KR20080004997A - A novel antimicrobial peptide pep-1-k designed from the cell-penetrating peptide pep-1 and uses thereof - Google Patents

Abstract

A novel synthetic or a recombinant antibacterial peptide is provided which shows strong antibacterial activity against various strains such as gram positive bacillus, gram negative bacillus and a strain having the resistance against antibiotics without any hemolytic activity in human erythrocyte cells, thereby being usefully used as a non-toxic antibacterial agent, a food preservative and a cosmetic additive. A synthetic or a recombinant antibacterial peptide is characterized in that it prepared by substituting at least one Glu at the 2nd, the 6th and the 11th of a cell-penetrating peptide Pep-1 with a positively charged basic amino acid such as arginine(Arg), histidine(His) and lysine(Lys) and it has the antibacterial activity against at least one strain selected from the group consisting of Bacillus subtilis KCTC 3068, Staphylococcus epidermidis KCTC 1917 and Staphylococcus aureus KCTC 1621. A non-toxic antibacterial agent comprises the antibacterial peptide as an effective ingredient.

Description

A novel antimicrobial peptide Pep-1-K designed from the cell-penetrating peptide Pep-1 and uses according to the present invention.

1 is a graph showing the hemolysis of human erythrocytes according to the peptide concentration in percentage (%) after Pep-1-K, Pep-1, and melittin (melittin), respectively. .

●: melittin,

○: Pep-1,

▼: Pep-1-K.

FIG. 2 shows EYPC / EYPG (7: 3, w / w) liposomes (A) mimicking bacterial cell membranes and EYPC / cholesterol (10: 1, w / w) liposomes (B) mimicking cell membranes of eukaryotic cells. Pep-1-K, Pep-1 and melittin by the graph showing the fluorescence emission quenching (fluorescence emission quenching) aspect.

●: melittin,

○: Pep-1,

▼: Pep-1-K.

3 is Dye release over time when PEP-1-K, Pep-1 and melittin were administered to EYPC / EYPG (7: 3, w / w) liposomes containing calcein, a fluorescent dye, respectively. A graph showing the dye leakage pattern.

The present invention is a novel antimicrobial peptide Pep-1-K using the cell permeable peptide Pep-1 And antimicrobial Pep-1-prepared by substituting lysine (Lys) for glutamic acid (Glu), which is the second, sixth and eleventh amino acids of Pep-1, known as cell permeable peptides. K and its use.

To date, the emergence of various resistant strains resistant to antibiotics has made it more difficult to treat infectious diseases. In order to combat such antibiotic resistance strains, immune factors specific for antibiotic resistance are required, and antimicrobial peptides, immune cells, phagocytic cells and antibodies are known as such immune factors (Boman, HG, Annu. Rev. Immunol., 13 , 61-92, 1995; Hancock, RE and Scott, MG, Proc. Natl. Acad. Sci. USA., 97, 8856-8861, 2000; and Zasloff, M., Nature, 415, 389-395, 2002). . In particular, antimicrobial peptide (antimicrobial peptide) is attracting attention as a candidate for the next generation antibiotics due to the difference in the antibiotic mechanism, unlike the conventionally prepared antibiotics (Yeaman, MR and Yount, NY, Pharmacol. Rev., 55 , 27-55, 2003), as a result of research on the development of peptide antibiotics that have no toxicity in the human body by using antimicrobial peptides at home and abroad, and which can specifically kill pathogenic resistant bacteria, There was no report on the synthesis of antimicrobial peptides that are not toxic to host cells due to the lack of hemolytic activity against red blood cells (Shin, SY, et al., Biochem. Biophys. Res. Commun., 275, 904-909, 2000; and Biochim. Biophys. Acta, 1463, 209-218, 2000).

On the other hand, hydrophilic macromolecules such as oligonucleotides, peptides, proteins and antibodies are difficult to deliver intracellularly due to their weak permeability to cell membranes. Recently, however, several cell-penetrating peptides have been developed that act as carriers for the passage of hydrophilic macromolecules into cells, and have been applied to drug delivery systems that can deliver drugs into cells. (Derossi, D., et al., J. Biol. Chem., 269, 10444-10450, 1994; Prochiantz, A., Curr. Opion. Cell Biol., 12, 400-406, 2000; Hallbrink, M , et al., Biochim. Biophys.Acta, 1515, 101-109, 2001; and Langel, U., Cell-Penetrating Peptides: Processes and Applications, CRC Press, Boca Raton, 2002). Pep-1 is a cell-penetrating peptide. Nucleotide sequence of amino acid sequence (KETWWETWWTEW) and Simian virus-40 (SV-40) large T antigen derived from reverse transcriptase of human acquired immunodeficiency virus-1 It is known as a peptide that connects the (nuclear localization sequence; KKKRKV) with a spacer sequence (SQP) (Morris, MC, et al., Nat. Biotechnol., 19, 1173-1176, 2001; Henriques, ST and Castanho , MA, Biochemistry, 43, 9716-9724, 2004; and Gros, E., et al., Biochim. Biophys.Acta, 1758, 384-394, 2006). The amino terminus of Pep-1 consists of a hydrophobic Trp-rich domain and the carboxyl terminus consists of a hydrophilic lysine-rich domain and is cationic. And forms an amphipathic α-helical structure, a common feature seen in antimicrobial peptides.

On the other hand, Pep-1 exhibits antimicrobial activity against bacteria in addition to cell permeable effect, which is antimicrobial activity by pVEC (LLIILRRRIRKQAHAH or NH ₂ ) and TP10 (AGYLLGKINLKALAALAKKIL-NH ₂ ), which are previously reported cell permeable peptides (Nekhotiaeva N, et al., FASEB J., 18, 394-396, 2004) and Tat (47-58; YGRKKRRQRRRD) are similar to reports of antifungal activity in C. albicans (Jung, HJ et. al., Biochem.Biophys.Res.Commun., 345, 222-228, 2006), substituting a negatively charged acidic amino acid of a cell-permeable peptide with a positively charged basic amino acid as in the present invention. There is no example of increasing antimicrobial activity through electrostatic interactions with negatively charged phospholipids. In addition, most known antimicrobial peptides are known as membrane targets (Orn, Z. and Shai, Y., Biopolymers., 47, 451-463, 1998; and Shai, Y. and Oren, Z., Peptides, 22, 1629-1641, 2001) or has been reported to sterilize by intracellular target (Park, CB, et al., Biochem. Biophys. Res. Commun., 244, 253-257, 1998 ).

Therefore, the present inventors act on host cells (human red blood cells) by substituting the negatively charged glutamic acid (Glu) of the cell permeable Pep-1 with the positively charged lysine (Lys). The present invention has been accomplished by preparing a novel antimicrobial peptide that selectively kills only Gram-positive bacteria, Gram-negative bacteria and bacteria that are resistant to antibiotics.

An object of the present invention is to provide a novel antimicrobial peptide having improved antibacterial activity and non-toxic to human body by substituting a negatively charged acidic amino acid of a cell permeable peptide with a positively charged basic amino acid. It is to provide a non-toxic antibacterial, food preservatives and cosmetic additives containing as an active ingredient.

In order to achieve the above object, the present invention provides a synthetic or recombinant antimicrobial peptide characterized in that it is prepared by replacing the negatively charged acidic amino acid of the cell-permeable peptide with a positively charged basic amino acid.

In addition, the present invention provides a nontoxic antimicrobial agent, food preservative and cosmetic additives containing the antimicrobial peptide as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a synthetic or recombinant antimicrobial peptide, which is prepared by replacing a negatively charged acidic amino acid of a cell permeable peptide with a positively charged basic amino acid.

In the present invention, in order to prepare a novel antimicrobial peptide, Pep-1 was used among previously known cell permeable peptides. That is, in the present invention, in order to prepare Pep-1-K, which is an antibacterial peptide, glutamic acid, which is a second, sixth, and eleventh negatively charged acidic amino acids of Pep-1 ( SEQ ID NO: 1 ), a known cell permeable peptide ( Glu) basic positively charged It was synthesized by substituting the amino acid lysine (Lys), wherein the positively charged basic amino acid may include arginine (Arg) and histidine (His) in addition to lysine (Lys). In addition, as a synthesis method, solid phase synthesis (Fric (9-fluorenylmethyloxycarbonyl) -chemistry) was used (solid phase synthesis; Merrifield, Science, 232, 341-347, 1986), and a negative control for Pep-1-K. (Pep-1, SEQ ID NO: 1 ) and positive control (meltin, Melittin, SEQ ID NO: 3 ) were also prepared in the same manner as above. Peptides thus synthesized were analyzed using analytical reversed phase chromatography to confirm that the purity was 95% or higher. In addition, the molecular weight of the peptide prepared from the above was measured by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, and then the results were examined to determine whether the molecular weight measurements of the peptide and the calculated molecular weight calculated from the Pep-1-K amino acid sequence coincide with each other. In addition, it was confirmed that a peptide having the correct amino acid sequence in which the two peptides match each other was synthesized (see Table 1 ).

Next, the present inventors, in order to measure the antimicrobial activity of the prepared antimicrobial peptide Pep-1-K ( SEQ ID NO: 2 ), Pep-1-K and the control peptide Pep-1 and Meli The antimicrobial activity of Gram-positive bacteria (3 types), Gram-negative bacteria (3 types) and antibiotic-resistant bacteria (6 types) against tin was investigated. In this case, using the Gram-positive bacteria include Escherichia coli received pre-sale from the Korea Research Institute of Bioscience and Biotechnology (KCTC 1682), Pseudomonas her labor Rouge (Pseudomonas aeruginosa , KCTC 1637) and Salmonella typhimurium ( KCTC 1926) were used, and Bacillus subtilis ( Bacillus) as gram-negative bacteria. subtilis, KCTC 3068), Rhodococcus epi deolmi discharge (as Staphylococcus Streptomyces epidermidis , KCTC 1917) and Staphylococcus aureus ( KCTC 1621) were used as antibiotic resistant strains.The Culture Collection of Antibiotic-Resistant Microbes, Seoul Women's University, CCARM Methicillin-resistant Staphylococcus aureus , MRSA), CCARM 3001, CCARM 3089 and CCARM 3543, and CCARM 2092, CCARM 2095 and CCARM 2109, which are multidrug-resistant Pseudomonas aeruginosa ( MDRPA). As a result, for Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant bacteria, Pep-1-K of the present invention is almost similar to melittin (the strongest antimicrobial peptide present in nature), which is a control peptide. It was confirmed that it exhibits a stronger antimicrobial activity (see Table 2 , Table 3 , Table 4 and Table 5 ).

In addition, the present invention provides a nontoxic antimicrobial agent, food preservative and cosmetic additives containing the antimicrobial peptide as an active ingredient.

The present inventors measured the hemolysis of human erythrocytes in order to test the toxicity of Pep-1-K of the present invention with strong antibacterial activity. As a result, as shown in Figure 1 Pep-1-K did not show any hemolytic activity at high concentrations unlike melittin, a control peptide. In addition, Pep-1-K of the present invention showed a low hemolytic activity similar to the existing Pep-1, suggesting that both Pep-1-K and Pep-1 of the present invention can be used as a safe antibacterial agent to the human body do.

Next, the present inventors measured tryptophan fluorescence blue shift and tryptophan fluorescence emission quenching in order to investigate whether Pep-1-K selectively showed antimicrobial activity only in bacteria. It was confirmed that Pep-1-K selectively sterilizes only bacterial cells by selective affinity for negative phospholipids of bacterial cell membranes. (See Table 6 and FIG. 2 ). In addition, existing Pep-1 also had a similar or weaker selective bactericidal action than Pep-1-K of the present invention. In addition, the inventors of the present invention, in order to determine whether the antimicrobial action of Pep-1-K of the present invention targets the cell membrane or the cytoplasm of the bacteria, bacteria including a calcein (fluorescent dye) A negatively charged liposome that mimics the cell membrane was prepared and the calcein was released over time when a certain amount of peptide was administered. As a result, Pep-1-K of the present invention is 2 μM and When administered at 64 μM, it was found that no calcein was released over time (see FIG. 3 ). This suggests that Pep-1-K of the present invention does not target the cell membrane but targets the cytoplasm and destroys bacteria.

The pharmaceutical composition comprising Pep-1-K, an antimicrobial peptide of the present invention as an active ingredient, may be used as an antimicrobial agent, food preservative, and cosmetic additive. That is, as an antimicrobial agent, the composition may be administered in various formulations orally or parenterally during clinical administration, and may be prepared by including one or more pharmaceutically acceptable carriers in addition to the peptide. In this case, as a pharmaceutically acceptable carrier, saline solution, sterile water, Ringer's solution, buffered saline solution, dextose solution, maltodextrin solution, glycerol, ethanol and one or more of these components may be mixed and used as necessary. Other conventional additive components such as agents, buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mark Publishing Company, Easton PA. In addition, parenteral administration may be administered by subcutaneous injection, intravenous injection or intramuscular injection. Pep-1-K of the present invention may be mixed with a stabilizer or a buffer to prepare a formulation for parenteral administration. And can be formulated in unit dosage forms of ampoules or vials.

In this case, the pharmaceutical composition comprising the peptide as an active ingredient may be administered in an amount effective to treat the patient. For example, it may be administered once to several times, and the dosage is 0.5 to 5 mg / kg, preferably 1 to 2 mg / kg.

In addition, when used as a food preservative and cosmetic additive containing Pep-1-K of the present invention as an active ingredient, it is preferable that the composition comprises 0.0001 to 50% by weight, depending on the formulation to be prepared.

Food preservatives containing the composition as an active ingredient include additives used to prevent the deterioration, decay, discoloration and chemical changes of food, such as fungicides, antioxidants and functional antibiotics.

In particular, the cosmetic containing the composition as an active ingredient may be prepared in the form of a general emulsion formulation and solubilized formulation. Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible cosmetics. In addition to cosmetics containing Pep-1-K of the present invention, it can be prepared in the form of topical or systemically applicable auxiliaries commonly used in the field of dermatology by containing a dermatologically acceptable medium or base. In addition, suitable formulations of cosmetics include, for example, emulsions, suspensions, microemulsions, microcapsules, microcapsules obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase, to which Pep-1-K is added. It may be provided in the form of granulocytes or ionic (liposomes), nonionic vesicle dispersants, creams, skins, lotions, powders, ointments, sprays or cone sticks. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the cosmetics of the present invention, in addition to the composition comprising Pep-1-K, fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, Fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or It may contain adjuvants conventionally used in the cosmetic or dermatological field, such as any other ingredients conventionally used in cosmetics. In addition, the above components may be introduced in an amount generally used in the dermatology field.

Hereinafter, the present invention will be described in detail by way of examples.

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

Example 1 Peptide Synthesis

In order to synthesize the antimicrobial peptide Pep-1-K, the cell permeable peptide Pep-1, and the antimicrobial peptide melittin (melittin) derived from bee venom, 9-fluorenyl-methoxycarbonyl (9 It was prepared by the solid phase method (merrifield, RB, Science, 232, 341-347, 1986) using -fluorenyl-methoxycarbonyl, Fmoc) -chemistry. Specifically, Fmoc-Val-Wang-Resin was used as a starting material because the carboxyl ends of Pep-1-K and Pep-1 were -COOH and valine, and the carboxyl end of maltin was -NH. since the _two types was used Fmoc-Amide-Resin Rinked as a starting material. In addition, peptide chain extension by each Fmoc-amino acid linkage used N-hydroxybenzotriazole (HOBt) and dicyclohexycarbodiimide (DCC) which are linking reagents. Finally, Fmoc-Lys (Boc) -OH, which is the Fmoc-amino acid of the amino terminal of Pep-1-K and Pep-1, was connected, and the Fmoc group was removed with 20% piperidine / N-methyl pyrolidone (NMP) solution. After washing several times with NMP and dichoromethane (DCM) and drying with nitrogen gas. Trifluoroacetic acid (TFA) -phenol-thioanisole-ethanedithiol-H ₂ O (82.5: 5: 5: 2.5: 5, volume / volume) solution was then added. The reaction was carried out at room temperature for 2 hours to remove the protecting group and to separate the peptide from the resin, and then the peptide was precipitated with diethyl ether. Subsequently, chromatography (HPLC) using acetonitrile (acetonitrile (CH3CN) to which the peptide obtained from the above was attached with a purified reverse phase column (Vydac C18, 300 μs, 15 μm, 2.2 cm × 25 cm) containing 0.05% TFA was performed. Purified). The purified peptide was measured by molecular mass spectrometry (Milldi et al., Rapid Commun. Mass Spectrometry, 5, 395-OOO, 1991) by matrix-assisted laser desorption ionization (MALDI-TOF-MS). By confirming that the measured values coincide with each other, the molecular weight calculated from the amino acid sequence of Pep-1-K, it was confirmed that a peptide having the correct amino acid sequence of interest was synthesized ( Table 1 ).

Molecular Weight of Synthetic Peptides Determined by MALDI-TOF-MS Peptide Amino acid sequence Molecular Weight ( Da ) Calculation Measure Pep-1 KETWWETWWTEWSQPKKKRKV 2848.3 2847.9 Pep-1-K KKTWWKTWWTKWSQPKKKRKV 2845.4 2845.7 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ-NH ₂ 2846.5 2846.4

Example 2 Determination of Antimicrobial Activity of the Peptides

The present inventors, in order to determine the antimicrobial activity of Pep-1-K ( SEQ ID NO: 2 ), Pep-1 ( SEQ ID NO: 1 ) and melittin ( SEQ ID NO: 3 ) determined from <Example 1>, Gram-positive bacteria, Gram -Antimicrobial activity was compared after treatment of the peptides to negative bacteria and antibiotic resistant strains, respectively. Specifically, Gram-negative bacteria are E. coli (KCTC 1682), Pseudomonas aeruginosa ( KCTC 1637), and Salmonella typhi, which were distributed from the Korea Collection for Type Cultures (KCTC). flock over (Salmonella typhimurium, KCTC 1926) was used for Gram-negative bacteria (Gram-negative bacteria) is Bacillus subtilis (Bacillus subtilis, KCTC 3068), Rhodococcus epi deolmi discharge (as Staphylococcus Streptomyces epidermidis , KCTC 1917) and Staphylococcus aureus ( KCTC 1621) were used, and the antibiotic resistant strains were The Culture Collection of Antibiotic-Resistant Microbes, Seoul Women's University, CCARM. Methicillin-resistant Staphylococcus aureus ( MRSA) CCARM 3001, CCARM 3089 and CCARM 3543, which were sold from the US, were used and multidrug-resistant Pseudomonas aeruginosa , MDRPA), CCARM 2092, CCARM 2095 and CCARM 2109.

Each strain was diluted with 1% peptone to 2 × 10 ⁶ colony-forming units (CFUs) per ml, then 100 μl in 96-well microtiter plates. After dispensing, 100 μl of the peptide solution (2: 1 step diluted solution) diluted with 1% peptone was added to each well. Subsequently, the plate was incubated at 37 ° C. for 18 hours, and then the absorbance of each well was measured at a wavelength of 620 nm using an ELISA reader (Bio-Tek, USA), followed by the minimal inhibitory concentration of each peptide. , MIC) was determined. In this case, the peptide used as a control was a cell permeable peptide, Pep-1, and an antimicrobial peptide, melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH ₂ ) derived from bee venom having a relatively strong antimicrobial activity among natural peptides.

As a result, as shown in Table 2 and Table 3, it was confirmed that Pep-1-K in the Gram-negative bacteria and Gram-positive bacteria showed a very increased antimicrobial activity compared to Pep-1. That is, the MIC value of Pep-1 is 8 μM to 64 μM or less, while the MIC value of Pep-1-K is 1 μM to 2 μM. In particular, Pep-1-K showed a strong antimicrobial activity similar to the control melittin. In addition, as shown in Table 4 and Table 5, it was confirmed that Pep-1-K showed a strong antimicrobial activity showing an MIC value of 1μM ~ 8μM against antibiotic resistance strains.

Antimicrobial Activity of Each Peptide against Gram-negative Bacteria Peptide Minimum growth inhibitory concentration (μM) Escherichia coli (KCTC 1682) Salmonella typhimurium (KCTC 1926) Pseudomonas aeruginosa (KCTC 1637) Pep-1 32 64 < 64 < Pep-1-K 2 One 2 Melittin 2 2 4

Antimicrobial Activity of Each Peptide against Gram-positive Bacteria Peptide Minimum growth inhibitory concentration (μM) Bacillus subtilis (KCTC 3068) Staphylococcus aureus (KCTC 1261) Staphylococcus Epidermidis (KCTC 1917) Pep-1 8 64 64 Pep-1-K 2 2 One Melittin 2 One One

Antimicrobial Activity of Each Peptide against Methicillin-Resistant Staphylococcus aureus Peptide Minimum growth inhibitory concentration (μM) MRSA 1 (CCARM 3001) MRSA 2 (CCARM 3089) MRSA 3 (CCARM 3543) Pep-1 64 < 64 < 64 < Pep-1-K 2 2 One Melittin One 2 One

Antimicrobial Activity of Each Peptide against Combination Drug-Resistant Pseudomonas aeruginosa Peptide Minimum growth inhibitory concentration (μM) MDRPA 1 (CCARM 2092) MDRPA 2 (CCARM 2095) MDRPA 3 (CCARM 2109) Pep-1 32 64 < 32 Pep-1-K 8 8 8 Melittin 16 8 8

Example 3 Measurement of Hemolysis of Hemocytosis of Pep-1-K

In order to measure the hemolytic activity of human erythrocyte cells against Pep-1-K of the present invention set forth in SEQ ID NO: 2 , the present inventors have applied the peptide and the control group Pep-1 and melittin to human erythrocytes, respectively. Hemolytic activity was compared after treatment. Human erythrocytes were diluted with phosphate buffered saline (35 mM phosphate buffer / 150 mM NaCl solution, pH 7.4), centrifuged at 1000 g for 10 minutes, and then washed three times. Subsequently, 100 μl of 8.0% red blood cells diluted with phosphate buffered saline were dispensed into a 96-well microtiter plate, and 100 μl of each peptide was added thereto, followed by incubation at 37 ° C. for 1 hour. Subsequently, the cultured 96-well microtiter plate was centrifuged at 1000 g for 5 minutes to obtain a supernatant, and then 100 μl of the supernatant was transferred to another 96-well microtiter plate to measure absorbance at a wavelength of 405 nm. In addition, melittin (melittin) was used as a control peptide, and the measured value was calculated as 100% hemolysis when treated with 0.1% Triton X-100 to determine the hemolytic activity (degree of cell destruction) of each peptide. It was calculated by the hemolysis (% hemolysis) as shown in equation (1 ).

Hemolysis also (% hemolysis) = [(OD peptide -OD zero - lysis) / (OD 100% - lysis -OD zero - lysis)] × 100

                            = [(A-C) / (B-C)] × 100

At this time, A is the absorbance of the peptide solution measured at 405nm, B is the absorbance of 0.1% Triton X-100 measured at 405nm, C is the absorbance of phosphate buffered saline measured at 405nm. As a control of the peptide of the present invention, melittin, an antimicrobial peptide showing strong hemolytic activity, was used.

As a result, as shown in Figure 1, melittin showed a very strong hemolytic activity, while Pep-1 and Pep-1-K of the present invention did not show any hemolytic activity at a high concentration of 100 ~ 200μM.

Example 4 Selectivity Analysis of Pep-1-K to Bacterial Cells

<4-1> Tryptophan fluorescence emission wavelength shift measurement

The present inventors prepared phospholipids that mimic the lipid membranes of bacteria and eukaryotic cells, respectively, in order to measure the selectivity of bacterial cells of Pep-1-K, and then examined the tryptophan fluorescence blue shift. Specifically, EGPE (egg yolk L-phosphatidylethanolamine) / EYPG (egg yolk L-α-phosphatidyl-DL-glycerol; 7: 3, w / w), which is a phospholipid composition that mimics the lipid membrane of bacteria (Sigma Chemical, USA) And EYPC (egg yolk phosphatidylcholine) / cholesterol (10: 1, w / w) (Sigma Chemical Co. USA), each of which is a phospholipid composition that mimics the lipid membrane of eukaryotic cells, was dissolved in chloroform, and then rotated into a rotary evaporator. evaporator, EYELA N-1000, Tokyo Rikakikai, Japan) was used to remove the solvent and a thin film was formed on the glass plate wall. Subsequently, Tris buffer solution (10 mM Tris, 0.1 mM EDTA, 150 mM NaCl, pH 7.4) was added to the dried thin film and mixed well. The lipid solution thus produced was ground using a titanium-tip ultrasonicator (Microson, USA) for 10-20 minutes at 0 ° C. until the solution became clear, and then EYPE / EYPG (7: 3, w / w) and small unilamellar vesicles (SUV) of EYPC / cholesterol (10: 1, w / w) were prepared.

Next, tryptophan fluorescence blue shift of the lipid membrane mimetic was measured using a model RF-5301PC spectrometer (Shimadzu, Japan). At this time, each peptide was added to 3 ml of Tris buffer solution containing 0.6mM liposomes (peptide: liposome = 1: 200), left for 10 minutes at 20 ℃, and then fluorescence was excited at 280nm After scanning the emission from 300nm to 400nm, the wavelength representing the maximum absorbance of each peptide was determined from the fluorescence spectrum.

As a result, as shown in Table 5, the Tris buffer solution showed the maximum value of tryptophan fluorescence emission for each peptide at a wavelength of 350 nm. This means that the tryptophan of the peptide is located in the hydrophilic environment of Tris buffer. In addition, in EYPE / EYPG (7: 3, w / w) liposome solutions that mimic bacterial lipid membranes, the maximum value of tryptophan fluorescence for Pep-1, Pep-1-K and melittin was the maximum in Tris buffer. Compared to the values, a short blue shift occurred at nearly similar maximum absorption wavelengths of 8 nm, 9 nm and 11 nm, respectively. This result means that the tryptophan of each peptide is located in the hydrophobic lipid duplex center of EYPE / EYPG (7: 3, w / w) liposomes. In contrast, in the EYPC / cholesterol (10: 1, w / w) liposome solution, melittin showed a short wavelength of 13 nm at the maximum absorption wavelength, whereas Pep-1 and Pep-1-K were only 1 nm short. It can be seen that the shift to the wavelength is shown. From these results, melittin is located in the hydrophobicity of the hydrophobic lipid bilayer of EYPC / cholesterol (10: 1, w / w) liposomes, while Pep-1 and Pep-1-K do not penetrate into the hydrophobic center and are hydrophilic Means to be located in a position on.

Peptide tryptophan emission maxima in Tris buffer, EYPE / EYPG (7: 3, w / w) liposome solution and EYPC / cholesterol (10: 1, w / w) liposome solution Peptide Tris  Buffer ( nm ) EYPE Of EYPG  (7: 3, w / w) Liposomes  Solution ( nm ) EYPC / Cholesterol (10: 1, w / w) Liposomes  Solution ( nm ) Pep-1 350 342 (8) ^a 349 (1) ^a Pep-1-K 350 341 (9) 349 (1) Melittin 350 339 (11) 337 (13)

a: blue shift at the maximum absorption wavelength (emission maxima).

<4-2> tryptophan fluorescence emission quenching measurement

The present inventors used acrylamide as a water-soluble quencher of fluorescence measurement as an additional method for verifying the result of the tryptophan fluorescence emission wavelength shift of Example 4-1. At this time, to reduce the absorption of tryptophan to acrylamide, tryptophan was excited at 295 nm instead of 280 nm, and 3M acrylamide solution was gradually added to a 0.6 mM liposome solution having a mole ratio of peptide / phospholipids of 1/200. Afterwards, the effect of acrylamide on the fluorescence of each peptide tryptophan was analyzed using the Stern-Volmer equation as shown in Equation 2 below.

F ₀ / F _{extinction factor (quencher) = 1 + Ksv [} Q]

In this case, F ₀ means fluorescence intensity when acrylamide is not present, and F _{quench factor (Fquencher)} means fluorescence intensity when acrylamide is present. In addition, Ksv is Stern-Volmer quenching constant, and [Q] means acrylamide concentration, respectively.

As a result, in EYPE / EYPG (7: 3, w / w) liposome solution as shown in Figure 2A it was confirmed that the effective fluorescence quenching effect of the similar tryptophan according to the concentration of acrylamide as a destructive factor in all peptides It was. This means that all peptides can effectively penetrate negatively charged phospholipid membranes that mimic bacterial cell membranes. On the other hand, only melittin in EYPC / cholesterol (10: 1, w / w) liposome solution, as shown in FIG. 2B, showed an effective fluorescence quenching effect of tryptophan depending on the concentration of acrylamide. Pep-1 and Pep-1-K Showed no fluorescence quenching effect. This means that Pep-1 and Pep-1-K can effectively penetrate negatively charged phospholipid membranes that mimic bacterial cell membranes rather than neutral lipids that mimic eukaryotic cell membranes. From the above results, the inventors confirmed that Pep-1 and Pep-1-K selectively bactericidal only in bacterial cells.

Example 5 Determination of Destructive Capacity of Artificial Lipid Membrane Mimicking Bacterial Cell Membrane by Pep-1-K

The inventors of the present invention described in SEQ ID NO: 2 , Pep-1-K of the present invention to determine whether the bactericidal action by targeting the cell membrane of the bacterium or the cytoplasm, a dye that is a fluorescent dye (fluorescent dye) Positive charge consisting of EYPE (egg yolk L-phosphatidylethanolamine) / EYPG (egg yolk L-α-phosphatidyl-DL-glycerol; 7: 3, w / w), an artificial lipid membrane that mimics the cell membranes of bacteria, including calcein Liposomes of large single lamellae vesicles (LUVs) were prepared and subjected to a fluorescent dye leakage assay. Specifically, the phospholipid consisting of EYPC / EYPG (7: 3, w / w) is dissolved in chloroform (CHCl3) to 10 mM per ml, and then a rotary evaporator (EYELA N-1000, Tokyo Rikakikai, Japan) Chloroform was removed and lyophilized for one day. Subsequently, the dried lipid film was dissolved in 1 ml of Tris buffer (10 mM Tris-HCl, 0.1 mM EDTA, 150 mM NaCl, pH 7.4) so that the concentration of calcein was 70 mM, and then the liposome solution was dissolved in liquid nitrogen. LiposoFast injection molding machine (Avestin, Inc.) equipped with a polycarbonate filter (two overlapping filters with a micropore size of 100 nm) after 11 freezing-thawing cycles. 10 times) to produce EYPC / EYPG (7: 3, w / w) LUV including Calcein. Subsequently, calcein containing no LUV was removed using a Sephadex G-50 column, and only liposomes containing calcein were received. Subsequently, the excitation wavelength of the spectrofluorometer (Shimadzu RF 5301 PC spectrofluorimeter, Japan) was set at 490 nm, the maximum emission wavelength was set at 520 nm, and then 3 ml of Tris buffer solution was added to a quartz cuvette (1 cm). After the optical path length), an appropriate amount of LUV solution was added thereto, and 20 μl of 10% Triton-X100 was added to adjust the amount of LUV so that the fluorescence intensity did not exceed 1000. Then, when the above conditions were established, each peptide was administered to Tris buffer solution of LUV. At this time, 100% dye leakage (dye leakage) was expressed by the fluorescence intensity when 0.1% Triton-X100 was added, and compared with melittin as a control peptide showing antimicrobial activity by targeting the bacterial cell membrane.

As a result, as shown in FIG. 3, when 2.0 μM of melittin, an antimicrobial peptide used as a control for targeting bacterium cell membranes, was used, a rapid calcein release of nearly 100% occurred, whereas Pep-1 Administration of Pep-1-K at 64 μM and 2.0 μM, respectively, did not cause calcein release. At this time, in order to use the concentration of the peptide close to the MIC value for each bacteria, Pep-1 was treated with 64μM, Pep-1-K was treated with 2μM. These results suggest that Pep-1 and Pep-1-K do bactericidal action by targeting the cytoplasm without targeting the bacterial cell membrane.

As described above, Pep-1-K, a novel antimicrobial peptide of the present invention, not only has strong antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and antibiotic resistant strains, but does not show any hemolysis in human erythrocytes. It can be usefully used as non-toxic antibacterial, food preservative and cosmetic additive.

<110> Chosun University <120> A novel antimicrobial peptide Pep-1-K designed from the          cell-penetrating peptide Pep-1 and uses <130> 6P-06-03 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> PRT <213> Amino acid sequence for Pep-1 <400> 1 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Val              20 <210> 2 <211> 21 <212> PRT <213> Amino acid sequence for Pep-1-K <400> 2 Lys Lys Thr Trp Trp Lys Thr Trp Trp Thr Lys Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 3 <211> 26 <212> PRT <213> Amino acid sequence for Melittin <400> 3 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 (11)

A synthetic or recombinant antimicrobial peptide, which is prepared by substituting any one or more of the second, sixth and eleventh glutamic acid (Glu) of Pep-1, which is a cell permeable peptide, with a positively charged basic amino acid. 2. The synthetic or recombinant antimicrobial peptide according to claim 1, wherein the positively charged basic amino acid is arginine (Arg), histidine (His) or lysine (Lys). The method of claim 1, wherein the bacteria KCTC 1682, Pseudomonas her labor Rouge (Pseudomonas aeruginosa) KCTC 1637 and Salmonella typhimurium herd over (Salmonella typhimurium ) Synthetic or recombinant antimicrobial peptides having antimicrobial activity against any one or more strains selected from Gram-positive bacteria comprising KCTC 1926. According to claim 1, Bacillus subtilis ( Bacillus subtilis ) KCTC 3068, Staphylococcus epidermidis ) KCTC 1917 and Staphylococcus aureus ) Synthetic or recombinant antimicrobial peptides having antimicrobial activity against any one or more strains selected from Gram-negative bacteria comprising KCTC 1621. The method of claim 1, wherein the methicillin-resistant Staphylococcus aureus , MRSA), selected from antibiotic resistant strains including CCARM 3001, CCARM 3089 and CCARM 3543 and CCARM 2092, CCARM 2095 and CCARM 2109, which are multidrug-resistant Pseudomonas aeruginosa ( MDRPA). Synthetic or recombinant antimicrobial peptides having antimicrobial activity against one or more strains. Non-toxic antimicrobial agent comprising the antimicrobial peptide of claim 1 as an active ingredient. The non-toxic antimicrobial agent according to claim 6, which does not destroy human red blood cells at all. 7. A nontoxic antimicrobial agent according to claim 6 having a selective affinity for negative phospholipids. 7. A non-toxic antimicrobial agent according to claim 6, characterized in that it targets and sterilizes the cytoplasm. Food preservatives comprising the antimicrobial peptide of claim 1 as an active ingredient. Cosmetic additive comprising the antimicrobial peptide of claim 1 as an active ingredient.

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