KR20110139947A - Peptide analogue with high antibacterial and anti-inflammatory activities designed from papiliocin peptide - Google Patents

Abstract

PURPOSE: A novel derivative of papiliocin isoalted from Papilio xuthus larva is provided to ensure antibacterial activity against gram negative bacteria and gram positive bacteria. CONSTITUTION: A peptide is selected from sequence numbers 1-2. A pharmaceutical composition contains the peptide as an active ingredient without toxicity to human erythrocytes or mammal normal cells. An antibacterial composition against microorganisms contains a peptide selected among sequence numbers 1 and 2 as an active ingredient. The microorganisms are gram positive bacteria, gram negative bacteria, or antibiotics resistant strain. An anti-inflammatory composition contains the peptide of sequence number 1 as an active ingredient.

Description

Peptide analogue with high antibacterial and anti-inflammatory activities designed from Papiliocin peptide}

The present invention relates to a novel peptide, more specifically, a swallowtail butterfly ( Papilio) xuthus ) relates to an antibiotic peptide derivative having the N-terminal sequence of Papiliocin 1 isolated from larva and its use as an antibiotic and / or anti-inflammatory agent.

Since penicillin, many kinds of antibiotics have been developed and used to combat external invasive bacteria. Recently, however, strains resistant to these antibiotics have emerged, which is considered to be a big problem. That may pose a threat to life, Enterococcus faecalis nose Syracuse (Enterococcus faecalis), Mycobacterium-to M. tuberculosis (Mycobacterium tuberculosis) and bacterial species such as Pseudomonas her rugi Labor (Pseudomonas aeruginosa) have raised resistance to all antibiotics known so far (Stuart B. Levy, Scientific American, 46-53, 1998).

Conventional chemical antibiotics such as penicillin, cephalosporin and the like exhibit antibiotic activity by inhibiting the cell wall or protein synthesis of microorganisms. The development of antibiotics with new mechanisms of action to combat these resistant strains is urgent. Antibiotic peptides, which exhibit different antibiotic mechanisms than conventional chemical antibiotics, are attracting attention as a new concept of next-generation antibiotics ( Zasloff M. Curr). Opin Immunol 1992 4 , 3-7; Boman, HG, Cell , 65: 205, 1991; Boman, HG J Intern Med. 2003, 254 (3), 197-215; Hancock, RE, & Scott, MG, Proc . Natl . Acad . Sci . USA 2000, 97, 8856-8861, Zasloff, M., Nature 2002, 415, 389-395).

In a broad sense, antigen-antibody responses, called immune responses, have played a major role in biological defense in invertebrates, including humans. But insects protect themselves by strengthening innate immune mechanisms, which are different from acquired immune mechanisms such as antigenic antibody responses. As such, innate immunity plays an important role as an important defense against early infection in vertebrates including humans as well as insects. A very important means of the innate immune mechanism of insects is to obtain substances with bactericidal and inactivating effects on various microorganisms. Among them, peptides and proteins having antimicrobial activity centered on antimicrobial peptides are highly applicable to disease control such as domestic animals and livestock. Such insect-derived anti-pathogenic protein may be very useful for the prevention and treatment of damage caused by the infection of various pathogens as well as the value of diseases of humans and livestock.

Antibiotic peptides can be divided into three groups depending on their structure. The first is a cysteine-rich beta-sheet peptide, the second is an alpha-helical amphiphilic peptide molecule, and the third is rich in certain amino acids such as proline or tryptophan. -rich, Trp-rich) peptide (Mayasaki et. al ., Int . J. Antimicrob . Agents , 9, 269-280, 1998; Andreu , D .; Rivas , L. Biopolymers 1998, 47, 415-433 ). These antimicrobial peptides are known to play an important role in host defense and the innate immune system.

In addition, the antimicrobial peptides have various structures according to the amino acid sequence, the most common of these structures is the absence of cysteine residues such as melittin or cecropin, antimicrobial peptides found in insects Mars is a structure that forms an alpha-helix.

The antimicrobial proteins and peptides of insects discovered to date have been described by Dr. Boman's team. About 200 species have been isolated from insects since they were first reported in the cecropia moth, which can be largely divided into peptides containing a large number of amino acids, such as cecropins, defensins, florins, and glycine, depending on their structure and size. .

Secropins are basic proteins composed of 31 -39 amino acids and have a stable, broad antimicrobial activity with alpha-helix-hinge-alpha-helix structures, including N-terminal amphiphilic helixes (Boman and Hultmark, Annu). Rev. Micrbiol., 41: 1003-126, 1987).

Major insect-derived antimicrobial proteins include sacotoxin (Baba et al., Biochem., 102: 69-74, 1987) found in Sarcophaga peregrina and diptericin (Wicker et al., Biol) found in Drosophila melanogaster . Chem., 265: 2249322498, 1990) holotricin 3 (Lee et al., Biol. Pharm. Bull., 18: 1049-1052, 1995), brown edible ( Tenebrio molitor ), found in the Holotrichia diomphalia Tenecin 3 (Lee et al., Biochem. Biophys. Res. Commun., 218: 6-11, 1996), Defensin, Coleoptericin A and B (Miyanoshita et al., Found in Allomirina dichotoma ) .. Biochem Biophys Res Commun, 220 : 526-531, 1996; Sakisaca et al, Insect Mol Biol, 10:..... 293-302, 2001) in tobacco and bangahgwa Heliothis Heliomicin found in virescens (Lamberty et al., J. Biol. Chem. 274: 9320-9326, 1999), Rhinocerosin, Scarabaecin (Yang J et al., European Journal of Biochemistry) found in the southern beetle ( Oryctes rhinoceros ) , 255 (3): 734-738, 1998; Tomie et al., Biochemical Biophysical Research Communications, 307 (2): 261-266, 2003), Cicada, Cicada cicadin (Wang and Ng, Peptides, 23: 7-11, 2002) found in flammata, Cecropin (saito et al., Comp. Biochem. Physiol., B142: 317323, 2005) found in the large velvet sky cow ( Acalolepta luxuriosa ) ), Moricin analogue (Oizumi Y et al., Biochimica Biophysica Acta., 1752 (1): 83-92, 2005) found in Spodoptera litura .

The present inventors have filed a patent for the high antibacterial and anti-inflammatory activity of papyliocin 1 peptide isolated from the larva butterfly (Korean Patent Application No. 10-2009-0051608). However, papillosin has a long residue sequence, which is a disadvantage in that the synthetic unit is expensive.

The present invention solves the problem that the residue sequence is too long because the antimicrobial activity is high, and the object of the present invention is devised by the necessity of developing short peptides consisting of residue sequences of active sites from papyliocin. It is to provide a peptide having.

Still another object of the present invention is to provide a peptide having anti-inflammatory activity.

The present invention provides a peptide selected from SEQ ID NO: 1 to 2 to achieve the above object.

The present invention also provides a pharmaceutical composition comprising the peptide of the present invention as an active ingredient.

In one embodiment of the present invention, the composition of the present invention is preferably not toxic to human red blood cells or stray normal cells, but is not limited thereto.

The present invention also provides an antimicrobial composition against a microorganism comprising the peptide selected from SEQ ID NOS: 1-2 as an active ingredient.

In one embodiment of the invention, the microorganism is preferably Gram-positive bacteria, Gram-negative bacteria, or antibiotic-resistant strains, The Gram-positive bacteria is B. subtilis, S. aureus, or S. epidermidis, Gram-negative bacteria is E. coli , P. aeruginosa, or S. typhimurium, the antibiotic resistant strain is more preferably antibiotic resistant strain is vancoamicin-resistant Staphylococcus aureus (MRSA), multidrug-resistant S. typhimurium (MDRST), or antibiotic-resistant E. coli (MDREC) It is not limited to this.

In another aspect, the present invention provides an anti-inflammatory composition for inflammatory substances comprising the peptide described in SEQ ID NO: 1 as an active ingredient.

In one embodiment of the invention, the anti-inflammatory activity is characterized in that it is carried out by inhibiting the production of inducer nitrate oxide and cytokines.

In one embodiment of the present invention, the anti-inflammatory composition is preferably not toxic to human red blood cells or stray normal cells.

In another aspect, the present invention provides a feed composition comprising the peptide of the present invention as an active ingredient.

Hereinafter, the present invention will be described.

In order to isolate the antimicrobial peptide gene from the larvae larvae, the present inventors first identified gram-negative bacteria ( Esherichia larvae). After inducing immunity by injecting coli JM109), a cDNA gene bank was constructed to select antifungal peptide genes highly homologous to previously reported antimicrobial genes and to reveal the amino acid sequence of papillosin antimicrobial peptides. Application No. 10-2009-19022). The present inventors also chemically synthesize the antimicrobial peptide papiliosin to predict secondary structure, to determine the antimicrobial activity of various standard strains (6 species) and resistant strains (9 species) and to determine whether they are toxic in normal human cells, and to inflammation. It has been shown to inhibit the production of nitrite oxide and cytokines by inducing substances (Korean Patent Application No. 10-2009-0051608). In the present invention, a derivative PapN-1 having an N-terminal helix residue sequence 22 is removed based on the helix-hinge-helix structure of the antimicrobial peptide papillosin having 38 residues, and the previous two residues are removed from the N-terminal residue sequence. PapN-2 having 20 residue sequences and derivative PapC having 14 C-terminal helix residues were chemically synthesized. This is designed to develop high functional antimicrobial peptides with shorter residue sequences because papillocin has high antimicrobial activity but is expensive to use as a high functional peptide antibiotic because of its high synthetic cost. To determine the antimicrobial activity of various standard strains (6 species) and resistant strains (9 species) and toxicity in normal cells of human erythrocytes or strays, and to confirm the inhibitory activity of nitrite oxide and cytokines. By doing so, an important role in influencing the activity was identified.

Hereinafter, the present invention will be described in more detail according to the steps.

In the present invention, a swallowtail butterfly ( Papilio) belonging to the Lepidoptera insect xuthus ) Three fragments based on the antimicrobial peptide Papiliocin 1 structure isolated from larvae were synthesized and tested for antimicrobial activity against six bacteria. As a result, the antimicrobial peptides effectively inhibited the growth of 6 strains, which were reported by PapN-1 and PapN-2 having residue sequences of N-terminal helix. In contrast, PapC with C-terminal helix residues showed no antimicrobial activity. In particular, PapN-1 showed higher antimicrobial activity against Gram-positive bacteria than its parent, Papiliocin 1, and confirmed that it had antimicrobial activity in various bacteria. On the other hand, as a result of examining the destructive ability of erythrocytes to confirm that the peptides having antimicrobial activity are cytotoxic, PapN-1 and PapN-2 showed no hemolytic activity at 100 μM of peptide concentration, but had excellent antimicrobial activity. It was confirmed that the cytotoxicity showed low selectivity. PapC, on the other hand, showed high toxicity without antimicrobial activity.

Peptide Amino acid sequence SEQ ID NO: 1: PapN-1 RWKIFKKIEKVGRNVRDGIIKA-NH ₂    SEQ ID NO: 2: PapN-2 KIFKKIEKVGRNVRDGIIKA-NH ₂ SEQ ID NO: 3 PapC AVAVVGQAATVVKG-NH ₂ Comparative Peptide SEQ ID NO: 4 Papiliocin 1 RWKIFKKIEKVGRNVRDGIIKAGPAVAVVGQAATVVKG-NH ₂

Table 1 is a residue sequence of the three high-functional peptide derivatives described in SEQ ID NOs 1-3 of the present invention developed based on the residue sequence of Papiliocin 1 of the present invention and the residue sequence of the comparative peptide.

First, in order to confirm whether three antimicrobial peptides can be used for the above purposes, the present inventors measured antimicrobial activity against Gram-negative bacteria (three strains) and Gram-positive bacteria (three strains) for three antimicrobial peptides and control peptides. It was. Antimicrobial activity was observed by measuring the minimum inhibitory concentration (MIC, hereinafter abbreviated as MIC) of peptides that did not divide cells. Bacillus as Gram-positive bacteria subtilis , Staphylococcus aureus , Staphylococcus epidermidis as gram-negative bacteria Escherichia coli , Psedomonas aeruginosa and Salmonella typhimurium were included. Melittin activity was also observed as a control peptide.

PapN-1 and PapN-2 antibiotic peptides had antimicrobial activity against all bacteria. Especially, PapN-1 antibiotic peptide showed higher antimicrobial activity than Papiliocin 1, a control peptide against Gram-positive bacteria. (See Table 2).

Antimicrobial peptides PapN-1, PapN-2 and PapC were tested for toxicity to human erythrocytes, and control melittin was highly toxic, whereas PapN-1 and PapN-2 peptides except PapC showed no hemolytic activity at high concentrations ( See FIG. 1).

As in the above results, PapN-1 and PapN-2 antibiotic peptides corresponding to SEQ ID NOS: 1 and 2 are not toxic at a range of concentrations that show antimicrobial activity against macrophages (RAW264.7 Cell) of normal cells. The survival rate was very high even at high concentrations. PapC, on the other hand, had a very low survival rate (see Figure 2).

In addition, PapN-1, which has excellent antimicrobial activity in both Gram-negative and benign bacteria, is induced by the pathogen LPS-stimulated mouse macrophage RAW264.7 cells, the nitric oxide (NO) and inflammation-inducing cytokines mMIP-2 and mTNF. -Alpha was excellently inhibited, and mRNA expression encoding these cytokines was also effectively suppressed, which proved to be excellent as an anti-inflammatory peptide (see FIGS. 3, 4 and 5).

On the other hand, PapN-2 and C-terminal PapC, which have removed two residues from the N-terminus, show no anti-inflammatory activity, and the N-terminal alpha-helix moiety plays an important role in antimicrobial activity. Arg) and tryptophan (Trp) are expected to play an important role in anti-inflammatory activity.

The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable excipients, carriers, diluents and the like.

Carriers usable in the present invention include macromolecules that are slowly metabolized, such as liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Salts of inorganic acids such as, for example, hydrochloride, hydrobromide, phosphate and sulfate; Pharmaceutically acceptable salts such as salts of organic acids such as acetate, propionate, malonate and benzoate; Liquids such as water, saline, glycerol and ethanol, and auxiliary materials such as wetting agents, emulsifying agents or pH buffering substances may be used.

Pharmaceutically acceptable carriers are described in Remingtion's Pharmaceutical Sciences, Mack Publishing Company, 1991.

In addition, the composition may be formulated in a unit dosage form suitable for intrabody administration of a patient according to a conventional method in the pharmaceutical field, preferably in the form of a formulation useful for the administration of a peptide medicine, and is commonly used in the art. Oral or skin, intravenous, intramuscular, intraarterial, intramedullary, intramedullary, intraventricular, lung, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal tract, topical, sublingual, intravaginal or rectal Administration may be by parenteral administration routes including, but not limited to, routes.

That is, the peptide of the present invention can be administered in various parenteral formulations, and when formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the peptide may be used in admixture with various carriers (pharmaceutically acceptable carriers) such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran (ascorbic acid) to increase stability or absorption. Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

The effective dose of the peptide is 0.001 to 20mg / kg body weight, preferably 0.01 to 10mg / kg body weight, may be administered 1 to 3 times a day.

In the pharmaceutical composition of the present invention, the total effective amount of the peptide and its derivatives set forth in SEQ ID NO: 1 or 2 is administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. It can be administered, and multiple doses can be administered by a fractionated treatment protocol with long term administration. Since the concentration of the peptide is determined by taking into consideration various factors such as the age and health status of the patient as well as the route of administration and the number of treatments of the drug, in consideration of this point, a person having ordinary knowledge in the art Appropriate effective dosages for the particular use of the peptides as pharmaceutical compositions may be determined.

The feed obtained by adding the antibiotic peptide of the present invention is used as a feed for animals such as mammals, birds, reptiles, amphibians, fish, preferably mammals. For example, the feed of the present invention is used as feed for pets such as dogs, cats, rats, cattle feed such as cows and pigs, feed for poultry such as chickens and turkeys, feed for breeding fish such as sea breams and defenses , Preferably used as feed for pets or feed for livestock.

The feed of the present invention can be made by appropriately blending the peptide of the present invention with feed material. Examples of feed materials include cereal grains, crude steel, vegetable oils, animal feedstuffs, other feedstuffs, and refined products.

Examples of cereals include maize, wheat, barley, oats, rye, brown rice, buckwheat, buckwheat, sorghum, blood, corn, soybeans and the like.

Examples of the crude steel include rice bran, defatted rice bran, wheat bran, horse meal, wheat, embryo, barley bran, screening, pellets, corn bran, and corn embryo.

Examples of vegetable oils include soybean meal, soybean meal, flaxseed meal, cottonseed meal, groundnut meal meal, safflower meal, palm oil palm, palm oil, fomarak, sunflower oil, rapeseed meal, cowpea oil and mustard oil.

As animal feed materials, for example, fish meal (North American wheat, imported wheat, whole wheat, coastal wheat), fish bulrush, meat meal, fish bone meal, blood meal, decomposing hair, bone meal, animal by-products, feather mill, silkworm, skim milk powder, Casein, dried whey, and the like.

Other feedstocks include plant foliage (alfalfa, haycube, alfalfa leaf powder, pseudo acacia powder, etc.), corn processing industrial by-products (corn gluten, wheat, corn gluten feed, corn staple, etc.), starch processed products, sugar, fermentation. Industrial products (yeast, beer foil, malt sprouts, alcohol foil, walnuts, etc.), agricultural production by-products (citrus fruits, tofu, coffee, cocoa, etc.), others (cassava, bean, guam, seaweed, Krill, spirulina, chlorella, minerals, etc.) etc. are mentioned.

Typical products include proteins (casein, albumin, etc.), amino acids, sugars (starch, cellulose, sucrose, glucose, etc.), minerals, vitamins, and the like.

By routinely ingesting the animal feed of the present invention in animals, it is possible to prevent infections against pathogenic microorganisms.

As described above, the peptide described in SEQ ID NO: 1 or 2 of the present invention exhibits antimicrobial activity in Gram-positive bacteria, Gram-negative bacteria, and in particular, PapN-1 peptides have improved antimicrobial activity than Papiliocin 1, a control peptide against Gram-positive bacteria. Indicates. Therefore, the peptides PapN-1 and PapN-2 showed antimicrobial activity, thereby confirming that the N-terminal a-helix region was an important part for antimicrobial activity. In addition, PapN-1 has a superior antimicrobial activity and a broader antimicrobial spectrum compared to the control peptide Papiliocin 1 peptide. Seemed. As a result, the peptide PapN-1 shows little cytotoxicity and can be usefully used as an antibiotic or anti-inflammatory agent that is safe for human body having antibacterial or anti-inflammatory action. In addition, PapN-2 has a slightly lower antimicrobial activity compared to Papiliocin 1 peptide and high antimicrobial activity only to Gram-negative bacteria.

1 shows the hemolytic activity of peptides according to the peptide concentration in% hemolysis after treatment with the antibiotic peptides of SEQ ID NOS. 1 to 3, wherein the antimicrobial peptide melittin is used as a control (peptide showing hemolytic activity). It was.
FIG. 2 shows the% survival rate of the peptides according to the peptide concentrations by treating the antibiotic peptides of SEQ ID NOS. 1 to 3 against mouse macrophages (RAW264.7 cell). Herein, antimicrobial peptide melittin was used as a control. .
Figure 3 shows the amount of inflammation-triggered nitric oxide (NO) produced by stimulating with LPS (lipopolysaccharide) known as a pathogen that causes inflammation in mouse macrophage RAW264.7 cells treated with the antibiotic peptides of SEQ ID NOS: 1-3 Measured accordingly.
4 is a sandwich ELISA method for the amount of the induction cytokines mMIP-2 and mTNF-a secreted by stimulation of LPS, a pathogen that causes inflammation in mouse macrophage RAW264.7 cells treated with the antibiotic peptides of SEQ ID NOS. 1-3 Measured by.
Figure 5 shows the degree of inhibiting the mRNA expression encoding the inflammation-induced cytokines secreted by the stimulation of LPS, a pathogen causing inflammation in the mouse macrophage (RAW264.7 cell) treated with the antibiotic peptide PapN-1 SEQ ID NO: As shown, the control group used GAPDH.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

However, the following examples are merely to illustrate the invention, the scope of the present invention is not limited by the following examples.

Example  One: Of peptides  Synthesis and Separation Purification

The antimicrobial peptide set forth in SEQ ID NOs 1-3 was prepared by solid phase peptide synthesis (Merrifield, R. B. Science 232: 341-347) using Fmoc (9-fluorenyl-methoxycarbonyl) -chemistry. Coupling reagents, N-hydroxybenzotriazole (HOBt) and dicyclohexycarbodiimide (DCC), were used to extend the peptide chain by coupling each Fmoc-amino acid and Fmoc-Nlys (Boc) -OH. After coupling Fmoc-Val-OH, an amino-terminal Fmoc-amino acid of each peptide, remove the Fmoc group with 20% piperidine / N-methyl pyrolidone (NMP) solution and wash it several times with NMP and dichoromethane (DCM). Dried with gas. A solution of trifluoroacetic acid (TFA) -phenol-thioanisole-ethanedithiol-H2O (82.5: 5: 5: 2.5: 5, vol./vol.) Was added thereto, followed by reaction at room temperature for 2 hours to remove the protecting group and the resin. Peptides were separated from and precipitated with diethylether. The crude peptide thus obtained was purified by chromatography (HPLC) with a CH3CN (acetonitrile) gradient containing 0.05% TFA attached to a purified reversed phase column (Vydac C18, 300 μs 15 μm, 2.2 cm × 25 cm). The purified pure peptide is a peptide having the correct amino acid sequence because the molecular weight obtained using MALDI mass spectrometry (Hill, et al. Rapid Commun. Mass Spectrometry, 5, 395, 1991) matches the molecular weight obtained from the amino acid sequence. It was confirmed that was synthesized.

Experimental Example  1: Determination of antimicrobial activity

In order to measure the antimicrobial activity of the peptide represented by SEQ ID NOS: 1 to 3 was carried out as follows. Bacillus subtilis (KCTC 3068), Staphylococcus aureus (KCTC 1621), Staphylococcus epidermidis (KCTC1917) as Gram-negative bacteria from Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology , Escherichia coli (KCTC 1682)) , Psedomonas aeruginosa (KCTC 1637), Salmonella typhimurium ( KCTC 1926) was the subject. Peptide solution diluted with 1% peptone to a bacterial count of 2x10 ⁶ colony-forming units (CFUs) per ml, 50 μl aliquots in 96-well microtiter plates and diluted with 1% peptone (2: 50 μl of each diluted solution) is added to each well. After incubating the plate for 18 hours at 37 ° C, the absorbance of each well was measured at 620 nm using an ELISA reader (Bio-Tek Instruments) to determine the minimum inhibitory concentration (MIC) of the peptide. Papiliocin 1 peptide and antimicrobial peptide melittin were used as control peptides. As a result, PaN-1 and PapN-2 peptides in the N-terminal helix region showed excellent activity against 6 standard strains. Especially, PapN-1 with 22 residues showed slightly lower antimicrobial activity than gram positive bacteria, melittin, but improved antimicrobial activity than parent, Papiliocin 1. Gram-negative bacteria showed similar or two to four times better antimicrobial activity than melittin, and similar or two times lower activity than Papiliocin 1. On the other hand, PapN-2, which has removed the first two residues, arginine (Arg) and tryptophan (Trp) from the N-terminal helix region, has a lower activity compared to PapN-1, and thus plays an important role in the activity. It was confirmed that. On the other hand, the C-terminal helix region did not show antimicrobial activity, so it was confirmed that it is not an active site showing antimicrobial activity.

MRSA (CCARM3089, 3090, 3108, 3114, 3126), MDRST (CCARM8003, 8007,), which was distributed by the Seoul Women's University Culture Collection of Antibiotic-Resistant Microbes (CCARM) resistant strain bank to measure the activity against the resistant strains . 8009), activity was measured against MDREC (CCARM1229) .

As a result, PapN-1 peptide showed 2 ~ 4 times higher activity in Gram-positive bacteria than control peptide Papiliocin 1 in Gram-positive bacteria. PapN-2 was found to have slightly lower activity than PapN-1 and Papiliocin 1.

Peptides MIC (μM) Standard bacterial strain E. coli S. typhimurium P. aeruginosa S. aureus B. subtilis S. epidermidis SEQ ID NO: 1: PapN-1 One One 2 8 8 One SEQ ID NO: 2: PapN-2 One 4 8 > 32 > 32 8 SEQ ID NO: 3 PapC > 32 > 32 > 32 > 32 > 32 > 32 Comparative Peptide SEQ ID NO: 4 Papiliocin 1 0.5 0.5 One 16 16 2 meliittin One 4 4 4 2 0.5

Table 2 is a table showing the antimicrobial activity of the antibiotic peptide against gram positive bacteria and gram negative bacteria.

Minimum growth inhibitory concentration MIC (μM) Peptides S. typhimurium 8003 (R) S. typhimurium 8007 ( R) S. typhimurium 8009 ( r) E. coli 1229 ( R) S. aureus 3089 (R) S. aureus3090 (R) S. aureus 3108 (R) S. aureus3114 (R) S. aureus3126 (R) SEQ ID NO: 1: PapN-1 4 4 2 4 16 16 8 8 16 SEQ ID NO: 2: PapN-2 8 8 8 4 > 32 > 32 > 32 > 32 > 32 SEQ ID NO: 3 PapC > 8 > 8 > 8 > 8 > 32 16 > 32 > 32 > 30 Comparative Peptide SEQ ID NO: 4 Papiliocin 1 0.5 One 0.5 0.5 32 32 32 16 32 Melittin 8 4 4 4 2 2 2 4 2

Table 3 shows the antimicrobial activity of antibiotic peptides against resistant strains.

Experimental Example 2: Measurement of Hemolytic Activity

Peptides represented by SEQ ID NOS: 1 to 3 were carried out as follows to determine cytotoxicity through human erythrocyte hemolytic activity. Human erythrocytes are diluted three times by diluting with PBS (phosphate-buffered saline: 35 mM phosphate buffer / 150 mM NaCl solution, pH 7.4) and centrifuging at 1000 g for 10 minutes. 100 μl of the 8.0% erythrocyte solution diluted with PBS was loaded into a 96-well microtiter plate, and then mixed with 100 μl of the peptide solution, followed by incubation at 37 ° C. for 1 hour. Centrifuge the titration plate for 5 min at 1000 g centrifugal force. 100 μl of supernatant was taken and transferred to another 96-well microtiter plate, and the absorbance at 405 nm was measured. In addition, melittin was used as a control peptide. At this time, the value when treated with 0.1% Triton X-100 was calculated as 100% hemolysis, and the hemolytic activity (degree of cell destruction) of the peptide was calculated by Equation 1 as% hemolysis. Where A is the absorbance of 405 nm in the peptide solution, B is the absorbance of 405 nm in 0.1% Triton X-100, and C represents the absorbance of 405 nm in the PBS solution. At this time, melittin was used as an antimicrobial peptide showing strong hemolytic activity as a peptide of the control group.

As shown in FIG. 1, the peptides PapN-1 and PapN-2 represented by SEQ ID NO: 1 or 2 did not show hemolytic activity at a high concentration of 100 uM, which is much higher than MIC, and thus showed high bacterial cell selectivity. PapC, on the other hand, was found to have lower hemolytic activity than melittin.

Experimental Example 3: Measurement of Cytotoxicity on Mouse Macrophages

The cytotoxicity of the peptides corresponding to SEQ ID NOS: 1 to 3 was measured by the following method. Mouse macrophages (RAW264.7 cell line) was cultured in RPMI1640 culture medium containing 10% fetal bivine serum (FBS) in a 5% CO ₂ incubator at 37 ℃. Dispense 100 μl of RPMI1640 culture into each well of a 96-well microtiter plate to give 2 × 10 ⁴ cell counts. After the cells were plated incubated overnight at 37 ° C. in a 5% CO ₂ incubator, the plate was removed from the culture medium and plated at 100 μM in 2-fold concentration, followed by 37 5% CO _2. Incubate overnight in the incubator. At this time, the negative control group was compared with the culture solution without the peptide. Dispense 20 μl of MTT [3- (4, 5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide] solution (solution of MTT dissolved in PBS at 5 mg / ml) into each well of the plate and 37 Incubate for 4 hours at ℃. After treatment with 100 μl of DMSO (dimethyl sulfoxide) in the MTT formazan produced by the mitochondrial enzyme of the viable cells, the absorbance was measured at 570 nm with a viable cell rate (% Survival) of each well using an ELISA reader. The cytotoxicity of the peptides was measured as a percentage obtained by dividing the absorbance values of the peptide-treated wells by the absorbance values containing control cells not treated with the peptides. As shown in Figure 2 it was confirmed that the peptide represented by SEQ ID NO: 1 or 2 is not toxic even at a much higher concentration than the concentration showing the antimicrobial activity. PapN-1 showed less virulence at higher concentrations than the peptide concentration showing anti-inflammatory activity as well as antimicrobial activity. PapC, as well as hemolytic activity, was toxic at low concentrations in mouse macrophages.

Experimental Example  4: Raw264 .7 cell in Pathogen LPS  NO produced by the stimulus nitrite  production inhibition measurement of

In order to determine whether the peptides represented by SEQ ID NOS: 1 to 3 are effective for anti-inflammatory activity, they were carried out as follows. Of mouse macrophages with 10% Raw264.7 cells, fetal bovine serum (FBS: fetal bovine serum), 1% antibiotics 5% CO 2 in RPMI1640 culture medium containing (100U / mL penicillin, 100ug / mL streptomycin) Incubated at 37 ° C. in the presence. Dispense 200 μl of RPMI 1640 culture solution into each well of a 96-well microtiter plate to 1 × 10 ⁵ cell counts. At this time, only the culture medium was used as the negative control, and the positive control was used only the culture solution treated with LPS. After 96-well microtiter plates were incubated overnight at 37 ° C. in a 5% CO ₂ incubator, Papiliocin 1 was confirmed to have no cytotoxicity at the highest peptide concentration (10 uM) by cell proliferation assay. RAW264.7 cells) were reacted for one hour, and then cultured for 24 hours with stimulation with LPS (lipopolysaccharide), which is a constituent of Gram-negative bacterial cell wall, known as an inflammatory pathogen. After 24 cultures, the absorbance was measured at 540 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA) by reacting 50 ul of the culture supernatant with the same amount of Griess Reagent reagent. The amount of NO is quantified by comparing the absorbance values of standard (NaNO ₂ ).

As shown in FIG. 3, PapC-2 having the sequences of PapN-2 and C-terminal helix region from which two N-terminal residues were removed, had no anti-inflammatory activity, while PapN-1 was compared with the NO value of the control without LPS treatment. The antimicrobial peptide PapN-1 was found to be an excellent peptide for inflammatory reactions by nitrite oxide because it effectively inhibited NO production from 1μM to 20μM concentrations and showed similar activity at 10μM or more when compared to the parental Papiliocin 1, an excellent anti-inflammatory activity. It can be predicted.

Experimental Example  5: Raw264 .7 cell in Pathogen LPS  Produced by the stimulus Inflammation  Inhibition Measurement of Cytokine Production

In order to determine whether the peptides represented by SEQ ID NOS: 1 to 3 specifically inhibited the inflammation-induced cytokines mMIP-2 and mTNF-alpha. Of mouse macrophages with 10% Raw264.7 cells, fetal bovine serum (FBS: fetal bovine serum), 1% antibiotics 5% CO 2 in RPMI1640 culture medium containing (100U / mL penicillin, 100ug / mL streptomycin) Incubated at 37 ° C. in the presence. Dispense 200 μl of RPMI 1640 culture solution into each well of a 96-well microtiter plate to 1 × 10 ⁵ cell counts. At this time, only the culture medium was used as the negative control, and the positive control was used only the culture solution treated with LPS. After 96-well microtiter plates were incubated overnight at 37 ° C. in a 5% CO ₂ incubator, Papiliocin was confirmed to have no cytotoxicity at the highest peptide concentration (10 uM) by cell proliferation assay. After 7 hours of reaction in 7 cells, the constituents of the Gram-negative bacterial cell wall are incubated for 18 to 24 hours by stimulating LPS (lipopolysaccharide), known as an inflammatory pathogen, by concentration. The immunoplate was then coated overnight with each primary antibody recognizing mMIP-2 and mTNF-alpha and washed at 4 ° C with a blocking buffer (5% BSA in PBS) to stop non-specific reactions after washing. do. The culture supernatant of Raw264.7 cells subjected to LPS stimulation after 18 to 24 hours of incubation on the plate coated with the primary antibody was treated with 50 ul each plate and incubated for 2 hours. Subsequently, two hours after the development of a secondary antibody capable of recognizing mMIP-2 and mTNF-alpha, a chromosome (streptavidin HRP) was added, followed by 40 minutes of TMB (3,3`, 5,5`-tetramethybensidine) solution. Color development After 10-15 minutes of color development, the color was stopped with H ₂ SO ₄ , and the absorbance at 540 nm was measured by ELISA reader (Molecular Devices, Sunnyvale, CA) in the amount of mMIP-2 and mTNF-alpha dissolved in the culture supernatant. The amount of mMIP-2 and mTNF-alpha is quantified by comparing the absorbance values of the standard.

As can be seen in Figure 4 the LPS 20ng / ml concentration of inflammation-induced cytokines mMIP-2 and mTNF-alpha was effectively inhibited only by PapN-1, thereby inducing inflammation with cytokines mMIP-2 and mTNF-alpha was specifically inhibited by PapN-1, resulting in 22 amino acids of PapN-1 containing two N-terminal residues of the N-terminal helix region, arginine (Arg ¹ ) and tryptophan (Trp ² ). It shows that the peptide consisting of can be used as a high functional anti-inflammatory peptide.

Experimental Example  6: Raw264 .7 cell in Pathogen LPS  Produced by the stimulus Inflammation  Cytokine mRNA  control

In order to determine whether PapN-1 represented by SEQ ID NO: 1 fundamentally inhibits mRNA encoding an inflammation-induced cytokine, it was performed as follows. Macrophage Raw264.7 cells of mouse were treated with RPMI1640 culture medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (100U / mL penicillin, 100ug / mL streptomycin) at 37 ° C in the presence of 5% CO _2. Incubated at. Dispense 2 ml of RPMI1640 culture into each well of a 6-well plate to 1 × 10 ⁶ cell count. At this time, only the culture medium was used as the negative control, and the positive control was used only the culture solution treated with LPS. 6-well plates were replaced with 5% CO ₂ After overnight incubation at 37 ° C in an incubator, Papiliocin 1 was confirmed to have no cytotoxicity at a peptide concentration of 5 uM by cell proliferation assay for 3 hours in mouse macrophage RAW264.7 cells. It is a constituent ingredient and stimulated with LPS (lipopolysaccharide), which is known as an inflammatory pathogen, and cultured for 6 hours. Then, mouse macrophage Raw264.7 cells were harvested and total RNA extracted using Superscript III Kit (invitrogen). 50 ~ 250ng oligo dT 15mer primer was added to the extracted RNA and placed at 70 ° C for 10 minutes. After centrifugation, the samples were collected and 5X first strand buffer, 0.1M DTT, 10mM dNTP, and RTase reagent were added. Thereafter, the reaction is carried out at 42 ° C. for 50 to 60 minutes, and 15 minutes at 70 ° C. to inactivate the enzyme. Using the synthesized cDNA as a template, add taq polymerase, 10X buffer, dNTP, gene specific primer, 5min, 40cycle (denaturation 94 ℃, 1min, Annealing 55 ℃, 1.5min, elongation 72 ℃, 1min) at 94 ℃ of predenaturation step. Step PCR is performed at 72 ° C. for 5 min. Samples obtained by PCR were electrophoresed on 1% agarose gel to confirm mRNA expression.

As can be seen in Figure 5 PapN-1 compared to the comparative peptide Papiliocin 1, mTNF-alpha, mMIP-2, mIL-6, mIL-1 beta cytokine mRNA expression encoding a little weak all but one Inhibition of PapN-1 induces cytokine inhibitory activity.

<110> Konkuk University Industrial Cooperation Corp. <120> Peptide analogue with high antibacterial and anti-inflammatory          activities designed from Papiliocin peptide <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 1 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              20 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 2 Lys Ile Phe Lys Lys Ile Glu Lys Val Gly Arg Asn Val Arg Asp Gly   1 5 10 15 Ile Ile Lys Ala              20 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 3 Ala Val Ala Val Val Gly Gln Ala Ala Thr Val Val Lys Gly   1 5 10 <210> 4 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 4 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 Pro Ala Val Ala Val Val Gly Gln Ala              20 25 30 Ala Thr Val Val Lys Gly          35

Claims (8)

Peptide selected from SEQ ID NO: 1-2. A pharmaceutical composition comprising the peptide of claim 1 as an active ingredient. The pharmaceutical composition according to claim 2, wherein the composition is not toxic to human red blood cells or stray normal cells. An antimicrobial composition against a microorganism comprising one peptide selected from SEQ ID NOs: 1 to 2 as an active ingredient. The antimicrobial composition of claim 4, wherein the microorganism is Gram-positive bacteria, Gram-negative bacteria, or antibiotic-resistant strains. The method according to claim 5, wherein the Gram-positive bacteria is B. subtilis, S. aureus, or S. epidermidis, Gram-negative bacteria are E. coli, P. aeruginosa, or S. typhimurium, and the antibiotic resistant strain is vancoa. Antimicrobial composition against microorganisms, characterized in that it is superstition resistant Staphylococcus aureus (MRSA), multidrug-resistant S. typhimurium (MDRST), or antibiotic-resistant E. coli (MDREC). An anti-inflammatory composition for an inflammatory substance comprising the peptide described in SEQ ID NO: 1 as an active ingredient. Feed composition comprising the peptide of claim 1 as an active ingredient.

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