KR101184008B1 - Antifungal coprisin peptide derived from Copris tripartitus and its use - Google Patents

Abstract

The present invention relates to a coprisin peptide (43 amino acids of the mature protein region of the coprisin gene) having antifungal activity isolated from Copris tripartitus belonging to the coleoptera .

The antifungal peptide according to the present invention has no cytotoxicity and exhibits excellent antifungal activity and is safe for the human body. Therefore, the antifungal peptide may be used as an antibiotic including an antifungal agent, a food preservative, a cosmetic preservative, a pharmaceutical preservative, and the like.

Description

Antifungal coprisin peptide derived from Copris tripartitus and its use

The present invention relates to a coprisin peptide having antifungal activity isolated from Copris tripartitus belonging to the coleoptera, and its use.

The antifungal peptide according to the present invention has no cytotoxicity and exhibits excellent antifungal activity and is safe for the human body. Therefore, the antifungal peptide may be used as an antibiotic including an antifungal agent, a food preservative, a cosmetic preservative, a pharmaceutical preservative, and the like.

Infection of 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 are resistant to new antibiotics is much faster than the rate at which new antibiotic analogs are developed. For example, which may pose a threat to life, Enterococcus faecalis kusu nose (Enterococcus faecalis ), Mycobacterium tuberculosis) and pathogenic micro-organisms such as Pseudomonas Aru whether labor (Pseudomonas aeruginosa) species have been brought up resistance to all antibiotics known so far (Stuart B. Levy, Scientific American , 46-53, 1998).

Tolerance to antibiotics is a distinct phenomenon from resistance to antibiotics. In the 1970s, Pneumococcus sp.) was first discovered and provided important clues 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. The 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. Activation kills bacteria and bacteria also inhibit their activity, resulting in survival during antibiotic treatment.

It is important, very clinical, if it becomes impossible to eradicate resistant microorganisms is because the effectiveness of antibiotics in clinical infections dropped (Handwerger and Tomasz, Rev. Infec . Dis. Pathogenic microorganisms having a resistance to antibiotics, 7 , 368-385, 1985).

Resistance is also considered a prerequisite to resistance to antibiotics, because strains survive even antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics. Virtually all resistant pathogenic microorganisms are known to have resistance (Liu and Tomasz, J. Infect . Dis ., 152, 365-372, 1985), and therefore, new pathogens capable of killing these antibiotic-resistant pathogenic microorganisms. The development of antibiotics is urgent.

As described above, in order to prevent damage caused by pathogenic microorganisms resistant to antibiotics, the development of new antibiotics is required, and the development of new antibiotics that act independently of autolysine activity is necessary. There is also a need to provide such new antibiotics with pharmacological compositions for effectively treating infections of pathogenic microorganisms.

On the other hand, some of the substances that play an important role in the process of maintaining the homeostasis of organisms are bioactive substances derived from various organisms.

Many studies have been conducted on a number of physiologically active substances, and among them, antimicrobial peptides isolated from various organisms are known to work in various ways from bacteria, fungi and viruses. There are also antibacterial peptides are known to play an important role in the host defense and innate immune system (Boman, HG, Cell, 65 , 205, 1991;... Boman, HG, Annu Rev Microbiol, 13, 61, 1995).

Accordingly, the present invention relates to a coprisine peptide having antifungal activity isolated from Copris tripartitus belonging to the coleoptera, and its use, which has no cytotoxicity and exhibits excellent antifungal activity and is therefore safe for the human body and thus contains an antifungal agent. It can be used as antibiotics, food preservatives, cosmetic preservatives, and pharmaceutical preservatives.

OBJECT OF THE INVENTION The present invention relates to a coprisin peptide having antifungal activity isolated from cedar beetle, which can be usefully used as a natural antibiotic for the prevention and treatment of fungal diseases in the human body and its use. It is an object of the present invention to provide a coprisin peptide having no cytotoxicity and having excellent antifungal activity which can be used as an antibiotic and its use.

In order to achieve the above object, the present invention provides a peptide (43 amino acids as the mature protein region of the coprisin gene) represented by SEQ ID NO: 1 having antifungal activity isolated from cedar beetle.

The present invention also provides an antifungal pharmaceutical composition comprising the peptide having the antifungal activity as an active ingredient.

As described above, the present invention relates to a copricin peptide having antifungal activity isolated from Copris tripartitus belonging to Coleoptera and its use. The antifungal peptide according to the present invention has no cytotoxicity and exhibits excellent antifungal activity and is safe for the human body. Therefore, the antifungal peptide may be used as an antibiotic including an antifungal agent, a food preservative, a cosmetic preservative, a pharmaceutical preservative, and the like.

The present invention will be described in more detail with reference to the following Examples. These examples are only to specifically describe the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples. In addition, although not shown in specific examples, it will be apparent to those skilled in the art that a pharmaceutical composition for an antifungal agent containing an antifungal peptide according to the present invention having an antifungal action as an active ingredient is possible.

Example 1 Synthesis and Purification of Antifungal Peptides

In Example 1, antifungal peptides derived from cedar beetles were synthesized and purified. At this time, the antifungal peptide is as shown in Table 1 below.

[Table 1]

Peptides Amino acid sequence Coprisin VTCDVLSFEAKGIAVNHSACALHCIALRKKGGSCQN-GVCVCRN

First, in order to synthesize antifungal peptides, the inventors used Merrifield's liquid phase solidification method using a Fmoc amino group protective container (Merrifield, RB., J. Am . Chem . Soc ., 85 , 2149, 1963). ) Antibacterial peptides were prepared. The antimicrobial peptide synthesis method was synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group of the Nα-amino group of amino acids. Specifically, the peptide having the carboxyl terminus of -NH ₂ form Rink Amide MBHA-Resin as the starting material, and the peptide of the carboxyl terminus -OH form was used as the starting material Fmoc-amino acid-Wang Resin. Elongation of peptide chains by coupling of Fmoc-amino acids was performed by N -hydroxybenzotriazole (HOBt) -dicyclo-hexylcar-bodiimide (DCC) method. After coupling the Fmoc-amino acid at the amino terminus of each peptide, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM), followed by nitrogen. Dried with gas. To this was added a solution of trifluoroacetic acid (phenol-thioanisole-H ₂ O-triisop-ropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) And reacted for 3 hours to remove the protecting group and The peptide was isolated and then the peptide was precipitated with diethyl ether. The crude peptide thus obtained was purified by reverse phase-HPLC column (Delta Pak, C18 300Å, 15μ, 19.0 mm × 30) with an acetonitrile gradient containing 0.1% TFA. Cm, Waters). The synthetic peptide was hydrolyzed with 6 N-HCl at 110 ° C. for 24 hours, and the residue thus obtained was concentrated under reduced pressure, dissolved in 0.02 N-HCl, and the amino acid composition was measured by 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 a matrix-assisted laser desorption ionization mass spectrometer. As a result, it was confirmed that the antimicrobial peptide having the correct amino acid sequence was synthesized because the measured molecular weight and the calculated molecular weight matched.

Example 2 Investigation of Antifungal Activity of Coprisin Against Pathogenic Fungi

In order to investigate the antifungal activity of the antimicrobial peptides prepared in Example 1 was performed as follows. At this time, melittin, a powerful antimicrobial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antifungal peptide was stored at -20 ° C and used by dissolving in sterile tertiary distilled water.

[Antifungal Activity Measurement]

For the measurement of antifungal activity of the antifungal peptide pathogenic fungus of Aspergillus Plastic bus (Aspergillus flavus), Aspergillus Fu Micah Tuscan (Aspergillus fumigatus), Aspergillus para City carcass (Aspergillus parasticus ), Candida albicans ), Candida parapsilosis ), Malassezia furfur , Trichosporon beigelii ), Trichophyton rubrum , were incubated in YM medium supplemented with YPD medium, PD medium and 1% olive oil. After diluting to 2 x 10 < 3 >

After shaking for 24 hours in an incubator at 28 ° C., MTT [3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide] solution [5 mg of MTT / ml of PBS (pH 7.4)] to each well and incubated for 4 hours in a 37 ℃ incubator. Subsequently, 20 μl of 20% SDS containing 0.02N HCl was added to dissolve Formazan produced by MTT, followed by reaction at 37 ° C. for 16 hours.

Next, the minimum growth inhibition concentration (MIC) was measured by measuring the absorbance of each well under a wavelength of 570 nm with an ELISA reader. The results obtained are shown in Table 2 below.

TABLE 2 Minimum growth inhibition concentration (MIC) for pathogenic fungi of antifungal peptides

Fungus
MIC for pathogenic microorganisms (μM) Coprisin Melitin Aspergillus Flavers 10 2.5 Aspergillus fumigatus 5 2.5 Aspergillus paracitus 10 5 Candida Albicans (TIMM 1768) 5-10 2.5 Candida Albicans
(ATCC 90028) 10 5 Candida paracylosis 10 5 Marasegia Pearl 5 1.25 Tricosporon Beige 10 2.5 Tricophyton Louvroom 20 5

MIC: Concentration of Antifungal Peptides That Are No Growth of Pathogenic Microorganisms

[Antifungal activity]

Referring to Table 2, as a result of measuring the antifungal activity of the present antifungal peptide, it showed a minimum growth inhibition concentration (MIC) of 5 ~ 20μM against pathogenic fungi. This is slightly weaker than melittin, a potent antifungal agent used as a control, but relatively strong antifungal activity, which means that the present antimicrobial peptides exhibit strong antifungal activity.

Example 3 Investigation of Coprisin's Activity on Dysplasia of Pathogenic Fungi

In order to investigate the activity of the antifungal peptides to the dimorphism of the pathogenic fungi was performed as follows. At this time, melittin, a powerful antimicrobial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antimicrobial peptide was stored at -20 ° C and dissolved in sterile tertiary distilled water.

[Measurement of activity on dysplasia of pathogenic fungi]

Candida albicans , a representative pathogenic fungus, was used as the target strain, and 20% FBS (fetal bovine serum) was added to form hyphae to obtain pathogenicity. The peptide was treated to the pathogenic fungi and incubated at 37 ° C. for 24 hours, and then observed under a microscope.

Referring to Figure 1 (A) is a yeast form of fungi (B) is made to show the pathogenicity with FBS (C) is to treat the fungi having a pathogenic peptide to aggregate the cells to visualize antifungal activity.

Example 4 Investigation of the Mechanism of Antifungal Activity of Coprisin

To investigate the mechanism of antifungal activity of coprisin was performed as follows. At this time, melittin, a powerful antimicrobial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antimicrobial peptide was stored at -20 ° C and dissolved in sterile tertiary distilled water.

[Investigation of mechanism with antifungal activity]

Candida albicans , a representative pathogenic fungus, was used, and after treatment with peptides, plated on a YPD agar plate by time and incubated at 28 ° C. for 24 hours to measure colony forming units (CFUs). It was determined whether the mechanism with antifungal activity was cidal (killed) or static (fungal).

[Antifungal Activity Through Killing Mechanism]

Referring to Figure 2 it can be seen that copricin effectively kills (cidal) like melittin used as a positive control. This shows that the present antifungal peptides have potent antifungal activity.

Example 5 Investigation of Cytotoxicity of Coprisin on Human Erythrocytes

The cytotoxicity of human erythrocytes was measured as follows.

Human erythrocytes were washed three times with phosphate-sodium chloride buffer, pH 7.4 (PBS) and then diluted with phosphate-sodium chloride buffer to prepare 8.0% erythrocyte cell solutions. Then, 100 μl of the 8.0% erythrocyte cell solution was dispensed into 96-well plates, followed by mixing the diluted solution of the present antimicrobial peptide.

Subsequently, physiological saline (Saline 0.85% NaCl) was added to the wells so that the total amount was 200 μl, and the cells were incubated for 90 minutes in a 37 ° C. incubator. After the incubation, the supernatant was transferred to the side well by centrifugation for 10 minutes at a rotational speed of 1500 rpm in a centrifuge.

Destructive capacity for erythrocytes was analyzed by measuring absorbance under a wavelength of 414 nm with an ELISA reader. And when treated with 0.1% Triton X-100 as a control, the value was calculated as 100% destructive capacity, and only the erythrocyte cells were added as 0% destructive capacity. At this time, the breaking ability of the antimicrobial peptide was calculated by the following Equation 1, and the obtained results are shown in Table 3 below.

[Formula 1]

TABLE 3 Measurement of cytotoxicity of the present antimicrobial peptides on human erythrocytes

% Human erythrocyte destructive capacity Peptide Concentration (μM) 100 50 25 12.5 6.25 3.13 Copricin 0 0 0 0 0 0 Melitin 100 100 100 100 98 98

% Human erythrocyte destructive capacity: Cytotoxicity to human is measured by the antimicrobial peptide's destructive ability on human erythrocyte cells

Referring to Table 3, it was confirmed that the present antifungal peptide does not show any cytotoxicity even at high concentrations. In contrast, melittin, a potent antimicrobial peptide, was found to be highly cytotoxic even at low concentrations. These results mean that the antifungal peptides found in the present invention can be used against humans without harming the human body.

FIG. 1 shows Candida albicans, a representative pathogenic fungus, having antifungal activity (43 amino acids as a mature protein region of the coprisin gene) having antifungal activity isolated from P. pylori albicans ) shows effective aggregation of hyphae.

Figure 2 is the same manner as the melittin (Melittin) of bee venom-derived peptides used as positive control Corp. lysine having the antifungal activity isolated from Arabidopsis horn cow dung copper pathogenic fungus Candida albicans (Candida albicans ) have the activity of killing.

<110> Republic of Korea <120> Antifungal coprisin peptide derived from Copris tripartitus and          its use <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 43 <212> PRT <213> Copris tripartitus <400> 1 Val Thr Cys Asp Val Leu Ser Phe Glu Ala Lys Gly Ile Ala Val Asn   1 5 10 15 His Ser Ala Cys Ala Leu His Cys Ile Ala Leu Arg Lys Lys Gly Gly              20 25 30 Ser Cys Gln Asn Gly Val Cys Val Cys Arg Asn          35 40  

Claims (2)

delete An antifungal pharmaceutical composition comprising an antifungal peptide represented by SEQ ID NO: 1 having antifungal activity isolated from Copris tripartitus .

Images