KR101653141B1

KR101653141B1 - Novel analogues of antibacterial peptide derived from halocynthia aurantium and the use thereof

Info

Publication number: KR101653141B1
Application number: KR1020140064369A
Authority: KR
Inventors: 이인희
Original assignee: 주식회사 베네팜
Priority date: 2014-05-28
Filing date: 2014-05-28
Publication date: 2016-09-01
Also published as: WO2015183002A3; KR20150136834A; WO2015183002A2

Abstract

The present invention relates to an antimicrobial peptide. More particularly, the present invention relates to an antimicrobial peptide, and more particularly, to an antimicrobial peptide isolated from a halocynthia aurantium body fluid cell by modifying a partial amino acid of halocodin, The present invention relates to a mutant antimicrobial peptide whose activity is greatly improved and an antimicrobial agent containing the above-mentioned antimicrobial peptide as an active ingredient. The antimicrobial peptide of the present invention has excellent antifungal activity against gram-positive and gram-negative bacteria, especially antibiotic-resistant bacteria, and also has a strong antifungal activity against fungi. The antimicrobial peptide is useful for hydrolysis and proteolysis by proteolytic enzymes, It is an antimicrobial peptide that overcomes the problem of loss of antimicrobial activity due to non-specific binding with proteins. Therefore, it can be usefully used as a novel peptide antibiotic.

Description

Novel analogues of antibacterial peptides derived from silkworm-derived antimicrobial peptides and their uses

More particularly, the present invention relates to an antimicrobial peptide, which is derived from an antimicrobial peptide isolated from a nematode, and has improved resistance to proteolytic enzymes and anti-microbial (bacterial, fungal) activity in the presence of serum, And to an antimicrobial peptide applicable to wound infection in which wound exudates are present.

A small protein with bactericidal activity against microorganisms is defined as an antimicrobial peptide. Antibacterial peptides exist in almost all organisms in nature and play important host defense roles in the body. Since its discovery in insect hemolymph and rabbit white blood cells for the first time before 1980, more than 1,500 antimicrobial peptides have been isolated from a wide variety of organisms.

The antimicrobial peptide is a small protein composed of 20 kinds of standard amino acids, usually composed of 12 to 50 amino acid residues. Antibacterial peptides often conjugated with carbohydrates have also been reported, but in most cases they consist only of standard amino acids. Most antimicrobial peptides are characterized by having a large number of positive charge amino acids (Arg, Lys) and several hydrophobic amino acids, which contribute to the amphipathicity of the antimicrobial peptides in the secondary and tertiary structures. Due to these structural characteristics of the antimicrobial peptide, the antimicrobial peptides bind to the surface of the phospholipid membrane of the microorganism, and then are inserted into the membrane to form pores. These antimicrobial peptides can be classified on the basis of structure as follows.

① helical peptides (magainin, cecropin, LL-37, etc.)

② Peptides (PR39, indolicidin, etc.) that contain specific amino acids intensively

③ Peptide (defensin, tachyplesin, protegrin, etc.) of β-sheet structure with disulfide bond (SS bond) between cysteine residues in molecule

Most of the antimicrobial peptides found so far have structures belonging to the above category, the structure itself being very vulnerable to proteolytic enzymes such as trypsin. In other words, Lys and Arg, which are important basic amino acids for the antimicrobial peptide to exert its antimicrobial activity, are the acting sites of trypsin. In order to overcome this problem, the amino acid substitution of D-form [Stromstedt AA, Pasupuleti M, Schmidtchen A, Malmsten M (2009) Evaluation of strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, an internal segment of LL- 37. Antimicrob Agents Chemother. 53 (2): 593-602.], Modification of amino acid residues [Meng H, Kumar K (2007) Antimicrobial activity and protease stability of peptides containing fluorinated amino acids. J Am Chem Soc. 129 (50): 15615-15622.], The Cys amino acid was added to the primary structure and then dimerization [Dalla Serra M, Cirioni O, Vitale RM, Renzone G, Coraiola M, Giacometti A, Potrich C, Baroni E, Guella G, Sanseverino M, De Luca S, Scalise G, Amodeo P, Scalonia (2008) Structural features of distinctive affecting peptide biological and biochemical properties. Biochemistry. 47 (30): 7888-7899.] Or conversion to circular form peptides [Rozek A, Powers JP, Friedrich CL, Hancock RE (2003) Structure-based design of an indolicidin peptide analogue with increased protease stability. Biochemistry. 42 (48): 14130-14138]. However, problems such as decrease in activity due to structural conversion, synthesis yield and cost increase have occurred. Therefore, antimicrobial peptides designed with the basic structure of a natural antimicrobial peptide resistant to proteolytic enzymes have been regarded as the most potent antibiotic candidates. The functional characteristics of antimicrobial peptides so far known are summarized as follows.

① Different methods of action than conventional antibiotics: Effects on resistant bacteria

② Broad spectrum spectrum

③ Rapid action mechanism

④ Binding to pathogen surface molecules such as LPS and LTA:

⑤ Target selectivity (ability to distinguish between human cells and microorganisms)

⑥ Various immune response modulating activity: immuno-modulatory activity

Clinical application of new drugs based on antimicrobial peptides The disease was mainly a topical antibiotic for treating topical infections (external medicine for the treatment of diabetic foot infections and oral mucosal infections of cancer patients) , While no toxicity was ever observed for external preparations. However, in all cases, it was omitted from the second or third clinical trial because the therapeutic effect was not significant when the antimicrobial peptide was applied to the actual treatment site. Therefore, it can be seen that there are various problems in terms of active or structural use of the antimicrobial peptide as a therapeutic agent. The critical limitations of antimicrobial peptides unsuitable for being developed as new external-use antibiotics can be summarized in two ways.

① Many AMPs lose their activity in physiological environments, ie, in environments with high concentrations of NaCl or in the presence of divalent cations (Ca ²⁺ , Mg ^2+, etc.).

② It is degraded by protease derived from pathogen or human epithelial tissue and loses its activity.

Therefore, even though it showed sufficient activity as an antibiotic in vitro , no effective therapeutic effect was observed in actual clinical application. In particular, the problem of instability for trypsin-, chymotrypsin-like proteases and matrix metalloproteases is very difficult to solve. To cope with this problem, many researchers have substituted antimicrobial peptide-forming amino acids with unusual or modified amino acids To try to devise new antimicrobial peptides. However, in this case, the activity of the antimicrobial peptide composed of standard amino acids may be weakened or changed, and unexpected problems may occur in pharmacokinetics or toxicity.

Recently, a new class of four antimicrobial peptides that do not fall into the categories described above have been reported, with one Cys amino acid in the antimicrobial peptide sequence, taking a dimer form through a disulfide bond (SS) A novel heterodimeric antimicrobial peptide from the tree-frog Phyllomedusa distincta ., Which was found in frogs [Batista CV, Scalonia, Rigden DJ, Silva LR, Rodrigues Romeroa, Dukor R, Sebben A, Talamo F, FEBS Lett. 494 (1-2): 85-89.), The CRS peptide (Hornef MW, PK, Karlsson J, Refai E, and Andersson M (2004) Increased diversity of synthetic secreted CRS peptide in rat Paneth cell intestinal antimicrobial peptides by covalent dimer formation. Nat Immunol. 5 (8): 836-843.), Dicynthaurin found in the somatic cells of murine leukocytes [Lee IH, Lee JS, Kim CH, Kim CR, Hong T, Menzel L, Boo LM, Pohl J, Sherman MA, Lehrer RI (2001) Dicynthaurin: an antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium . Biochim Biophys Acta. (2002) Halocidin: a new antimicrobial peptide from the solitary tunicate, Halocynthia aurantium . (Korean J Thorac Cardiovasc Surg 2000; FEBS Lett. 521 (1-3): 81-86.].

It has been reported that these dimeric antimicrobial peptides can be resistant to proteolytic enzymes [Hornef MW, Putsep K, Karlsson J, Refai E, and Anderson M (2004) Increased diversity of intestinal antimicrobial peptides by covalent dimer formation . Nat Immunol. 5 (8): 836-843., Lehrer RI (2004) Paradise lost and paradigm found. Nat Immunol. 5 (8): 775-776.). Of the four antimicrobial peptides, halocidin is the smallest in size, and is an antibacterial peptide purified from hematopoietic cells of the silkworm moth, Donghae, 10 years ago by the present inventors [Jang WS, Kim MH, Lee IH ) Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium . FEBS Lett. 521 (1-3): 81-86.). Based on the basic structure of 'Halocidin', amino acids were substituted, inserted and cleaved to produce various derivatives. Through comparative studies of these derivatives, efforts to derive optimal active derivatives have been continued for many years. As a result, it has been reported that, while possessing a broad spectrum of anti-microbial (bacterial, fungal) activity [Jang WS, Kim KN, Lee YS, Nam MH, Lee IH (2002) Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium . FEBS Lett. (2003) Biological activities of synthetic analogs of halocidin, an antimicrobial peptide from the tunicate Halocynthia (2001), Jang WS, Kim CH, Kim SY, Lee JH, aurantium . Antimicrob Agents Chemother. Antifungal activity of synthetic peptides derived from halocidin, antimicrobial peptides from the tunicate, Halocynthia aurantiu (2006), Jang WS, Kim HK, Lee KY, Kim SA, Han YS Lee SH Lee IH . FEBS Lett. 580: 1490-1496.], And devised a derivative with resistance to proteolytic enzymes, which is regarded as the biggest obstacle to the practical use of antimicrobial peptides [Shin YP, Park HJ, Shin SH, Lee YS, Park S, , Lee YH, Lee IH (2010) Antimicrobial activity of a halocidin-derived peptide to attack by proteases. Antimicrob Agents Chemother. 54 (7): 2855-2866.], Which is named HG1 (Di-K19Hc) and is under development as a drug for treating oral mucositis.

HG1 is limited in its application range due to its non-specific binding with its derivatives and human serum proteins in terms of antimicrobial activity and resistance to proteolytic enzymes. In other words, since infectious diseases such as wound and pressure ulcer exuding blood plasma components can not be used for therapeutic effects due to loss of activity, they can be used only for infectious diseases in which blood components do not exist, .

Disclosure of the Invention The present invention has been conceived to solve the problems of the conventional antibacterial peptide derived from halocynthia aurantium as described above, and it has been found that by replacing some amino acid residues constituting the antibacterial peptide halocodin with other amino acids, The present invention provides a mutant antimicrobial peptide that significantly alleviates the problem of the disappearance of anti-microbial activity in the blood of the cytidine peptide derivative (HG1) and an anti-bacterial and anti-fungus agent containing the above-mentioned antimicrobial peptide as an active ingredient The purpose.

In order to accomplish the above object, the present invention provides an antimicrobial peptide, wherein Leucine (L) located at the third amino acid from the N-terminal in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine (Q) Alanine (A) located at the first amino acid from the terminal is substituted with lysine (K).

Here, it is preferable that the peptide has the amino acid sequence of SEQ ID NO: 2.

The present invention also relates to an antimicrobial peptide represented by the following formula (1) wherein the cysteine (C) residue of the amino acid sequence of SEQ ID NO: 2 is connected to a disulfide bond to form a dimer form.

[Chemical Formula 1]

In addition, the peptide may have antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria.

In addition, the peptide can be resistant to proteolytic enzymes.

It is also preferred that the peptide has antimicrobial activity in human serum.

Further, it is more preferable that the peptide has antibacterial activity in a human wound fluid (HWF).

On the other hand, another embodiment of the present invention is an antimicrobial or noxious composition comprising the above-mentioned peptide as an active ingredient.

The present invention can also be a pharmaceutical composition for antibiotics, which comprises the above-mentioned peptide as an active ingredient.

In addition, the present invention may be an antibiotic food additive characterized by containing the above-mentioned peptide as an active ingredient.

The details of other embodiments are included in the detailed description and drawings.

The present invention relates to a mutant antimicrobial peptide in which some amino acids of halosidine, which is an antimicrobial peptide isolated from silkworm cells, are modified to greatly improve resistance to proteolytic enzymes and activity in serum, and antimicrobial peptides, As an antimicrobial agent.

The antimicrobial peptide according to the present invention has excellent antifungal activity against gram-positive and gram-negative bacteria, particularly antibiotic-resistant bacteria, and also has a strong antifungal activity against fungi. The antimicrobial peptide has a strong inhibitory effect on proteolytic enzymes It is an antimicrobial peptide that overcomes the problem of loss of antimicrobial activity due to nonspecific binding with serum proteins and can be usefully used as a novel peptide antibiotic.

FIG. 1 is an example graph showing hemolytic toxicity of HG1 and HG1 derivatives according to an embodiment of the present invention.
FIG. 2 is an example of a graph showing antimicrobial activity of human HG1 and HG1 derivatives in human serum according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of reverse-phase HPLC (High Pressure Liquid Chromatography) of halophenan according to a preferred embodiment of the present invention.
4 is a graph showing hemolytic toxicity of halothane according to a preferred embodiment of the present invention.
FIG. 5 is a graph showing the resistance according to the protease concentration of halopenan according to a preferred embodiment of the present invention. FIG.
FIG. 6 is a graph showing the resistance of halopanger to proteolytic enzyme according to a preferred embodiment of the present invention. FIG.
FIG. 7 is a graph showing antibacterial activity in human serum of halopenan according to a preferred embodiment of the present invention. FIG.
8 is a graph showing antimicrobial activity in halothane human wound exudate (HWF) according to a preferred embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprises" or "having" and the like are used to specify that there is a feature, a number, a step, an operation, an element, a component or a combination thereof described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

The terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.

The antimicrobial peptide according to the present invention is characterized in that leucine (L) located at the third amino acid from the N-terminal in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine (Q) and located at the first amino acid from the C- Alanine (A) is replaced by lysine (K).

The amino acid sequence of SEQ ID NO: 1 of the present invention can be prepared by adding and / or substituting some amino acid residues to halocodin, which is an antimicrobial peptide derived from halocynthia aurantium . Specifically, It can be produced by a conventional peptide synthesis method known in the art, and the production method is not particularly limited.

Generally, halothiazine, a natural antimicrobial peptide isolated from humoral cells of silkworm, is composed of 18 amino acid long chains and 15 short amino acid chains containing cysteine (C) The peptide is a heterodimer formed by a disulfide bond formed by an amino acid, and the C-terminal thereof is C-terminal amidated (see Table 1 below).

Then, a homo-dimer of a chain obtained by adding one cationic amino acid, lysine (K), to the N-terminus of the long chain having 18 amino acid sequences of halocidin was designed Peptide has Di-K19Hc (hereinafter referred to as HG1), which has greatly improved anti-microbial activity and resistance to proteolytic enzymes, but has the disadvantage of losing anti-microbial activity in serum as described above. It is the haloganan peptide of the present invention which solves this problem.

In addition, the halothane according to the present invention is a homodimer peptide of a peptide in which one amino acid is added to the N-terminus of the long chain of halocidin, and two amino acids in the sequence are substituted. More specifically, the cationic amino acid lysine (K) is added to the N-terminus of the halocidin long chain (18mer) and the first leucine (L) amino acid at the N-terminal side is replaced with glutamine (Glutamine, Q) , A homomeric form of the 19 mer peptide in which alanine (Alanine, A) amino acid at the C-terminus is replaced with lysine (K) amino acid.

The reason why the amino acid sequence of halothane is devised as follows is as follows. As mentioned above, halocidines are antimicrobial peptides found in the fluid cells of the East Sea, tunicates and are hetero- dimers in which 18 amino acid chains and 15 amino acid chains are linked by disulfide bonds .

In the initial study, a 19 mer peptide was synthesized by adding a lysine (K) residue to the N-terminus of 18 amino acid chains to increase the activity of halocidin, and 19-residue homodimeric-dimeric form of HG1 was developed. Lysine (K), a cationic amino acid, is known to play an important role in the activity of antimicrobial peptides [Hancock RE, Diamond G (2000). Trends Microbiol. 8 (9): 402-410.), Particularly in the case of an antimicrobial peptide starting at the N-terminus with an amino acid tryptophan (W), the activity is further increased by adding lysine (K) before tryptophan . A representative antimicrobial peptide cecropin of insects [Moore AJ, Beazley WD, Bibby MC, Devine DA (1996) Antimicrobial activity of cecropins. J Antimicrob Chemother. 37 (6): 1077-1089.), While the D-type of the secretin peptide group starts at the N-terminus with tryptophan (W), while the A- and B- In addition, it is known that the antimicrobial activity is higher than that of D-type secretin (KW) [Hultmark D, EngstrA, Bennich H, Kapur R, and Boman HG 1982) Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. Eur J Biochem. 127 (1): 207-217.).

For this reason, we synthesized 19 residues by adding lysine (K) residues to 18 residues beginning with tryptophan (W). Thus synthesized HG1 has strong antimicrobial activity against not only microorganisms but also fungi. However, HG1 has a problem of high toxicity and its activity is lost by binding with certain components of human serum. Therefore, a new peptide, haloganan according to the present invention, was synthesized to compensate for this.

The present inventors have paid attention to the ionicity of amino acids in antimicrobial peptides such as HG1. That is, hydrophobic amino acids play an important role in the activity of antimicrobial peptides with cationic amino acids. Hydrophobic amino acids also affect the solubility of peptides in human body fluids as well as their activity.

HG1 is a Leucine-rich antimicrobial peptide with five residues of leucine (L) at 19 residues. Therefore, we assumed that the main hydrophobic amino acid in HG1 would be the leucine (L) residue, and that the leucine (L) residue would contribute to not only the antimicrobial activity of HG1, but also the serum binding to serum proteins. Thus, the leucine (L) residue was replaced with glutamine (Q). Glutamine is a non-charged polar amino acid that will solve the problem of binding to serum and is thought to reduce toxicity because it is the most abundant amino acid in our body.

Thus, five leucines (L) were each replaced with glutamine (Q). The results are shown in Figs. 1 and 2, and Tables 2 to 4 below.

Table 2 shows the nucleotide sequences of HG1 and HG1 derivatives and the characteristics of each peptide. Table 3 shows the anti-bacterial activities of HG1 and HG1 derivatives. Table 4 shows anti-fungal activities of HG1 and HG1 derivatives . As shown in the figure, when the third leucine (L) was replaced with glutamine (Q), the activity against the microorganism was maintained, but the toxicity was lowered, and the problem of loss of activity due to non- . However, it was confirmed that anti-fungal activity against fungi was lowered.

Accordingly, the present inventors substituted alanine (A) at the C-terminal with lysine (K) amino acid to increase activity against fungi. The lysine (K) present at the C-terminus not only increases the activity but is also stable to hydrolysis by proteolytic enzymes.

Accordingly, the present invention is also applicable to an antimicrobial peptide represented by the following formula (1) wherein the cysteine (C) residue of the amino acid sequence of SEQ ID NO: 2 is linked to a disulfide bond to form a dimer form.

[Chemical Formula 1]

As a result, it was confirmed that the modified halopanger of the present invention does not cause loss of activity due to nonspecific binding with human body fluid components including serum, and that the activity against bacteria and fungi is improved.

The present invention relates to a mutant antimicrobial peptide which significantly improves resistance to proteolytic enzymes and activity in serum by modifying a part of amino acids of halosidine, which is an antimicrobial peptide isolated from silkworm fluid cells, As an antimicrobial agent.

In addition, the antimicrobial peptide according to the present invention has excellent antifungal activity against gram-positive and gram-negative bacteria, particularly antibiotic resistant bacteria, as well as against fungi. The antimicrobial peptide has a strong antifungal activity against fungi, Which is an antimicrobial peptide that overcomes the problems of degradation and loss of antimicrobial activity due to nonspecific binding with serum proteins, can be usefully used as a novel peptide antibiotic.

On the other hand, the present invention is an antimicrobial or noxious composition comprising the above peptide as an active ingredient.

In a specific embodiment of the present invention, the antimicrobial peptide according to the present invention has no cytotoxicity and has an excellent antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria as well as fungi. Therefore, in all the substances and foods in which microorganisms can proliferate, And can be usefully used as an anti-adhering composition. The above use is not limited to the composition for the noodle of food, but it can be used as a preservative for inhibiting the growth of microorganisms in all materials requiring antimicrobial activity such as cosmetic preservatives and medicine preservatives.

The present invention also provides a pharmaceutical composition for preventing and treating a pathogenic bacterial infection containing the above-mentioned antimicrobial peptide as an active ingredient.

In a specific embodiment of the present invention, the antimicrobial peptide according to the present invention has excellent antimicrobial activity not only against gram-negative and gram-positive bacteria but also against fungi, has no cytotoxicity, is hydrolyzed by proteolytic enzymes, which is a major obstacle to commercialization of antimicrobial peptides Can overcome the problem of loss of antimicrobial activity due to nonspecific binding between serum proteins and serum proteins, and thus can be usefully used as a pharmaceutical composition for antibiotic or pharmaceutical composition for prevention and treatment of pathogenic bacterial infection.

The composition containing the antimicrobial peptide of the present invention preferably comprises 0.1 to 50% by weight of the antimicrobial peptide based on the total weight of the composition, but is not limited thereto.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention can be administered orally or parenterally, and any of parenteral administration methods can be used, and systemic administration or local administration is possible. However, topical administration is more preferable, and the characteristics of the antimicrobial peptide Stability, optimum efficacy), it is most preferable to administer it as a topical external preparation.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the effective dose of the antimicrobial peptide of the present invention is 1 to 2 mg / kg, preferably 0.5 to 1 mg / kg, and can be administered 1 to 3 times a day. The dose is not intended to limit the scope of the invention in any way.

Antibiotics containing the antimicrobial peptide of the present invention as an active ingredient can be administered to a patient in a single dose by bolus administration or by infusion for a relatively short period of time, dose may be administered by a fractionated treatment protocol administered over a prolonged period of time. The effective dose of the antimicrobial peptide of the present invention is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient as well as the administration route of the antimicrobial peptide. The appropriate effective dose can be determined.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in any form suitable for pharmaceutical preparations including oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations such as ointments and creams, suppositories and sterile injectable solutions, , Dispersants, or stabilizers.

In a specific embodiment of the present invention, the antimicrobial peptide according to the present invention has an excellent antimicrobial activity against gram-negative and gram-positive bacteria as well as fungi, and has no cytotoxicity, and thus can be usefully used as an antibiotic food additive. In addition, the antimicrobial peptide of the present invention can be used not only as a food additive but also as a feed additive containing an active ingredient thereof.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products such as drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The antimicrobial peptide of the present invention can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of the antimicrobial peptide in the food may be 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The beverage composition of the present invention is not particularly limited to other ingredients other than those containing the above-mentioned antimicrobial peptide as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the antimicrobial peptide of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and heavy stabilizers (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the antimicrobial peptides of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the antimicrobial peptide of the present invention.

The present invention may be better understood by the following examples, which are for the purpose of illustrating the invention and are not intended to limit the scope of protection defined by the appended claims.

Example : Synthesis of Haloganan

First, monomer peptides were synthesized using an automatic solid phase peptide synthesizer and purified 19 mer monomer peptides that were synthesized correctly using C18 reverse-phase high-pressure liquid chromatography (RP-HPLC). The purified monomer peptides were mixed in an aqueous solution of 0.1 M ammonium bicarbonate, and the mixture was reacted at room temperature for 72 hours or more to synthesize a homodimer-dimer. After the reaction, the halonane synthesized by the homodimer was purified by RP-HPLC (FIG. 3).

Halogenane is a white or almost white powder in the completely dried state, and there is no particular crystalline polymorph. DMSO, 1% (V / V) acetic acid, or trifluoroacetic acid, at least 10% aqueous acetonitrile at a concentration of 1 mg / mL.

Molecular weight was measured with a MALDI mass spectrometer to confirm whether the halonan peptide was synthesized correctly. As a result, it was confirmed that the expected mass of halothane and the mass measured by MALDI were in agreement (Table 5).

Experimental Example 1: Anti-bacterial activity of haloganan

Analysis of the anti-bacterial activity of halothane was carried out using the method of M7-A7 (Clinical and Laboratory Standards Institute, USA) prescribed by CLSI (Clinical and Laboratory Standard Institute). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed. Approved standard M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA. 2006.] were measured for various pathogenic bacteria by microbroth dilution analysis method. For this purpose, the bacteria were first incubated in MHB (Mueller-Hilton broth) at 37 ° C and 200 rpm overnight to form a stationary phase. The culture was diluted with fresh MHB to a final concentration of 2 x 10 ⁴ to 10 ⁵ colony forming units (CFU) / ml. Stock solutions of each peptide were prepared at a concentration of 640 [mu] g / ml in 0.01% acetic acid. Then, the peptide solution was sequentially diluted with 0.01% acetic acid twice, to a concentration of 10 / / ml. 100 [mu] l equivalent of the bacterial suspension was dispensed into each well of a 96-well plate (Costar 3790, Corning, USA) and then 11 [mu] l of the peptide solution was added. After culturing at 37 DEG C for 18 hours, the antimicrobial activity of the peptides was evaluated by confirming the turbidity of each well. The minimum inhibitory concentration (MIC) refers to the minimum concentration that completely inhibits the growth of the test bacteria.

The reference material used for the test was the well known two antimicrobial peptides (MSI-78 [Jacob, L. and Zasloff, M. Potential therapeutic applications of maginins and other antimicrobial agents of animal origin, Ciba Found Symp. 1994. 186: 197-223.), LL-37 [Gudmundsson, GH, Agerberth, B., Odeberg, J., Bergman, T., Olsson, B. and Salcedo, R. The human gene FALL39 and processing (Ceftazidine, Teicoplanin, Aztreonam), which is currently being used in clinical trials, has been reported to be effective in the treatment of osteoporosis, , Ceftriaxone) were used.

The bacterium used in the test was purchased from the Culture Collection of Antimicrobial Resistance Microbes (CCARM) and KCTC (Korean Collection for Type Cultures) of Seoul Women's University, and the gram- aureus (methicillin resistant staphylococcus aureus CCARM 3696: MRSA), Bacillus subtilis (Bacilus subtilis KCTC 2213: Bs) , micro Cocos base Proteus (Micrococus luteus KCTC 9341: Ml) , vancomycin-resistant enterococci (vancomycin-resistant Enterococcus faecium CCARM 5028: VRE), Listeria monocytogenes ATCC 19111 ( Lm ), multidrug resistance pseudomonas aeruginosa CCARM 2161 (MDRPA), Escherichia coli KCTC 1039: Ec , Klebsiella oxytoca KCTC 1686: Ko ), Citrobacter freundii KCTC 2359: Cf ), and Salmonella enterica (KCTC 2930: Cf ).

As shown in Table 6, the halogens of the present invention exhibited stronger antimicrobial activity than the antimicrobial peptides and antibiotics used as a control group for anti-bacterial phlegm controlling the bacterium used in the test at a concentration of 4 μg / ml. The four most commonly used antibiotics used in patients at hospitals are biologically active only in certain pathogens. In particular, all four antibiotics in vancomycin-resistant enterococci (VRE) have a maximum concentration of 64 μg / ml ). &Lt; / RTI > On the other hand, the halogens of the present invention showed an even anti-bacterial activity against the antibiotic resistant strains (MRAS, VRE, MDRPA) used in the test.

Experimental Example 2: Anti-fungal activity of halopenan

Analysis of the anti-fungal activity of halothane was performed in the same manner as the microbroth dilution assay described above. Candida spp. , A representative skin fungus inducing strain frequently infected to mucous membranes and skin of the human body, was used as a test strain. exam The strain The clinically detached antifungal resistance to fungi in from resistant week the Bank of Seoul Women's University, antibiotics ((Candida albicans, CCARM 14024) , (Candida albicans, CCARM 50651), (Candida albicans, CCARM 50582), (Candida glabrata, CCARM 50701), ( Candida krusei, CCARM 50633) were purchased and used. The cultures were incubated at 30 ° C. and 200 rpm for 18 hours using a Sabouraud dextrose broth (SDB; Difco, USA) medium and counted with a hemocytometer to give 2 × 10 ³ colony forming units (CFU) / ml . Thereafter, the test procedure was carried out in the same manner as the anti-bacterial test method described above.

As a result of the test, as shown in the following Table 7, when the two control peptides had weak anti-fungal activity (MSI-78) or little (LL-37), the halopants of the present invention had strong anti- And exhibited anti-fungal activity equivalent to that of the other HCl peptide, which is reported to be the other halocidin derivative.

Experimental Example 3: Hemolytic toxicity of halopenan

To analyze the erythrocyte hemolytic activity of the antimicrobial peptide of the present invention, 100 μl of the peptide diluted to a predetermined concentration and 100 μl of 10% (v / v) human erythrocyte suspension were mixed in PBS (phosphate-buffered saline). The mixed solution was reacted at 37 DEG C for 30 minutes, and 600 mu l of PBS was added to each tube. The solution was centrifuged at 10,000 g for 3 minutes, and the supernatant was separated. The absorbance at 540 nm was measured, and hemolysis activity (%) was calculated according to the following equation.

At this time, 1% Triton-X100 was used as a positive control for 100% hemolytic activity, and 0.01% acetic acid was used as a negative control for 0% hemolytic activity.

Hemolytic toxicity to erythrocytes was determined at diluted concentrations of test peptides at two-fold dilutions from a concentration of 200 μg / ml. As a result of the test, as shown in Fig. 4, HG1 used as a control group showed hemolytic toxicity close to about 100%, but halohane of the present invention was about 30% even at the maximum concentration (200 / / Of hemolytic activity.

Experimental Example 4: Resistance to proteolytic enzymes of halothane

Infectious tissue caused by pathogenic microorganisms has various proteolytic enzymes derived from pathogens or human epithelial tissues. Antimicrobial peptides that do not have resistance to these proteolytic enzymes can not be expected to be clinically effective. Thus, the test for resistance to haloperase proteolytic enzymes of the present invention was performed on trypsin and chymotrypsin, which are representative protein hydrolytic enzymes.

50 μg of a solution of trypsin and 400 μM of chymotrypsin diluted in duplicate at a maximum concentration of 1200 nM, and 40 μg of a diluted peptide solution at a concentration of 20 μg / ml were mixed and reacted at 37 ° C. for 10 minutes, To the mixed solution was added 10 占 퐇 of a solution of bacteria (MRSA) at a concentration of 10 ⁸ CFU / ml. After reacting at 37 DEG C for 10 minutes, a part of the reaction solution was plated on a plate medium. The plated culture media were counted for colonies of bacteria grown after incubation at 37 ° C for 18 hours to analyze the effect of peptides on proteolytic enzymes.

As a result, as shown in FIG. 5, the MSI-78 peptide used as the control group showed a rapid disappearance of the antimicrobial activity, while the HG1 and halonane peptides showed partial loss of activity only at the maximum trypsin concentration (200 nM) But retained almost the original activity. Therefore, in order to analyze the resistance of the peptides according to the reaction time with the proteolytic enzyme, a further test was carried out by partially modifying the test method described above. In detail, a mixture of protease (50 nM for trypsin and 200 nM chymotrypsin) and peptide (8 μg / ml) were mixed at a constant concentration and incubated at 37 ° C. for 5, 10, 20, 30 and 60 Min). Then, 10 μl of 10 ⁸ CFU / ml bacterium (MRSA) solution was added and reacted at 37 ° C. for 10 minutes, and a part of the reaction solution was plated on a plate medium. The platelet culture media were counted for colonies grown after culturing at 37 ° C for 18 hours, and the effect of peptidadid on the proteolytic enzyme reaction time was analyzed.

As a result, as shown in FIG. 6, the MSI-78 peptide used as the control group showed rapid loss of activity after 10 minutes of reaction with trypsin and 5 minutes of chymotrypsin, while HG1 and halonane peptide showed a The original activity was maintained.

Experimental Example 5: Analysis of antimicrobial activity in serum of halofanan

Experiments on the reduction of antimicrobial activity of antimicrobial peptides by human serum protein binding were carried out as follows.

The antimicrobial peptides were prepared using PBS (Phosphate-buffered saline) buffer solution so that the diluted concentration was doubled from 200 μg / ml to 6.25 μg / ml. 100 μl of the peptide solution was mixed with 100 μl of human serum, Lt; / RTI > for 30 minutes. The sample mixture was centrifuged at 10,000 g for 10 minutes at 4 ° C, and the binding activity of the antimicrobial peptide and human serum proteins was analyzed by measuring the antimicrobial activity by radial diffusion analysis of 5 μl of the supernatant.

A positive control sample was prepared by mixing the same amount of PBS buffer solution instead of human serum. The clear zone diameter in which the bacteria did not grow was expressed as units (0.1 mm = 1 unit).

As a result, as shown in FIG. 7, the HG1 peptide, which is a comparative group, lost the antimicrobial activity in the serum, whereas the halonane peptide of the present invention maintained an excellent antimicrobial activity in the serum. Therefore, the HG1 peptide has excellent antimicrobial activity. However, as shown in the above test results, the HG1 peptide completely inhibits the antibacterial activity by binding to human serum proteins, Have. On the other hand, it was confirmed that the halopangerine peptide of the present invention can be applied to a variety of lesions in clinical situations because nonspecific binding reaction with such serum proteins does not occur.

Experimental Example 6: Analysis of antimicrobial activity in human wound exudate of halothane

Human wound fluid (HWF) was obtained from 4 patients with open wound caused by serum species. Suspensions such as hemocyte of wound exudate were removed by centrifugation (12,000g, 10 minutes). The test was conducted by measuring the number of bacteria that survived by mixing peptides and bacteria in human wound exudates. The details are as follows.

50 μl of human wound exudate was mixed with 40 μl of a peptide solution at a concentration of 250 μg / ml, 10 μl of MRSA bacteria at a concentration of 1 × 10 ⁸ cfu / ml was added and reacted at 37 ° C. for 10 minutes. Were plated on a plate medium. The plated medium was counted for bacterial colonies grown after incubation at 37 ° C for 18 hours to analyze the effect of peptides on human wound exudates. In the present test, a sample to which no buffer was added and a buffer solution was added was used as a negative control, and HG1 peptide and MSI-78 peptide were used as control peptides.

Wound effusion (HWF) from 4 patients were each individually tested and the results are as shown in FIG. In comparison with the antimicrobial activity test in the previous serum, the comparative groups HG1 peptide and MSI-78 peptide showed almost no antimicrobial activity, whereas the present halohane peptide showed excellent antimicrobial activity even in the presence of human wound exudate .

&Lt; Preparation Example 1 > Preparation of pharmaceutical preparations

<1-1> Purification (direct pressurization)

After 5.0 mg of the antibacterial peptide was sieved, 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate were mixed and pressed to prepare tablets.

<1-2> Purification (wet assembly)

After 5.0 mg of the antibacterial peptide was sieved, 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of Polysorbate 80 was dissolved in pure water, and an appropriate amount of this solution was added, followed by atomization. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The fine particles were pressed to prepare tablets.

<1-3> Powders and capsules

After 5.0 mg of the antibacterial peptide was sieved, 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate were mixed. The mixture was extruded through a hard No. 5 gelatin capsules.

<1-4> Injection

An injection was prepared by adding 100 mg of the antibacterial peptide, 180 mg of mannitol, 26 mg of Na2HPO412H2O and 2974 mg of distilled water.

&Lt; Preparation Example 2 > Production of food

<2-2> Production of antimicrobial food

100 mg of the antimicrobial peptide

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed according to a conventional method for producing antibacterial food Next, granules can be prepared and used in the manufacture of food compositions for antimicrobial use according to a conventional method.

<2-2> Preparation of antibacterial auxiliary drinks

100 mg of the antimicrobial peptide

Citric acid 100 mg

100 mg of oligosaccharide

2 mg of plum concentrate

100 mg taurine

Purified water was added to 500 ml

The above components were mixed according to the usual method for preparing an auxiliary drink, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution was filtered to obtain a sterilized 1 liter container, sealed sterilized, It is used in the production of the health beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Although the present invention has been shown and described with respect to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the following claims It will be apparent to those skilled in the art.

<110> LEE, In Hee <120> Novel analogues of antibacterial peptide derived from halocynthia aurantium and the use thereof <130> P140142PP <140> 10-2014-0064369 <141> 2014-05-28 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> halocidin <400> 1 Lys Trp Leu Asn Ala Leu Leu His His Gly Leu Asn Cys Ala Lys Gly 1 5 10 15 Val Leu Ala <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> halocidin <400> 2 Lys Trp Gln Asn Ala Leu Leu His His Gly Leu Asn Cys Ala Lys Gly 1 5 10 15 Val Leu Lys

Claims

(Alanine, A) located in the first amino acid from the C-terminus is substituted with glutamine (Glutamine, Q), which is located in the third amino acid from the N-terminal in the amino acid sequence of SEQ ID NO: (Lysine, K).

The method according to claim 1,
Wherein said peptide has the amino acid sequence of SEQ ID NO: 2.

An antimicrobial peptide according to claim 2, wherein the cysteine (C) residue of the amino acid sequence of SEQ ID NO: 2 is linked by a disulfide bond to form a dimer form.
[Chemical Formula 1]

The method according to claim 1,
Wherein said peptide has antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria.

The method according to claim 1,
Wherein said peptide is resistant to trypsin or chymotrypsin.

The method according to claim 1,
Wherein said peptide has an antimicrobial activity in human serum.

The method according to claim 1,
Wherein the peptide has an antimicrobial activity in a human wound fluid (HWF).

delete

A pharmaceutical composition for preventing and treating an infectious disease caused by a bacterium, which comprises the peptide according to any one of claims 1 to 7 as an active ingredient.

An antibiotic food additive characterized by comprising the peptide according to any one of claims 1 to 7 as an active ingredient.