KR102146930B1 - An anti-microbial peptide, Teleogryllusine 3 isolated from Teleogryllus emma and its synthetic composition - Google Patents

Abstract

The present invention relates to a novel tereogryllusine 3 peptide isolated from cricket and a composition thereof. The tereogrileucine 3 peptide exhibits antimicrobial action against Gram-positive bacteria, Gram-negative bacteria and fungi, has minimal cytotoxicity, and has strong antibacterial activity, so antibiotics including antibacterial or antifungal agents, food preservatives, cosmetic preservatives, pharmaceutical preservatives, etc. Can be used as

Description

Anti-microbial peptide tereogrileucine 3 derived from crickets and its composition {An anti-microbial peptide, Teleogryllusine 3 isolated from Teleogryllus emma and its synthetic composition}

The present invention relates to a novel antimicrobial peptide tereogrileucine 3 derived from a cricket and a composition thereof, and more particularly, a novel tereogrileucine 3 peptide derived from a cricket and exhibiting antibacterial and antifungal activity while having minimal cytotoxicity and It relates to an antimicrobial agent or antibiotic comprising.

Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease, unfortunately, the abuse of antibiotics has resulted in antibiotic resistance of pathogenic microorganisms. Indeed, the rate at which pathogenic microorganisms become resistant to new antibiotics is much faster than the rate at which analogs of new antibiotics are developed. For example, pathogenic microbial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa , which can pose a threat to life, are known to date. It has developed resistance to all antibiotics.

Tolerance to antibiotics is a phenomenon that is distinct from resistance to antibiotics, and was first discovered in Pneumococcus sp. in the 1970s and provided important clues about the mechanism of action of penicillin. Species that show resistance stop growing in the presence of conventional concentrations of antibiotics, but do not die as a result. This resistance is due to the fact that when antibiotics inhibit cell wall synthase, autolytic enzymes such as autolysin do not activate. This is because penicillin uses endogenous hydrolytic enzymes. By activating it, bacteria are killed, and the bacteria also inhibit their activity, resulting in survival even when treated with antibiotics.

It is of great importance clinically that pathogenic microorganisms have resistance to antibiotics, because if it becomes impossible to eradicate resistant microorganisms, the effectiveness of antibiotic treatment in clinical infections decreases. In addition, the development of resistance is considered to be a prerequisite for the development of resistance to antibiotics, because a strain that survives despite antibiotic treatment occurs. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics.

Practically, all resistant pathogenic microorganisms are known to have resistance, so the development of a novel antibiotic capable of killing pathogenic microorganisms having such antibiotic resistance is urgent.

Accordingly, in order to prevent damage by pathogenic microorganisms that are resistant to antibiotics, it is necessary to develop new antibiotics, and to develop new antibiotics that act independently of autolysine activity. In addition, there is a need to provide a pharmaceutical composition for effectively treating infection of pathogenic microorganisms with such novel antibiotics.

Meanwhile, some of the substances that play an important role in the process of maintaining the homeostasis of living organisms are physiologically active substances derived from various organisms. Many studies have been conducted on numerous physiologically active substances so far, and among them, antimicrobial peptides isolated from various organisms are known to act in various ways, including bacteria, fungi and viruses. In addition, antimicrobial peptides are known to play an important role in host defense and innate immune system.

As related prior art, Korean Patent Registration No. 10-0625875 (a new peptide isolated from Aspergillus nidulans and a pharmaceutical composition containing it as an active ingredient), Korean Patent No. 10-0964136 (separated from Uldo skyworm Antibacterial and antifungal peptide genes and synthetic peptides having antibacterial and antifungal activity), Korean Patent Laid-Open Patent No. 10-2011-0013074 (New use and manufacturing method of insect glycozaminoglycan), Korean Patent No. 10-1660381 (Insect N-Glycan and its use), etc. are known, but until now, studies on novel peptides isolated from crickets and their new uses have not been conducted.

Korean Patent Registration No. 10-0625875 (New peptide isolated from Aspergillus nidulans and pharmaceutical composition containing it as an active ingredient) Korean Patent Registration No. 10-0964136 (Antibacterial and antifungal peptide gene isolated from Uldo sky larva and synthetic peptide having antibacterial and antifungal activity) Korean Patent Publication No. 10-2011-0013074 (New use and manufacturing method of insect glycozaminoglycan) Korean Patent Registration No. 10-1660381 (Insect N-Glycan and its use)

An object of the present invention is to provide an amino acid sequence of tereogrileucine 3, an antibacterial peptide isolated from gram-negative bacteria, positive bacteria, and human fungal disease prevention and treatment, and a composition thereof, which can be usefully used in the prevention and treatment of human fungal diseases.

The present invention relates to an antibacterial or antifungal peptide (SEQ ID NO: 1) derived from a king cricket ( Teleogryllus emma ) consisting of the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1: MFWKNKLFFIVKK-NH ₂

The peptide is characterized by having antimicrobial activity against any one or more of Staphylococcus aureus and Escherichia coli 0-157.

The peptide is characterized by having antifungal activity against Candida albicans .

The peptide is characterized by insignificant toxicity to rat red blood cells.

The present invention is to provide a pharmaceutical composition for antibacterial or antifungal preparations, antibiotics, food preservatives, cosmetic preservatives or pharmaceutical preservatives containing the above peptides as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an antibacterial or antifungal peptide derived from Teleogryllus emma consisting of the amino acid sequence of SEQ ID NO: 1.

In the present invention, in order to identify the peptide gene derived from the king cricket ( Teleogryllus emma ), first, the king cricket was rapidly frozen with liquid nitrogen to isolate total RNA, and this was confirmed by using the RNA seq analysis method to identify the gene information on the king cricket transcripts. And, based on the amino acid sequence translated from each of the transcripts, using the properties of the previously discovered antibacterial peptides, Unigene is selected as a peptide that exhibits antibacterial and antifungal activity.

The amino acid sequence of this peptide is MFWKNKLFFIVKK-NH ₂ and consists of 13 amino acids, which is called Teleogryllusine 3.

In addition, the present invention provides a pharmaceutical composition for antibacterial and antifungal preparations or antibiotics or food preservatives or cosmetic preservatives or pharmaceutical preservatives comprising the same as an active ingredient.

Specifically, in order to measure the antibacterial and antifungal activity of the tereogrileucine 3 peptide, Staphylococcus aureus , Escherichia coli 0-157), and antifungal bacteria RDA assay (radial diffusion assay) was performed on Candida albicans as an active target bacterium, which has antibacterial and antifungal activity (see FIG. 1).

In addition, the novel tereogrileucine 3 peptide of the present invention was confirmed to have no cytotoxicity as a result of confirming the ability to destroy red blood cells, whereas melittin, a bee venom used as a positive control, showed very high cytotoxicity (see Table 2). ).

Therefore, the pharmaceutical composition containing the novel tereogrileucine 3 peptide of the present invention as an active ingredient not only exhibits antifungal and antibacterial effects, but also exhibits insignificant cytotoxicity, and thus can be usefully used as a safe antibacterial agent or antifungal agent.

Such, the pharmaceutical composition can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation.

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

In addition, the novel peptide of the present invention may be used in combination with various carriers permitted as drugs such as physiological saline or an organic solvent, and carbohydrates such as glucose, sucrose or dextran, in order to increase stability or absorption, Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers may be used as pharmaceuticals.

In the pharmaceutical composition of the present invention, the total effective amount of the novel peptide of the present invention and the peptide having more than 95% homology thereto is in the form of a bolus or a single dose by diffusion for a relatively short period of time. It may be administered to a patient by using multiple doses, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. Since the concentration is determined by taking into account various factors such as the patient's age and health condition, as well as the route of administration and the number of treatments of the drug, the effective dosage of the patient is determined. It will be possible to determine an appropriate effective dosage according to the specific use of the novel peptide of the pharmaceutical composition.

In addition, the present invention provides an antibiotic, food preservative, cosmetic preservative, or pharmaceutical preservative containing a novel peptide as an active ingredient.

At this time, food preservatives, cosmetic preservatives, and pharmaceutical preservatives are additives used to prevent deterioration, spoilage, discoloration, and chemical change of food or pharmaceuticals, and fungicides and antioxidants may be included therein, and microorganisms such as bacteria, mold, and yeast Functional antibiotics such as inhibiting proliferation and inhibiting the growth of spoiled microorganisms in foods and medicines or sterilizing them are also included. These food preservatives, cosmetics, and pharmaceutical preservatives should not be toxic under ideal conditions, and should be effective in trace amounts.

The novel tereogrileucine 3 peptide of the present invention exhibits antibacterial and antifungal activity as shown in FIGS. 1 and 2 and has minimal toxicity, so it can be usefully used as a food preservative, cosmetics, and pharmaceutical preservative.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

According to the present invention, as the antibacterial peptide tereogrileucine 3 derived from the cricket and its composition is provided, the cytotoxicity is insignificant and exhibits antibacterial and antifungal activity, so that it can be provided as an antibiotic, food preservative, cosmetic preservative, medicine, etc. have.

1 is a view showing the antibacterial and antifungal activity of the novel tereogrileucine 3 peptide of the present invention derived from the cricket.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

Example 1: Selection of antibacterial peptide genes derived from crickets

In order to identify the peptide gene that exhibits antibacterial or antifungal activity from the cricket, the total RNA was first quickly frozen with liquid nitrogen to isolate the total RNA, and the genetic information on the cricket transcripts was confirmed using RNA seq analysis. The entire Unigene was filtered based on the existing antibacterial peptide properties based on the amino acid sequence translated from the cadavers, and as a result, a gene presumed to be a new antibacterial peptide was selected. The selected amino acid sequence was MFWKNKLFFIVKK-NH ₂ consisting of 13 amino acids and named as Tereogryllusine 3 (see Table 1).

Peptide Amino acid sequence Teleogryllusine 3 MFWKNKLFFIVKK-NH ₂

Example 2: Tereogrileucine 3 synthesis and separation and purification

The tereogrileucine 3 peptide derived from the cricket found in Example 1 was synthesized and separated and purified.

First, in order to synthesize tereogrileucine 3 peptide, the present inventors have conducted a liquid solid phase method (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, of Merrifield) using a Fmoc amino group protective container. 1963) was used to prepare a peptide.

The method of synthesizing the peptide was synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group of the N _α -amino group of amino acids.

Specifically, Rink Amide MBHA-Resin was used as a starting material for a peptide having a carboxyl terminal in the form of -NH ₂ , and Fmoc-amino acid-Wang Resin was used as a starting material for a peptide having a carboxyl end in the -OH form.

The elongation of the peptide chain by Fmoc-amino acid coupling was performed by the DCC ( N -hydroxybenzo-triazole (HOBt)-dicyclo-hexylcar-bodiimide) method.

After coupling the Fmoc-amino acid at the amino terminal of each peptide, remove the Fmoc group with 20% piperidine/N-methylpyrrolidone (NMP) solution, wash several times with NMP and dichloromethane (DCM), and then nitrogen Dried with gas.

TFA (trifluoroacetic acid)-phenol-thioanisole-H ₂ O-triisop-ropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) solution was added and reacted for 3 hours to remove the protecting group and to remove the protective group from the resin. After separating the peptide, the peptide was precipitated with diethyl ether. The crude peptide thus obtained was purified by using an acetonitrile gradient containing 0.1% TFA in a reverse phase-HPLC column (Delta Pak, C ₁₈ 300Å, 15μm, 19.0 mm× 30, Waters).

The synthetic peptide was hydrolyzed with 6 N-HCl at 110° C. for 24 hours, the obtained residue was concentrated under reduced pressure, dissolved in 0.02 N-HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A).

In addition, the molecular weight was calculated based on the sequence of the synthesized peptide, and the exact molecular weight was measured using MALDI mass spectrometry (matrix-assisted laser desorption ionization mass spectrometer).

As a result, it was confirmed that an antimicrobial peptide having an exact amino acid sequence was synthesized because the measured molecular weight and the calculated molecular weight match.

Example 3: Investigation of antibacterial activity of tereogrileucine 3 antibacterial peptide against pathogenic microorganisms

RDA (radial diffusion assay) was performed to investigate the antibacterial activity against tereogrileucine 3 peptide. At this time, melittin, a strong antibacterial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antibacterial peptide was stored at -20°C and dissolved in sterilized tertiary distilled water and used.

[Antibacterial activity measurement]

To measure the antimicrobial activity of the antimicrobial peptide, first, pathogenic bacteria Staphylococcus aureus and Escherichia coli 0-157 were added to 3% (w/v) TSB (Tryptic Soy Broth). ) At 37° C. and 200 rpm for 18 hours, inoculated into a new medium, and grown for 2 hours to become logarithmic.

Then, a sterilized undercoat consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4), 1% (w/v) type I (low electroendosmosis) agarose, 0.03% TSB. Cultured bacteria (4×10 ⁶ colony forming units/ml) are added to the underlying gel, mixed, and poured into a square plate. When the underlay gel is hardened, a hole of 3 mm in diameter is made to obtain peptides by concentration. Each 5 µl was put into the hole.

After incubation at 37°C for 3 hours to spread the peptide, 10 ml of overlay gel (6% TSB, 1% agarose) was poured and incubated again at 37°C to confirm the formation of a clear zone. The results are shown in FIG. 1.

[Measurement of antifungal activity]

To measure the antifungal activity of the antimicrobial peptide, the pathogenic fungus Candida albicans was grown overnight at 30°C and 200 rpm in YPD medium (1% yeast extract, 2% peptone, 2% dextrose), and then it was fresh medium. Was inoculated and grown for 2 hours to become logarithmic.

After that, bacteria cultured on a sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4), 1% (w/v) type I (low electroendosmosis) agarose, and 0.03% TSB ( 4×10 ⁶ colony forming units/ml) was added, mixed, and poured into a square plate. When the underlay gel was hardened, a hole with a diameter of 3 mm was made, and 5 µl of peptide for each concentration was put into the hole.

After incubating for 3 hours at 37°C to diffuse the peptide, 10 ml of overlay gel (6% TSB, 1% agarose) was poured and incubated again at 37°C to confirm the formation of a clear zone. It is shown in 1.

[Antibacterial activity result]

As shown in FIG. 1, it was confirmed that the tereogrileucine 3 peptide had activity against Gram-negative bacteria and positive bacteria, and this was found to increase in a concentration-dependent manner. This shows that it can be used as a therapeutic agent for existing harmful strains.

[Antifungal activity result]

As shown in FIG. 1, as a result of measuring the antifungal activity of tereogrileucine 3 peptide, a clear zone was formed, and it was confirmed that the activity increased in a concentration-dependent manner as in the gram-negative bacteria positive bacteria.

Example 4: Measurement of cytotoxicity of tereogrileucine 3 peptide to rat red blood cells

The measurement of cytotoxicity to rat red blood cells was performed as follows.

Red blood cells (rat erythrocytes) were washed three times with phosphate-sodium chloride buffer, pH 7.4 (PBS), and then diluted with phosphate-sodium chloride buffer to prepare a 5.0% red blood cell solution.

Thereafter, 100 µl of a 5.0% red blood cell solution was dispensed into a 96-well plate, and a stepwise diluted solution of the antimicrobial peptide was mixed.

Subsequently, phosphate-sodium chloride buffer, pH 7.4 (PBS) was added to all the tubes so that the total amount was 200 µl, and incubated in an incubator at 37° C. for 30 minutes.

After the cultivation was completed, the supernatant was transferred to a 96-well plate by centrifuging for 10 minutes at a rotation speed of 1000 rpm in a centrifuge.

The disruption ability for red blood cells was analyzed by measuring the absorbance under a wavelength of 550 nm with an ELISA reader.

And the value when treated with 0.1% Triton X-100 as a control was calculated as 100% destructive ability, and when only red blood cells were added, it was calculated as 0% destructive ability.

At this time, the breaking capacity of the peptide was calculated by the following equation 1, and the obtained results are shown in Table 2 below.

[Equation 1]

% Destruction ability = (absorbance of the solution treated with peptide 550 nm-absorbance of phosphate-sodium chloride buffer solution 550 nm)/(absorbance of solution treated with Triton X-100 550 nm-absorbance of phosphate-sodium chloride buffer solution 550 nm) × 100

Measurement of cytotoxicity of the antibacterial peptide of tereogrileucine 3 on rat erythrocytes
% Rat red blood cell destruction ability ^a (㎍/ml) 0 12.5 25 50 100 200 Teleogryllusine 3 0 0 0.09 0.28 0 1.22 Melittin 0 75.8 83.1 89.8 88.3 91.4 [Note] a: Cytotoxicity is measured by the ability of antibacterial peptides to destroy red blood cells.

As shown in Table 2, it was confirmed that the tereogrileucine 3 peptide had insignificant ability to destroy red blood cells when treated at a concentration of up to 200 μg/ml. On the other hand, it can be seen that melittin, a strong antibacterial peptide as a control, has high cytotoxicity even at low concentrations.

These results suggest that the antimicrobial peptides found in the present invention are not only insignificant in cytotoxicity, but also exhibit antibacterial and antifungal activities, so that they can be used as safe antibiotics, food preservatives, cosmetic preservatives, and pharmaceuticals.

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> An anti-microbial peptide, Teleogryllusine 3 isolated from Teleogryllus emma and its synthetic composition <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 13 <212> PRT <213> Unknown <220> <223> Teleogryllus emma <400> 1 Met Phe Trp Lys Asn Lys Leu Phe Phe Ile Val Lys Lys 1 5 10

Claims (9)

delete delete delete delete In the pharmaceutical composition for an antibacterial or antifungal agent, characterized in that it contains a peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The antibacterial is an antimicrobial activity against Staphylococcus aureus or Escherichia coli 0-157,
The antifungal is antifungal activity against Candida albicans , the composition. In the antibiotic characterized in that it contains the peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The antibiotic is Staphylococcus aureus ( Staphylococcus aureus ) or E. coli 0-157 ( Escherichia coli 0-157) antibacterial activity; Or Candida albicans ( Candida albicans ) Antifungal activity against; that is, antibiotics. In a food preservative comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The food preservative has antibacterial activity against Staphylococcus aureus or Escherichia coli 0-157; Or Candida albicans ( Candida albicans ) antifungal activity; that is, food preservatives. In the cosmetic preservative, characterized in that it contains a peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The cosmetic preservative has antimicrobial activity against Staphylococcus aureus or Escherichia coli 0-157; Or Candida albicans ( Candida albicans ) antifungal activity against; that is, cosmetic preservatives. In the pharmaceutical preservative, characterized in that it contains a peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The pharmaceutical preservative may have antimicrobial activity against Staphylococcus aureus or Escherichia coli 0-157; Or Candida albicans ( Candida albicans ) antifungal activity against; that is, pharmaceutical preservatives.

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