KR100883516B1 - A pleurocidin peptide derivatives having an antifungal activity - Google Patents

Abstract

A pleurocidin peptide derivative having antifungal activity is provided to increase hydrophile region, to reduce cytotoxin, to be harmless in human body and to be used safely as antibiotic by substituting an amino acid written as sequence number 1 of pleurocidin into an amino acid written as sequence number 2. A synthetic peptide written as sequence number 2 is manufactured by substituting an aliphatic amino acid into an amino acid having a positive charge. The aliphatic amino acid are G^1(glysine), G^3(glysine), V^12(valine), A^19(alanine) and L^21(leucine) in an amino acid written as sequence number 1. The amino acid having a positive charge is R(arginine). The synthetic peptide is used in a pharmaceutical composition for antifungal.

Description

A pleurocidin peptide derivatives having an antifungal activity

The present invention is a flounder ( Pleuronectes) The present invention relates to a peptide having an increased positively charged amino acid region by substituting an aliphatic amino acid region of Pleurocidin, an antimicrobial peptide isolated from americanus ), and specifically, a pharmaceutical composition comprising the same. Relates to a synthetic peptide set forth in SEQ ID NO: 2 prepared by substituting an aliphatic amino acid portion of a Pleurocidin peptide described in SEQ ID NO: 1 with another positively charged amino acid, and an antifungal pharmaceutical composition comprising the same .

Infection of pathogenic microorganisms is one of the most common and deadly causes of human disease, and humans have developed antibiotics to overcome these diseases. Unfortunately, however, human abuse of antibiotics has made pathogenic microorganisms resistant to antibiotics. The resistance of these microorganisms is distinguished from resistance, which means that the microorganism is changed into a strain resistant to various drugs.

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, which is due to the fact that penicillin does not endogenous hydrolytic enzymes. 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 for resistance to antibiotics, because of the surviving strains despite antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics. Since virtually all resistant pathogenic microorganisms are known to have resistance (Liu and Tomasz, J. Infect. Dis ., 152 , 365-372, 1985), novel antibiotics that can kill these antibiotic-resistant pathogenic microorganisms The development of antibiotics began.

The prior art attempts to use the antimicrobial peptides produced by insects as antibiotics, but there are problems in that the antimicrobial and antifungal activity of the antibiotics are deteriorated and the range of microorganisms showing antimicrobial activity is limited. In order to overcome this problem, Pleurocidin was selected as the next-generation antibiotic. Pleurocidin is a Pleuronectes. This is because the antimicrobial peptide isolated from americanus ) had a wide range of antimicrobial activity against Gram-positive and Gram-negative bacteria. In addition, pleurocidin was valuable as an constituent of antibiotics because of its antifungal activity against pathogenic fungi.

However, the flucidin peptide (Pleurocidin peptide) has a problem that the cytotoxicity is strong because it has a hemolytic activity that destroys red blood cells in the human body when injected into the human body. Due to the problems described above, pleurocidin was recognized as a candidate peptide as an antimicrobial agent due to its strong and wide range of antimicrobial activity, but there was a problem in that its utilization as an antibiotic was inferior to the human body.

The present invention has excellent antimicrobial effect, but it is harmless to human body by replacing flucidin, which was toxic to human body and could not be used as antibiotic or antifungal agent, by replacing the amino acid sequence of flucidin with other amino acid sequence, and having strong antibacterial effect. It is an object to provide antifungal peptides and uses thereof.

In order to achieve the above object, the present invention provides a synthetic peptide as set forth in SEQ ID NO: 2. The synthetic peptides of the present invention can be prepared using the liquid phase solidification method of Merrifield using a Fmoc amino group protective container (Merrifield, R. B., J. Am. Chem. Soc., 1963, 85, 2149).

Preferably, the synthetic peptides of the invention are all synthetic peptides having increased hydrophilic regions by substituting aliphatic amino acids of the Pleurocidin peptide set forth in SEQ ID NO: 1 with other positively charged amino acids.

Preferably, the synthetic peptide of the present invention selectively substitutes the 1st, 3rd, 12th, 19th and 21st amino acids present in the aliphatic amino acid region of the Pleurocidin peptide set forth in SEQ ID NO: 1, and is positively charged. The amino acid is independently selected from the group consisting of R (arginine, Arginine), K (lysine, Lysine), and H (histidine, Histidine). In addition, the most preferred as the synthetic peptide in the present invention is a synthetic peptide described in SEQ ID NO: 2 prepared by replacing an aliphatic amino acid with arginine (see Fig. 1).

Preferably, the synthetic peptide set forth in SEQ ID NO: 2 of the present invention comprises a pharmaceutically acceptable excipient, to provide a pharmaceutical composition for treating or preventing infection with fungi. As a result of investigating the antifungal activity of the antifungal peptide of SEQ ID NO: 2 against the pathogenic fungi through the preferred embodiment 2 of the present invention, it was weaker than the strong antifungal peptide melittin (Melittin), but similar to the template peptide Pleurocidin. Antifungal effect was shown. Specifically, the antifungal peptides according to the present invention have antifungal activity with a minimum growth inhibition concentration (MIC) of pathogenic fungi of 2.5-5 μg.

Preferably, the synthetic peptide according to the present invention is Candida sp . , Trichosporon sp.), and Saccharomyces sp., a pharmaceutical composition having antifungal activity against fungi selected from the group consisting of.

More preferably, the synthetic peptide according to the present invention is a pathogenic fungus Candida albicans ( Candida albicans ), Trichosporon beigelii ), Saccharomyces cerevisiae ) and the like is characterized by excellent antifungal activity (see Table 2).

As described above, the synthetic peptides used in the antifungal pharmaceutical composition according to the present invention are hydrophilic by replacing the amino acid of SEQ ID NO: 1 with Pleurocidin with the amino acid of SEQ ID NO: 2 Increasing the area decreases cytotoxicity, so it is safe for antibiotic use because it is harmless to human body.

In addition, since the aliphatic amino acid portion of Pleurocidin is replaced with a positively charged amino acid, the Pleurocidin is positively increased as a whole. On the contrary, since fungi have a negative charge on the cell membranes, flucidin substituted by the positively charged amino acids increases accessibility to the fungal cell membranes. Due to this, the fluoridated (Pleurocidin) substituted with the amino acid ( Pleuronectes) americanus ) Pleurocidin derived from Pleurocidin is effective for use in pharmaceutical compositions as an antifungal substance because it maintains its original strong antifungal activity and is harmless to humans.

Therefore, the present invention can prevent the disease against pathogenic fungi due to the above effects, it is possible to prevent the overdose of antibiotics to shorten the treatment period, thereby increasing the economic efficiency and the effect of extending the life of human.

1 is a schematic diagram of a synthetic peptide derived from Pleurocidin of the present invention,

Figure 2 is a synthetic membrane cell membrane of synthetic peptides by treating the synthetic peptides on the microcapsules prepared by the lipid composition of the cell membranes of higher organisms, including fluorescent materials, to determine how the synthetic peptides affect the cell membranes of higher organisms, including humans This graph shows the destructive power of.

●: Pleurocidin peptide having the amino acid sequence of SEQ ID NO: 1

○: synthetic peptide having the amino acid sequence of SEQ ID NO: 2

▼: melittin

Figure 3 is to confirm the effect of the synthetic peptide on the cell membrane of the fungus, by treating the synthetic peptide to the fine membrane particles prepared by the lipid composition of the fungal cell membrane including the fluorescent material to confirm the breaking capacity of the cell membrane of the synthetic peptide It is a graph.

●: Pleurocidin peptide having the amino acid sequence of SEQ ID NO: 1

○: synthetic peptide having the amino acid sequence of SEQ ID NO: 2

▼: melittin

In the present invention, the synthetic peptide set forth in SEQ ID NO: 2 is obtained by substituting another amino acid for the amino acid of the Pleurocidin peptide described in SEQ ID NO: 1. There are 20 types of amino acids, and according to their structure, they are classified into nonpolar aliphatic amino acids, polar and uncharged amino acids, aromatic amino acids, positively charged amino acids, and negatively charged amino acids. Aliphatic amino acids are hydrophobic and do not mix with water. Positively charged amino acids have hydrophilic properties and mix with water. Aliphatic amino acids include glycine, alanine, valine, leucine, methionine, isoleucine, and positively charged amino acids such as lysine and egg. There are Arginine and Histidine.

In the present invention, amino acids having hydrophobicity as aliphatic amino acids among the amino acids of the Pleurocidin peptide described in SEQ ID NO: 1 are selectively substituted with amino acids having positive charge and hydrophilicity. In addition, the aliphatic amino acids at positions 1, 3, 12, 19, and 21 of the pleurocidin peptide described in SEQ ID NO: 1 are substituted with arginine among the positively charged amino acids to SEQ ID NO: To prepare a synthetic peptide described in 2. However, in the present invention, the synthetic peptide is not limited to the synthetic peptide described in SEQ ID NO: 2, but is an amino acid having at least one aliphatic amino acid at the position in the Pleurocidin peptide described in SEQ ID NO: 1 as a positively charged amino acid. It is kindness. In addition, the present invention is not limited to arginine for replacing amino acids.

In the present invention, synthetic peptides can be used as pharmaceutical compositions for treating or preventing infection with fungi, including pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include commonly used fillers, extenders, binders, wetting agents, disintegrants, diluents such as surfactants, and the like. In addition, sterilized aqueous solution, water-insoluble solvents, suspension solvents, emulsions, lyophilized formulations, suppositories, and non-aqueous solvents, suspension solvents, such as propylene glycol (polyethylene glycol), polyethylene glycol, olive oil Oils, injectable esters such as ethyloleate, and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, and the like may be used as the base material of the suppository.

In addition, the pharmaceutical composition of the synthetic peptide of the present invention can be used in combination with a variety of carriers, such as physiological saline or an organic solvent, including the synthetic peptide, can be used to increase the stability or absorption, glucose (glucose), Carbohydrates such as sucrose or dexrine, antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers Can be used with (stabilisers).

The synthetic peptides of the present invention have antifungal activity against fungi belonging to the genus Candida, Tricosphoron, and Saccharomyces. Candida in Candida albicans , Candida glabrata , Candida krusei , Candida parapsilosis , Candida lusitaniae , Candida tropicalis , Candida guillier There are many fungi besides mondii . Trichosporon pullulans and Trichosporon are fungi distributed in refrigerated meat and the like. melibiosaceum , Trichosporon There are many fungi besides cutaneum and Trichosporon barssicae . Also, Saccharomyces cerevisiae and Saccharomyces formosensis , Saccharomyces ellipsoideus , Saccharomyces sake , Saccharomyces manchuricus , Saccharomyces carlsbergensis , Saccharomyces coreanus, Saccharomyces rouxii , Saccharomyces mellis, Saccharomyces diasticus , Saccharomyces There are many fungi besides pastorianus . The synthetic peptide set forth in SEQ ID NO: 2 in the present invention is particularly Candida albicans ( Candida albicans ), Trichosporon beigelii ), Saccharomyces cerevisiae ) has antifungal activity against pathogenic fungi, but is not limited thereto. Candida albicans (Candida albicans ) causes candidiasis, Trichosporon beigelii ) causes peritonitis, pneumonia, and skin mycosis. Saccharomyces cerevisiae causes respiratory and skin diseases.

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

Example  1: antifungal of SEQ ID NO: 2 Peptide  synthesis

In Example 1, fish ( Pleuronectes) americanus ) was synthesized from the antimicrobial peptide (Pleurocidin peptide) and the synthetic peptide of the present invention. At this time, the antimicrobial peptide Flucidin (Pleurocidin) and the synthetic peptide are shown in Table 1 below.

Peptide Amino acid sequence SEQ ID NO: 1: Pleurocidin GWGSFFKKAAHVGKHVGKAALTHYL-NH ₂ SEQ ID NO 2: Synthetic peptide R W R SFFKKAAH R GKHVGK R A R THYL-NH ₂

First, in order to synthesize the Pleurocidin peptide and the synthetic peptide, the present inventors have used a liquid phase solidification method of Merrifield using a Fmoc amino group protecting group (Merrifield, RB., J. Am . Chem) . Soc., 85, 2149, 1963) was prepared in the antimicrobial peptides described in SEQ ID NO: 1 and 2.

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

Specifically, the peptide having the carboxyl terminus of -NH ₂ form Rink Amide MBHA-Resin as a starting material, and the carboxyl terminus of the -OH type peptide used Fmoc-amino acid-Wang Resin as a starting material. Elongation of peptide chains by coupling of Fmoc-amino acids was performed by N -hydroxybenzotriazole (HOBt) -dicyclo-hexylcarbodibodiide (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 (TFA): phenol: thioanisole: H ₂ O: triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) And reacted for 3 hours to remove the protecting group and remove the peptide from the resin. After separation, the peptide was precipitated with diethyl ether.

The crude peptide thus obtained was purified by reverse phase-HPLC column (Delta Pak, C ₁₈ 300 Å, 15μ, 19.0 mm ×) with an acetonitrile gradient containing 0.05% TFA. 30 cm, Waters). The concentration gradient conditions were A buffer containing 0.01% TFA (Trifluoroacetic acid) in tertiary distilled water and B buffer containing 0.01% TFA in acetonitrile (ACN; Aceronitrile) as shown in Table 2.

Time (min) A buffer concentration (%) B buffer concentration (%) 0.001 100 0 5 100 0 20 0 100 30 0 100 40 100 0 45 stop stop

Each synthetic peptide was hydrolyzed at 110 ° C. for 24 hours with 6 N-HCl, 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 accurate molecular weight was determined using a matrix-assisted laser desorption ionization mass spectrometer. As a result, the anticipated molecular weight of the peptide described in SEQ ID NO: 2 was 3093.6 g, and the measured molecular weight was 3092.5 g.

Example  2: antibacterial of SEQ ID NO: 2 Peptide  Investigation of antifungal activity against pathogenic fungi

In order to investigate the antifungal activity of the synthetic peptide prepared in Example 1 was carried out as follows. At this time, melittin, a powerful antifungal peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antifungal peptides of SEQ ID NOs: 1 and 2 were stored at -20 ° C and dissolved in sterile tertiary distilled water.

[Antifungal Activity Measurement]

The antifungal activity of the synthetic peptide of SEQ ID NO: 2 can be used to determine the pathogenic fungus Candida albicans ( Candida). albicans ), Trichosporon beigelii ), Saccharomyces cerevisiae ) was incubated in YPD complete medium (2% glucose, 1% peptone, 0.5% yeast extract, pH 5,5). In addition, dilute to 2 × 10 ³ cells / 1 mL of the bacteria in YPD liquid medium, and dispense 100 μl into 96-well plate, and then the solution of the antimicrobial peptides of SEQ ID NO: 1 and 2 in a stepwise dilution Treated.

After shaking for 24 hours in an incubator at 28 ℃, 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 0.02 N 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 3 below.

Minimum Growth Inhibitory Concentration (MIC) for Pathogenic Fungi of Antimicrobial Peptides of SEQ ID NO: 2 MIC ^a (μg / mL) against pathogenic fungi Pleurocidin SEQ ID NO: 1 Synthetic peptide SEQ ID NO: 2 Melittin Fungus Candida Albicans 5 5 1.25 Tricosporon Beiglai 2.5 2.5 0.63 Saccharomyces cerevisiae 5 5 1.25 [Note] a: Concentration of the antimicrobial peptide of SEQ ID NO: 2 which does not grow pathogenic fungi at all

Referring to Table 3, the antifungal activity of the antimicrobial peptide of SEQ ID NO: 2 showed a minimum growth inhibition concentration (MIC) of 2.5 ~ 5 ㎍ against pathogenic fungi. This is weaker than melittin, a potent antifungal agent used as a control, but with strong antifungal activity similar to the template peptide Pleurocidin, which means that the antifungal peptide of SEQ ID NO: 2 exhibits strong antifungal activity.

Example  3: antifungal of SEQ ID NO: 2 Peptide  Review of the effects on fungal and higher organisms' cell membranes

The composition of the fungal cell membrane lipids is composed of apolar cholinephosphatidyl choline and a negatively charged phosphatidyl serine. In contrast, lipid composition of the cell membrane of higher organisms is composed of phosphatidyl choline and cholesterol (Cholesterol). Influence on the cell membrane of fungi and higher organisms of Pleurocidin described in SEQ ID NO: 2 in the present invention was measured as follows.

Similar to fungal cell lipid composition, phosphatidyl choline and phosphatidyl serine were used as 3: 1 (w / w), and phosphatidyl choline and cholesterol (Cholesterol) were similar to those of higher organisms. Large unilamellar vesicles containing fluorescent material (Calcein) were prepared using 10: 1 (w / w) and used for the experiment. After mixing, drying and mixing the respective lipids, dissolve in fluorescent material buffer solution (70 mM calcein, 10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, pH 7.4), and then freeze and dissolve 10 times. After passing this through a polycarbonate filter (pore size: 100 nm) using LiposoFast extruder (Avestin, Inc. Canada), the fluorescent material not included in the vesicles was separated and separated using a column filled with Sephadex G-50. Make one large vesicle.

Destructive capacity of peptides on cell membranes of fungi and higher organisms was excitation with a fluorescence photometer (Jasco FP-6500); 490 nm emission; Absorptivity was analyzed by measuring absorbance at a wavelength of 520 nm. And the value when treated with 20 μl of 10% Triton X-100 as a control was calculated to be 100% destructive capacity, 0% destructive capacity was calculated when no material was treated in large uniform vesicles. At this time, the destruction ability of the antifungal peptide was calculated by the following Equation 1, and the results obtained are shown in the graphs of FIGS. 2 and 3.

% Fracture Capacity = [absorbance of solution treated with test substance-absorbance only of large uniform vesicles / absorbance of solution treated with Triton X-100-absorbance only of large uniform vesicles] ⅹ100.

 Figure 2 is a graph that calculates the destructive capacity by measuring the released amount of the fluorescent substance contained in the vesicles after treating the antifungal peptides in large uniform vesicles to determine how the antifungal peptides affect the cell membranes of higher organisms. In this case, ● is Pleurocidin having the amino acid sequence of SEQ ID NO: 1, ○ is a synthetic peptide having the amino acid sequence of SEQ ID NO: 2, and ▼ represents melittin.

2, in the case of the synthetic peptide comprising the amino acid sequence of SEQ ID NO: 2 according to the present invention does not have a destructive ability on the cell membrane of higher organisms, melittin and a template peptide used as a positive control (Pleurocidin) is It was found to have a strong cell membrane destruction ability.

Figure 3 is a graph that calculates the destructive capacity by measuring the released amount of the fluorescent substance contained in the vesicles treated with the antifungal peptides in large uniform vesicles to determine how the antifungal peptides affect the fungal cell membrane. In this case, ● is Pleurocidin having the amino acid sequence of SEQ ID NO: 1, ○ is a synthetic peptide having the amino acid sequence of SEQ ID NO: 2, and ▼ represents melittin.

Referring to FIG. 3, the synthetic peptide comprising the amino acid sequence of SEQ ID NO: 2 according to the present invention does not have a destructive ability on the cell membrane of higher organisms, but has a destructive ability on the fungal cell membrane. I could confirm it. On the other hand, melittin and the template peptide, Pleurocidin, used as a positive control, were found to have destructive ability in both cell membranes of higher organisms and fungi.

Example  4: antifungal of SEQ ID NO: 2 Peptide  Cytotoxicity Measurements on Human Red Blood Cells

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 antifungal peptide of SEQ ID NO: 2.

Then, 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 1 hour in a 37 ° C. incubator. After the incubation, the supernatant was transferred to the side wells by centrifugation for 10 minutes at a rotational speed of 1000 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 red blood cells were counted as 0% destructive capacity. At this time, the destructive ability of the antifungal peptide was calculated by the following Equation 2, and the results obtained are shown in Table 4 below.

% Cytotoxicity = [absorbance of treated antifungal peptide of SEQ ID NO: 2 solution _414nm - phosphate - sodium chloride absorbance of the buffer processes the absorbance _414nm / Triton X-100 solution of _414nm - phosphate - sodium chloride buffer solution absorbance at _414nm] ⅹ100.

Measurement of Cytotoxicity of Antifungal Peptides of SEQ ID NO: 2 on Human Red Blood Cells (Human erythrocytes) % Human erythrocyte destructive capacity ^a (㎍ / ml) 100 50 25 12.5 6.25 3.12 Pleurocidin SEQ ID NO: 1 77 29 6 0 0 0 Synthetic peptide SEQ ID NO: 2 0 0 0 0 0 0 Melittin 100 100 100 100 100 98 A: Cytotoxicity in humans is determined by the destructive ability of the antimicrobial peptide of SEQ ID NO: 2 on human erythrocytes.

Referring to Table 4, it was confirmed that the antifungal peptide of SEQ ID NO: 2 does not exhibit cytotoxicity even at high concentrations. In contrast, melittin, a potent antifungal peptide, showed high cytotoxicity even at low concentrations, and the template peptide, flusidine (Pleurocidin), also showed high cytotoxicity at high concentrations. This result means that the antifungal peptide of SEQ ID NO: 2 found in the present invention can be used for humans without harming the human body.

On the other hand, in the present invention, flucidin (Pleurocidin) has a positive charge similar to the characteristic structure of the existing antimicrobial peptide. In addition, the flucidin (Pleurocidin) is divided into both sites of positively charged amino acids and aliphatic amino acids, and has an antimicrobial activity in the structure of alpha-helix (α-helix). However, pleurocidin has a hemolytic activity and cannot be used as an antibiotic due to its harmfulness to humans.

In order to overcome this difficulty, in the present invention, the amino acid sequence shown in SEQ ID NO: 1 of Pleurocidin was replaced with the amino acid sequence shown in SEQ ID NO: 2. The amino acid to be substituted here is an aliphatic amino acid at the boundary between the positively charged amino acid and the aliphatic amino acid region among the amino acids of SEQ ID NO: 1, wherein the aliphatic amino acid is substituted with an amino acid having positive charge and hydrophilic properties.

Hydrophilic amino acids include aspartic acid, glutamic acid, histidine, lysine, asparagine, proline, glutamine, arginine, and serine. (Serine), Threonine (Threonine), Tyrosine (Tyrosine). Among the positively charged amino acids are histidine, arginine, and lysine.

Increasing aliphatic amino acids in Pleurocidin increases antimicrobial activity but at the same time increases cytotoxicity, if the substitution with hydrophilic amino acids has the effect of reducing the cytotoxicity. However, when hydrophilic amino acids are increased, cytotoxicity is reduced but antimicrobial activity is inferior.

Therefore, in the present invention, the accessibility to the cell membrane of the fungus having negative charge was increased by increasing the positive charge of the synthesized Pleurocidin peptide by selecting an amino acid having a hydrophilicity and a positive charge. Synthetic flucidin (Pleurocidin) with increased accessibility as described above increases the antimicrobial activity to destroy bacterial cell membranes. In addition, the synthesized flucidine described in SEQ ID NO: 2 is substituted with arginine, which has a strong positive charge among the amino acids, and is excellent in antibacterial activity. However, the present invention is not limited to the number and type of amino acids that are identically substituted with the synthetic peptide set forth in SEQ ID NO: 2 to Arginine.

As described above, the synthesized flucidin (Pleurocidin) of the present invention has excellent efficacy as an antifungal agent and is harmless to the human body, and thus is useful as a pharmaceutical composition for preventing or treating infection by pathogenic fungi.

<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> A Pleurocidin peptide derivatives having an antibiotic and antifungal activity <130> dp20070235 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> PRT <213> Pleuronectes americanus <400> 1 Gly Trp Gly Ser Phe Phe Lys Lys Ala Ala His Val Gly Lys His Val 1 5 10 15 Gly Lys Ala Ala Leu Thr His Tyr Leu 20 25 <210> 2 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> A Pleurocidin peptide derivatives <400> 2 Arg Trp Arg Ser Phe Phe Lys Lys Ala Ala His Arg Gly Lys His Val 1 5 10 15 Gly Lys Arg Ala Arg Thr His Tyr Leu 20 25

Claims (7)

Among the amino acids of SEQ ID NO: 1, the aliphatic amino acids of G ¹ (glycine, Glycine), G ³ (glycine, Glycine), V ¹² (valine, Valine), A ¹⁹ (alanine, Alanine) and L ²¹ (leucine, Leucine) are positively charged. An antifungal peptide having improved human safety having an amino acid sequence as set forth in SEQ ID NO: 2 substituted with R (arginine, Arginine). delete delete delete A pharmaceutical composition for treating or preventing infection with fungi comprising the antifungal peptide of claim 1 as an active ingredient. The method of claim 5, The fungus is selected from the group consisting of Candida sp . , Trichosporon sp., And Saccharomyces sp. The method of claim 5, The fungus is selected from the group consisting of Candida albicans , Trichosporon beigelii and Saccharomyces cerevisiae .