WO2006080625A1

WO2006080625A1 - Novel peptide isolated from aspergillus nidulans and pharmaceutical composition containing the same

Info

Publication number: WO2006080625A1
Application number: PCT/KR2005/003120
Authority: WO
Inventors: Kyung-Soo Hahm; Yoonkyung Park; Ji-Sun Kwak; Seong-Cheol Park
Original assignee: Chosun University
Priority date: 2004-11-02
Filing date: 2005-09-21
Publication date: 2006-08-03
Also published as: KR100625875B1; KR20060039317A

Abstract

This invention relates to a novel peptide of Nidulin having antibiotic and antifungal activities, which is identified and isolated from Aspergillus nidulans , and relates to a pharmaceutical composition comprising the peptide as an effective component. Since the novel peptide of Nidulin has potent antibiotic and antifungal activities and has no toxicity, it can be used as preparations of a pesticide, which is non-toxic to human body, a food preservative, a cosmetics preservative, or preparations of a drug preservative.

Description

NOVEL PEPTIDE ISOLATED FROM ASPERGILLUS NIDULANS AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME

TECHNICAL FIELD

The present invention relates to a novel peptide and a pharmaceutical composition containing the same as an active ingredient , and more particularly to novel peptide Niduline having antibiotic and antifungal activities , which is isolated and puri fied from fungus Aspergill us nidulans , as well as a pharmaceutical composition containing the same as an active ingredient .

BACKGROUND ART Bacterial infection is one of the most common fatal causes of various diseases . Since Fleming discovered penicillin in 1929 , studies to develop antibiotics , which can treat bacterial infection, using substances i solated from nature have been continued . The term "antibiotics" was coined since Selman A . Waksman , who discovered streptomycin by isolating microorganisms from soil samples , proposed to the American Society for Bacteriology that substances inhibiting the growth or metabolism of other microorganisms would be called "antibiotics" . Antibiotics are generally defined as chemical substances produced by microorganisms , which inhibit the growth of other microorganisms even at low concentrations , but the term also encompasses those isolated from microorganisms and the case where the microbial products can be chemically synthesized or their derivatives are chemically prepared from the microbial products . Furthermore , the term also encompasses the case where the growth or metabolism of not only microorganisms but also cancer cells is inhibited .

Meanwhile , in the case of antibiotics for use in the treatment of diseases , many kinds of antibiotic substances have been discovered and developed so far and are widely used . However, microorganisms tolerant to antibiotics are appearing and the number thereof is gradually increasing .

Tolerance to antibiotics is a phenomenon distinguished from resistance to antibiotics , and was first found in Pneumococcus sp . m 1970s (Tomasz et al . , Na ture, 227 : 138- 140 , 1970 ) . Microbial species showing tolerance to antibiotics stop growing m the presence of a general concentration of the antibiotics but are not killed . This is because , when the antibiotics suppress cell wall synthetases , an autolytic enzyme ( e . g . , autolysm) of the bacteria themselves is not activated .

Tolerance consists broadly of two pathways in terms of its mechanism; the first is phenotypic tolerance occurring in all bacteria when the growth rate decreases (Tuomanen E . , Revs . Infect . Dis . , 3 , S279-S291 , 1986 ) , and the second is genetic tolerance caused by mutation of certain bacteria . In the two cases , down regulation of autolysm activity commonly occurs , where the down regulation is temporary for phenotypic tolerance , but is permanent for genetic tolerance caused by mutation . The simplest case of genetic tolerance is where deletions occur m the autolysm enzyme , but a bacterial strain having tolerance caused by deletions in the enzyme has not yet been clinically found . Rather , strains having tolerance caused by the regulation of autolysm activity have been mainly reported (Tuomanen et al . , J. infect . Dis . , 158 : 36-43 , 1988 ) .

Clinically, it is highly important that bacteria have tolerance to various antibiotics ; because the effect of antibiotic treatment m clinical infection will be reduced, when it is impossible to exterminate tolerant bacteria

(Handwerger and Tomasz , Rev. Infec . Dis . , 7 ; 368-386 , 1985 ) .

In addition, when tolerance occurs , viable bacterial strains will occur despite antibiotic treatment , and such strains — A —

will acquire new genetic factors making them resistant to antibiotics , and so continue to grow even in the presence of antibiotics . Thus , tolerance is a precondition for bacteria becoming resistant to antibiotics . Currently, VANCOMYCIN ( trademark) and TEICOPLANIN ( trademark) , which are potent antibiotics against penicillin and methicillm antibiotic-resistant bacterial strains , such as MRSA (methicillm-resistant Staphylococcus aureus ) , MRSE (methicillin-resistant Staphylococcus epidermidis ) and the li ke , have been developed and the use thereof is increasing . However , due to the excessive use of antibiotics , enterococci , that is a pathogenic bacterial strain having tolerance even to Vancomycin, commonly called "the last antibiotics" , has been found, and Enterococcus faecalis, Mycobacterium tuberculosis , Pseudomonas aeruginosa , etc . , show resistance to all the antibiotics currently known ( Stuart B . Levy, Scientific American, 46-53 , 1998 ) . For this reason, there is an urgent need for the development of novel antibiotics capable of killing bacteria having antibiotic tolerance and resistance .

Accordingly, the present inventors have isolated and purified novel peptide Niduline from a culture medium of fungus Aspergill us nidulans , and have observed whether the peptide Niduline has antibiotic and antifungal activities , and as a result , have found that the peptide Niduline shows potent antibiotic activity, and thus will be useful not only as an antibiotic agent and an antifungal agent , but also as a biopesticide , a food preservative , a cosmetics preservative and a drug preservative , thereby completing the present invention .

TECHNICAL PROBLEM It is an obj ect of the present invention to provide a novel peptide Niduline having antibiotic and antifungal activities , and a pharmaceutical composition containing the same as an active ingredient .

DESCRIPTION OF DRAWINGS

FIG . 1 is a graphic diagram showing the result of isolation and purification of novel peptide Niduline by reverse phase-high performance liquid chromatography (RP- HPLC ) . FIG . 2 is a SDS-PAGE photograph of novel peptide Niduline isolated and purified from Aspergill us nidulans .

FIG . 3 is the amino acid sequence of novel peptide Niduline , which is set forth in SEQ ID NO : 1. FIG . 4 shows the antifungal activity of novel peptide Niduline , visualized after culturing fungus Candida albicans in a medium containing novel peptide Niduline .

FIG . 5 shows the antibiotic activity of novel peptide Niduline , visuali zed after culturing bacteria Staphylococcus a ureus in a medium containing novel peptide Niduline .

MODE FOR INVENTION

To achieve the above obj ect , in one aspect, the present invention provides novel peptide Niduline having the ammo acid sequence set forth in SEQ I D NO : 1 , or peptides having a homology of at least 95% to the ammo acid sequence of said peptide Niduline .

In another aspect , the present invention provides an antifungal pharmaceutical composition comprising , as an active ingredient , novel peptide Niduline having the ammo acid sequence set forth in SEQ ID NO : 1 , or peptides having a homology of at least 95% to the amino acid sequence of said peptide Niduline . In still another aspect , the present invention provides a biopesticide , a food preservative , a cosmetics preservative or a drug preservative , which contains , as the active ingredient , novel peptide Niduline having an ammo acid sequence set forth in SEQ ID NO : 1 , or peptides having a homology of at least 95% to the amino acid sequence of said peptide Nidulme .

Hereinafter , the present invention will be described in detail .

The present invention provides novel peptide Nidulme having the amino acid sequence set forth in SEQ ID NO : 1 , or peptides having a homology of at least 95% to the amino acid sequence of said peptide Nidulme . The present inventors isolated a novel protein from a culture medium of Aspergill us nidulans by ion exchange chromatography and reverse-phase high-performance liquid chromatography ( RP HPLC ) . As shown in FIGS . 2 and 3 , the isolated protein was confirmed to be a peptide having the ammo acid sequence set forth m SEQ ID NO : 1 and a molecular weight of 17 kDa . The present inventors named the novel peptide "Nidulme" .

Also, the present invention provides an antifungal pharmaceutical composition containing , as an active ingredient , a novel peptide Nidulme having the ammo acid sequence set forth m SEQ ID NO : 1 , or peptides having a homology of at least 95% to the ammo acid sequence of the said peptide Nidulme . In order to measure the antifungal activity of Niduline , the present inventors performed MTT assays for pathogenic fungi C. albicans, T. begelii , and S . cerevisiae . As a result , it was found that the minimal inhibitory concentration (MIC ) of the novel peptide Niduline of the present invention was higher than that of amphotericin B , a commercially available antibiotics , but was 2-8 times lower than that of melittm having potent antifungal activity, indicating that the inventive peptide has potent antifungal activity ( see Table 1 ) . The MTT assay plates were visualized by observation with a microscope , and as a result , it was found that the growth of C. albicans was inhibited only in the test group to which the novel peptide Nidulin was added, so that the strain did not substantially grow . Thus , the antifungal activity of Niduline according to the present invention was visually confirmed ( see FIG . 4 ) .

Also , the present invention provides an antibiotic pharmaceutical composition containing , as an active ingredient , novel peptide Niduline having the ammo acid sequence set forth in SEQ ID NO : 1 , or peptides having a homology of at least 95% to the ammo acid sequence of said peptide Niduline . In order to measure the antibiotic activity of the novel peptide Niduline , the present inventors performed tests on Staphylococcus a ureus ( KCTC 1621 ) . As a result , as shown in FIG . 5 , it was found that Staphylococcus aureus was actively grown in a negative control plate with no peptide Niduline of the present invention added, whereas no colony formation of Staphylococcus a ureus occurred when Niduline of the present invention was added . This indicates that the peptide Nidulm of the present invention has antibiotic activity . Also , in order to examine whether the novel peptide Niduline of the present invention shows cytotoxicity, the present inventors examined the erythrocyte destruction capability of the peptide Niduline . As a result , it was found that the inventive peptide Niduline did not show cytotoxicity, whereas melittm , a bee venom used as the positive control group, showed a very high cytotoxicity ( see Table 2 ) .

From the above results , it was found that the pharmaceutical composition containing the novel peptide Niduline of the present invention as an active ingredient shows excellent antifungal and antibiotic effects and has no cytotoxicity, and thus will be useful as an antibiotic agent and antifungal agent safe to the human body . The novel peptide Niduline having the amino acid sequence set forth in SEQ ID NO : 1 , or peptides having a homology of at least 95% to the ammo acid sequence of said peptide Niduline may be parenterally administered in clinical trials and can be used in the form of general drug preparations .

Namely, the novel peptide of the present invention and peptides having a homology of at least 95% thereto can be actually administered as various parenteral formulations and can be formulated with commonly used diluents or excipients , such as fillers , extenders , binders , wetting agents , disintegrants , and surfactants . Examples of preparations for parenteral administration include sterilized aqueous solutions , non-aqueous solutions , suspensions , emulsions , lyophilized preparations and suppositories . Non-aqueous solutions and suspensions can be prepared using, e . g . , propylene glycol , polyethylene glycol , vegetable oil such as olive oil , and inj ectable ester such as ethyl oleate . Examples of bases of suppositories may include Witepsol , macrogol , tween 61 , cacao butter, laurin fat and glycerogelatm fat .

Also , the novel peptide of the present invention or peptides having a homology of at least 95% thereto can be used in a mixture with various pharmaceutically acceptable carriers , such as physiological saline or organic solvents , and can be formulated together with carbohydrates , such as glucose , sucrose or dextran, antioxidants , such as ascorbic acid or glutathione , chelating agents , low-molecular proteins , or other stabilizers , in order to increase stability or absorption properties .

The effective dose of the novel peptide of the present invention or peptides having a homology of at least 95% thereto is 0.01-100 mg/kg, and preferably 0.1-10 mg/kg , and the inventive peptide may be administered 1-3 times a day .

A total effective amount of the novel peptide of the present invention or peptides having a homology of at least 95% thereto in the inventive pharmaceutical composition may be administered to a patient in a single dose in a bolus form or by infusion for a relatively short period of time . Alternatively, a multiple dose of the inventive peptide can be administered to a patient by a fractionated treatment protocol . Since the effective dose of the inventive peptide is determined m view of various factors , including the administration route of the drug and treatment times , as well as the age and health condition of a patient , an appropriate effective dose for a particular application of the novel peptide of the present invention or peptides having a homology of at least 95% thereto can be determined by any person s killed in the art .

In addition, the present invention provides a biopesticide , a food preservative , a cosmetics preservative and a drug preservative , which contain , as an active ingredient , novel peptide Niduline having the amino acid sequence set forth in SEQ ID NO : 1 or peptides having a homology of at least 95% to the amino acid sequence of said peptide Niduline .

Food preservatives , cosmetics preservatives and drug preservatives are additives used to prevent the deterioration , decomposition, discoloration or chemical change of foods or drugs , and include fungicides and antioxidants , and also functional antibiotics , which have effects on the growth inhibition or disinfection of decomposing microorganisms in foods and drugs by inhibiting the proliferation of microorganisms , such as bacteria , fungi , yeasts , etc . Such food, cosmetics and drug preservatives ideally must have no toxicity and are effective even in trace amounts . Also , pesticides for eliminating damage to crops by blight and pest must inhibit the proliferation of hazardous microorganisms and must be harmless to the human body, such that the crops sprayed with the pesticides can be ingested safely . As shown in Tables 1 and 2 below, the novel peptide Niduline of the present invention , and peptides having a sequence homology of at least 95% thereto, show potent antibiotic activity and have no toxicity, and thus will be useful as a food preservative , a cosmetics preservative and a drug preservative .

Hereinafter , the present invention will be described m detail by example . It is to be understood, however , that these examples are for illustrative purposes only and should not be construed to limit the scope of the present invention .

EXAMPLES Example 1 : Extraction of protein from culture medium of Aspergillus nidalans

The present inventors isolated a protein from a culture medium of Aspergillus nidulans by ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP HPLC ) . For this purpose , Aspergillus nidulans was first cultured with shaking, and the culture medium was filtered through filter papers (Whatman, No . 2 ) . The filtrate was loaded onto an ion exchange chromatography column equipped with a carboxymethyl ( CM) -sepharose column . Each of the fractions obtained by the ion exchange chromatography was loaded onto a reverse phase ( RP ) -high performance liquid chromatography (HPLC) column equipped with a Shim-pack VP-ODS C18 column ( 4.6 X 250 mm; 12.0 ± 1.0 nm pore , Shimadzu ) . The protein bound to the column was eluted by a linear gradient of acetonitrile concentration in a buffer containing 0.1% trifluoroacetic acid from 5% to 65% at a rate of 2% /mm for 30 minutes . Then , a peak fraction was collected ( FIG . 1 ) .

Example 2 : Protein electrophoresis

The present inventors subj ected the fraction obtained m Example 1 to SDS-PAGE ( sodium dodecyl sulfate polyacrylamide gel electrophoresis ) using slab gel according to the method of Laemmli ( Laemmli et al . 1972 ) . For this purpose , the protein sample obtained in Example 1 and standard proteins were loaded onto 15% acrylamide gel , followed by electrophoresis . The gel was stained with coomassie brilliant blue R-250 and destained with destaining buffer (MeOH : glacial acetic acid : distilled water = 30 : 10 : 60 ) to confirm the resulting protein bands . The molecular weight of the isolated protein was measured using the following molecular weight standard proteins belonging to a class of low-molecular-weight proteins ( FIG . 2 ) : 66 kDa bovine serum albumin , 45 kDa chicken egg ovalbumin, 36 kDa GAPDH ( rabbit muscle glyceraldehyde-3-phosphate dehydrogenase ) , 29 kDa bovine erythrocytes carbonic anhydrase , 24 kDa bovine pancreas trypsmogen, 20 kDa soybean trypsin inhibitor , 14.2 kDa bovine milk α -lactalbumm, and 6.5 kDa bovine lung aprotmin . As a result , the novel protein isolated from the culture medium of Aspergillus nidulans was found to be a peptide having a molecular weight between 14.2 kDa and 20 kDa .

Example 3 : Analysis of amino acid sequence and molecular weight

The peptide isolated in Example 1 was dissolved in 6N HCl and hydrolyzed at 110 ⁰C for 22 hours , followed by drying . The dried peptide was analyzed for its amino acid sequence with an ammo acid analyzer , and analyzed for its molecular weight by mass spectra . As a result , as shown in

FIG . 3 , the above peptide was found to be a peptide having the ammo acid sequence set forth m SEQ I D NO : 1 and a molecular weight of 17 kDa . The present inventors named the peptide "Niduline" . Example 4 : Measurement of antibiotic activxty

<Example 4- l> MTT assay and microscopic observation

In order to measure the antifungal activity of the peptide Nidulme of the present invention, Niduline was added to each of pathogenic fungus C. albicans, T. begelii , and S . cerevisiae, followed by MTT assay . For this purpose, a YPD medium containing each of C. albicans, T. begelii , and S . cerevisiae was diluted, and 100 μl of the dilution was spread into each well of a 96-well microtiter plate at a concentration of 2 X 10³ cells /well . To the plate , each of the inventive peptide Nidulme , the prior Melittm peptide (Anygen) known to have potent antifungal activity, and antifungal antibiotics Amphotericin B ( Sigma ) was added at various concentrations . Then , each of the fungal mixtures was left to stand at 37 ^°C overnight , after which the absorbance at 620 nm was measured by ELISA (Enzyme-Linked Immunosorbent Assay) , and a graph of absorbance as a function of concentration was plotted . Then, the minimal inhibitory concentration (MIC ; concentration at which the absorbance is not shown ) was determined . As a result , as shown in Table 1 below, the minimal inhibitory concentration (MIC ) of the peptide Nidulme of the present invention was higher than that of Amphotericin B , a commercially available antibiotics , but was 2-8 times lower than that of melittin having potent antifungal activity, indicating that the inventive peptide has potent antifungal activity . Table 1

<Example 4-2> Microscopic observation

The well containing C. albicans alone , the well containing C, albicans together with the inventive Nidulin, and the well containing C. albicans together with Amphotericin B, which have been provided m <Example 4 -l>, were set as a negative control , a test group and a positive control , respectively, and visualized by observation with a microscope . As a result , as shown in FIG . 4 , the negative control with nothing added showed active growth of C. albicans, and the test group with the inventive Niduline added showed little or no growth of C. albicans in a similar manner to the positive control . Thus , the potent antifungal activity of the inventive Nidulme was visually confirmed .

Example 5 : Examination of antibiotic 1 ⁽

The present inventors obtained bacteria Staphylococcus aureus ( KCTC 1621 ) from Korean Collection for Type Culture

( KCTC ) and cultured the bacteria in LB medium to the mid-log phase . 100 μl of the culture medium of Staphylococcus aureus was evenly plated on LB plates , and then the negative control plate was treated with nothing, and the test group was treated with 100 μg of the inventive peptide Nidulme . The plates were incubated at 37 ^°C for 16 hours , followed by observation . As a result , as shown in FIG . 5 , Staphylococcus aureus was actively grown in the negative control plate containing no Nidulme of the present invention , whereas no colony of Staphylococcus a ureus was formed m the test group containing the inventive Nidulme . This suggests that the inventive Nidulme shows potent antibiotic activity .

Example 6 : Measurement of cytotoxicxty of Nidulme

In order to examine whether the Nidulme shows cytotoxicity, the present inventors examined the erythrocyte destruction capability of Nidulme . For this purpose , human erythrocytes were diluted in phosphate buffered saline ( PBS , pH 7.0 ) to a concentration of 8 % . In the dilution, the inventive peptide Nidulme was continuously diluted at a concentration of 1/2 starting with 12.5 μM/well , and allowed to react at 37 ^°C for 1 hour . The resulting product was centri fuged at 1 , 00Ox g to collect the supernatant , and the amount of hemoglobin contained in the supernatant was examined by measuring the absorbance at 414 nm. To examine the degree of cell destruction, 1% Triton X-100 was added to human erythrocytes , and the absorbance of the supernatant was calculated, and percent erythrocyte destruction capability was measured according to following Equation 1 by comparing the erythrocyte destruction capability of each of the antibiotic conj ugated peptides to the cell destruction capability of 1% Triton X-100 taken as 100% .

[Equation 1 ]

Erythrocyte destruction capability ( % ) =

( absorbance A x absorbance B/absorbance C x absorbance B) x 100

In the equation, absorbance A represents the absorbance of Niduline solution at a wavelength of 414 nm, absorbance B represents the absorbance of PBS at a wavelength of 414 nm, and absorbance C represents the absorbance of 1% Triton X-100 at a wavelength of 414 nm. The results of erythrocyte destruction capability obtained by the above equation are shown in Table 2 below . Table 2 : Measurement of hemolytic activity of Niduline

As a result , the peptide Niduline of the present invention showed little or no cytotoxicity, whereas the Melittin peptide and Magainin peptide used as positive controls showed a significantly high cytotoxicity .

INDUSTRIAL APPLICABILITY

As described above , the novel peptide Niduline according to the present invention shows excellent antibiotic effects against hazardous fungi and bacteria . Thus , a pharmaceutical composition containing the peptide Niduline will be useful not only as an antibiotic agent and an antifungal agent , but also as a biopesticide , a food preservative , a cosmetics preservative and a drug preservative .

Claims

What is claimed is :

1. Peptide Niduline having the amino acid sequence set forth m SEQ ID NO : 1 , or peptides having a homology of at least 95% to the amino acid sequence of said peptide Niduline .

2. The peptide of Claim 1 , wherein the peptide is derived from Aspergillus nidulans .

3. An antifungal pharmaceutical composition containing the peptide of Claim 1 as an active ingredient .

4. The pharmaceutical composition of Claim 3 , which has antifungal activities against Candida albicans, Trichosporon begeln, and Saccharomyces cerevisiae .

5. An antibiotic pharmaceutical composition containing the peptide of Claim 1 as an active ingredient .

6. The pharmaceutical composition of Claim 5 , which has antibiotic activity against Staphylococcus aureus .

7. A biopesticide , food preservative , cosmetics preservative or drug preservative containing the peptide of Claim 1 as an active ingredient .