KR102415734B1 - Novel antimicrobial peptide and uses thereof - Google Patents

Abstract

The present invention relates to a novel antibacterial peptide and uses thereof. The novel antibacterial peptide of the present invention consisting of the amino acid sequence of SEQ ID NO: 6 not only has excellent antibacterial activity against gram-positive bacteria, gram-negative bacteria and antibiotic-resistant bacteria, but also is effective in regenerating cells. Therefore, the novel antibacterial peptide is expected to be effectively used in various fields including antibacterial compositions, cosmetic compositions, preservative compositions, and the prevention and treatment of infectious diseases.

Description

Novel antimicrobial peptide and uses thereof

The present invention relates to novel antibacterial peptides and uses thereof.

It is known that most living things on Earth make antimicrobial peptides (hereinafter referred to as AMPs) to prevent invasion from external microorganisms and to protect themselves. Antibacterial peptide is a basic, positively-charged, basic peptide composed of 10-50 amino acids with a molecular weight of 2-6 KDa. It is a host defense factor. Antibacterial peptides are the only defense system for invertebrates that do not have an immune system, such as insects and crustaceans. (G. Wang, (2014) Pharmaceuticals, 7:545-594; L. Zhang. et al., (2016) Current Biology 26:R1-R21).

The first antibiotic discovered was penicillin, discovered by Alexander Fleming in 1928, but before that, in 1922, Fleming discovered lysozyme with antibacterial activity in human saliva. Then, peptide antibiotics such as gramicidin were developed and used for treatment in the 1940s, and as science and technology developed after the 1980s, scientific technology for separation and structural analysis of peptides developed, and many peptides were discovered. In particular, the medical community began to draw attention from the US National Institutes of Health, Dr. Zaslov, in 1987, an antibacterial peptide called magainin from African frogs for the treatment of diabetic food ulcers. After completing clinical trials and trying to obtain new drug approval, it began to receive a lot of attention. After that, in 1996, the existence of various antibacterial peptides was revealed from various organisms, including the discovery of a peptide called buforin from the stomach of a Korean toad ( Bufo bufo gargarizans ), and many studies on characteristics and functions were conducted. Antibacterial peptides have been isolated from about 1,500 different organisms so far. However, when looking at the studies on many antibacterial peptides discovered so far, they have general tendencies such as alpha-helix or beta-sheet and basicity in terms of structure and activity, but almost similar in amino acid sequence. is not visible This is thought to be because each organism has evolved its own peptide structure and sequence differently to suit the environment in which it lives. Various mechanistic models have been proposed, and as an example, it is known that an antibacterial peptide having an alpha helix structure acts on the cell membrane of bacteria and destroys the cell membrane, thereby killing cells.

The excellent points of these antibacterial peptides as new pharmaceuticals and cosmetic raw materials are summarized as follows.

First, due to the misuse of antibiotics, strains resistant to antibiotics continue to appear, which is emerging as a serious threat to human health. Representative resistant bacteria include gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), gram-negative bacteria Pseudomonas aeruginosa , acine ( Acinetobacter baumannii ), etc., and as new multidrug-resistant bacteria continue to be discovered, antibiotic-resistant bacteria survive in all parts and organs of the body, such as acute sepsis, respiratory infections, cystic fibrosis, and urinary tract infections. have. However, antimicrobial peptides have a mechanism of action that physically destroys microbial cell membranes, and thus can prevent the emergence of antibiotic-resistant bacteria. Unlike general chemically synthesized peptides that stop bacterial growth by interfering with cell wall or nucleic acid synthesis, antibacterial peptides directly destroy cells while penetrating cell membranes, thereby avoiding the adaptation mechanism of antibiotic resistance.

Second, the antimicrobial peptide is rapid, effective and has a broad antimicrobial spectrum.

Third, the antibacterial peptide selectively acts only on pathogens, so it is effective for the human body. This is because, in the case of human eukaryotic cells, a large amount of cholesterol having a neutral charge exists in the cell membrane, which prevents the passage of antibacterial peptides.

Fourth, since the antimicrobial peptide does not have secondary modifications such as glycosylation, it can be mass-produced using the process of genetic engineering and has a low production cost during development.

Fifth, since most antibacterial peptides have cell regeneration, that is, wound healing activity, they can be used for skin care along with antibacterial activity (Ana Gomes et. al., (2017) Molecules, 22:1743- 1761).

Antibacterial peptide research on plants and terrestrial organisms in nature is being actively conducted, and has been isolated from various tissues ranging from vertebrates to invertebrates so far. Studies have been conducted to prepare an antibacterial peptide library based on physicochemical properties and to search for peptides with improved shortcomings therefrom, or to search for and improve new peptides in nature. In particular, the potency of antibacterial peptides is greatly affected by various physical properties, such as length and electrical properties. In general, the length of amino acids should be less than 50, cationic amino acids should be large, hydrophobic, and should have an amphipathic structure. In particular, the distribution and hydrophobicity of cationic amino acids are important because the surface of the bacterial cell membrane is negatively charged, making it easy to bind. In the case of the alpha helix structure, since the number, structure, and properties of helix rotations are affected according to the amino acid composition, a software program can be used as a means to visually express it. Using these programs, hydrophobic amino acids are concentrated in any part of the helix, and the sequence of cationic amino acids that are easy to bind to the cell membrane of bacteria can be analyzed. In addition, it was found that the antimicrobial activity of the antibacterial peptide of the alpha helix structure is increased when proline is present to act as a structural hinge in the middle part (Ruth Ann Veach. et al., (2004) J. Biol. Chem. 279:(3), 11425-11431). Therefore, it is necessary to improve the peptide that increases the antibacterial activity while reflecting these characteristics.

Meanwhile, Korea Patent No. 1734331 discloses 'a novel antibacterial active peptide isolated from Korean sea cucumber and its use', and Korean Patent No. 2039400 discloses 'a novel antibacterial peptide derived from mBjAMP1 peptide and its use'. However, there is no description of the novel antibacterial peptide of the present invention and its use.

The present invention has been derived from the above needs, and the present inventors have some residues in the amino acid sequence of a novel antibacterial peptide (SEQ ID NO: 5) prepared by combining the amino acid sequences of known antibacterial peptides (SEQ ID NO: 1 and SEQ ID NO: 2) It was confirmed that the novel antibacterial peptide (SEQ ID NO: 6) prepared by replacing By confirming that shows an excellent cell regeneration effect, the present invention was completed.

In order to solve the above problems, the present invention provides an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 6.

In addition, the present invention provides a polynucleotide encoding the antibacterial peptide.

In addition, the present invention provides an antibiotic containing the antibacterial peptide as an active ingredient.

In addition, the present invention provides an antibacterial quasi-drug composition containing the antibacterial peptide as an active ingredient.

In addition, the present invention provides an antibacterial cosmetic composition containing the antibacterial peptide as an active ingredient.

In addition, the present invention provides an antibacterial food additive containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antibacterial feed additive containing the antibacterial peptide as an active ingredient.

In addition, the present invention provides an antibacterial biological pesticide containing the antibacterial peptide as an active ingredient.

In addition, the present invention provides an antiseptic composition containing the antibacterial peptide as an active ingredient.

In addition, the present invention provides an antibacterial method in a subject comprising administering to the subject a pharmaceutically effective amount of the antimicrobial peptide.

The novel antibacterial peptide of the present invention has excellent antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant bacteria, and has a cell regeneration effect, so it can be used in various fields such as antibacterial compositions, prevention and treatment of infectious diseases, cosmetic compositions, and antiseptic compositions. It may be useful.

1 is a helical wheel diagram of an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 6.
Figure 2 is a helical wheel diagram of the antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 4 (helical wheel diagram).
3 is a result showing the results of scratch wound healing assay (A), cell density (B) and average interval (C) for measuring the cell regeneration effect of the antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 4 . *, *** means that there is a statistically significant difference in the results of the peptide-treated group compared to the untreated group, * is p<0.05, *** is p<0.001. Scale bar = 200 μm.
4 is a result showing the results of scratch wound healing assay (A), cell density (B) and average interval (C) for measuring the cell regeneration effect of the antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 6 . *, **, *** means that there is a statistically significant difference in the results of the peptide-treated group compared to the untreated group, * is p<0.05, ** is p<0.01, *** is p<0.001. Scale bar = 200 μm.

In order to achieve the object of the present invention, the present invention provides an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 6.

As used herein, the term "antibacterial peptide" refers to a polymer composed of amino acids linked by an amide bond (or peptide bond), and has growth inhibitory activity against microorganisms.

The novel antibacterial peptide having the amino acid sequence of SEQ ID NO: 6 according to the present invention is the amino acid sequence (RLLRRLLRPK) of the antibacterial peptide (SEQ ID NO: 5) prepared by combining the amino acid sequences of known antibacterial peptides (SEQ ID NO: 1 and SEQ ID NO: 2) KF GK A FVKILK K ) in the 11th, 12th, 15th, and 22nd amino acids (underlined) were prepared by substituting the same replacement amino acids as in each characteristic. Specifically, SEQ ID NO: 5 is an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RLLRRLLR) with known antibacterial activity and the amino acid sequence of SEQ ID NO: 2 (GIGKFLKKA KKFGKAFVKILKK ) from the 10th to the 22nd (underlined) rear part It was prepared by splicing the sequences, and in order to double the effect of increasing the antibacterial activity, it was prepared by inserting proline (P) to serve as a structurally bent part (hinge) between the two sequences. A novel antibacterial peptide (RLLRRLLRPK RW GK L FVKILK R , SEQ ID NO: 6) was prepared by substituting R, W, L and R for the 11th, 12th, 15th, and 22nd amino acids in SEQ ID NO: 5, respectively. In addition, SEQ ID NO: 4 was prepared by splicing the back partial sequences from the 4th to the 14th of the antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 and the antibacterial peptide known as LK peptide (SEQ ID NO: 3, LKKLLKLLKKLLKL). SEQ ID NO: 4 was similarly prepared by inserting proline (P) to serve as a structurally broken part (hinge) between the two sequences (see Tables 1 and 2).

In the present invention, the amino acid of the antibacterial peptide preferably has an L form, but substituted with a D-form is not excluded from the present invention.

The antibacterial peptide can be prepared by a conventional peptide synthesis method known in the art. As a method for the synthesis, it is preferable to synthesize by a chemical synthesis method (W. H. Freeman and Co., Proteins; structures and molecular principles, 1983), and specifically, a solution phase peptide synthesis method, a solid phase peptide synthesis method ( Solid-phase peptide synthesis), fragment condensation, and F-moc or T-BOC chemistry may be more preferable, but are not limited thereto. In addition, there is a method for biologically producing a polynucleotide encoding an antibacterial peptide by expressing it in genetically engineered E. coli (Cheng KT et al., (2018) Molecules, 23(4):800-811), but is not limited thereto.

The antibacterial peptide preferably has antibacterial activity against Gram-positive bacteria, Gram-negative bacteria or antibiotic-resistant bacteria, but is not limited thereto.

The Gram-positive bacteria include all Gram-positive bacteria including Staphylococcus, Enterococcus, Bacillus, Listeria, or Lactobacillus. It may be a benign bacterium, preferably a gram-positive bacterium of the genus Staphylococcus, Enterococcus or Bacillus, most preferably Staphylococcus aureus , Enterococcus faecium or Bacillus Cereus ( Bacillus cereus ) may be, but is not limited thereto.

The Gram-negative bacteria are Escherichia, Pseudomonas, Acinetobacter, Klebsiella, Salmonella, Leptospira or Rickettsia. It may be any Gram-negative bacteria known in the art as Gram-negative bacteria, including, preferably, Gram-negative bacteria of the genus Escherichia coli, Pseudomonas, Acinetobacter or Klebsiella, and most preferably Escherichia coli , Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Acinetobacter baumannii ) and Klebsiella pneumoniae ) It may be any one or more selected from the group consisting of, but is not limited thereto.

In addition to the gram-positive and gram-negative bacteria, the antibacterial peptide of the present invention may have antibacterial activity against antibiotic-resistant bacteria. The antibiotic is not limited thereto, but aminoglycoside (aminoglycoside, gentamicin, neomycin, etc.), penicillin (ampicillin, etc.), sulfonamide, beta-lactam (beta-lactam, amoxicillin/clar) fluoranic acid), chloramphenicol, erythromycin, florfenicol, fosfomycin, kanamycin, lincomycin, methicillin, quinolone, streptomycin, tetracycline, trimesoprim, and vancomycin class of antibiotics.

In one embodiment of the present invention, the antibiotic-resistant bacteria are vancomycin-resistant Enterococcus faecium , methicillin-resistant Staphylococcus aureus , ESBL (Extended-spectrum β) -lactamases) producing Escherichia coli , carbapenem-resistant E. coli, carbapenem-resistant Pseudomonas aeruginosa , carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Klebsi It may be any one or more selected from the group consisting of Klebsiella pneumoniae , but is not limited thereto.

The antibacterial peptide of the present invention has low cytotoxicity to human-derived cells, and is characterized in that it has a cell regeneration effect.

In addition, the present invention provides a polynucleotide encoding the antibacterial peptide.

In addition, the present invention provides an antibiotic containing the antibacterial peptide as an active ingredient.

The antibacterial peptide of the present invention is a peptide having the amino acid sequence of SEQ ID NO: 6 as described above. Since the antibacterial peptide of the present invention exhibits strong antibacterial activity against gram-negative bacteria, gram-positive bacteria and antibiotic-resistant bacteria, the antibacterial peptide of the present invention can be usefully used as an active ingredient of antibacterial antibiotics.

The peptide of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations. Parenteral administration may refer to administration via a route other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular, and subcutaneous administration. When the antibacterial peptide of the present invention is used as a pharmaceutical, it may further contain one or more active ingredients exhibiting the same or similar function.

The antibacterial peptide of the present invention can be prepared in various parenteral formulations, and in the case of formulation, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, and surfactant. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspension solution, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base of the suppository, Witepsol, macrogol, Tween 61, cacao butter, liulinji, glycerogelatin, etc. may be used.

In addition, the antibacterial peptide of the present invention can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, to increase stability or absorbency; Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used as pharmaceuticals.

In the antibiotic of the present invention, the total effective amount of the novel peptides of the present invention may be administered to a patient in a single dose in the form of a bolus or by infusion for a relatively short period of time, and multiple administrations Multiple doses may be administered by a fractionated treatment protocol in which they are administered for a long period of time. Since the concentration is determined by considering various factors such as the patient's age and health status, as well as the administration route and number of treatments of the drug, the effective dosage of the patient is determined. It will be possible to determine an appropriate effective dosage according to the specific use of the novel peptide as an antibiotic.

In addition, the present invention provides an antibacterial quasi-drug composition containing the antibacterial peptide as an active ingredient.

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

The antibacterial quasi-drug of the present invention is not limited thereto, but preferably disinfectant, shower foam, gargrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler.

In addition, the present invention provides an antibacterial cosmetic composition containing the antibacterial peptide as an active ingredient.

The antibacterial peptide has excellent antibacterial activity, has low cytotoxicity to human-derived cells, and has a cell regeneration effect, so it can be very usefully used as an antibacterial cosmetic composition.

The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the antibacterial peptide, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

In the cosmetic composition of the present invention, the peptide of the present invention may be added in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight, in the cosmetic composition usually contained therein.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion (skin), a nourishing lotion (milk lotion), a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder. .

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. are used as carrier components. can be

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like may be used.

In addition, the present invention provides an antibacterial food additive containing the antimicrobial peptide as an active ingredient.

The antibacterial peptide has excellent antibacterial activity, has low cytotoxicity to human-derived cells, and has a cell regeneration effect, so it can be very usefully used as an antibacterial food additive.

When the peptide of the present invention is used as a food additive, the peptide may be added as it is or used together with other food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of its use. In general, the peptide of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, 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 above the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all foods in a conventional sense.

In addition, the present invention provides an antibacterial feed additive containing the antibacterial peptide as an active ingredient.

The feed composition of the present invention replaces the existing antibiotics and suppresses the growth of harmful food pathogens to improve the health of animals, improve the weight gain and quality of livestock, and increase milk production and immunity. The feed composition of the present invention may be prepared in the form of fermented feed, compound feed, pellet form, silage, and the like.

The fermented feed may be prepared by fermenting organic matter by adding various microbial groups or enzymes other than the peptide of the present invention, and the compounded feed may be prepared by mixing several types of general feed and the peptide of the present invention. Feed in the form of pellets can be prepared by applying heat and pressure to the compounded feed, etc. in a pellet machine, and silage can be prepared by fermenting Cheongye feed with microorganisms. Wet fermented feed collects and transports organic matter such as food waste, mixes excipients for sterilization process and moisture control in a certain ratio, and ferments it at a temperature suitable for fermentation for 24 hours or more so that the moisture content is about 70%. It can be adjusted and manufactured. Fermented dry feed can be prepared by adjusting the moisture content of wet fermented feed to 30% to 40% through an additional drying process.

In addition, the present invention provides an antibacterial biological pesticide containing the antibacterial peptide as an active ingredient.

In addition, the present invention provides an antiseptic composition containing the antibacterial peptide as an active ingredient.

The antibacterial peptide of the present invention is a peptide having the amino acid sequence of SEQ ID NO: 6, and exhibits strong antibacterial activity against Gram-negative bacteria, Gram-positive bacteria and antibiotic-resistant bacteria. It can be usefully used as an ingredient.

The preservative composition includes a cosmetic preservative or a pharmaceutical preservative. The food preservatives, cosmetic preservatives and pharmaceutical preservatives are additives used to prevent deterioration, spoilage, discoloration and chemical changes of pharmaceuticals. These include sterilizing agents and antioxidants, and inhibiting the growth of microorganisms such as bacteria, mold, yeast, etc. and functional antibiotics, such as inhibiting the growth or sterilization of putrefactive microorganisms in pharmaceuticals. Ideal conditions for such a preservative composition should be non-toxic and effective even in a small amount.

In addition, the present invention provides an antibacterial method in a subject comprising administering to the subject a pharmaceutically effective amount of the antimicrobial peptide. The subject may be a mammal other than a human, but is not limited thereto.

In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type, severity, drug activity, and drug of the patient. It can be determined according to factors including sensitivity, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The antimicrobial peptide of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the antimicrobial peptide of the present invention can be administered in various ways depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, but the administration route, obesity The dosage is not intended to limit the scope of the present invention in any way because it may increase or decrease depending on the severity, sex, weight, age, and the like.

The amino acid sequences used herein are abbreviated as follows according to the IUPAC-IUB nomenclature: alanine A, arginine R, asparagine N, aspartic acid D, cysteine C, glutamic acid E, glutamine Q, glycine G, histidine H, Isoleucine I, leucine L, lysine K, methionine M, phenylalanine F, proline P, serine S, threonine T, tryptophan W, tyrosine Y, valine V. In addition, the amino acids may include both D-type amino acids and L-type amino acids. can, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Peptide Synthesis

In the present invention, the peptides of Table 1 were synthesized through the solid/solution phase using the Fmoc chemistry method, a peptide synthesis technology, and purified by high-performance liquid chromatography to a purity of 95% or more. .

Synthesized peptide information SEQ ID NO: antibacterial peptide number of amino acids note One RLLRRLLR 8 Korean Patent No. 1341210 2 GIGKFLKKA KKFGKAFVKILKK 22 magainin, msi 78 3 LKKLLKLLKKLLKL 14 LK peptide 4 RLLRRLLRPILLKLLKKLLKL 21 Novel derivative peptide (H10) 5 RLLRRLLRPK KF GK A FVKILK K 22 Novel derivative peptide (H11) 6 RLLRRLLRPK RW GK L FVKILK R 22 Novel derivative peptide (H12)

A peptide of SEQ ID NO: 4 (H10) was synthesized by combining the peptides of SEQ ID NO: 1 and SEQ ID NO: 3, and a peptide of SEQ ID NO: 5 (H11) was synthesized by combining the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (underlined). , The peptide of SEQ ID NO: 6 (H12) was synthesized by substituting the 11th, 12th, 15th, and 22nd amino acids (underlined) in the peptide of SEQ ID NO: 5 with replacement amino acids identical to each characteristic (non-polar or positive charge). (Table 2).

(MW: molecular weight)

All of the peptides in the table above have the L-form, but those substituted with the D-form are not excluded from the present invention.

Example 2. Measurement of antibacterial activity of novel derivative peptides

In order to compare the antibacterial activity of the synthesized novel derivative peptide, the antibacterial activity was measured against non-resistant or antibiotic-resistant strains of gram-negative and gram-positive bacteria, respectively. Antibacterial activity was measured by measuring the minimum growth inhibitory concentration (MIC) of the peptide that does not divide the cells in MH (Mueller Hinton) medium with sufficient nutrients. Obtain the strains shown in Table 3 below, dilute with MH medium so that the number of bacteria per ml becomes 2 × 10 ⁶ CFUs (colony-forming units), and then dispense 100 μl each in a 96-well microtiter plate, 100 μl of the peptide solution diluted with MH medium was added to each well. The MIC was determined by measuring the absorbance at 620 nm after incubating the plate at 37° C. for 16 hours.

Antimicrobial activity analysis of novel derivative peptides target strain Peptide MIC (μg/ml) Bacteria strain Antibiotic resistance characteristics SEQ ID NO: 4
(H10) SEQ ID NO: 5
(H11) SEQ ID NO: 6
(H12)




gram
positivity Enterococcus faecium BM 4147 intolerant (susceptible) 128 >128 32 KHIDI 70 Vancomycin Resistance (VRE) 8 8 8 KHIDI 73 Vancomycin Resistance (VRE) 8 16 4 Stapylococcus aureus ATCC 29213 intolerant (susceptible) 64 >128 64 KHIDI 25 methicillin resistance (MRSA) 64 >128 128 KHIDI 29 methicillin resistance (MRSA) 64 >128 64





gram negative Escherichia coli ATCC 25922 intolerant (susceptible) 64 >128 32 KHIDI 77 ESBL ¹⁾ 32 64 32 KHIDI 7 carbapenem resistance 32 128 16 Pseudomonas aeruginosa
ATCC 27853 intolerant (susceptible) 128 >128 64 KHIDI 9 carbapenem resistance 128 >128 32 KHIDI 16 carbapenem resistance 128 >128 32 Acinetobacter baumannii KHIDI 61 intolerant (susceptible) 64 32 32 KHIDI 58 carbapenem resistance 64 32 8 Klebsiella pneumoniae KHIDI 2 carbapenem resistance 64 128 >128 1) ESBL: Extended-spectrum β-lactamase

As a result, it was found that the novel derivative peptide of SEQ ID NO: 6 of the present invention showed strong antibacterial activity compared to the novel derivative peptides of SEQ ID NO: 4 and SEQ ID NO: 5. In addition, the antibacterial peptide of SEQ ID NO: 1 has been reported to exhibit a MIC of 1 mg/ml, that is, 1,000 μg/ml, against both Gram-positive and Gram-negative bacteria (refer to Korean Patent No. 1341210), in the present invention It was confirmed that the newly synthesized antibacterial peptide (SEQ ID NO: 6) exhibited significantly increased antimicrobial activity compared to the novel derivative peptide (SEQ ID NO: 4, H10) and the parent peptide (SEQ ID NO: 5, H11) (Table 3).

Vancomycin-resistance Enterococcus faecium (VRE), according to a report from the Korea Centers for Disease Control and Prevention (KCDC), has shown a significant increase in the range of 12-23% depending on the region over the past three years. It was confirmed that the antibacterial activity of the antimicrobial peptide (SEQ ID NO: 6) of the present invention against VRE was superior to that of the non-resistant (susceptible) strain.

In the case of methicillin-resistance Staphylococcus aureus (MRSA), which is famous for multi-resistant bacteria, it was found that the antibacterial peptide (SEQ ID NO: 6) of the present invention showed a MIC value similar to that of the non-resistant strain.

For carbapenem-resistant Escherichia coli strains, the antibacterial peptide (SEQ ID NO: 6) of the present invention showed superior antibacterial activity compared to SEQ ID NO: 4 and SEQ ID NO: 5. And Extended-spectrum β-lactamase (ESBL) is a β-lactamase propagated by a plasmid, and the frequency of nosocomial infections by Gram-negative strains that produce ESBL is gradually increasing. Penicillin, cephalosporin , it has been reported to show multiple resistance to carbapenems, etc., making it difficult to treat infection (Pilhoon Yoon et al. (2014) Korean J Nosocomial Infect Control 19(2):45-51). There is also a report that they have carbapenem resistance as a last resort, resulting in a mortality rate of 12.9%. The novel antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 6 showed antibacterial activity even in these ESBL multi-resistant strains.

Pseudomonas aeruginosa is a major medical-related causative agent that causes skin infections, pneumonia, bedsores, sepsis, and meningitis. This also shows versatility to antibiotics such as carbapenems and amicoglycosides. The antibacterial peptide (SEQ ID NO: 6) of the present invention shows rather superior antibacterial activity in carbapenem-resistant Pseudomonas aeruginosa compared to carbapenem-resistant strains. gave.

Acinetobacter baumannii ( Acinetobacter baumannii ) has increased the number of hospital-acquired infections worldwide since 2007, and the multidrug resistance rate including carbapenem antibiotics has also started to increase significantly. In 2010, 46 people were infected with Acinetobacter pylori infection at the University of Tokyo Hospital in Japan, and 10 of them died. The resistance rate to carbapenem antibiotics is showing a rapid increase from 27% in 2007 to 71.7% in 2010 (Jungwook Cha et al. (2012), PUBLIC HEALTH WEEKLY REPORT, KCDC Vol. 7 No. 29, 630-632). Multidrug-resistant Acinetobacter baumani is a resistant bacterium that hospitals should pay particular attention to because it can cause infections in patients with reduced immunity, chronic lung disease, or long-term hospitalization. The antibacterial peptide (SEQ ID NO: 6) of the present invention showed superior antibacterial activity against carbapenem-resistant Acinetobacter baumani compared to non-resistant bacteria.

It also showed strong antibacterial activity against carbapenem-resistant bacteria of Klebsiella pneumoniae , called St. pneumoniae. From the above results, since the antibacterial peptide (SEQ ID NO: 6) of the present invention shows excellent antibacterial activity against various antibiotic-resistant bacteria, including gram-positive and gram-negative bacteria, an antibacterial composition or an infectious disease prevention and treatment and cosmetic composition, preservative composition It is expected to be useful in various fields such as

Example 4. Characterization of novel antibacterial peptides

In order to analyze the excellent antimicrobial activity of the novel antimicrobial peptide (SEQ ID NO: 6), the structural characteristics were identified using the helical wheel diagrams technique. Comparing the helical wheel structure of SEQ ID NO: 6 (FIG. 1) and the helical wheel structure of SEQ ID NO: 4 (FIG. 2), SEQ ID NO: 4 is the position of the amino acid P (proline) serving as the folding structure (hinge) is a hydrophobicity face (hydrophobicity face) ), but SEQ ID NO: 6 was judged to have more enhanced hydrophobicity in the hydrophobicity face. In addition, as in SEQ ID NO: 6, hydrophobic amino acids are uniformly arranged at the bottom of the peptide like the helical wheel structure of SEQ ID NO: 1 and the helical wheel structure of SEQ ID NO: 2, so it was determined that the basic characteristics that the antibacterial peptide should have were well equipped. In addition, the peptide of SEQ ID NO: 6 has an average hydrophobicity of 0.362, which is higher than 0.280 of SEQ ID NO: 5, thereby supporting excellent antibacterial activity.

Example 5. Measurement of hemolytic activity

In order to compare the cytotoxicity of the peptides prepared by the method of Example 1, the erythrocyte hemolytic activity of the synthesized peptides was measured.

Specifically, human red blood cells were diluted with PBS (pH 7.0) to a concentration of 8%, and the peptides of SEQ ID NOs: 2, 4, or 6 were treated at a concentration of 1, 5, 10, 20 or 100 μg/ml per well, respectively. , and reacted at 37°C for 1 hour. Then, the amount of hemoglobin contained in the supernatant obtained by centrifugation at 1,000 x g was confirmed by measuring the absorbance at a wavelength of 414 nm. As a control that serves as a standard for the degree of cell destruction, the absorbance of the supernatant obtained after treatment with 0.2% Triton and reacting at 37° C. for 1 hour was measured. The hemolysis activity (hemolysis) of each peptide was calculated using the formula.

Hemolytic activity = (absorbance A-absorbance B)/(absorbance C-absorbance B)

(Absorbance A: Absorbance of each peptide-treated reaction solution measured at a wavelength of 414 nm, Absorbance B: Absorbance of a reaction solution treated with PBS measured at a wavelength of 414 nm, Absorbance C: 0.2% Triton measured at a wavelength of 414 nm Absorbance of the reaction solution treated with

As a result of comparing the hemolytic activity (%) expressing the hemolytic activity as a percentage, the hemolytic activity of the peptide of SEQ ID NO: 2 was 0.5±0.4% and 1.2±0.4%, respectively, at concentrations of 1, 5, 10, 20 and 100 μg/ml, respectively; 1.8±0.7%, 2.9±1.0%, and 9.2±2.9%. The hemolytic activity of the peptide of SEQ ID NO: 4 (H10) was 7.2±0.6%, 34.4±2.2%, 53.8±3.9%, 75.7±4.3% and 95.7±1.9 at concentrations of 1, 5, 10, 20 and 100 μg/ml, respectively. % was shown. The hemolytic activity of the peptide of SEQ ID NO: 6 (H12) was -0.9±0.6%, 0±0.9%, 0.7±0.4%, 1.2±0.8% and 6± at concentrations 1, 5, 10, 20 and 100 μg/ml, respectively. 1.5% (Table 4). All hemolytic activities (%) were expressed as mean ± standard deviation.

Based on the above results, it was confirmed that the hemolytic activity of the novel antibacterial peptide (SEQ ID NO: 6) of the present invention was less than 1% at low concentrations (1, 5 and 10 μg/ml), so it is the most stable compared to other antibacterial peptides and human It was found that there was no cytotoxicity to red blood cells.

Hemolytic activity of antibacterial peptides test sample PBS Peptide concentration (μg/ml) Triton One 5 10 20 100 SEQ ID NO: 2
(msi78) One 0 0.000 0.008 0.014 0.019 0.065 One 2 0 0.006 0.010 0.012 0.024 0.071 One 3 0 0.004 0.013 0.019 0.031 0.107 One 4 0 0.010 0.018 0.028 0.043 0.126 One SEQ ID NO: 4
(H10) One 0 0.073 0.340 0.534 0.735 0.955 One 2 0 0.064 0.323 0.500 0.723 0.932 One 3 0 0.074 0.375 0.591 0.820 0.971 One 4 0 0.078 0.339 0.526 0.750 0.973 One SEQ ID NO: 6
(H12) One 0 -0.011 -0.011 0.002 0.002 0.042 One 2 0 -0.004 -0.002 0.007 0.014 0.070 One 3 0 -0.015 0.002 0.007 0.012 0.075 One 4 0 -0.003 0.010 0.012 0.021 0.067 One

* Hemolytic activity (%) is a value obtained by multiplying the value in the table above by 100.

Example 6. Measurement of cell regeneration effect

In order to measure the cell regeneration efficacy of the antimicrobial peptide of the present invention, a scratch wound healing assay was performed. HaCaT cells were seeded in a 12-well plate at 5×10 ⁴ cells/ml and cultured for 24 hours in DMEM medium containing 10% FBS for stabilization. proceeded. When the cells in the wells showed 100% confluency, the center of each well was constantly scraped using a 200 μl tip, and then all the medium was removed and washed with PBS. Thereafter, peptides were treated at a concentration of 1, 5, 10, 25 or 50 μg/ml, respectively, and incubated for 24 hours, then observed using a microscope and artificially created gaps between cells ( The change in whether the gap) was filled was confirmed.

As a result, it was confirmed that the peptides of SEQ ID NO: 4 and SEQ ID NO: 6 had no cell regeneration effect at a high concentration of 50 μg/ml. The peptide of SEQ ID NO: 4 showed the best cell regeneration effect at a concentration of 10 μg/ml, whereas the peptide of SEQ ID NO: 6 showed the best cell regeneration effect at a concentration of 5 to 25 μg/ml (Fig. 3, Fig. 4).

<110> PepXGen Inc. <120> Novel antimicrobial peptide and uses thereof <130> PN21301 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> antimicrobial peptide <400> 1 Arg Leu Leu Arg Arg Leu Leu Arg 1 5 <210> 2 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> antimicrobial peptide <400> 2 Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe 1 5 10 15 Val Lys Ile Leu Lys Lys 20 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> antimicrobial peptide <400> 3 Leu Lys Lys Leu Leu Lys Leu Leu Lys Lys Leu Leu Lys Leu 1 5 10 <210> 4 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> antimicrobial peptide <400> 4 Arg Leu Leu Arg Arg Leu Leu Arg Pro Ile Leu Leu Lys Leu Leu Lys 1 5 10 15 Lys Leu Leu Lys Leu 20 <210> 5 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> antimicrobial peptide <400> 5 Arg Leu Leu Arg Arg Leu Leu Arg Pro Lys Lys Phe Gly Lys Ala Phe 1 5 10 15 Val Lys Ile Leu Lys Lys 20 <210> 6 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> antimicrobial peptide <400> 6 Arg Leu Leu Arg Arg Leu Leu Arg Pro Lys Arg Trp Gly Lys Leu Phe 1 5 10 15 Val Lys Ile Leu Lys Arg 20

Claims (14)

Antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 6. The antibacterial peptide according to claim 1, wherein the antibacterial peptide has antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, or antibiotic-resistant bacteria. According to claim 2, wherein the Gram-positive bacteria is Staphylococcus aureus ( Staphylococcus aureus ), Enterococcus faecium ( Enterococcus faecium ) and Bacillus cereus ( Bacillus cereus ) Characterized in that at least one selected from the group consisting of antibacterial peptides. According to claim 2, wherein the gram-negative bacteria is Escherichia coli , Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Acinetobacter baumannii ( Acinetobacter baumannii ) and Klebsiella pneumoniae selected from the group consisting of Antibacterial peptide, characterized in that any one or more that is. The method of claim 2, wherein the antibiotic-resistant bacteria is vancomycin-resistant Enterococcus faecium , methicillin-resistant Staphylococcus aureus , ESBL (Extended-spectrum β-lactamase) Escherichia coli , carbapenem-resistant E. coli, carbapenem-resistant Pseudomonas aeruginosa , carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Klebsiella Antibacterial peptide, characterized in that any one or more selected from the group consisting of Ae ( Klebsiella pneumoniae ). A polynucleotide encoding the antibacterial peptide of claim 1. An antibiotic comprising the antimicrobial peptide of claim 1 as an active ingredient. An antibacterial quasi-drug composition comprising the antibacterial peptide of claim 1 as an active ingredient. An antibacterial cosmetic composition comprising the antibacterial peptide of claim 1 as an active ingredient. An antibacterial food additive containing the antimicrobial peptide of claim 1 as an active ingredient. An antibacterial feed additive containing the antimicrobial peptide of claim 1 as an active ingredient. An antibacterial biological pesticide containing the antibacterial peptide of claim 1 as an active ingredient. An antiseptic composition comprising the antibacterial peptide of claim 1 as an active ingredient. An antibacterial method in a non-human subject, comprising administering a pharmaceutically effective amount of the antibacterial peptide of claim 1 to a subject other than a human.

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