KR100849162B1 - Antimicrobial Peptide Having Reduced Hemolytic Activity - Google Patents

Abstract

The present invention relates to an antimicrobial peptide, and more particularly, to a variant antimicrobial peptide that significantly improves cell hemolytic activity by modifying some amino acids of halosidine, an antimicrobial peptide isolated from humoral cells of silkworms, and effectively It relates to an antimicrobial agent containing as a component. The antimicrobial peptides of the present invention not only have excellent resistance to Gram-positive and Gram-negative bacteria, in particular antibiotic-resistant bacteria, but also greatly reduce hemolytic activity against human cells, and also have excellent stability in serum, thereby reducing side effects. It can be usefully used as a novel peptide antibiotic.

Antimicrobial Peptides, Silkworms, Cell Hemolytic Activity

Description

Antimicrobial Peptide Having Reduced Hemolytic Activity

Figure 1 is a graph of the mass analysis of the antimicrobial peptide of the present invention MALDI (Matrx-assised Laser Desorption / Ionisation).

2 is a graph showing the results of performing reversed phase HPLC (High Pressure Liquid Chromatography) by purifying the antimicrobial peptide of the present invention.

Figure 3 is for the methicillin resistant Staphylococcus aureus (hereinafter abbreviated as "MRSA") 3695 strain in the absence of salt (A) and in the presence of 150 mM NaCl (B). It is a graph showing the antimicrobial activity of each antimicrobial peptide.

●; DiK19Hc, ■; Khal, ▲; KWKK-hetero, ▼; KWKK-Homo

4 shows the antimicrobial activity of each antimicrobial peptide against the multidrug resistant Pseudomonas aeruginosa (hereinafter abbreviated as "MDRPA") 2171 strain in the absence of salt (A) and in the presence of 150 mM NaCl (B). Is a graph showing

●; DiK19Hc, ■; Khal, ▲; KWKK-hetero, ▼; KWKK-Homo

FIG. 5 shows each antimicrobial peptide for the vancomycin resistant Enterococcus faecium (hereinafter abbreviated as “VRE”) 5029 strain in the absence of salt (A) and in the presence of 150 mM NaCl (B). It is a graph showing the antimicrobial activity of the.

●; DiK19Hc, ■; Khal, ▲; KWKK-hetero, ▼; KWKK-Homo

Figure 6 is a graph comparing the hemolytic activity of each antimicrobial peptide against human red blood cells according to the peptide concentration.

●; DiK19Hc, ■; Khal, ▲; KWKK-Homo, ▼; KWKK-Hetero

Figure 7 is a photograph (A) and graph (B) showing the stability of each of the antimicrobial peptides, and electrophoresis (C) showing whether the antimicrobial peptides are precipitated with proteins in the serum.

8A-8E show Escherichia coli 1039, Escherichia coli ML35, Pseudomonas aeruginosa 2171, Pseudomonas aeruginosa 2161 and Salmonella, respectively, in the absence of salt (A) and in the presence of 150 mM NaCl (B). It is a graph showing the antimicrobial activity of each antimicrobial peptide against Salmonella choleraesuis ATCC 2930.

●; KWKK-hetero, ■; KWKK-Homo, ▲; DiK19Hc,

▼; Khal, ◆; Halocidin

9A-9E show respective antimicrobial peptides for MRSA 3696, MRSA 3697, VRE 5028, VRE 5029 and Enterococcus faecalis KCTC 40450 in the absence of salt (A) and the presence of 150 mM NaCl (B), respectively. It is a graph showing the antimicrobial activity of the.

●; KWKK-hetero, ■; KWKK-Homo, ▲; RWRR-Homo,

▼; DiK19Hc, ◆; Khal, ○; Halocidin

10A is a graph showing the antimicrobial activity of each antimicrobial peptide against the fungus Candida albiucans 7122 in the absence of salt (A) and in the presence of 150 mM NaCl (B).

●; KWKK-Homo, ■; DiK19Hc, ▲; Khal

Figure 10b is a photograph showing the antimicrobial activity of each antimicrobial peptide against Candida albiucans 7122 in the absence of salt and the radial diffusion assay method in the presence of 150 mM NaCl.

The present invention relates to an antimicrobial peptide, and more particularly, to a variant antimicrobial peptide derived from an antimicrobial peptide isolated from silkworms, which has significantly reduced hemolytic activity on cells, thereby significantly reducing side effects.

In modern immunity research, innate immunity has not received much attention compared to adaptive immunity with memory and specificity. However, in the following aspects, the role of innate immunity in the system of self-defense in the animal kingdom is considered to be very important. First, innate immune cells naturally inhibit the invasion of microorganisms through epithelial cells of the skin or intestine. Second, pathogens that invade blood or body fluids are controlled by phagocytosis of innate immune cells. Third, because innate immunity does not have specificity, it preferentially inhibits the growth of various invading microorganisms in body fluids immediately after infection before the immune system or phagocytosis is activated.

Cells responsible for innate immunity utilize various antimicrobial agents such as simple inorganic compounds (such as H ₂ O ₂ and NO), antimicrobial peptides and proteins to perform the above functions. Antimicrobial peptides and proteins are mainly present in the mucosal epithelial surface, body fluids and intracellular organelles of phagocytes, and have been reported to vary widely in size, structure and activity (Hancock, RE et al. , Proc. Natl. Acad. Sci., 2000, 97, 8856-8861), the majority of which are complementary to the negative charge of microbial cell membranes, and are characterized by antibacterial proteins (proteases or muramidases) with enzymatic activity. Mainly hydrolyze bacterial membranes, and antimicrobial peptides are also believed to be a cell membrane that is a common target site for microorganisms (Zhang, L. et al., J. Biol. Chem., 2001, 276, 35714-35722). This mechanism of action gives the possibility that antimicrobial peptides could be developed into a new type of antibiotic that could be effectively used against resistant strains of existing antibiotics. The emergence has further increased interest in these antimicrobial peptides.

The antimicrobial peptides found so far can be broadly classified into those having a β-sheet structure that forms a stable structure with bipolar α-helix structures and disulfide bonds. Antimicrobial peptides with cysteine residues usually have 2 to 8 even cysteine residues, most of which stabilize the structure by forming intramolecular disulfide bonds.

The mechanism of action and selectivity of antimicrobial peptides depends on how they interact with the bacterial membrane. Peptides are generally accepted through a self-promoted uptake pathway by interacting with lipopolysaccharide (LPS) on Gram-negative bacterial surfaces. The first step of the process is that the peptide acts on the binding site of divalent cations present in the LPS on the cell surface, and the second step is inserted into the cell membrane to form a channel.

In the first step, since the peptide can bind to LPS with at least three times higher affinity than divalent ions such as Mn ²⁺ or Mg ²⁺ , it is possible to competitively replace divalent ions, thus characterizing the normal cell membrane of the outer membrane. To break down. Bacterial membranes affected in this way temporarily create crevices through which hydrophobic substances, small proteins or antibiotics can pass (Piers, KL et al., Antimicrob. Agents Chemother., 1994, 38, 2311-2316). In particular, the effect of accepting the peptide itself is increased.

In the second step, when the peptide is inserted into the cell membrane to form a channel, when the cationic peptide itself acts on the negatively charged bacterial membrane, the next action is to direct the hydrophobic region toward the membrane and the hydrophilic region to the inside to form a channel. (Hancock, RE et al., Adv. Microbial Physiol., 1995, 37, 135-175). At this time, when the membrane potential difference is large, the amount of negatively charged lipids and the amount of cholesterol decreases, the channel is formed well, and the membrane structure collapses, causing bacteria to die (Falla, T. et al., J. Biol. Chem. , 1996, 271, 19298-19303). In contrast, in eukaryotic cells with large amounts of cholesterol and some anionic lipids, peptides do not work effectively, resulting in high selective activity against bacteria.

For these reasons, antimicrobial peptides are attracting attention as antibiotics with very low cytotoxic effects, and in addition, the advantages of antimicrobial peptides as new antibiotics are summarized as follows.

1. A mechanism of physical destruction of pathogen membranes that can prevent the emergence of resistant bacteria.

2. It works faster than bacteria's life cycle.

3. It works effectively against existing antibiotic-resistant bacteria.

4. It has a broad antimicrobial spectrum.

5. It has anti-endotoxicity effect by binding force with LPS.

6. It is possible to mass-produce using the process of genetic engineering so that it can have low production cost when developing as a new drug product.

On the other hand, in animal phylogenetic aspects, tunicate belonging to a posterior animal is characterized by having notochord and dorsal tubular nerve cord during the tadpole larva period. protochordata). Thus, from a systematic evolutionary perspective, the sea squirrel can be classified as a pre-evolutionary group of vertebrates, and because of its taxonomic position, it is a very good way to identify the evolutionary origin of the animal's immune system. It has been considered to be an animal. In particular, the body cavity (hemocoel) of the sea squirrel has been found to contain many phagocytes with similar morphology and function to the neutrophils and macrophage found in the circulatory system of vertebrates (Bone, Q.). , The Origin of Chordates, 1979, 2nd edn). To date, studies to identify antimicrobial peptides in humoral humoral cells have been conducted in various ways, and clavanin (Lee, IH et al., FEBS Lett.) Isolated from humoral cells of Styela clava. , 1997, 400, 158-162; Lee, IH et al., Infection and Immunity, 1997, 65, 2898-2903; Zhao, C. et al., FEBS Lett., 1997, 410, 490-492) and Styelin (Lee, IH et al., Comp. Biochem. Physiol. B Biochem. Mol. Biol., 1997, 118, 515-521; Zhao, C. et al., FEBS Lett., 1997, 412, 144-148) is a representative antimicrobial peptide found in sea urchin.

It is known that two types of sea urchins live in Korea. Halocynthia roretzi, which lives or grows mainly on the southwest coast, and inhabited only in the Sokcho area of the east coast, and Halocynthia aurantium, called silk sea urchin, which is already Although many studies have been conducted in Japan for several years, it has been known that antibacterial substances in the form of modified peptides in the form of tetrapeptide such as halocyamine exist (Azumi, K. et al., Experientia, 1990). , 46, 1066-1068; K. Azumi et al., Biochemistry, 1990, 29, 159-165), little is known about the antimicrobial peptides isolated from halosinthia oranthium.

Accordingly, the present inventors have studied the antimicrobial peptides present in the humoral cells of halosinthia oranthium, and antimicrobial peptides named dicinthaurin (Lee, IH et al., Biochem. Biophys. Acta, 2001, 1527, 141-148) and a new antimicrobial protein called halosidin (Jang, WS et al., FEBS Letters, 2002, 521, 81-86; Jang, WS et al., Antimicrobial Agents and Chemotherapy , 2003, 47, 2481-2486) and its derivative Di-K19Hc peptide (Jang, WS et al., FEBS Letters, 2006, 580, 1490-1496) have been isolated and reported. However, although the peptides have excellent antimicrobial activity, hemolytic activity on human cells is still too high, making it difficult to apply in actual clinical practice.

The present invention has been made to improve the problems of the conventional silkworm-derived antimicrobial peptide as described above, by replacing some other amino acid residues constituting the antimicrobial peptide halosidine with another amino acid cell hemolysis that the conventional halosidine peptide had It is an object of the present invention to provide a variant antimicrobial peptide that significantly reduces its activity and an antimicrobial agent containing the antimicrobial peptide as an active ingredient.

In order to achieve the above object, the present invention is to replace some of the amino acid residues of the halosidin, an antimicrobial peptide isolated from the humoral cells of the sea urchin with another cationic amino acid residues, and to add one cationic amino acid at the N-terminus It provides an antimicrobial peptide represented by Formula 1 having a 19 amino acid sequence represented by the following one letter.

Wherein Z is at least one amino acid residue or derivative thereof selected from the group consisting of lysine, arginine, histidine; W is tryptophan or a derivative thereof; X is at least one amino acid residue or derivative thereof selected from the group consisting of tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan; B is one or more amino acid residues or derivatives thereof selected from the group consisting of arginine, lysine and histidine; B 'is one or more amino acid residues or derivatives thereof selected from the group consisting of arginine, lysine and histidine or asparagine and glutamine; U is at least one amino acid residue selected from the group consisting of glycine, serine, alanine and threonine or derivatives thereof; And C represents cysteine.

Preferably, the amino acid constituting the antimicrobial peptide of the present invention represented by Formula 1 is Z is lysine, arginine or histidine; W is tryptophan; X is leucine, isoleucine or valine; B is asparagine, glutamine, histidine, lysine or arginine; U is alanine, serine or glycine; And C is cysteine. Most preferably, the amino acids constituting the antimicrobial peptide of the present invention represented by Formula 1 are Z ₁ is lysine, W ₂ is tryptophan, Z ₃ is lysine, Z ₄ is lysine, U ₅ is alanine, X ₆ is leucine, X ₇ is leucine, B ₈ is histidine, B ₉ is histidine, U ₁₀ is glycine, X ₁₁ is leucine, B _'12 is asparagine, C ₁₃ is cysteine, U ₁₄ is alanine, B ₁₅ is lysine, U ₁₆ This glycine, X ₁₇ valine, X ₁₈ leucine and U ₁₉ are alanine, and therefore, the antimicrobial peptide of the present invention most preferably has the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment of the present invention synthesized the peptide described in SEQ ID NO: 1 and confirmed that the peptide has an antimicrobial activity, it was named 'KWKK-15'.

The present invention also provides an antimicrobial peptide represented by Formula 2 having a dimer form in which two cysteine residues represented by Formula 1 are linked by disulfide bonds.

In the above formula, the amino acid represented by one letter is the same as described in Formula 1, wherein the antimicrobial peptide is most preferably linked by disulfide bonds at the cysteine residue, which is the 13th amino acid of each of the two peptides of SEQ ID NO: 1. In addition, the antimicrobial peptide of the present invention is more preferable in terms of amidating the C-terminus to modify the NH ₂ group to prevent hydrolysis by carboxyl peptidase present in the body.

The present inventors named the antibacterial peptide represented by the formula (2a) in which two peptides of SEQ ID NO: 1 are linked by disulfide bonds as "KWKK-homo".

In addition, the present invention is an antimicrobial peptide represented by Formula 3 having a dimer form in which the cysteine residues of the peptide represented by Formula 1 and the N-terminal 4 amino acids of the peptide represented by Formula 1 are deleted by disulfide bonds. To provide.

Wherein the amino acid represented by one letter is the same as that described in Formula 1, wherein the peptide is cysteine which is the 13th amino acid of the peptide sequence described in SEQ ID NO: 1 and the ninth amino acid residue of the peptide sequence described in SEQ ID NO: 3 Most preferably linked by this disulfide bond. The present inventors named the antimicrobial peptide represented by Formula 3a in which the peptide described in SEQ ID NO: 1 and the peptide described in SEQ ID NO: 3 are linked by disulfide bonds as "KWKK-hetero", and the lysine residue of the KWKK-hetero. The antimicrobial peptide represented by the formula (3b) in which is substituted with an arginine residue was named "RWRR-hetero".

On the other hand, the present inventors, in order to compare the antibacterial activity, erythrocyte hemolytic activity, etc. of the KWKK-homo or KWKK-hetero antimicrobial peptide, the lysine residue at the N-terminal of the halosidine represented by the formula (4) Variant peptides represented by Formula 5 which were further bound were prepared and named "Di-K19Hc" (Jang, WS et al., FEBS Letters, 2006, 580, 1490-1496). In addition, a variant peptide represented by Chemical Formula 6, in which the N-terminal four amino acids of one of the amino acid sequences constituting Di-K19Hc was deleted, was prepared and named "Khal".

According to a preferred embodiment of the present invention, the antimicrobial peptides represented by Formulas 2a and 3a synthesized in the present invention have a molecular weight of 4171.50 Da and 3602.38 Da, respectively (see Table 1).

The antimicrobial peptides represented by Chemical Formulas 1 to 3 of the present invention have increased concentration-dependent antimicrobial activity against various gram-positive and gram-negative bacteria in a salt-free environment without any difference from the comparative groups Di-K19Hc and Khal peptides. However, when NaCl is present at the same concentration of 150 mM as the salt environment in the blood, the antimicrobial activity is significantly superior to the comparative peptide (see FIGS. 3 to 5).

In addition, as a result of analyzing the cell hemolytic activity using red blood cells, the antimicrobial peptide of the present invention showed a very low hemolytic activity compared to the comparative group peptide (see FIG. 6), thus the antimicrobial peptide of the present invention is a conventional halo In addition to the problem of cell hemolytic activity, which is a problem with the peptide derived from cydin, the problem of decreasing activity against Gram-negative bacteria in the presence of salt concentration has the advantage that it can be easily applied to clinical practice.

In addition, the comparative peptides lose antimicrobial activity in serum, while the antimicrobial peptide of the present invention maintains excellent antimicrobial activity in serum (see FIG. 7). Therefore, although the antimicrobial activity of Di-K19Hc, one of the comparative peptides, is relatively excellent, the peptide binds to human serum proteins, causing aggregation in the blood, which makes it difficult to apply to actual clinical practice. The antimicrobial peptide of the present invention can be more easily applied to the clinic because such an aggregation reaction does not occur.

On the other hand, as a result of measuring the minimum inhibition concentration (MIC) for various Gram-positive, Gram-negative bacteria and fungi, the antimicrobial peptides of the present invention in the presence of salts have significantly superior antimicrobial activity compared to the comparative peptides (Table 2 to Table 5), in particular, showed excellent antimicrobial activity against gram negative bacteria (see Table 3 and Figure 8).

From the above results, the antimicrobial peptides of the present invention represented by the formulas (1) to (3) have excellent antibacterial activity against various resistant strains belonging to gram-positive and gram-negative and stability in serum, and especially hemolytic activity against eukaryotic cells. Since it is significantly reduced compared to the halosidine-derived antimicrobial peptide, it can be seen that it is an antimicrobial peptide having excellent properties compared to conventional antibiotics.

The present invention also provides an antibacterial and antifungal agent containing the antimicrobial peptide as an active ingredient.

As described above, the antimicrobial peptide of the present invention has excellent antibacterial and antifungal activity against microorganisms, low hemolytic activity against cells, and can be used as an excellent antibacterial and antifungal agent because of its excellent antibacterial activity in serum.

The antimicrobial and antifungal agents of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations. That is, it can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. . Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . 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 the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the antimicrobial agent of the present invention is 2 to 10 mg / kg, preferably 4 to 8 mg / kg, and may be administered once or twice a day. The effective dose of the antifungal agent of the present invention is 2 to 8 mg / kg, preferably 4 to 16 mg / kg, and may be administered once or twice a day.

The antimicrobial peptide of the present invention can be widely used as an antimicrobial agent, an antiviral agent and an antifungal agent, and the applicable range includes viruses, gram-positive bacteria, gram-negative bacteria, and fungi that are widely harmful to plants, animals and humans. It can be applied to yeast, protozoa and the like. The antimicrobial peptides of the present invention may be used alone or in combination with other antibiotics, such as erythromycin, tetratracycline, azithromycin, vancomycin and cephalosporins, depending on the application. May be used. In addition, it can also be usefully used for food additives, cosmetics, ointments or injections.

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 to the following examples.

Example 1 Synthesis of Antimicrobial Peptides

The inventors have used an automated solid phase peptide synthesizer (Pioneer, Applied Biosystems, USA) to KWKK-15 artificially described as SEQ ID NO: 1 and to SEQ ID NO: 2 where the N-terminal lysine residue of KWKK-15 is substituted with an arginine residue. The peptide described by SEQ ID NO: 3, which lacks the described RWRR-15 and the N-terminal four amino acid residues of KWKK-15, was synthesized and reisolated by RP-HPLC. Homodimer consisting of the peptide monomer (KWKK-homo: peptide linked to two KWKK-15) or heterodimer (KWKK-hetero: KWKK-15 and a structure linked to the peptide described in SEQ ID NO: 3, RWRR-hetero: RWRR-15 And peptides of the structure described in SEQ ID NO: 3) were synthesized by mixing 1 mg / ml of each synthetic peptide in 0.1 M ammonium bicarbonate and leaving the mixture in air for 72 hours or longer. Finally, the synthesized KWKK-homo and KWKK-hetero were measured by MALDI mass spectrometry to confirm whether the antimicrobial peptide of the present invention was properly synthesized.

As a result, it was confirmed that the expected mass of the antimicrobial peptides of the present invention and the mass measured through MALDI match (FIG. 1 and Table 1).

Peptide Expected mass MALDI measured mass KWKK-15 2086.63 2086.75 KWKK-Homo 4171.12 4171.50 KWKK-Hetero 3602.46 3602.38 RWRR-Hetero 4255.29 4256.83

Example 2 Purification of Antimicrobial Peptides

The present inventors purified the antimicrobial peptide synthesized in Example 1 by reverse phase HPLC. To this end, the synthesized antimicrobial peptide was loaded on a C ₁₈ reversed phase high pressure liquid chromatography column (Vydac 218TP54: The Separation Group, Hesperia, CA). After loading the sample, the column was washed with 5% acetonitrile containing 0.1% trifluoroacetic acid (TFA) at a rate of 0.5 ml / min for the first 10 minutes, and then 5 ml / min. At a rate of 10 minutes in 5% SolB, 100% acetonitrile solution containing 0.1% TFA, 15 minutes in 5-20% SolB and 30 minutes in 20-30% Sol B.

As a result, it was confirmed that the antimicrobial peptide of the present invention was separated in about 38 minutes (Fig. 2).

Comparative Example 1 Synthesis of Di-K19Hc Antimicrobial Peptide

In the same manner as in Example 1, Di-K19Hc antimicrobial peptide represented by Formula 5 was prepared.

Comparative Example 2 Synthesis of Khal Antibacterial Peptide

In the same manner as in Example 1, Khal antimicrobial peptide represented by Formula 6 was prepared.

Experimental Example 1 Analysis of Antimicrobial Activity of Antimicrobial Peptides of the Present Invention

<1-1> Determination of antimicrobial activity against methicillin resistant Staphylococcus aureus strain

The present inventors measured the antimicrobial activity against methicillin resistant Staphylococcus aureus strain in order to analyze the antimicrobial activity of the antimicrobial peptide synthesized in Example 1. At this time, the antimicrobial activity was measured in the presence and absence of 150 mM NaCl, respectively.

Specifically, MRSA 3695 strain in mid-logarithmic growth phase (received from antibiotic resistant strain bank) was treated with 9 mM sodium phosphate, 1 mM sodium citrate buffer, 1% (w / v) agarose (Sigma). -Aldrich: A6013), trapped in a thin underlay gel containing 0.3 mg trypsin soy broth (TSB, Difco, MD, USA). The agarose gel solution was adjusted to pH 7.4 before sterilization. Stock peptide solutions ranging from 3.12 to 200 μg / ml concentrations and their sequential dilutions were prepared in 0.01% acetic acid solution, and then 5 μl of peptide samples were loaded onto 3 mm diameter discs. After 3 hours of incubation at 37 ° C, an overlay gel containing 1% agarose and 6% TSB was poured onto the lower gel. After incubating the plate overnight at 37 ° C., the diameter of the clear area was measured and graphically plotted against the logarithm of the peptide concentration. The diameter of the transparent region was 0.1 mm in one unit.

As a result, in the absence of NaCl, all of the KWKK-homo and KWKK-hetero peptides of the present invention and the Di-K19Hc and Khal peptides of the comparison group increased in a concentration-dependent transparent region without any difference (A in FIG. 3). When mM NaCl is present, it was confirmed that the KWKK-homo and KWKK-hetero peptides of the present invention were superior in antimicrobial activity to a significant degree compared to the comparative group peptide (FIG. 3B).

<1-2> Antimicrobial Activity of Multidrug-Resistant Pseudomonas aeruginosa Strains

The present inventors measured the antimicrobial activity against the multi-drug resistant Pseudomonas aeruginosa 2171 strain (prepared by the antibiotic resistant strain bank) in the same manner as in Experimental Example 1.

As a result, similar to the case of Staphylococcus aureus, the peptides of the present invention increased the concentration-dependent transparent region in the environment without NaCl without any difference compared to the comparative peptide (A of FIG. 4). , 150 mM NaCl concentration it was confirmed that the antimicrobial activity was superior to the comparative group Khal peptide (Fig. 4 B).

<1-3> Antibacterial activity of vancomycin resistant enterococcus fasium strain

The present inventors measured the antimicrobial activity against vancomycin resistant enterococcus passium 5029 strain (prepared by antibiotic resistant strain bank) in the same manner as in Experimental Example 1.

As a result, similar to the case of Staphylococcus aureus, especially the KWKK-homo peptide among the peptides of the present invention is not only control in the absence of NaCl (A in Fig. 5), but also at 150 mM NaCl concentration. It was confirmed that the antimicrobial activity is superior to the peptide (Fig. 5 B).

Experimental Example 2 Analysis of Hemolytic Activity of Red Blood Cells

In order to analyze the erythrocyte hemolytic activity of the antimicrobial peptide of the present invention, 20 μl of peptide diluted to a predetermined concentration and 180 μl of 2.5% (v / v) human erythrocyte suspension were mixed in phosphate-buffered saline (PBS). The mixture was reacted at 37 ° C. for 30 minutes, and then 600 μl of PBS was added to each tube. The solution was centrifuged at 10,000 g for 3 minutes to separate the supernatant, which was then measured for absorbance at 540 nm, and the hemolytic activity (%) was calculated according to Equation 1 below. At this time, 1% of Triton-X100 was used as a control for 100% hemolytic activity, and 0.01% acetic acid was used as a control for peptide removal.

As a result, it was confirmed that KWKK-hetero showed the lowest hemolytic activity, and the comparative group DiK19Hc showed very high hemolytic activity (FIG. 6).

Experimental Example 3 Measurement of Antimicrobial Activity in Serum

An experiment on the antimicrobial activity of the antimicrobial peptides by human serum protein binding was performed as follows. The antimicrobial peptide was added to 100% human serum to a concentration diluted twice from 400 μg / ml to 50 μg / ml, followed by ultracentrifugation at 295,000 μg g at 4 ° C. for 3 hours. 5 μl of the obtained supernatant was analyzed for antimicrobial activity by means of a radial diffusion assay to analyze the binding activity of the antimicrobial peptide and human serum proteins. The 100% control immediately used the peptides added to human serum just before the radial diffusion assay. % Binding of peptides to serum proteins was calculated as in Equation 2 below.

As a result, in the case of the comparative group Di-K19Hc lost antimicrobial activity in the serum, compared to Khal and KWKK-heteropeptide of the present invention maintained excellent antimicrobial activity in the serum (Fig. 7). Therefore, although the antimicrobial activity of Di-K19Hc is relatively excellent, the Di-K19Hc peptide binds to human serum proteins, causing precipitation reactions in the blood, making it difficult to be used in actual clinical practice. Heteropeptides were found to be more readily applicable to the clinic because such precipitation reactions do not occur.

Experimental Example 4 Measurement of Minimum Growth Concentration of Peptides

<4-1> MIC measurement for various Gram-positive and Gram-negative bacteria

The MIC of the antimicrobial peptides of the present invention against a variety of pathogenic bacteria was determined via a microbroth dilution assay method slightly modified from the method proposed by the US National Committee for Clinical Laboratory Standards (NCCLS). To this end, first, bacteria were incubated at 37 ° C. and 200 rpm overnight using Meller-Hilton broth (MHB) to make a stationary phase. The culture was diluted with fresh MHB to a final concentration of 2 × 10 ⁴ to 10 ⁵ colony forming units (CFU) / ml. Stock solutions of each peptide were prepared at a concentration of 640 μg / ml in 0.01% acetic acid. Thereafter, the peptide solution was serially diluted twice with 0.01% acetic acid until the concentration was 10 μg / ml. 100 μl of bacterial suspension was dispensed into each well of a 96-well plate (Costar 3790, Corning, USA), followed by addition of 11 μl of peptide solution. After 18 hours of incubation at 37 ° C., the antimicrobial activity of the peptide was evaluated by checking the turbidity of each well. The minimum developmental concentration is shown in the range (ab), where a indicates the highest concentration at which the bacteria are still growing and b indicates the minimum concentration that completely inhibits growth. In this experimental example, in order to confirm the effect of salt concentration on the antimicrobial activity, MHB containing 150 mM NaCl was used, and bacterium Gram-positive bacteria Enterococcus faecalis KCTC 40882, Bacillus subtilis ( Bacilus subtilis, KCTC 2213), Listeria monocytogens (ATCC 19111), MRSA 3696, MRSA 3697, VRE 5028, VRE 5029, Enterococcus faecalis 40450 and Klebsiella oxytoca (Glebsiella oxytoca) , KCTC 1686), Citrobacter freundii (KCTC 2359), Pseudomonas aeruginosa KCTC 1636, E. coli ML35 and E. coli 1039, Salmonella choleraaceus ATCC 2930, Pseudomonas aeruginosa 2161, and Pseudomonas aeruginosa 171 Was used.

As a result, it was confirmed that the antimicrobial peptide of the present invention had superior antimicrobial activity in comparison to Khal, Di-K19Hc and halosidine peptides in the presence of 150 mM NaCl (Tables 2 to 4, 8A to 8E). 9a-9e).

Minimum growth concentration (㎍ / ㎖) NaCl (mM) Gram positive Gram voice E.f B.s L.m K.o C.f P.a Di-K19Hc 0 2-4 1-2 2-4 2-4 2-4 2-4 150 2-4 2-4 4-8 4-8 4-8 2-4 Khal 0 32-64 2-4 4-8 4-8 4-8 4-8 150 32-64 2-4 16-32 16-32 16-32 16-32 KWKK-Hetero 0 32-64 1-2 1-2 1-2 1-2 1-2 150 32-64 1-2 4-8 2-4 4-8 4-8

In the above, Ef represents enterococcus faecalis, Bs represents Bacillus subtilis, Lm represents Listeria monocytogens, Ko represents Klebsiella oxytoca, Cf represents Citrobacter preundi, and Pa represents Pseudomonas aeruginosa. .

Minimum growth concentration (㎍ / ㎖) NaCl (mM) ML35 1039 2930 2161 2171 KWKK-Hetero 0 1-2 1-2 1-2 1-2 1-2 150 2-4 2-4 8-16 4-8 4-8 KWKK-Homo 0 1-2 <1 1-2 1-2 1-2 150 1-2 1-2 1-2 1-2 1-2 Di-K19Hc 0 1-2 1-2 1-2 1-2 2-4 150 2-4 2-4 2-4 4-8 2-4 Khal 0 4-8 2-4 4-8 4-8 8-16 150 16-32 8-16 16-32 16-32 16-32 Halocidin 0 16-32 16-32 32-64 32-64 32-64 150 > 64 > 64 > 64 > 64 > 64

Minimum growth concentration (㎍ / ㎖) NaCl (mM) 3696 3697 5028 5029 40450 KWKK-Hetero 0 2-4 2-4 2-4 4-8 16-32 150 8-16 16-32 8-16 8-16 32-64 KWKK-Homo 0 2-4 1-2 1-2 1-2 1-2 150 2-4 2-4 1-2 2-4 4-8 Di-K19Hc 0 2-4 2-4 1-2 1-2 2-4 150 2-4 2-4 1-2 1-2 4-8 Khal 0 4-8 4-8 4-8 4-8 16-32 150 8-16 8-16 8-16 8-16 > 64 RWRR-Homo 0 2-4 1-2 1-2 2-4 4-8 150 2-4 1-2 1-2 2-4 4-8 Halocidin 0 8-16 4-8 8-16 8-16 32-64 150 32-64 16-32 16-32 16-32 > 64

<4-2> MIC Measurement of the Fungus Candida albicans

The same experiment as in Experimental Example <4-1> was performed except that fungi Candida albicans 7122 and 7278 were used.

As a result, it was confirmed that the KWKK-homo peptide of the present invention in the presence of 150 mM NaCl has a superior antimicrobial activity compared to the comparative group Khal, Di-K19Hc peptide (Table 5, Figure 10a and Figure 10b).

Minimum growth concentration (㎍ / ㎖) of Candida albicans 7278 NaCl (mM) Di-K19Hc Khal KWKK-Homo 0 2-4 8-16 2-4 150 > 64 > 64 16-32

<4-3> MIC measurement of major resistant strains

MIC was measured in the same manner as in Experimental Example <4-1> using three kinds of resistant strains used in Experimental Example 1.

As a result, it was confirmed that the KWKK-hetero peptide and KWKK-homo peptide of the present invention showed superior antimicrobial activity to the Gram-negative bacterium MDRPA than the comparative Khal peptide (Table 6).

MRSA 3695 NaCl concentration Di-K19Hc Khal KWKK-Homo KWKK-Hetero Not included 2-4 4-8 4-8 4-8 150 mM 4-8 16-32 4-8 16-32 MDRPA 2171 NaCl concentration Di-K19Hc Khal KWKK-Homo KWKK-Hetero Not included 2-4 4-8 4-8 2-4 150 mM 4-8 32-64 4-8 8-16 VRE 5029 NaCl concentration Di-K19Hc Khal KWKK-Homo KWKK-Hetero Not included 1-2 4-8 1-2 4-8 150 mM 1-2 8-16 2-4 8-16

<4-3> MIC measurement of various MRSA and MDRPA strains in salt-free environment

MIC was measured in the same manner as in Experimental Example <4-1>, except that MHB containing no salt was used. In this case, various strains belonging to MRSA and MDRPA were used as strains.

As a result, it was confirmed that the KWKK-heteropeptide of the present invention showed superior antimicrobial activity to the Gram-negative bacteria MDRPA than the comparative groups Di-K19Hc and Khal peptides (Table 7 and Table 8).

MRSA MIC (µg / mL) 3695 3696 3697 3698 3699 Di-K19Hc 4-8 2-4 2-4 2-4 2-4 Khal 4-8 4-8 4-8 4-8 2-4 KWKK-Hetero 4-8 2-4 2-4 4-8 4-8

MDRPA MIC (µg / mL) 2171 2165 2137 2161 2126 Di-K19Hc 4-8 2-4 4-8 4-8 4-8 Khal 8-16 4-8 4-8 4-8 4-8 KWKK-Hetero 2-4 2-4 2-4 2-4 2-4

<4-4> MIC measurement of various MRSA and MDRPA strains in salt-containing environments

MIC was measured in the same manner as in Experimental Example <4-3>, except that MHB containing 150 mM NaCl was used.

As a result, it was confirmed that the KWKK-heteropeptide of the present invention showed superior antimicrobial activity to the Gram-negative bacterium MDRPA than the comparative Khal peptide (Table 9 and Table 10).

MRSA MIC (µg / mL) 3695 3696 3697 3698 3699 Di-K19Hc 4-8 2-4 2-4 4-8 4-8 Khal 16-32 8-16 8-16 8-16 8-16 KWKK-Hetero 16-32 8-16 16-32 16-32 16-32

MDRPA MIC (µg / mL) 2171 2165 2137 2161 2126 Di-K19Hc 4-8 4-8 4-8 4-8 4-8 Khal 32-64 32-64 32-64 32-64 32-64 KWKK-Hetero 8-16 8-16 8-16 8-16 8-16

As described above, the antimicrobial peptides of the present invention not only exhibit antimicrobial activity against various Gram-positive and negative bacteria, but also have excellent antimicrobial activity against resistant bacteria, and also have excellent stability and serum hemolytic activity in serum. It can be usefully used as an antibiotic or antifungal agent against various bacteria.

Attach sequence list electronic file  

Claims (9)

An antimicrobial peptide having the amino acid sequence set forth in SEQ ID NO: 1. delete delete An antimicrobial peptide represented by Formula 2a, wherein the cysteine residue of the antimicrobial peptide set forth in SEQ ID NO: 1 of claim 1 has a dimer form linked by disulfide bonds: [Formula 2a]

delete The antimicrobial peptide set forth in SEQ ID NO: 1 of claim 1 and the N-terminal four amino acids of the peptide set forth in SEQ ID NO: 1 cysteine residue of the peptide is represented by the formula 3a or 3b having a dimer form linked by disulfide bonds Antibacterial Peptides: [Formula 3a]

[Formula 3b]

delete An antimicrobial agent comprising the peptide of any one of claims 1, 4 and 6 as an active ingredient. An antifungal agent containing the peptide of any one of claims 1, 4 and 6 as an active ingredient.

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