KR100459808B1 - Novel antibiotic peptide derived from ribosomal protein L1 of Helicobacter pylori and use thereof - Google Patents

Abstract

The present invention relates to an antibiotic peptide synthesized by substituting an amino-terminal region of ribosomal protein L1 (RPL1) produced by Helicobacter pylori bacteria with other amino acids, and specifically, a use thereof. SEQ ID NO: 2 synthesized with an amphipathic structure that increases the hydrophobic region by substituting the hydrophilic region of HP (2-20), an amphiphilic peptide having an affinity among the amino terminal regions of the RPL1 protein, with another amino acid. It relates to an antibiotic peptide described herein and its use. Antibiotic peptides and derivatives thereof of the present invention have no cytotoxicity and exhibit excellent antibacterial, antifungal and anticancer activity, and thus can be usefully used as antibiotics and anticancer agents that are safe for humans.

Description

Novel antibiotic peptide derived from ribosomal protein L1 of Helicobacter pylori and use according to Helicobacter pylori bacteria

The present invention relates to an antibiotic peptide synthesized by substituting the amino terminal portion of ribosomal protein L1 (hereinafter, referred to as "RPL1") produced by Helicobacter pylori with other amino acids, and its use Specifically, as described in SEQ ID NO: 2 synthesized with an amphiphilic structure that increases the hydrophobic region by substituting the hydrophilic region of HP (2-20), a peptide having an affinity and anti-activity among the amino terminal portions of the RPL1 protein, with another amino acid. Antimicrobial peptides and their use.

Bacterial infections are one of the most common and fatal causes of human disease. Unfortunately, the abuse of antibiotics has resulted in antibiotic resistance of bacteria. Indeed, the rate at which bacteria are resistant to new antibiotics occurs much faster than the rate at which new antibiotic analogs are developed. For example, bacterial species such as Enterococcus faecalis , Mycobacterium tuberculosis, and Pseudomonas aeruginosa, which can pose a life threat, are all known. It has developed resistance to antibiotics (Stuart B. Levy, Scientific American , 46-53, 1998).

Tolerance to antibiotics is a distinct phenomenon from antibiotic resistance, first discovered in Pneumococcus sp. In the 1970s and providing an important clue to the mechanism of action of penicillin (Tomasz et al., Nature , 227, 138-140, 1970). Tolerant species stop growing in the presence of the usual concentration of antibiotics but do not die as a result. Resistance is due to the fact that when antibiotics inhibit cell wall synthase, the activity of bacterial autolytic enzymes, such as autolysin, does not occur, which is why penicillin activates endogenous hydrolytic enzymes. By killing bacteria, bacteria also inhibit their activity, resulting in survival during antibiotic therapy.

It is clinically very important for bacteria to be resistant to antibiotics, because the inability to eradicate resistant bacteria can reduce the effectiveness of antibiotic therapy in clinical infections (Handwerger and Tomasz, Rev. Infec. Dis. , 7 , 368-386, 1985). In addition, developing resistance is considered a prerequisite for resistance to antibiotics, because the resulting strains survive. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics. Practically all resistant bacteria are known to have resistance (Liu and Tomasz, J. Infect. Dis. , 152, 365-372, 1985), so that new antibiotics that can kill these antibiotic resistant bacteria Need development

In terms of mechanisms of action, resistance consists of two main pathways: the first is the phenotypic resistance that occurs when growth rate decreases in all bacteria (Tuomanen E., Revs. Infect. Dis. , 3, S279). -S291, 1986), the second is genetic resistance by mutations in certain bacteria. In both cases, the basic phenomenon is that down regulation of autolysine activity occurs, which is transient when it is externally resistant to external stimuli and causes changes in the pathways that regulate cellular hemolysis. It is permanent in the case of genetic resistance to which mutations have occurred. Obviously, the simplest case of genetic resistance is a defect in the autolysine enzyme, which has not been clinically found to be resistant to the deficiency of this suicide enzyme for a variety of uncertain reasons. By modulating the activity of autolysine (Tuomanen et al., J. infect. Dis. , 158, 36-43, 1988).

As discussed above, new antibiotics are needed to fight antibiotics resistant bacteria, and new antibiotics that act independently of autolysine activity are required. There is also a need to provide pharmaceutical compositions for the effective treatment of such new antibiotics for the treatment of bacterial infections and inflammation.

On the other hand, bacteria can kill neighboring bacteria by synthesizing peptides or small organic molecules, and these bacteriocins are structurally classified. The first is lantibiotics, the second is nonlantibiotics and the third is secreted by signal peptides (Cintas et al.,J. Bad., 180, 1988-1994, 1998). Animals, including insects, also produce naturally occurring peptide antibiotics (Bevins et al.,Ann. Rev. Biochem., 59, 395-414, 1990). The antibiotics are structurally divided into three groups. The first is a cysteine-rich β-sheet peptide, the second is an α-helical amphiphilic molecule, and the third is a proline-rich peptide ( Mayasaki et al.,Int. J. Antimicrob. Agents, 9, 269-280, 1998). These antimicrobial peptides are known to play an important role in host defense and the innate immune system (Boman, H. G.,Cell, 65: 205, 1991; Boman, H. G.,Annu. Rev. Microbiol., 13:61, 1995). These antimicrobial peptides have a variety of structures depending on the amino acid sequence, the most of these structures are free of cysteine residues such as cecropin, an antimicrobial peptide found in insects, and form an amphiphilic alpha helical. .

The idea that peptic ulcers are predominantly caused by stress and gastric acid overproduction in the last century, but recently it has been found that these peptic ulcers are caused by Helicobacter pylori (Blaser, MJ.,Trends Microbiol., 1, 255-260, 1991). Helicobacter pylori is an anaerobic microorganism belonging to gram-negative bacteria and having a very slow-growing spiral and flagella. Of the many proteins produced by Helicobacter pylori, the RPL1 protein is composed of 230 amino acids, and the amino-terminal portion of the protein has been found to be similar in structure to cecropin, and in particular, eight amino acids are known to be identical. The RPL1 amino terminus of Helicobacter pylori has a perfect amphiphilic helix structure (Putsep, K. et al.,Nature, 398, 671-672, 1999), these amphiphilic peptides have a structure similar to the lipid component of the cell membrane, so that they bind to the cell membrane lipids of the microorganisms and destroy the cell membranes of the microorganisms or affect the potential of the cell membranes to change them. The mechanism of action of destroying has been reported. In addition, the amino terminal region of the RPL1 protein of Helicobacter pylori also has antimicrobial activity (Putsep K. et al.,Nature, 398, 671-672, 1999).

Accordingly, the present inventors designed and synthesized peptide derivatives with an amphipathic structure that increases the hydrophobic region by substituting a specific site sequence among the amino terminal sites of the RPL1 protein of Helicobacter pylori having amphiphile with other amino acids, By confirming the antimicrobial, antifungal, anticancer activity and cytotoxicity (hemolytic activity) of the peptide derivatives thus synthesized, the present invention was completed by revealing that the peptide derivative is an antibiotic peptide that can be used as an antibiotic and an anticancer agent.

It is an object of the present invention to provide an antibiotic peptide and derivatives thereof which are non-cytotoxic and exhibit excellent antibacterial, antifungal and anticancer activity which can be used as antibiotics and anticancer agents.

1 shows a diagram of a design of an analog peptide derived from an amino terminal HP (2-20) peptide of ribosomal protein L1 of the present invention,

2 is a photograph showing the number of viable cells by treating the antibiotic peptide of the present invention to Bacillus subtilis cells and then smearing on LB agar medium.

A: no treatment

B: HP (2-20)

C: antibiotic peptide set forth in SEQ ID NO: 2

3 is a photograph showing the number of viable cells by treating the antibiotic peptide of the present invention to Escherichia coli cells and then smearing on LB agar medium.

A: no treatment

B: HP (2-20)

C: antibiotic peptide set forth in SEQ ID NO: 2

4 is a photograph showing the number of living cells by treating the antibiotic peptide of the present invention to Candida albicans cells and then spreading on PDB agar medium.

A: HP (2-20)

B: Melittin

C: antibiotic peptide set forth in SEQ ID NO: 2

5 is a graph showing the anticancer activity of cells of the antibiotic peptide of the present invention.

A: anticancer activity against AML-2 / WT

B: anticancer activity against SNU 638

C: anticancer activity against SNU 668

●: HP (2-20)

○: antibiotic peptide set forth in SEQ ID NO: 2

▼: melittin

In order to achieve the above object, the present invention provides an antibiotic peptide and derivatives thereof synthesized by replacing the amino-terminal region of the RPL1 protein produced by the Helicobacter pylori bacteria with other amino acids.

In addition, the present invention provides a use of the antibiotic peptide and its derivatives for antibacterial, antifungal and anticancer.

In addition, the present invention provides a pharmaceutical composition for antimicrobial, antifungal and anticancer activity using the antibiotic peptide and derivatives thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an antibiotic peptide and derivatives thereof synthesized by substituting the amino terminal portion of the RPL1 protein produced by Helicobacter pylori with other amino acids.

The present invention provides an antibiotic peptide having an amphiphilic structure by increasing the hydrophobic region by substituting the hydrophilic region of HP (2-20), which is a peptide having an affinity and antibiotic activity, in the amino terminal region of the Helicobacter pylori-derived RPL1 protein with another amino acid. Was synthesized.

In order to synthesize the antibiotic peptide, the inventors used a liquid phase solidification method of Merrifield using a Fmoc amino group protective container (Merrifield, RB., J. Am. Chem. Soc. , 85, 2149, 1963). antibiotic peptides described in HP (2-20) 17 amino acid glutamine is the 19th amino acid is substituted with an aspartic acid, respectively tryptophan SEQ ID NO: 2 of the peptide described in SEQ ID NO: 1 was prepared (see Fig. 1). The antibiotic peptide thus prepared was isolated and purified, and its purity was found to be 95% or higher. The molecular weight obtained using the Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry was consistent with the molecular weight obtained from the amino acid sequence. Therefore, it was confirmed that the antibiotic peptide having the correct amino acid sequence was synthesized.

Antimicrobial peptides of the present invention consists of an amino acid sequence described in SEQ ID NO: 2 is to be substituted for the 17th amino acids of the HP (2-20) peptide described in SEQ ID NO: 1, glutamine and aspartic acid to the 19th amino acid, another hydrophobic amino acid other than tryptophan And preferably substituted with alanine, isoleucine, methionine, phenylalanine, proline, valine and leucine.

In addition, the antibiotic peptide of the present invention can be synthesized with an amphiphilic structure in which the hydrophobic region is increased by substituting specific amino acids of HP (2-20) peptides with other amino acids. It can be synthesized by substituting phosphorous acid and lysine, the 20th amino acid, with other hydrophobic amino acids described above.

In addition, the present invention provides a use of the antibiotic peptide and its derivatives for antibacterial, antifungal and anticancer.

First, in order to confirm whether the antibiotic peptide of the present invention can be used for the above purposes, the present inventors measured the antimicrobial activity of the antibiotic peptide synthesized in the present invention. Antimicrobial activity was observed by measuring the minimal inhibitory concentration (hereinafter, abbreviated as "MIC"), which is the minimum concentration of peptides that do not divide cells.

As Gram-positive bacteria Bacillus subtilis (Bacillus subtilis) and Stability Philo nose kusu aureus (Staphylococcus aureus) for, by Gram-negative bacteria was used for E. coli (Escherichia coli) and Proteus vulgaris (Proteus vulgaris), described in SEQ ID NO: 2 As a result of measuring the MIC value of each strain using melittin as the antibiotic peptide of the present invention and a positive control, the antibiotic peptide of the present invention is 10 times higher antibacterial activity depending on the strain than the HP (2-20) peptide. It showed activity, and showed similar antimicrobial activity as melittin used as a positive control (see Table 1 ).

In addition, E. coli and B. As a result of confirming the antimicrobial activity of the antibiotic peptide of the present invention in LB agar medium for subtilis, it was confirmed that the antibiotic peptide of the present invention exhibited excellent antimicrobial activity compared to the HP (2-20) peptide ( FIG. 2 and FIG. 3 ).

In order to measure the antifungal activity of the antibiotic peptides of the present invention, the present inventors have identified the pathogenic fungi Candida albicans , Trichosporon beigelii and Saccharomyces cerevisiae . As a result of measuring the MIC value of each strain by MTT assay, the antibiotic peptide described in SEQ ID NO: 2 of the present invention showed more than four times higher antifungal activity than the HP (2-20) peptide, and was used as a positive control. It showed antifungal activity similar to melittin (see Table 2 ).

In addition, as a result of confirming the antifungal activity of the antibiotic peptide of the present invention in the PDB agar plate against the strain, the antibiotic peptide described in SEQ ID NO: 2 of the present invention shows excellent antifungal activity compared to the HP (2-20) peptide It was confirmed (see FIG. 4 ).

In addition, the present inventors measured anticancer activity in leukemia cell lines AML-2 / WT and gastric cancer cell lines SNU 638 and SNU 668 to determine whether the antibiotic peptide of the present invention exhibits anticancer activity against cancer cells. HP (2-20) did not show anticancer activity, but the antimicrobial peptide of the present invention described in SEQ ID NO: 2 and melittin used as a positive control showed strong anticancer activity (see FIG. 5 ).

In addition, the present inventors examined the erythrocyte destructive ability of the peptide to confirm whether the peptide of the present invention exhibits cytotoxicity, the antibiotic peptide described in HP (2-20) and SEQ ID NO: 2 of the present invention is almost cytotoxic On the other hand, melittin, a bee venom used as a positive control, was found to be highly cytotoxic (see Table 3 ).

From the above results, the antibiotic peptide described in SEQ ID NO: 2 of the present invention not only exhibits excellent antibacterial, antifungal and anticancer effects, but also can be usefully used as a safe antibacterial, antifungal and anticancer agent for humans because it is not cytotoxic.

In addition, the present invention provides a pharmaceutical composition for antimicrobial, antifungal and anticancer activity using the antibiotic peptide and derivatives thereof as an active ingredient.

The antibiotic peptide described in SEQ ID NO: 2 and its derivatives can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the antibiotic peptide described in SEQ ID NO: 2 of the present invention and derivatives thereof may be administered in various parenteral formulations, and when formulated, fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used It is prepared using diluent or excipient. 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 antibiotic peptide described in SEQ ID NO: 2 and derivatives thereof may be used in admixture with various carriers accepted as a medicament such as physiological saline or an organic solvent, and glucose, sucrose or Carbohydrates such as dextran, ascorbic acid or antioxidants such as glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

The effective dose of the antibiotic peptide described in SEQ ID NO: 2 and derivatives thereof is 0.1 to 2 mg / kg, preferably 0.5 to 1 mg / kg, and may be administered 1 to 3 times a day.

The total effective amount of the antibiotic peptide and its derivatives set forth in SEQ ID NO: 2 in the pharmaceutical composition of the present invention is administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may be administered by a fractionated treatment protocol with long term administration. Since the concentration of the antibiotic peptide and its derivative described in SEQ ID NO: 2 is determined by the effective dose of the patient in consideration of various factors such as the age and health of the patient as well as the route of administration and the number of treatment of the drug, when considering this point One of ordinary skill in the art will be able to determine the appropriate effective dosage for the particular use of the antibiotic peptide and its derivatives set forth in SEQ ID NO: 2 as pharmaceutical compositions.

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 and Separation of Peptides

In order to synthesize an antibiotic peptide having an amphiphilic structure with an increased hydrophobic region, the present inventors have used the hydrophilic portion of the HP (2-20) peptide described in SEQ ID NO: 1 as a hydrophobic amino acid in the amino terminal portion of the RPL1 protein derived from Helicobacter pylori. Substituted. Specifically, an antibiotic peptide described in SEQ ID NO: 2 in which glutamine, the 17th amino acid, and 19th amino acid, asparagine, of the HP (2-20) peptide described in SEQ ID NO: 1 was substituted with tryptophan, respectively, was prepared. Peptide synthesis was performed using Mermofield's liquid phase solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as an amino group protecting vessel (Merrifield, RB., J. Am. Chem. Soc. , 85, 2149, 1963) ( 1 ).

Specifically, the peptide having the carboxyl terminus of -NH ₂ form of the peptide designed in the present invention used Rink Amide MBHA-Resin as a starting material, and the carboxyl terminus of the -OH type peptide was Fmoc-amino acid-Wang Resin. Used as starting material. The extension of the peptide chain by the coupling of Fmoc-amino acids was carried out by DCC (N-hydroxybenzo triazole (HOBt) -dicyclo-hexycarbodiimide) method. After coupling the Fmoc-amino acid at the amino terminal of each peptide, the Fmoc group was removed with 20% NMP (piperidine / N-methyl pyrolidone) solution, washed several times with NMP and DCM (dichoromethane) and dried with nitrogen gas. A solution of trifluoroacetic acid (TFA) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) Was added thereto and reacted for 2 to 3 hours to remove the protecting group and to remove the peptide from the resin. After separation, the peptide was precipitated with diethylether. Crude peptides obtained by the above method were purified by reverse phase (RP) -HPLC column (Delta Pak, C ₁₈ 300Å, 15, 19.0) in an acetonitrile gradient containing 0.05% TFA. mm x 30 cm, Waters). The synthetic peptide was hydrolyzed at 110 ° C. using 6 N HCl, and the residue was concentrated under reduced pressure and dissolved in 0.02 N HCl to measure amino acid composition using an amino acid analyzer (Hitachi 8500 A).

As a result of confirming the purity of the peptide prepared by the above method, the purity was more than 95%, and the molecular weight obtained using MALDI mass spectrometry (Hill, et al., Rapid Commun. Mass Spectrometry , 5, 395, 1991) is amino acid sequence. It was confirmed that the antibiotic peptide having the correct amino acid sequence was synthesized because it is consistent with the molecular weight obtained from the calculation.

Example 2 Antibacterial Activity of Antibiotic Peptides

<2-1> Measurement of growth minimum inhibition concentration

In order to measure the antimicrobial activity of the antibiotic peptide prepared in Example 1, the present inventors first measured the MIC value, which is the minimum concentration of the peptide cells are not cleaved.

We measured Bacillus subtilis (KCTC 1918) and Staphylococcus aureus (KCTC 1621) as Gram-positive bacteria and Escherichia coli (KCTC 1682) and Proteus vulgaris (KCTC 2433) as Gram-negative bacteria. Each strain was incubated in LB medium (1% Bakto tryptone, 0.5% Bakto yeast extract, 1% sodium chloride) to the mid-log phase and then 1% Bakto. Diluted to a cell concentration of 1 × 10 ⁴ cells / 100 μl in peptone medium and inoculated into micro titrate plates. The antibiotic peptide synthesized in Example 1 and melittin, which were used as a positive control, were diluted 1/2 fold from 25 μM / well, respectively, and added to the plate, followed by incubation at 37 ° C. for 6 hours, and microtight. The absorbance was measured at a wavelength of 620 nm using a rate plate reader to determine the MIC value of each strain, and the results are shown in Table 1 below.

Antimicrobial Activity of Antibiotic Peptide against Gram-positive and Gram-negative Bacteria Peptide Growth Inhibitory Concentration (μM) Gram-positive bacteria Gram-negative bacteria B. Subtilis S. Aureus Escherichia coli P. Bulgarias HP (2-20) 3.12 12.5 12.5 6.25 Antibiotic Peptides of SEQ ID NO: 2 0.39 0.78 1.56 0.39 Melittin 0.19 0.78 1.56 1.56

As a result, the antibiotic peptide set forth in SEQ ID NO: 2 of the present invention showed more than 10 times higher antimicrobial activity according to strain than HP (2-20) peptide, and showed similar antimicrobial activity as melittin used as a positive control. It was confirmed. Specifically, the antibiotic peptide of the present invention showed excellent antimicrobial activity in both Gram-positive and Gram-negative bacteria, in particular P. In Bulgaria, antibacterial activity was superior to HP (2-20) peptide and melittin.

<2-2> Visualization of Antimicrobial Activity

In addition, in order to visualize the antimicrobial activity of the antibiotic peptide of the present invention on the medium, E. coli and B. Subtilis was inoculated in LB medium (1% bacto tryptone, 0.5% yeast extract, 1% sodium chloride) and incubated until mid-log phase. Escherichia coli and B of 4 × 10 ⁵ cells. Antimicrobial peptides at 4 μM and 1 μM concentrations were mixed in the subtilis, and then incubated at 37 ° C. for 2 hours, and the culture was plated on LB plates to visualize the strains.

As a result, B. Gram-positive bacteria. In the positive control group treated with nothing in the subtilis bacteria, a large number of colonies were found (A in FIG. 2 ) and even when HP (2-20) was added, the bacteria grew slightly (B in FIG. 2 ) . When the antibiotic peptide of the present invention was added, it was confirmed that the growth of the bacteria was completely inhibited and colonies were not seen ( FIG . 2C). In addition, a large number of colonies were found in the positive control group treated with E. coli, which is Gram-negative bacteria (A in FIG. 3 ), and even when HP (2-20) was added ( FIG. 3). B), when the antibiotic peptide of the present invention was added, it was confirmed that the growth of the bacteria was completely inhibited and colonies were not seen ( FIG . 3C).

From the above results, it was confirmed that the antibiotic peptide of the present invention shows excellent antimicrobial activity compared to the HP (2-20) peptide.

Example 3 Determination of Antifungal Activity of Antibiotic Peptides

<3-1> MTT analysis

In order to determine the antifungal activity of the antibiotic peptides of the present invention, the present inventors have determined the pathogenic fungi Candida albicans (TIMM 1768), Tricosporone Beiglai (KCTC 7707) and Saccharomyces cerbiziae (KCTC 7296). MTT analysis was performed on the subjects. Specifically, 100 μl of PDB medium (20% potato infusion frum, 2% bactodextrose) containing various fungi was dispensed in a 96-well plate and 50 μM / well of the antibiotic peptide of the present invention was added to 1 / After diluting twice, adding to the plate and incubating again, 10 μl of MTT solution (3- [4,5-dimethyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazolium bromide) at a concentration of 5 mg / ml was added. Add to all wells and incubated again 5-6 hours. Subsequently, formazan produced by mitochondrial enzymes in living cells was well dissolved with 100 μl of 0.04 N HCl-isopropanol solution, and then absorbance was measured at 570 nm using an ELISA reader. The MIC value was measured to the extent of color development, and the results are shown in Table 2 below.

Determination of Antifungal Activity of Antibiotic Peptides Peptide Growth Inhibitory Concentration (μM) C. Albicans S. Serbian T. Beigeilai HP (2-20) 25.0 25.0 12.5-25.0 Antibiotic Peptides of SEQ ID NO: 2 6.25 3.12-6.25 3.12 Melittin 3.12 3.12 3.12-6.25

As a result, as compared to the antifungal activity of the antimicrobial peptides described in SEQ ID NO: 2 of the present invention and HP (2-20) peptide C. For albicans it has showed a high antifungal activity of 4 times that of HP (2-20) peptide S. Serbia and T. In case of Beiglai, it showed high antifungal activity of about 4 to 8 times, and showed antifungal activity similar to that of melittin used as a positive control.

<3-2> Visualization of Antifungal Activity

In addition, to visualize the antifungal activity of the antibiotic peptides of the present invention, C. 2 × 10 ³ cells obtained by culturing Albicans strains in PDB medium and 20 μM of antibiotic peptide were mixed and incubated at 28 ° C. for 3 hours, and then the cultures were plated on PDB agar plates to visualize the strains.

As a result, C. On the plate added with HP (2-20) to the albicans strain, a large number of colonies could be found (A in FIG. 4 ) . However, when the melittin, which is a positive control, and the antibiotic peptide of the present invention, the growth of bacteria was completely inhibited colony was confirmed to have not shown (B and C in Fig. 4 in Fig. 4).

From the above results, it was confirmed that the antibiotic peptide of the present invention exhibited superior antifungal activity compared to HP (2-20) peptide and similar antifungal activity as melittin used as a positive control.

Example 4 Anticancer Activity of Antibiotic Peptides

In order to measure the anticancer activity of the antibiotic peptide of the present invention, the present inventors performed MTT analysis on AML-2 / WT, a leukemia cell line, and SNU 638 and SNU 668, a gastric cancer cell line. First, 90 ml of AML-2 / WT cell line at 2 × 10 ⁵ cells / ml and SNU 638 and SNU 668 cell lines at 5 × 10 ⁴ cells / ml were dispensed into 96-well plates. At this time, only the culture solution was used as a negative control. The plate was shaken well and incubated in a CO ₂ incubator for 3 days, and the formazan produced by the mitochondrial enzyme of living cells was dissolved in 100 μl of 0.04 N HCl-isopropanol solution, and the absorbance was measured at 540 nm using an ELISA reader. The anti-cancer activity of the antibiotic peptide of the present invention was shown as a percentage obtained by dividing the absorbance value of the well treated with antibiotic peptide by the absorbance value containing control cells not treated with antibiotic peptide.

As a result, as shown in Figure 5 HP (2-20) did not show anti-cancer activity against all the cell lines tested. Antibiotic peptide of the present invention did not show anticancer activity up to 1 μM concentration, but showed a rapid anticancer activity as the concentration was increased, completely inhibited the growth of cancer cells at a concentration of 10 μM or more, strong anticancer activity similar to the melittin used as a positive control It was confirmed that it represents.

Example 5 Cytotoxicity of Antibiotic Peptides

In order to confirm whether the antibiotic peptide of the present invention is cytotoxic, the present inventors examined the erythrocyte destructive capacity of the antibiotic peptide.

Human red blood cells were diluted with phosphate buffer (PBS, pH 7.0) to 8% concentration, and antibiotic peptides were continuously diluted at 12.5 μM / well to 1/2 concentration and reacted at 37 ° C for 1 hour. Then, the amount of hemoglobin contained in the supernatant by centrifugation at 1,000 g was investigated by measuring the absorbance at 414 nm wavelength. In order to investigate the degree of cell destruction was measured for absorbance by the addition of 1% Triton -X100, 1% Triton, compared to the cell death function of X-100 to make 100% of red blood cells and this destruction function of each antimicrobial peptide expression (1) Calculated according to.

In the above formula, absorbance A is the absorbance of the peptide solution at 414 nm wavelength, absorbance B is the absorbance of PBS at 414 nm wavelength and absorbance C is the absorbance of 1% Triton X-100 at 414 nm wavelength.

The results of erythrocyte destructive ability according to the above formula are shown in Table 3 below.

Cytotoxicity of Antibiotic Peptides Peptide % Erythrocyte destructive capacity (μM) 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.097 HP (2-20) 0 0 0 0 0 0 0 0 Antibiotic Peptides of SEQ ID NO: 2 0 0 0 0 0 0 0 0 Melittin 100 100 95 93 31 0 0 0

As a result, the antibiotic peptide described in HP (2-20) and SEQ ID NO: 2 of the present invention showed little cytotoxicity, whereas melittin, a bee venom used as a positive control, showed very high cytotoxicity.

Example 6 Acute Toxicity of Parenteral Administration in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended parenterally with an antibiotic peptide set forth in SEQ ID NO: 2 of the present invention in 0.5% methylcellulose solution at a dose of 1 g / kg / 15 ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, the antibiotic peptide of the present invention tested did not show a toxicity change in rats up to 5 mg / kg and the parenteral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of 10 mg / kg or more.

From the above results, the antibiotic peptide described in SEQ ID NO: 2 of the present invention showed excellent antimicrobial activity in both Gram-positive bacteria and Gram-negative bacteria, high antifungal activity against fungi, and excellent anti-cancer activity against cancer cells and cytotoxicity. Since it does not represent, it can be usefully used as a safe antibiotic and anticancer agent for the human body.

<110> HAHM, Kyung-Soo LEE, Dong Gun <120> Novel antibiotic peptide derived from ribosomal protein L1 of Helicobacter pylori and use approximately <160> 2 <170> KopatentIn 1.55 <210> 1 <211> 19 <212> PRT <213> Helicobacter pylori <220> <221> PEPTIDE <222> (1) .. (19) 2-20 amino acid sequence of RPL1 protein from amino-terminal end <400> 1 Ala Lys Lys Val Phe Lys Arg Leu Glu Lys Leu Phe Ser Lys Ile Gln 1 5 10 15 Asn Asp Lys <210> 2 <211> 19 <212> PRT <213> Helicobacter pylori <400> 2 Ala Lys Lys Val Phe Lys Arg Leu Glu Lys Leu Phe Ser Lys Ile Trp 1 5 10 15 Asn Trp Lys

Claims (9)

Characterized by having an amphiphilic structure with increased hydrophobic sites by substituting glutamine, the 17th amino acid, and asparagine, the 19th amino acid, of tryptophan, the peptide HP (2-20), having the amino acid sequence set forth in SEQ ID NO: 1 An antibiotic peptide according to SEQ ID NO: 2 . delete delete delete delete delete Antimicrobial and antifungal pharmaceutical composition comprising the antibiotic peptide of claim 1 as an active ingredient. A pharmaceutical composition for anticancer comprising the antibiotic peptide of claim 1 as an active ingredient. delete