a result, it has been found that the N-terminal region of the antipathic peptide papyriloxine and the N PapMA-2, PapMA-3 and PapMA-4 antibiotic peptides were prepared using the PapMA-1 antibiotic peptide conjugated with the end-region residues as the template. The synthesized antibiotic peptides showed antimicrobial activity against Gram-positive and Gram- The antibiotic peptide of the present invention can be used as an active ingredient of a composition for antimicrobial use as a combination with a conventional antibiotic because it exhibits a synergistic synergistic effect with respect to Gram- The present invention has been completed.

KR 10-1150281 B1

 Kim, Jin-Kyoung, et al. "Structure and function of papilocin with antimicrobial and anti-inflammatory activities from the swallowtail butterfly, Papilio xuthus." Journal of Biological Chemistry 286.48 (2011): 41296-41311.  Lee, Eunjung, et al. "Functional roles of aromatic residues and helices of papilocin in its antimicrobial and anti-inflammatory activities." Scientific reports 5 (2015).  Oh, Donghoon, et al. "Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A (1-8) -magainin 2 (1-12) and its analogues, on their antibiotic activities and structures." Biochemistry 39.39 (2000): 11855-11864.  Shin, Areum, et al. &Quot; Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity. &Quot; Biochemistry 54.25 (2015): 3921-3931.

Thus, the present inventors have found that the Papami-1 antibiotic peptide conjugated with the residue of the N-terminal region of papilleriosin, an antibiotic peptide, and the residue of the N-terminal region of maganin is used as a template, Replace the 15th amino acid alanine (A) with tryptophan (W); PapMA-2, PapMA-3 and PapMA-4 antibiotic peptides with higher antimicrobial activity were prepared by replacing the 18th amino acid phenylalanine (F) with tryptophan (W). The antibiotic peptides exhibit excellent antimicrobial activity against Gram-negative bacteria such as Escherichia coli and Acinetobacter bacteria and their antibiotic-resistant bacteria, and they can exhibit significant synergistic antimicrobial activity when they are administered together with antibiotics, .

Accordingly, an object of the present invention is to provide a novel antibiotic peptide showing excellent antimicrobial activity against Gram negative multidrug-resistant bacteria.

Still another object of the present invention is to provide an antimicrobial composition which exhibits antimicrobial activity of an excellent synergistic effect against Gram negative multidrug resistant bacteria by administering the antibiotic peptide together with an antibiotic as a combined agent.

In order to achieve the above object, the present invention provides a novel peptide consisting of any one of the amino acid sequences shown in SEQ ID NOS: 2 to 4.

The present invention also relates to a method for producing a peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the amino acid of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) An antibiotic peptide consisting of a substituted amino acid sequence.

In one preferred embodiment of the present invention, the antibiotic peptide may be a peptide consisting of any one of the amino acid sequences shown in SEQ ID NOS: 2 to 4.

The present invention also relates to a method for producing a protein having an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) An antimicrobial pharmaceutical composition comprising an antimicrobial peptide consisting of an amino acid sequence as an active ingredient.

The present invention also provides a food additive comprising the antibiotic peptide as an active ingredient.

The present invention also provides a feed additive comprising the antibiotic peptide as an active ingredient.

In addition, the present invention provides a composition for oral cavity comprising the antibiotic peptide as an active ingredient.

In addition, the present invention provides a quasi-drug composition comprising the antibiotic peptide as an active ingredient.

(A) an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of any one or both of alanine (A), which is the 15th amino acid and phenylalanine (F) An antimicrobial peptide consisting of an amino acid sequence substituted with an amino acid sequence; And (b) an antibiotic as an active ingredient.
The present invention also provides a food additive comprising (a) an antimicrobial peptide and (b) an antibiotic as an active ingredient. The present invention also provides a feed additive comprising (a) an antimicrobial peptide and (b) an antibiotic as an active ingredient.
In addition, the present invention provides a composition for oral cavity comprising (a) an antimicrobial peptide and (b) an antibiotic as an active ingredient.
The present invention also provides a quasi-drug composition comprising (a) an antimicrobial peptide and (b) an antibiotic as an active ingredient.

In one preferred embodiment of the present invention, the antibiotic peptide may be composed of any one of the amino acid sequences of SEQ ID NOS: 1 to 4.

In another preferred embodiment of the present invention, the antibiotic is selected from the group consisting of Erythromycin, Ampicillin, Vancomycin, Linezolid, Methicillin, Oxacillin, It has been found that the compounds of the present invention can be used in combination with other drugs such as Cefotaxime, Rifampicin, Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, (Eg, Ertapenem, Doripenem, Imipenem / Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, A drug such as Ceftobiprole, Aztreonam, Piperacillin, Polymyxin B, Colistin, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Tigecycline, < RTI ID = 0.0 > And a derivative thereof. In the present invention,

In another preferred embodiment of the present invention, the antimicrobial pharmaceutical composition may have antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant bacteria thereof.

In another preferred embodiment of the present invention, the Gram positive bacteria are selected from the group consisting of Bacillus subtilis , Staphylococcus aureus and Staphylococcus epidermidis . Lt; / RTI >

In a preferred embodiment of the invention, the gram negative bacteria are selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa , Acinetobacter baumannii and Salmonella typhimurium It can be any one or more selected

Therefore, the present invention relates to a papilliferin hybrid peptide made by linking the N-terminal region of an antimicrobial peptide papillocin (Pap) isolated from a larva of a tiger butterfly with the N-terminal region residues of magainin 2 (MA) (A), which is the 15th amino acid in the sequence of PapMA-1, is substituted with tryptophan (W); PapMA-2, PapMA-3 and PapMA-4 in which phenylalanine (F) as the 18th amino acid is replaced with tryptophan (W).
The antibiotic peptide of the present invention not only exhibits significant antimicrobial activity against Gram-negative bacteria but also exhibits a strong antimicrobial activity only against Gram-positive bacteria and an antibiotic which does not have antimicrobial activity against gram-negative bacteria or has a low antimicrobial activity and the antibiotic peptide of the present invention , It is possible to exhibit an excellent antimicrobial effect against gram-negative bacteria and their antibiotic-resistant bacteria as well as Gram-positive bacteria. Therefore, the antimicrobial composition comprising the antibiotic peptide of the present invention and the antibiotic peptide-antibiotic composition of the present invention can be used as a pharmaceutical composition for antimicrobial use, a food additive, a feed additive, It contains a small amount of chemical antibiotics and can exhibit increased antimicrobial activity.

FIG. 1 shows the synergistic effect of the antimicrobial activity against E. coli and its resistant bacteria against 8 μg / ml of the peptide of the present invention and commercial antibiotics.
FIG. 2 is a graph showing the synergistic effect of the antimicrobial activity against A. baumannii fungus and resistant fungus against 8 μg / ml of the peptide of the present invention and known antibiotics.

Hereinafter, the present invention will be described in detail.

As described above, the development of antibiotics exhibiting antimicrobial activity against multidrug-resistant bacteria having antimicrobial resistance has been continuously required. Antimicrobial peptides thus produced have been reported, but they are mixed with antibiotics in the past to produce synergistic effects Antigenic peptides that can be expressed need to be studied further.

The antibiotic peptides PapMA-1, PapMA-2, PapMA-3 and PapMA-4 of the present invention exhibited significant antimicrobial activity against Gram-negative bacteria, and when mixed with an antibiotic exhibiting an antibacterial activity that is effective only for Gram- , Antimicrobial activity against antibiotics resistant to gram-negative bacteria and their antibiotics due to synergistic effect of antibiotic peptides and antibiotics. Therefore, even when a small amount of antibiotics is administered, the antimicrobial effect against Gram-negative bacteria, particularly Escherichia coli and Acinetobacter bacteria and their multidrug- It is effective.

Accordingly, the present invention is characterized in that the amino acid sequence of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) from the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is tryptophan (W) A novel synthetic peptide consisting of a substituted amino acid sequence.
The present invention also relates to a method for producing a peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the amino acid of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) An antibiotic peptide consisting of a substituted amino acid sequence.

The "peptide consisting of the amino acid sequence of SEQ ID NO: 1" of the present invention is a peptide consisting of 8 residues of the 1-8 region of the N-terminal region of Papiliocin (Pap) (SEQ ID NO: 5) Papama-1, a synthetic peptide consisting of 18 residues made by linking 9 peptides of residues 4 to 12 of the N-terminal region of SEQ ID NO: 2 (SEQ ID NO: 6) with proline.

The amino acid of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) is replaced with tryptophan (W) from the above-mentioned "peptide consisting of the amino acid sequence of SEQ ID NO: And preferably an antibiotic peptide of the amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 4.
By substituting "alanine (A) and / or phenylalanine (F) with tryptophan (W)" in the sequence of the above-mentioned "antibiotic peptide", an increased antimicrobial activity over the peptide consisting of the amino acid sequence of SEQ ID NO: 1 The sequence can be optimized so as to have a higher synergistic effect when expressed or mixed with conventional antibiotics.
More specifically, of the antibiotic peptides, the antibiotic peptide consisting of the amino acid sequence of SEQ ID NO: 2 is PapMA-2, and the 18th amino acid phenylalanine from the amino acid sequence of SEQ ID NO: 1 is substituted with tryptophan.
The antibiotic peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 is PapMA-3, and alanine, which is the 15th amino acid from the amino acid sequence shown in SEQ ID NO: 1, is substituted with tryptophan.
The antibiotic peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is PapMA-4, and alanine, which is the 15th amino acid from the amino acid sequence shown in SEQ ID NO: 1, and phenylalanine, which is the 18th amino acid, are substituted with tryptophan.
However, the antibiotic peptide of the present invention is not limited to the amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 4 but may include the functional equivalent of amino acid sequence of SEQ ID NO: 2-4.

Is at least 70%, preferably 80% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, Refers to a peptide having a homology of at least 95% and exhibiting substantially the same physiological activity as a novel antibiotic peptide.

The novel antimicrobial peptide of the present invention is a synthetic peptide, and it is preferable to synthesize it by a chemical synthesis method (WH Freeman and Co., Proteins; structures and molecular principles (1983)) of a conventional peptide in the art as a method for the above synthesis Specifically, it is more preferable to synthesize by solution phase peptide synthesis, solid-phase peptide syntheses, fractional condensation and F-moc or T-BOC chemical methods, and more specifically, Is most preferably synthesized by the liquid phase solid phase method (Merrifield, RB., J. Am. Chem . Soc ., 85.2149: 196), but is not limited thereto.

The "novel antibiotic peptide" of the present invention is preferably, but not limited to, those having antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and their antibiotic-resistant bacteria. Specifically, the Gram-positive bacteria are Staphylococcus genus (Staphylococcus), Listeria genus (Listeria), Streptococcus genus (Streptococcus), the genus Corynebacterium (Corynebacterium), Lactobacillus genus (Lactobacillus), genus Clostridium ( Clostridium), Enterobacter nose kusu genus (Enterococcus), Erie when fellow matrix in (Erysipelothrix) and Bacillus (Bacillus) gram and positive bacteria preferably a positive bacteria of all known Gram in the art, including, Bacillus subtilis (Bacillus subtilis , Staphylococcus aureus or Staphylococcus epidermidis , but is not limited thereto. The gram-negative bacteria is E. coli in (Escherichia), Pseudomonas species (Pseudomonas), Salmonella genus (Salmonella), Leptospira in (Leptospira), rikechiah in (Rickettsia) industry preferably all gram-negative bacteria is known in the art as gram-negative bacteria, including And more preferably Escherichia coli , Pseudomonas aeruginosa , Acinetobacter baumannii or Salmonella typhimurium , and more preferably Escherichia coli or Acetobacter spp. It is most preferable to use one of Acinetobacter baumannii , but the present invention is not limited thereto.

In a specific example of the present invention, the inventors of the present invention have found that, in order to synthesize a novel antibiotic peptide exhibiting excellent antimicrobial activity, the N-terminal region of the antimicrobial peptide papillocin (Pap) isolated from the larval butterfly larva 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 23) is replaced with tryptophan by replacing alanine and / or 18th amino acid phenylalanine, which is the 15th amino acid of Papami- The peptides were named PapMA-2, PapMA-3 and PapMA-4 (Table 1).

In addition, the present inventors confirmed that the antimicrobial activity of PapMA-1, PapMA-2, PapMA-3 and PapMA-4 showed high antimicrobial activity against all Gram-negative bacteria, (Table 2 and Table 3). The results are shown in Table 2 and Table 3, respectively. In addition, it has been confirmed that erythromycin, vancomycin, and linzolide, which are conventionally used as antibiotics, have antimicrobial activity against gram-positive bacteria and very low antimicrobial activity against gram-negative bacteria (Table 4).

In order to confirm whether PapMA-1, PapMA-2, PapMA-3 and PapMA-4 can exhibit synergistic antimicrobial activity with existing antibiotics, the inventors of the present invention found that Gram-negative bacteria and antibiotic-resistant strains together with erythromycin, vancomycin or linezolid The antimicrobial activity of the antimicrobial agent of the present invention was significantly elevated as compared with the antimicrobial activity of the antimicrobial agent alone (Table 5 to Table 14) (Fig. 1, Fig. 2 and Tables 15 to 19). ≪ tb > < TABLE >

Therefore, the antibiotic peptides PapMA-1, PapMA-2, PapMA-3 and PapMA-4 of the present invention not only show significant antimicrobial activity against Gram-negative bacteria but also have significant antibacterial activity only against Gram- When the antibiotic having a low antimicrobial activity and the antibiotic peptide of the present invention are co-administered, the antimicrobial peptide of the present invention or the combination comprising the antibiotic of the present invention exhibits an excellent synergistic antimicrobial activity against Gram-negative bacteria and its antibiotic- And can be usefully used as an effective ingredient of the composition.

The present invention also provides a method for producing a protein having an amino acid sequence of SEQ ID NO: 1 or amino acid sequence of alanine (A) which is the 15th amino acid thereof and phenylalanine (F) which is the 18th amino acid thereof is substituted with tryptophan (W) An antimicrobial pharmaceutical composition comprising an antimicrobial peptide consisting of an amino acid sequence as an active ingredient.
(A) an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of any one or both of alanine (A), which is the 15th amino acid and phenylalanine (F) An antimicrobial peptide consisting of an amino acid sequence substituted with an amino acid sequence; And (b) an antibiotic as an active ingredient.

The present invention also relates to a method for producing a protein having an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) An antimicrobial peptide consisting of an amino acid sequence; And an antibiotic as an active ingredient.

The "antibiotic peptide" of the present invention is preferably composed of any one of the amino acid sequences set forth in SEQ ID NOS: 1 to 4, but is not limited thereto, and derivatives and functional equivalents thereof. Is at least 70%, preferably 80% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, Refers to a peptide having a homology of at least 95% and exhibiting substantially the same physiological activity as a novel antibiotic peptide.

The "antibiotic" of the present invention may be any of those known in the art as having antimicrobial activity against Gram-positive bacteria and having low antimicrobial activity against gram-negative bacteria or having no antimicrobial activity. Specifically, the antibiotics of the present invention may be selected from the group consisting of Erythromycin, Ampicillin, Vancomycin, Linezolid, Methicillin, Oxacillin, Cefotaxime, But are not limited to, Rifampicin, Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, The compounds of the present invention may be used in combination with other drugs such as Doripenem, Imipenem / Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Piperacillin, Polymyxin B, Colistin, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, and the like. , Tigecycline, a combination thereof, and a combination thereof Body preferably at least one selected from the group consisting of and, more specifically, erythromycin, one or more preferably more than one selected from the group consisting of vancomycin, and Rhine Jolly draw, and the like.

The "antimicrobial pharmaceutical composition" of the present invention is preferably but not limited to: Acinetobacter baumannii , Actinomyces sp. (Example Actinomyces israelii and Actinomyces naeslundii ), Aeromonas sp. (For example, Aeromonas hydrophila , eromonas verona, Aeromonas sobria ( Aeromonas sobria ) and Aeromonas caviae ), Anaplasma phagocytophilum , Alcaligenes < RTI ID = 0.0 > xylosoxidans), Bacillus bad bad Martino Martino micro system comitán (Actinobacillus actinomycetemcomitans), in Bacillus (Bacillus sp.) (e.g. anthrax (Bacillus anthracis), cereus bacteria (Bacillus cereus), Bacillus subtilis (Bacillus subtilis), pitcher Lindsey N-Sys bacteria (Bacillus thuringiensis), and Bacillus styryl Summer pillar's (Bacillus stearothermophilus), in addition bacteria genus (Bacteroides sp.) (For example, (For example, Bacteroides fragilis ), Bartonella sp (for example, Bartonella bacilliformis and Bartonella henselae ), bifidobacteria in (Bifidobacterium sp.), Bode in telra (Bordetella sp.) (for example, whooping cough bacteria (Bordetella pertussis), para pertussis bacteria (Bordetella parapertussis) and Bode telra Braun Kai counts Mathematica (Bordetella bronchiseptica)) in Oh, borelri ( Such as Borrelia sp. (For example, Borrelia recurrentis and Borrelia burgdorferi ), Brucella sp. (For example, Brucella abortus , ( Brucella canis , Brucella melintensis and Brucella suis ), Burkholderia sp. (for example, Burkholderia pseudomallei and Burkholderia spp . ( E.g. , Burkholderia cepacia ), Campylobacter sp. ( E.g. , plant Campylobacter jejuni , Campylobacter coli , Campylobacter lari and Campylobacter sp . fetal gogyun (Campylobacter fetus)), Cobb nosayi topa belongs (Capnocytophaga sp.), carboxylic video tumefaciens hoe varnish (Cardiobacterium hominis), Chlamydia trachomatis (Chlamydia trachomatis), the Cloud shown pillar can monitor (Chlamydophila pneumonia), parrots Chlamydophila psittaci , Citrobacter sp. ≪ / RTI > Coxiella burnetii), Corey four bacterial genus (Corynebacterium sp.) (For example, diphtheria bacteria (Corynebacterium diphtheria), Corynebacterium Jay Kiwoom (Corynebacterium jeikeum) and Corynebacterium (Corynebacterium)), genus Clostridium ( Clostridium sp.) (e.g., Welch bacteria (Clostridium perfringens), Clostridium difficile silica bacteria (Clostridium difficile), Clostridium botulinum (Clostridium botulinum) and tetanus (Clostridium tetani)), the pond Nella nose Rhodes's Eikenella corrodens , Enterobacter sp. (For example, Enterobacter aerogenes , Enterobacter agglomerans , Enterobacter cloacae and enterotoxin, elements of E. coli (enterotoxigenic E. coli), jangchim foray into E. coli (enteroinvasive E. coli), Chapter E. coli (enteropathogenic E. coli), Chapter haemorrhagic Escherichia coli (enterohemorrhagic E. coli), the Minister Aggregation coli (enteroaggregative E. Coli) and E. coli urinary tract disease-induced (uropathogenic E. coli) and E. coli containing the opportunity infectious E. coli (Escherichia coli)), Enterococcus genus (Enterococcus sp.) (E.g. Ende Lokomotiv kusu Fe Enterococcus faecalis and Enterococcus faecium ), Ehrlichia sp. (For example, Ehrlichia chafeensia and Ehrlichia canis ) , Erysipelothrix rhusiopathiae , Eubacterium sp., Francisella tularensis , Fusobacterium nucleatum , Gardnerella vaginalis , Gemelamorvil, Rum ( Gemella morbillorum ), Haemophilus sp. ( Haemophilus sp. ) (E. G., Haemophilus influenzae (Haemophilus influenzae), Haemophilus Duke ray (Haemophilus ducreyi), Haemophilus this jipti mouse (Haemophilus aegyptius), H. fur loose hemo influenza (Haemophilus parainfluenzae), Haemophilus H. Mori T kusu (Haemophilus haemolyticus) and ( E.g. , Haemophilus parahaemolyticus ), Helicobacter sp. (For example, Helicobacter pylori , Helicobacter cinaedi and Helicobacter fennelliae ) King Gela kingge (Kingella kingii), keulrepeu when Ella in (Klebsiella sp.) (for example, Klebsiella pneumoniae (Klebsiella pneumoniae), as Klebsiella Gras pressed clematis (Klebsiella granulomatis) and Klebsiella oxy cytokines (Klebsiella oxytoca ), Lactobacillus sp., Listeria monocytogenes , Leptospira interrogans ( Lep tobacco interrogans , Legionella pneumophila , Leptospira interrogans , Peptostreptococcus sp. , Moraxella catarrhalis , Morganella sp. , tombstones Rune kusu in (Mobiluncus sp.), US aureus in (Micrococcus sp.), Mycobacterium genus (Mycobacterium sp.) (e.g., nabyeonggyun (Mycobacterium leprae), Mycobacterium intra cellulose rare (Mycobacterium intracellulare), plastic tuberculosis (Mycobacterium avium), bovine tuberculosis (Mycobacterium bovis) and Mycobacterium horses over (Mycobacterium marinum)), my algorithm in Copley (Mycoplasm sp. ( E.g. , Mycoplasma pneumoniae , Mycoplasma hominis and Mycoplasma genitalium ), Nocardia sp. ( E.g., Nocardia spp.), Nocardia asteroides , Nocardia cyriacigeorgica and Nocardia brasiliensis ), Neisseria sp. (For example Neisseria gonorrhoeae ) And Neisseria meningitidis ), Pasteurella multocida , Plesiomonas shigelloides , Prevotella sp., Porphyromonas sp., Prevore telra Carmelo Nino's Danica (Prevotella melaninogenica), in Proteus (Proteus sp.) (for example, Proteus vulgaris (Proteus vulgaris) and Proteus Billy's mum (Proteus mirabilis)), au Lobby den cyano in (Providencia sp.) (E.g., Providencia alkali Pacific yen (Providencia alcalifaciens), Providencia inlet Gary (Providencia rettgeri) and Providencia Stu Artemisia (Providencia stuartii)), Pseudomonas aeruginosa, (Pseudomonas for example, aeruginosa , Propionibacterium acnes , Rhodococcus equi , Salmonella sp. (for example, Salmonella enterica , Salmonella typhi , Salmonella paratyphi , Salmonella enteritidis , Salmonella cholerasuis and Salmonella typhimurium , Serratia sp. (For example, Serratia marcesans and Serratia limypsia ) Serratia liquifaciens ), Shigella sp. (For example, Shigella dysenteriae , Shigella flexneri , Shigella b oydii) and Shigella Sone yigyun (Shigella sonnei)), Staphylococcus Aspergillus genus (Staphylococcus sp.) (e.g., Staphylococcus aureus (Staphylococcus aureus), epidermal Staphylococcus (Staphylococcus epidermidis), Staphylococcus H. Mori T kusu Staphylococcus hemolyticus , Staphylococcus saprophyticus ), Streptococcus sp. (For example, Streptococcus pneumoniae , Spectinomycin-resistant serotype 6B pneumococcal strain Streptomycin-resistant serotype 6B Streptococcus pneumoniae , Streptomycin-resistant serotype 9V Streptococcus -resistant serotype 9V Streptococcus pneumoniae , Erythromycin-resistant serotype 14 Streptococcus pneumoniae , , Optochin-resistant serotype 14 pneumococcal streptococci (Optochin-resistant serotype 14 Streptococcus pneumoniae ), rifampicin-resistant serotype 18C Resistant serotype 18C Streptococcus pneumoniae , a tetracycline-resistant serotype 19F Tetracycline-resistant serotype 19F Streptococcus pneumoniae , a penicillin-resistant serotype 19F Streptococcus 19F Streptococcus pneumoniae) and trimesoyl Supreme-resistant serotype 23F pyeryeon Streptococcus (Trimethoprim-resistant serotype 23F Streptococcus pneumoniae ), chloramphenicol-resistant serotype 4 Streptococcus pneumoniae (chloramphenicol-resistant serotype 4 Streptococcus pneumoniae ), streptomycin-resistant serotype 9V Streptomycin-resistant serotype 9V Streptococcus pneumoniae , Optochin-resistant serotype 14 Streptococcus pneumoniae , rifampicin-resistant serotype 18C Rifampicin-resistant serotype 18C Streptococcus pneumoniae ), Penicillin-resistant serotype 19F pneumococcal strain Aureus (Penicillin-resistant serotype 19F Streptococcus pneumoniae ) or trimesoyl Supreme-resistant serotype 23F pyeryeon Streptococcus (Trimethoprim-resistant serotype 23F Streptococcus pneumoniae ), Streptococcus Agar lock thienyl (Streptococcus agalactiae), Streptococcus myutan (Streptococcus mutans ), Streptococcus pyogenes , Group A streptococci , Streptococcus pyogenes , Group B streptococci , Streptococcus agalactiae , Group C streptococci , Streptococcus anginosus , Streptococcus equismilis , Group D streptococci , Streptococcus bovis, Streptococcus equisilis , Streptococcus bovis , Group F streptococci and Streptococcus anginosus , Group G streptococcus , Hemolytic Streptococcus (Group G streptococci), RY rilrum negative (Spirillum minus), Mo nilri formate miss chain bacilli (Streptobacillus moniliformi), Trail Four nematic bacteria in (Treponema sp.) (E.g., TRE Four nematic color lithium (Treponema Caratoum , Treponema petenue , Treponema pallidum and Treponema endemicum , Tropheryma whippelii , Ureaplasma urealyticum, Ureaplasma urealyticum, ), meyo Nella in (Veillonella sp.), parahaemolyticus in (Vibrio sp.) (e.g., Asian cholera (Vibrio cholerae), Vibrio parahaemolyticus (Vibrio parahemolyticus), Vibrio vulnificus bacteria (Vibrio vulnificus), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Vibrio vulnificus (Vibrio vulnificus), Vibrio know nori Tea Syracuse (Vibrio alginolyticus), Vibrio Mimi Syracuse (Vibrio mimicus), the three Vibrio holly (Vibrio hollisae) , Vibrio flat ruby Alice (Vibrio fluvialis), Vibrio methoxy struck Kobe (Vibrio metchnikovii), Vibrio demsel La (Vibrio damsela) and Vibrio pureuni time (e. G. (Vibrio furnisii)), for example reusi in California (Yersinia sp.), Yersinia enterocolitica and Yersinia pestis ) and Xanthomonas maltophilia .

The Gram-positive bacteria are Staphylococcus genus (Staphylococcus), Listeria genus (Listeria), Streptococcus genus (Streptococcus), the genus Corynebacterium (Corynebacterium), Lactobacillus genus (Lactobacillus), Clostridium genus (Clostridium), Enterobacter nose kusu genus (Enterococcus), Erie when fellow matrix in (Erysipelothrix) and Bacillus (Bacillus) gram and positive bacteria preferably a positive bacteria of all known Gram in the art, including, Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus or Staphylococcus epidermidis is more preferable, but is not limited thereto, and preferably it can include antibiotic-resistant strains thereof.

The gram-negative bacteria is E. coli in (Escherichia), Pseudomonas species (Pseudomonas), Salmonella genus (Salmonella), Leptospira in (Leptospira), rikechiah in (Rickettsia) industry preferably all gram-negative bacteria is known in the art as gram-negative bacteria, including And more preferably Escherichia coli , Pseudomonas aeruginosa , Acinetobacter baumannii or Salmonella typhimurium . However, it is not limited to these, and preferably those of Escherichia coli , Pseudomonas aeruginosa , Antibiotic resistant strains.

Since the PapMA-1, PapMA-2, PapMA-3 and PapMA-4 peptides of the present invention exhibit significant synergistic antimicrobial effects against Gram-negative bacteria and their antibiotic-resistant strains when mixed with conventional antibiotics, Antibiotic peptides and antibiotics may be usefully used as an effective ingredient of a pharmaceutical composition for antimicrobial use.

The antimicrobial pharmaceutical composition of the present invention can be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration may refer to administration via routes other than oral, such as rectal, intravenous, peritoneal, muscular, arterial, percutaneous, nasal, inhalation, ocular and subcutaneous. When the pharmaceutical composition for antimicrobial use of the present invention is used as a medicine, it may further contain one or more active ingredients showing the same or similar functions.

That is, the antimicrobial pharmaceutical composition of the present invention may be administered in various forms of parenteral administration. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Witepsol, macrogol, Tween 61, cacao bean, Liu lingzhi, glycerogelatin and the like can be used as the base of the suppository.

In addition, the pharmaceutical composition for antimicrobial use of the present invention can be used by mixing with various carriers that are accepted as medicines such as physiological saline or organic solvents, and can be used as glucose, sucrose or dextran such as sucrose, Antioxidants such as carbohydrates, ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

The effective dose of the antibacterial pharmaceutical composition of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and can be administered once to three times a day.

In the pharmaceutical composition of the present invention, the total effective amount of the antibiotic peptide of the present invention or the combination of the antibiotic peptide and the antibiotic is in a Bolus form or a single dose by infusion for a relatively short period of time And may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, To determine the appropriate effective dose according to the particular use as a pharmaceutical composition of the invention.

The antimicrobial adjuvant of the present invention can be administered at the time of administering the antibiotic, together with the antibiotic, before the antibiotic treatment, after the antibiotic treatment, and as an adjuvant for enhancing the antibiotic activity of the antibiotic. Preferably, the adjuvant has a synergistic effect on the antimicrobial activity with antibiotics, and it is preferable to increase the antimicrobial activity of the antibiotic against Gram-negative bacteria and antibiotic-resistant strains.

The amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of alanine (A) which is the 15th amino acid and phenylalanine (F) which is the 18th amino acid are substituted with tryptophan (W) The present invention provides a food additive comprising an antibiotic peptide as an active ingredient.
(A) an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of any one or both of alanine (A), which is the 15th amino acid and phenylalanine (F) An antimicrobial peptide consisting of an amino acid sequence substituted with an amino acid sequence; And (b) an antibiotic as an active ingredient.

Since the PapMA-1, PapMA-2, PapMA-3 and PapMA-4 peptides of the present invention exhibit significant synergistic antimicrobial effects against Gram-negative bacteria and their antibiotic-resistant strains when mixed with conventional antibiotics, Antibiotic peptides, or a combination of antibiotic peptides and antibiotics, may be usefully used as an active ingredient in food additives.

When the antibiotic peptide or antibiotic peptide and antimicrobial agent of the present invention is used as a food additive, the antibiotic peptide, antibiotic peptide and antimicrobial agent may be added intact or used together with other food ingredients, Can be used. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use thereof. Generally, the antibiotic peptide or antibiotic peptide and antibacterial agent complex of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material. However, in the case of long-term ingestion, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount in the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, and includes foods in a conventional sense.

The amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of alanine (A) which is the 15th amino acid and phenylalanine (F) which is the 18th amino acid are substituted with tryptophan (W) The present invention provides a feed additive comprising an antibiotic peptide as an active ingredient.
(A) an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of any one or both of alanine (A), which is the 15th amino acid and phenylalanine (F) An antimicrobial peptide consisting of an amino acid sequence substituted with an amino acid sequence; And (b) an antibiotic as an active ingredient.

Since the PapMA-1, PapMA-2, PapMA-3 and PapMA-4 peptides of the present invention exhibit significant synergistic antimicrobial effects against Gram-negative bacteria and their antibiotic-resistant strains when mixed with conventional antibiotics, Antibiotic peptides, or combinations of antibiotic peptides and antibiotics, may be usefully employed as active ingredients in feed additives.

The feed composition comprising the antibiotic peptide or antibiotic peptide and antibacterial agent combination of the present invention replaces the existing antibiotics and inhibits the growth of harmful food pathogenic bacteria to improve the health condition of the animal and improve the body weight and meat quality of the livestock , Milk production and immunity. The feed composition of the present invention can be produced in the form of a fermented feed, a compound feed, a pellet form, a silage reed, or the like.

The fermented feed can be prepared by fermenting an organic material by adding various microbial groups or enzymes other than the antibiotic peptide of the present invention or the antibiotic peptide and antimicrobial combination agent. The compounded feed can be prepared by mixing various kinds of general feed, Antimicrobial agent combination can be prepared. The pellet-shaped feed can be produced by applying heat and pressure to the compound feed or the like in a pelletizer, and the silage can be produced by fermenting a chrysanthemum feed as a microorganism. The wet fermented feed is prepared by collecting and transporting organic matter such as food waste, mixing the excipient and the excipient for moisture control at a certain ratio, fermenting at a suitable temperature for fermentation for more than 24 hours, And the like. The fermented dried feed can be prepared by adjusting the wet fermented feed so that the moisture content is 30% to 40%.

The present invention also relates to a method for producing a protein having an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) Which comprises an antimicrobial peptide consisting of an amino acid sequence as an effective ingredient.
(A) an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of any one or both of alanine (A), which is the 15th amino acid and phenylalanine (F) An antimicrobial peptide consisting of an amino acid sequence substituted with an amino acid sequence; And (b) an antibiotic as an active ingredient.

Since the PapMA-1, PapMA-2, PapMA-3 and PapMA-4 peptides of the present invention exhibit significant synergistic antimicrobial effects against Gram-negative bacteria and their antibiotic-resistant strains when mixed with conventional antibiotics, An antibiotic peptide, or a combination of an antibiotic peptide and an antibiotic, may be usefully used as an effective ingredient of the composition for nonspecific use.

The preservative composition includes a food preservative, a cosmetic preservative, or a pharmaceutical preservative. Preservatives, cosmetic preservatives, and pharmaceutical preservatives of foods mentioned above are additives used to prevent deterioration, decay, discoloration and chemical change of medicines, including antiseptics and antioxidants, which inhibit the growth of microorganisms such as bacteria, fungi and yeast, And functional antibiotics such as inhibiting the growth of microorganisms or sterilizing microorganisms in pharmaceuticals. Ideal conditions for such preservative compositions should be non-toxic and effective in trace amounts.

The present invention also relates to a method for producing a protein having an amino acid sequence of SEQ ID NO: 1 or amino acid sequence of alanine (A) which is the 15th amino acid and phenylalanine (F) which is the 18th amino acid is substituted with tryptophan (W) An antimicrobial quasi-drug composition comprising an antimicrobial peptide consisting of an amino acid sequence as an active ingredient.
(A) an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of any one or both of alanine (A), which is the 15th amino acid and phenylalanine (F) An antimicrobial peptide consisting of an amino acid sequence substituted with an amino acid sequence; And (b) an antibiotic as an active ingredient.

Since the PapMA-1, PapMA-2, PapMA-3 and PapMA-4 peptides of the present invention exhibit significant synergistic antimicrobial effects against Gram-negative bacteria and their antibiotic-resistant strains when mixed with conventional antibiotics, Antibiotic peptide, or antibiotic peptide and antibiotic may be usefully used as an active ingredient of quasi-antimicrobial quasi-drugs.

When the composition of the present invention is used as a quasi-drug additive, the antibiotic peptide, antibiotic peptide and antibiotic combination of the present invention may be added as it is or may be used together with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment agent, a patch or a filter filler.

Hereinafter, the present invention will be described in more detail with reference to examples and preparation examples. It should be apparent to those skilled in the art that these examples and preparations are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these examples and preparations

Synthesis of antibiotic peptides

(N-terminal residue of Magiliin (SEQ ID NO: 6)) of the antibiotic peptide Papiliocin (SEQ ID NO: 5) and the N-terminal residue of Magainin (SEQ ID NO: 6) were linked to a peptide showing antimicrobial activity against Gram- One sequence was designed to design antibiotic peptides.

Specifically, peptides were synthesized by a solid phase synthesis method using a synthesis method of N- (9-fluorenyl) methoxycarbonyl (Fmoc) and purified ( Biochimica et Biophysica Acta (BBA ) -Biomembranes 1798.10 (2010): 1913-1925). The concentration of synthesized peptides was then quantitated using a UV spectrophotometer and the final peptide purity (> 98%) was analyzed by reverse-phase high performance liquid chromatography.

As a result, antibiotic peptides composed of the amino acid sequences of SEQ ID NOS: 1 to 4 were synthesized as shown in the following Table 1 (Non-Patent Document 4).

The sequence of the peptides synthesized in the present invention and the comparative peptide sequence Peptides
designation Amino acid sequence SEQ ID NO: net
charge Hydrophobicity Remarks Papa-1 RWKIFKKIPKFLHSAKKF-NH2 SEQ ID NO: 1 +8 -0.67
synthesis
Peptides
PapMA-2 RWKIFKKIPKFLHSAKKW-NH2 SEQ ID NO: 2 +8 -0.69 PapMA-3 RWKIFKKIPKFLHSWKKF-NH2 SEQ ID NO: 3 +8 -0.07 PapMA-4 RWKIFKKIPKFLHSWKKW-NH2 SEQ ID NO: 4 +8 -0.09 Papillie RWKIFKKIEKVGRNVRDGIIKAGPAVAVVGQAATVVK-NH2 SEQ ID NO: 5 +8 -1.48 compare
Peptides Maganin GIGKFLHSAKKFGKAFVGEIMNS SEQ ID NO: 6 +3 -0.58 Melitin GIGAVLKVLTTGLPALISWIKRKRQQQ SEQ ID NO: 7 +5 -0.02

Antibacterial activity of antibiotic peptides

<2-1> Confirmation of antimicrobial activity of antimicrobial peptides against Gram-positive and Gram-negative bacteria

In order to compare the antimicrobial activities exhibited by the antibiotic peptides of the present invention, antimicrobial activities of antimicrobial peptides or conventional antibiotics against Gram-negative bacteria and Gram-positive bacteria were measured. The antimicrobial activity was measured by measuring the minimum inhibitory concentration (MIC) of the peptides in which the cells were not cleaved in a nutrient-rich MH medium.

Specifically, the strains described in [Table 2] below were purchased, diluted with MH medium such that the number of bacteria was 2 × 10 ⁶ colony-forming units (CFUs) per ml, and 100 μl was added to a 96-well microtiter plate , And a peptide solution (2: 1 step diluted solution) diluted with MH medium was added to each well in an amount of 100 占 퐇. Plates were incubated at 37 for 16 hours and the absorbance of each well was measured at 620 nm with an ELISA reader (Bio-Tek Instruments) to determine the MIC of the peptide. When the MIC value is higher than the corresponding concentration, the MIC is assumed to be the corresponding concentration, and the experiment is performed thereafter.

The strains used in the present invention and their sources division Strain name Accession number source
Gram-
Positive bacteria

Bacillus subtilis
( Bacillus subtilis ) KCTC 3068
Korean Cell Line Bank
(Korean Collection for Type Cultures)

Staphylococcus aureus
( Staphylococcus aureus ) KCTC 1621 Starfilacocus epidermidis
( S. epidermidis ) KCTC 1917

Gram-
Negative bacteria
E. coli
( Escherichia coli ) KCTC 1682
Korean Cell Line Bank
(Korean Collection for Type Cultures)

Pseudomonas Maeruginosa
( Psedomonas aeruginosa ) KCTC 1637 Acinetobacter Baumanni
( Acinetobacter baumannii ) KCTC 2508 Salmonella typhimurium
( Salmonella typhimurium ) KCTC 1926 Antibiotic
Resistant bacteria MDREC CCARM 1229 Antibiotic resistant bacteria main bank
(Culture collection of antimicrobial resistant microbe) CCARM 1238 MDRAB CCARM 12035 CCARM 12035 MDRPA CCARM 2002 CCARM 2003 MDRST CCARM 8007 CCARM 8009 MRSA CCARM 3114 CCARM 3126

As a result, as shown in the following Table 3, it was confirmed that the peptides of SEQ ID NOS: 1 to 4 had a high antimicrobial activity against all Gram-negative bacteria. In particular, the peptides PapMA-2, PapMA-3, and PapMA-4 of SEQ ID NOs: 2-4 exhibited four times better antimicrobial activity against A. baumannii and its resistant bacteria in gram-negative bacteria than PapMA-1. In particular, PapMA-2, PapMA-3 and PapMA-4 peptides showed antimicrobial activity comparable to papilio sinus or melitin, a comparative peptide known to have a high antimicrobial activity against Gram-negative bacteria. These peptides and antibiotic peptide papyriosin used as a comparative control group were confirmed to have a very high antimicrobial activity selectively for all Gram-negative bacteria and very low antimicrobial activity for Gram-positive bacteria (Table 3).

Antimicrobial activity of antimicrobial peptides against Gram-negative bacteria, Gram-positive bacteria and antibiotic resistant bacteria Target strain Minimum growth inhibitory concentration MIC (占 퐂 / ml) SEQ ID NO: 1:
Papa-1 SEQ ID NO: 2:
PapMA-2 SEQ ID NO: 3:
PapMA-3 SEQ ID NO: 4:
PapMA-4 Comparative peptide
SEQ ID NO: 5:
Papiliocin Comparative peptide
SEQ ID NO: 7:
Melittin E. coli 64 64 32 16 8 16 MDREC 1229 64 64 32 32 8 16 MDREC 1238 64 64 64 32 16 32 P. aeruginosa 16 16 16 16 16 8 MDRPA 2002 16 16 16 16 16 16 MDRPA 2003 32 16 16 16 16 16 A. baumannii 64 16 16 16 8 8 MDRAB 12035 64 16 16 16 8 8 MDRAB 12036 64 16 16 16 16 16 S. typhimurium 64 32 32 32 16 32 MDRST 8007 64 32 32 32 16 32 MDRST 8009 64 64 64 32 16 32 B. subtilis > 128 > 128 > 128 > 128 > 128 32 S. epidermidis > 128 > 128 > 128 > 128 > 128 16 S. aureus > 128 > 128 > 128 > 128 > 128 8 MRSA 3114 > 128 > 128 > 128 > 128 > 128 8 MRSA 3126 > 128 > 128 > 128 > 128 > 128 16

<2-2> Identification of antimicrobial activity of existing antibiotics against Gram-positive and Gram-negative bacteria

In order to compare the antimicrobial activity of the antibiotic peptides of the present invention with those of conventional antibiotics, the MIC concentration of antibiotics was confirmed for the same Gram-positive bacteria and Gram-negative bacteria.

Specifically, the target strain was prepared in the same manner as in Example <2-1>, and the MIC of the antibiotic was confirmed by treating the antibiotic Erythromycin, Vancomycin, or Linezolid, respectively .

As a result, as shown in Table 4 below, antibiotics have a low antimicrobial activity with a MIC of 32 μg / ml or more for Gram-negative bacteria, while all antibiotics have a very high antimicrobial activity against Gram-positive bacteria (Table 4).

Antimicrobial activity of existing antibiotics in Gram-negative bacteria, Gram-positive bacteria and antibiotic-resistant bacteria division Target strain Minimum growth inhibitory concentration MIC (占 퐂 / ml) Erythromycin Vancomycin Linezolid
Gram-negative bacteria

E. coli 128 1024 256 P. aeruginosa 256 > 1024 > 1024 A. baumannii 32 1024 256 S. typhimurium 64 512 256
Gram-positive bacteria
B. subtilis 0.25 One One S. epidermidis One One One S. aureus 0.25 One One Antibiotic resistant bacteria
MDREC1229 256 > 1024 512 MDRAB12035 32 > 1024 512

Identification of synergistic effects of antibiotic peptide and antibiotic treatment

<3-1> Confirmation of MIC change by antibiotic or antibiotic peptide when mixed with antibiotic peptide and antibiotic

The antibiotic peptides of the present invention showed high antimicrobial activity against Gram-negative bacteria. Thus, when an antibiotic agent having a strong antimicrobial activity only against Gram-positive bacteria and having no significant antibacterial activity against Gram-negative bacteria was mixed with the synthetic peptide of the present invention Gt; synergistic activity &lt; / RTI &gt;

Specifically, each of the strains described in Table 2 was prepared, diluted with MH medium such that the number of bacteria was 2 × 10 ⁶ colony-forming units (CFUs) per ml, and 100 μl was added to a 96-well microtiter plate ), And then 50 [mu] l of a peptide solution (2: 1 step diluted solution) diluted from the MIC value with MH medium was added to each well. Then, the antibiotic solution was diluted in the MH medium, and 50 μl was added to each well. The MIC value of each mixed solution was determined in the same manner under the opposite conditions.

As a result, as shown in the following Tables 5 to 9, existing antibiotics erythromycin, vancomycin or linazolidine were used as the antibiotic peptides PapMA-1, PapMA-2, PapMA-3 or PapMA- -4 showed that the MIC of erythromycin was as low as 1024-fold for E. coli and its resistant bacteria, and against A.baumannii and its resistant bacteria when the peptide of the present invention was treated with 1/2 MIC (Table 5 to Table 9). Among the peptides of SEQ ID NOS: 2 to 4, which are derivative peptides designed with the PapA-1 peptide of SEQ ID NO: 1 as its parent, in particular the PapMA-2 peptide of SEQ ID NO: 2, when the same peptide concentration as PapMA- -1, the MIC value of the antibiotic mixed with PapMA-2 was found to be 2 to 4 times lower.

For example, when the peptide is mixed with an antibiotic at a concentration of 16 쨉 g / ml, which is a 1/4 concentration of the peptide MIC, and treated with Escherichia coli, when erythromycin or linazolidide is mixed with PapAm-1, Were found to be 0.5 and 16 ㎍ / ㎖, respectively (1/256 and 1/16 of antibiotic MIC, respectively). In contrast, when MIC values of erythromycin and linezolid were 0.25 and 8 쨉 g / ml, respectively, when PapMA-2 was treated with erythromycin or linezolide, the antibiotic MICs were 1/512 and 1/32 (Table 5).

For A. baumannii , PapM-1 (1/8 MIC) had a MIC value of 0.5 μg / ml (1/64) compared to 8 μg / ml of peptides, whereas PapMA-2 ) Was 0.125 占 퐂 / ml (1/256), confirming that the synergistic effect of PapMA-2 with the same amount of peptide was exhibited (Table 7).

In the antibiotic peptide treatment of the present invention, the antimicrobial activity of the antibiotic against Escherichia coli E. coli Peptide treatment concentration
(占 퐂 / ml) Antibiotic MIC (占 퐂 / ml) Erythromycin Vancomycin Linezolid Compound
Alone 128 1024 256

Papa-1
concentration
(Concentration / MIC) 64 (1) 0 0 0 32 (1/2) 0.125 4 8 16 (1/4) 0.5 16 16 8 (1/8) 2 32 16 6 (1/11) 4 32 16 4 (1/16) 16 > 128 64 2 (1/32) 32 > 128 128 1 (1/64) 64 > 128 128

PapMA-2
concentration
(Concentration / MIC) 64 (1) 0 0 0 32 (1/2) 0.125 4 4 16 (1/4) 0.25 8 8 8 (1/8) 0.5 32 8 6 (1/11) 2 64 16 4 (1/16) 16 > 128 16 2 (1/32) 32 > 128 64 1 (1/64) 64 > 128 128

PapMA-3
concentration
(Concentration / MIC) 64 (2) 0 0 0 32 (1) 0 0 0 16 (1/2) 0.125 8 2 8 (1/4) One 32 16 6 (1/5) 4 128 32 4 (1/8) 16 > 128 32 2 (1/16) 32 > 128 64 1 (1/32) 64 > 128 128

PapMA-4
concentration
(Concentration / MIC) 64 (4) 0 0 0 32 (2) 0 0 0 16 (1) 0 0 0 8 (1/2) 8 32 16 6 (1/3) 8 128 32 4 (1/4) 16 > 128 32 2 (1/8) 32 > 128 64 1 (1/16) 64 > 128 128

The antimicrobial activity of antibiotics against MDREC 1229 in the antibiotic peptide treatment of the present invention MDREC 1229 Peptide treatment concentration
(占 퐂 / ml) Antibiotic MIC (占 퐂 / ml) Erythromycin Vancomycin Linezolid Compound
Alone 256 > 1024 512

Papa-1
concentration
(Concentration / MIC)

64 (1) 0 0 0 32 (1/2) 0.25 8 8 16 (1/4) One 16 16 8 (1/8) 4 128 32 6 (1/11) 8 > 128 64 4 (1/16) 16 > 128 64 2 (1/32) 32 > 128 128 1 (1/64) 128 > 128 > 128

PapMA-2
concentration
(Concentration / MIC)
64 (1) 0 0 0 32 (1/2) 0.125 8 4 16 (1/4) 0.5 16 8 8 (1/8) 2 64 16 6 (1/11) 8 128 32 4 (1/16) 16 128 32 2 (1/32) 32 > 128 64 1 (1/64) 64 > 128 > 128

PapMA-3
concentration
(Concentration / MIC)
64 (2) 0 0 0 32 (1) 0 0 0 16 (1/2) 0.25 16 4 8 (1/4) 4 128 16 6 (1/5) 16 > 128 32 4 (1/8) 32 > 128 64 2 (1/16) 32 > 128 64 1 (1/32) 128 > 128 > 128

PapMA-4
concentration
(Concentration / MIC)
64 (2) 0 0 0 32 (1) 0 0 0 16 (1/2) 0.25 32 4 8 (1/4) 4 128 16 6 (1/5) 16 > 128 64 4 (1/8) 16 > 128 64 2 (1/16) 32 > 128 64 1 (1/32) 64 > 128 > 128

Antimicrobial activity of antibiotics against A. baumannii in the treatment of antibiotic peptides of the present invention A. baumannii Peptide treatment concentration
(占 퐂 / ml) Antibiotic MIC (占 퐂 / ml) Erythromycin Vancomycin Linezolid Compound
Alone 32 1024 256

Papa-1
concentration
(Concentration / MIC) 64 (1) 0 0 0 32 (1/2) 0.0625 2 4 16 (1/4) 0.125 8 4 8 (1/8) 0.5 8 8 6 (1/11) 2 16 32 4 (1/16) 8 32 32 2 (1/32) 16 128 128 1 (1/64) 32 > 128 > 128

PapMA-2
concentration
(Concentration / MIC) 64 (4) 0 0 0 32 (2) 0 0 0 16 (1) 0 0 0 8 (1/2) 0.125 8 8 6 (1/3) One 16 16 4 (1/4) 8 32 32 2 (1/8) 16 128 64 1 (1/16) 16 > 128 > 128

PapMA-3
concentration
(Concentration / MIC) 64 (4) 0 0 0 32 (2) 0 0 0 16 (1) 0 0 0 8 (1/2) 0.125 32 8 6 (1/3) One 32 16 4 (1/4) 8 64 32 2 (1/8) 16 128 64 1 (1/16) 32 > 128 > 128

PapMA-4
concentration
(Concentration / MIC) 64 (4) 0 0 0 32 (2) 0 0 0 16 (1) 0 0 0 8 (1/2) 4 32 16 6 (1/3) 4 64 32 4 (1/4) 8 64 64 2 (1/8) 16 128 64 1 (1/16) 32 > 128 > 128

The antimicrobial activity of antibiotics against MDRAB 12035 in the antibiotic peptide treatment of the present invention MDRAB 12035 Peptide treatment concentration
(占 퐂 / ml) Antibiotic MIC (占 퐂 / ml) Erythromycin Vancomycin Linezolid Compound
Alone 32 > 1024 512

Papa-1
concentration
(Concentration / MIC) 64 (1) 0 0 0 32 (1/2) 0.125 8 4 16 (1/4) 0.25 16 8 8 (1/8) 0.5 16 16 6 (1/11) 4 32 32 4 (1/16) 16 128 64 2 (1/32) 32 > 128 128 1 (1/64) 32 > 128 > 128

PapMA-2
concentration
(Concentration / MIC) 64 (4) 0 0 0 32 (2) 0 0 0 16 (1) 0 0 0 8 (1/2) 0.25 16 8 6 (1/3) 2 32 32 4 (1/4) 4 128 32 2 (1/8) 16 > 128 64 1 (1/16) 32 > 128 > 128

PapMA-3
concentration
(Concentration / MIC) 64 (4) 0 0 0 32 (2) 0 0 0 16 (1) 0 0 0 8 (1/2) 0.25 16 16 6 (1/3) 4 32 32 4 (1/4) 8 128 32 2 (1/8) 16 > 128 128 1 (1/16) 32 > 128 > 128

PapMA-4
concentration
(Concentration / MIC) 64 (4) 0 0 0 32 (2) 0 0 0 16 (1) 0 0 0 8 (1/2) 2 64 32 6 (1/3) 4 64 64 4 (1/4) 8 128 64 2 (1/8) 16 > 128 128 1 (1/16) 32 > 128 > 128

As shown in the following Table 9, the antibacterial activity of antibiotics against Escherichia coli and A. baumannii was confirmed by mixing papyriosin and antibiotics as a comparative control. As a result, papyriosin itself has excellent antimicrobial activity It can be seen that the antimicrobial activity does not have a synergistic effect when compared with the peptides of the present invention when mixed with antibiotics (Table 9).
&lt; b &gt; In the control group of papyrioxin, E. coli and &lt; i &gt; Antimicrobial activity of antibiotics against baumannii </ i></b> MIC Concentration (μg / ml) Peptide treatment concentration
(占 퐂 / ml)
E. coli A. baumannii Erythromycin Vancomycin Linezolid Erythromycin Vancomycin Linezolid Compound alone 128 1024 256 32 1024 256 Papiliocin
density
(Concentration / MIC) 8 (1) 0 0 0 0 0 0 4 (1/2) 64 256 128 16 256 64 2 (1/4) 64 256 128 16 256 64 1 (1/8) 64 512 128 32 512 128

In addition, to [Table 10] to [Table 14] The result of measuring, when the antibiotic was applied PapMA-1, PapMA-2, the antimicrobial activity of PaPMA-3 and 4-PapMA antimicrobial peptide as shown in, E. coli and It was confirmed that the antimicrobial peptide had an MIC lowered to 256 times by adding antibiotics to the resistant bacteria, A.baumannii and its resistant bacteria (Tables 10 to 14).

Antimicrobial activity of the antibiotic peptides of the present invention against E. coli during antibiotic treatment E. coli Antibiotic treatment concentration (占 퐂 / ml) Peptide MIC ([mu] g / ml) Papa-1 PapMA-2 PapMA-3 PapMA-4 피 peptide 64 64 32 16 Erythromycin
concentration
(Concentration / MIC) 128 (1) 0 0 0 0 64 (1/2) One One One One 32 (1/4) 2 2 2 2 16 (1/8) 4 4 2 4 Vancomycin
concentration
(Concentration / MIC) 1024 (1) 0 0 0 0 512 (1/2) 2 2 2 2 256 (1/4) 4 4 4 4 128 (1/8) 8 8 8 8 Linezolid
concentration
(Concentration / MIC) 256 (1) 0 0 0 0 128 (1/2) One One One One 64 (1/4) 4 2 2 2 32 (1/8) 4 4 4 4

Antimicrobial activity of the antibiotic peptides of the present invention against MDREC 1229 upon treatment with antibiotics MDREC 1229 Antibiotic treatment concentration (占 퐂 / ml) Peptide MIC ([mu] g / ml) Papa-1 PapMA-2 PapMA-3 PapMA-4 피 peptide 64 64 32 32 Erythromycin
concentration
(Concentration / MIC) 256 (1) 0 0 0 0 128 (1/2) One One 0.5 0.5 64 (1/4) One One One One 32 (1/8) 2 2 2 2 Vancomycin
concentration
(Concentration / MIC) 1024 (1) One One One One 512 (1/2) 2 2 2 2 256 (1/4) 4 4 4 4 128 (1/8) 8 8 8 8 Linezolid
concentration
(Concentration / MIC) 512 (1) 0 0 0 0 256 (1/2) One One One One 128 (1/4) 2 One 2 2 64 (1/8) 2 2 One 2

Antimicrobial activity of the antibiotic peptides of the present invention against A. baumannii during antibiotic treatment A. baumannii Antibiotic treatment concentration (占 퐂 / ml) Peptide MIC ([mu] g / ml) Papa-1 PapMA-2 PapMA-3 PapMA-4 피 peptide 64 16 16 16 Erythromycin
concentration
(Concentration / MIC) 32 (1) 0 0 0 0 16 (1/2) 2 One 2 2 8 (1/4) 4 4 4 4 4 (1/8) 4 4 4 8 Vancomycin
concentration
(Concentration / MIC) 1024 (1) 0 0 0 0 512 (1/2) 0.25 0.25 0.25 0.25 256 (1/4) 0.5 0.5 0.25 0.25 128 (1/8) 4 2 2 2 Linezolid
concentration
(Concentration / MIC) 256 (1) 0 0 0 0 128 (1/2) 0.5 0.5 One One 64 (1/4) 4 4 2 4 32 (1/8) 4 4 4 4

Antimicrobial activity of the antibiotic peptides of the present invention against MDRAB 12035 upon antibiotic treatment MDRAB 12035 Antibiotic treatment concentration (占 퐂 / ml) Peptide MIC ([mu] g / ml) Papa-1 PapMA-2 PapMA-3 PapMA-4 피 peptide 64 16 16 16 Erythromycin
concentration
(Concentration / MIC) 32 (1) 0 0 0 0 16 (1/2) 4 2 2 2 8 (1/4) 4 4 4 4 4 (1/8) 4 4 4 4 Vancomycin
concentration
(Concentration / MIC) 1024 (1) One 0.25 0.25 0.25 512 (1/2) 2 One 2 2 256 (1/4) 4 2 2 2 128 (1/8) 4 4 4 4 Linezolid
concentration
(Concentration / MIC) 512 (1) 0 0 0 0 256 (1/2) One One One One 128 (1/4) 4 2 2 2 64 (1/8) 4 4 4 4

As shown in the following Table 14, when antibiotics were treated, antibiotic activity of papyriosin was confirmed by mixing with comparative control papyriosin. As a result, antimicrobial activity was significantly enhanced compared to the peptides of the present invention (Table 14).
<b> Antibiotic treatment, E. coli and <i> A. &lt; / b &gt;&lt; / b &gt;&lt; / RTI &gt; Peptide MIC ([mu] g / ml) Bacteria Antibiotic treatment concentration Papiliocin Antibiotic alone treatment 8 E. coli Erythromycin
concentration
(Concentration / MIC) 128 (1) 0 64 (1/2) 4 32 (1/4) 4 16 (1/8) 4 Vancomycin
concentration
(Concentration / MIC) 1024 (1) 0 512 (1/2) 2 256 (1/4) 2 128 (1/8) 4 Linezolid
concentration
(Concentration / MIC) 256 (1) 0 128 (1/2) 4 64 (1/4) 4 32 (1/8) 8 A. baumannii Erythromycin
concentration
(Concentration / MIC) 32 (1) 0 16 (1/2) 4 8 (1/4) 4 4 (1/8) 8 Vancomycin
concentration
(Concentration / MIC) 1024 (1) 0 512 (1/2) 2 256 (1/4) 2 128 (1/8) 4 Linezolid
concentration
(Concentration / MIC) 256 (1) 0 128 (1/2) 4 64 (1/4) 4 32 (1/8) 8

<3-2> Confirmation of synergistic effect of antimicrobial activity using antimicrobial peptide and antibiotic mixture using MIC value

Using the determined MIC values for each mixture, the synergistic effect of the peptide and the antibiotic on the antimicrobial activity against the strain was evaluated. Fractional inhibitory concentration (FIC) values were used for the evaluation (Elion, Gertrude B., Samuel Singer, and George H. Hitchings. Journal of Biological Chemistry 208.2 (1954): 477-488.).

Specifically, the FIC value was calculated according to the following Equation 1 using the MIC value calculated in the above Example <3-1>, and was evaluated according to the calculated FIC value as follows: <0.5, Synergy effect; 0.5 to 1, partial synergy effect; 1.0, additive effect; 2.0 to 4.0, Indifference; > 4.0, Antagonism.

A commonly used method for determining synergy using FIC values is the Checkerboard assay, which selects the lowest FIC index value of the wells in which growth of the bacteria is inhibited. However, the selection of one FIC index in this method has a problem that the antibiotic activity of the antibiotic is calculated to have a large synergy effect when the antibacterial activity is lower, and it is not accurate and there are many experimental errors. Therefore, the FIC index calculated by the above-mentioned equation (1) was calculated again using the following formula (2): He, Jing, Charles G. Starr, and William C. Wimley. Biochimica et Biophysica Acta (BBA) -Biomembranes 1848.1 (2015): 8-15.).

As a result, as shown in the following Tables 15 to 19, the existing antibiotics erythromycin, vancomycin or linazolidine were used as the antibiotic peptides PapMA-1, PapMA-2, PapMA-3 or PapMA- 4, the synergistic effect of the antimicrobial activity of the present invention was confirmed using the FIC value. As a result, when the peptide of SEQ ID NO: 1 to 4 of the present invention was administered together with antibiotics having high antimicrobial activity only for Gram- We confirmed that the antimicrobial activity of Gram-negative bacteria, E. coli and its resistant strains, A. baumannii and its resistant strains, has an FIC index of 0.07-0.47 or less, which is much lower than 0.5. It was confirmed that Gram-negative bacteria which are social issues are antimicrobial agents having high synergy effect by using them as a combination with existing antibiotics Indicators 18, Figs. 1 and 2). A. baumannii PapM-2 had a synergistic effect at low concentrations compared to PapMA-1, but the FIC index was slightly higher in Papama-1 than in A. baumannii and its resistant strains (64 ㎍ / ㎖) / ㎖, which is 4 times lower than that of control.

Comparison of FIC index showing synergy of antimicrobial activity against Escherichia coli in antibiotic peptide and antibiotic mixture treatment E. coli Peptide name Average FIC index Erythromycin Vancomycin Linezolid Papa-1 0.14 0.16 0.22 PapMA-2 0.14 0.16 0.14 PapMA-3 0.15 0.22 0.20 PapMA-4 0.23 0.33 0.24

Comparison of FIC index showing the synergistic effect of antimicrobial activity against MDREC 1229, a multi-drug resistant bacterium mixed with antibiotic peptide and antibiotics MDREC 1229 Peptide name Average FIC index Erythromycin Vancomycin Linezolid Papa-1 0.11 0.20 0.16 PapMA-2 0.08 0.16 0.12 PapMA-3 0.13 0.26 0.14 PapMA-4 0.09 0.26 0.15

Comparison of FIC index showing synergy of antimicrobial activity against A.baumannii when mixed with antibiotic peptide and antibiotics A.baumannii Peptide name Average FIC index Erythromycin Vancomycin Linezolid Papa-1 0.27 0.07 0.26 PapMA-2 0.30 0.10 0.32 PapMA-3 0.38 0.10 0.29 PapMA-4 0.47 0.11 0.35

Comparison of FIC index showing synergistic effect of antimicrobial activity against MDRAB 12035, a multi-drug resistant strain, when antibiotic peptide and antibiotics were mixed MDRAB 12035 Peptide name Average FIC index Erythromycin Vancomycin Linezolid Papa-1 0.38 0.13 0.16 PapMA-2 0.37 0.20 0.21 PapMA-3 0.39 0.21 0.22 PapMA-4 0.40 0.22 0.24

Comparison of FIC index showing the synergy of antimicrobial activity against Escherichia coli and A.baumannii in the case of the control treatment with papyriosin and antibiotics Average FIC index E. coli A. baumannii Erythromycin Vancomycin Linezolid Erythromycin Vancomycin Linezolid Papiliocin One 0.67 1.17 1.33 0.67 One

From these results, it can be seen that the peptides of SEQ ID NOS: 1 to 4, which are peptides of the present invention, exhibit excellent synergy when used in combination with existing antibiotics. In particular, when peptides of the same concentration are treated, -2 peptide showed the best reduction of MIC of conventional antibiotics. On the other hand, as shown in the above Table 19, the peptides of the comparative peptides, papillosin, are excellent in antimicrobial effect only on peptides, but they are very low in synergy when they are used in combination with existing antibiotics (Table 19)

Hereinafter, a preparation example of a medicament containing the antibiotic peptide according to the present invention as an active ingredient will be described, but the present invention is not intended to be limited thereto but is specifically explained. Antibiotic peptides showing antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria according to the present invention were produced according to the conventional methods according to the following composition components and composition ratios.

&Lt; Preparation Example 1 > Preparation of pharmaceutical preparations

<1-1> Preparation of powder

2 g of the peptide and antibacterial agent combination of the present invention

Lactose 1g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of the peptide and antibacterial agent combination of the present invention

100 mg of corn starch

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of the peptide and antibacterial agent combination of the present invention

100 mg of corn starch

100 mg of milk

Stearin Magnesium 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of the peptide and antibacterial agent complex of the present invention

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the peptide and antibacterial agent combination of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

<1-6> Preparation of injections

500 ng of the peptide and antibacterial agent complex of the present invention

180 mg mannitol

Na2HPO42H2O 26 mg

Distilled water 2974 mg

According to the conventional method for preparing an injectable preparation, an injectable preparation was prepared by incorporating the aforementioned components in the amounts indicated.

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Antimicrobial peptides with high synergistic effect with          antibiotics against multidrug resistant gram-negative bacteria          and their uses <130> 1060795 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Papa-1 <400> 1 Arg Trp Lys Ile Phe Lys Lys Ile Pro Lys Phe Leu His Ser Ala Lys   1 5 10 15 Lys Phe         <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> PapMA-2 <400> 2 Arg Trp Lys Ile Phe Lys Lys Ile Pro Lys Phe Leu His Ser Ala Lys   1 5 10 15 Lys Trp         <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> PapMA-3 <400> 3 Arg Trp Lys Ile Phe Lys Lys Ile Pro Lys Phe Leu His Ser Trp Lys   1 5 10 15 Lys Phe         <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> PapMA-4 <400> 4 Arg Trp Lys Ile Phe Lys Lys Ile Pro Lys Phe Leu His Ser Trp Lys   1 5 10 15 Lys Trp         <210> 5 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Papiliocin <400> 5 Arg Trp Lys Ile Phe Lys Lys Ile Glu Lys Val Gly Arg Asn Val Arg   1 5 10 15 Asp Gly Ile Ile Lys Ala Gly Ala Val Ala Val Val Gly Gln Ala              20 25 30 Ala Thr Val Val Lys          35 <210> 6 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Magainin <400> 6 Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe   1 5 10 15 Val Gly Glu Ile Met Asn Ser              20 <210> 7 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Melittin <400> 7 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu   1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln              20 25

Claims (15)

From the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
An antibiotic peptide comprising an amino acid sequence in which the amino acid of any one or both of the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) is replaced with tryptophan (W).
The antibiotic peptide according to claim 1, wherein the antibiotic peptide is composed of any one of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 4.
An antibiotic peptide consisting of an amino acid sequence in which the amino acid of any one or both of alanine (A), which is the 15th amino acid from the amino acid sequence shown in SEQ ID NO: 1 and phenylalanine (F), which is the 18th amino acid, is substituted with tryptophan (W) As an active ingredient.
(a) an amino acid sequence in which the amino acid sequence of any one or both of the amino acid sequence of SEQ ID NO: 1, or the 15th amino acid alanine (A) and the 18th amino acid phenylalanine (F) An antimicrobial peptide consisting of And
(b) an antibiotic as an active ingredient,
The antibiotic may be selected from the group consisting of Erythromycin, Ampicillin, Vancomycin, Linezolid, Methicillin, Oxacillin, Cefotaxime, Rifampicin, Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Doripenem, Cefepime, Ceftaroline, Ceftobiprole, Aztreonam, and the like, as well as the compounds of the present invention, , Piperacillin, Polymyxin B, Colistin, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Tigecycline, ), A mixture thereof and a derivative thereof Wherein the antimicrobial composition is at least one selected from the group consisting of:
The antimicrobial composition according to claim 4, wherein the antibiotic peptide is composed of any one of the amino acid sequences of SEQ ID NOS: 1 to 4.
delete 5. The composition according to claim 3 or 4, wherein the antimicrobial composition is selected from the group consisting of Bacillus subtilis , Staphylococcus aureus , and Staphylococcus epidermidis . Any one or more Gram positive bacteria; One or more gram-negative bacteria selected from the group consisting of Escherichia coli , Pseudomonas aeruginosa , Acinetobacter baumannii , and Salmonella typhimurium ; And antimicrobial activity against the antibiotic resistant bacteria of the present invention.
delete delete The antimicrobial composition according to claim 3 or 4, wherein the antimicrobial composition is a pharmaceutical composition.
The antimicrobial composition according to claim 3 or 4, wherein the antimicrobial composition is a food additive composition.
The antimicrobial composition according to claim 3 or 4, wherein the antimicrobial composition is a feed additive composition.
The antimicrobial composition according to claim 3 or 4, wherein the antimicrobial composition is a composition for antimicrobial use.
The antimicrobial composition according to claim 3 or 4, wherein the antimicrobial composition is a quasi-drug composition.
A peptide consisting of any one of the amino acid sequences represented by SEQ ID NO: 2 to SEQ ID NO: 4.

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