KR102433665B1 - Antimicrobial peptide derived from indolicidin and uses thereof - Google Patents

Abstract

The present invention relates to a peptide selected from a group which comprises: i) a peptide consisting of sequence number 1; ii) a peptide in which all tryptophan (Trp) in the peptide of the i) is substituted with methylated phenylalanine (Phe) at carbon number 2; iii) a peptide in which all tryptophan (Trp) in the peptide of the i) is substituted with D-form phenylalanine (Phe); and iv) a peptide in which an N-terminal tryptophan (Trp) in the peptide of the i) is substituted with phenylalanine (Phe) with valeric acid bonded to carbon number 4. Accordingly, the peptide has higher or equal antibacterial activity than conventional peptides and is less toxic, and thus can be used as an antibacterial substance in various fields.

Description

Antimicrobial peptide derived from indolicidin and uses thereof

The present invention provides an antibacterial peptide derived from indolicidin and uses thereof.

Antimicrobial peptides are one of the important factors in the host defense system. Antimicrobial peptides have broad spectrum activity against microorganisms and perform important functions in host defense in eukaryotic cells. Antimicrobial peptides have been found in all classes of living organisms (eg, mammals, amphibians, insects, etc.), suggesting that antimicrobial peptides may be evolutionarily conserved components of the innate immune response.

However, most of the known antibacterial peptides are inactivated at physiological salt concentrations when injected into the body and are rapidly degraded by proteolytic enzymes either present in serum or secreted by pathogens, so their actual bioavailability is extremely low, and cell membranes rather than specific receptors Due to the direct action on the microbes, the problem of toxicity to human cells as well as microorganisms has been raised. Recently, many studies on antimicrobial peptides have been focused on the development of short analogues of naturally occurring antimicrobial peptides with increased antimicrobial activity and low cytotoxicity to mammalian cells. Some studies have investigated the physicochemical parameters of peptides to modulate selectivity, such as net charge, helicity, hydrophobicity per residue (H), hydrophobic moment (μ) and positively charged polar helices. An attempt was made to optimize the angle determined by the plane by modifying it. In addition, an antibacterial peptide is constructed using a peptoid, dimerization or cyclization of the peptide using a disulfide bond, and modification of both ends of the peptide (modification; N-terminal acetylation, C- terminal amidation) to enhance resistance to proteolytic enzymes. That is, existing studies have tried to solve the problems of the prior art by adding unusual amino acid residues to peptides or by adding secondary modifications to the structure. come.

1. Republic of Korea Patent No. 10-1329223.

An object of the present invention is i) a peptide consisting of SEQ ID NO: 1; ii) a peptide in which all tryptophan (Trp) in the peptide of i) is substituted with phenylalanine (Phe) in which carbon 2 is methylated; iii) a peptide in which all tryptophan (Trp) in the peptide of i) is substituted with D-form phenylalanine (Phe); iv) a peptide in which the N-terminal tryptophan (Trp) in the peptide of i) is substituted with phenylalanine (Phe) in which valeric acid or butyramide is bonded to the 4th carbon; And v) a peptide consisting of SEQ ID NO: 2; to provide a peptide selected from the group consisting of.

Another object of the present invention is to provide an antibacterial pharmaceutical composition, a coating composition for a medical device, a cosmetic composition, a feed additive and a hygiene product comprising the peptide as an active ingredient.

Another object of the present invention is to provide an antibacterial method in a non-human subject, comprising administering a veterinarily effective amount of the peptide to a non-human subject.

In order to achieve the above object, the present invention provides: i) a peptide consisting of SEQ ID NO: 1; ii) a peptide in which all phenylalanine (Phe) carbon 2 is methylated in the peptide consisting of SEQ ID NO: 3; iii) a peptide in which all phenylalanine (Phe) is D-form in the peptide consisting of SEQ ID NO: 3; iv) a peptide in which valeric acid or butyramide is bonded to the 4th carbon of the N-terminal phenylalanine (Phe) in the peptide consisting of SEQ ID NO: 4; And v) a peptide consisting of SEQ ID NO: 2; provides a peptide selected from the group consisting of.

In addition, the present invention provides an antibacterial pharmaceutical composition, a coating composition for a medical device, a cosmetic composition, a feed additive, and a hygiene product comprising the peptide as an active ingredient.

In addition, the present invention provides an antibacterial method in a non-human subject, comprising administering a veterinarily effective amount of the peptide to a non-human subject.

The present invention relates to a peptide consisting of a sequence consisting of 9 amino acids derived from indolicidin, and has antibacterial activity against both gram-positive and gram-negative bacteria, and has significantly low cytotoxicity, treatment for skin diseases, medical device coatings and cosmetics, etc. It can be used as an antibacterial material in various fields.

1 shows the erythrocyte hemolysis rate (%) for each concentration of the peptide.

Hereinafter, the present invention will be described in more detail.

Existing antibacterial peptides have a problem in that they have not been commercialized due to their high cytotoxicity. , completed the present invention with low toxicity.

The present invention provides: i) a peptide consisting of SEQ ID NO: 1; ii) a peptide in which all phenylalanine (Phe) carbon 2 is methylated in the peptide consisting of SEQ ID NO: 3; iii) a peptide in which all phenylalanine (Phe) is D-form in the peptide consisting of SEQ ID NO: 3; iv) a peptide in which valeric acid or butyramide is bonded to the 4th carbon of the N-terminal phenylalanine (Phe) in the peptide consisting of SEQ ID NO: 4; and v) a peptide consisting of SEQ ID NO: 2; provides a peptide selected from the group consisting of, and may further include a peptide in which a hydrophobic residue is bound to an amino acid of the peptide consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

The peptide may be amidated at the C-terminus.

The peptide may be an antibacterial peptide.

The peptide may have antibacterial activity against both Gram-positive and Gram-negative bacteria, and preferably, the Gram-positive bacteria are Bacillus subtilis , Enterococcus faecalis . faecalis ), any one selected from the group consisting of Staphylococcus aureus ; And the gram-negative bacteria is Escherichia coli ), Cutibacterium acnes , Staphylococcus epidermidis , and Proteus mirabilis may be any one selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides an antibacterial pharmaceutical composition comprising the peptide as an active ingredient.

The concentration of the peptide in the pharmaceutical composition may be 1 to 150 μM, preferably 1 to 50 μM.

In one embodiment of the antibacterial pharmaceutical composition comprising the peptide as an active ingredient, the pharmaceutical composition is an injection, granule, powder, tablet, pill, capsule, suppository, gel, suspension, emulsion, Any one formulation selected from the group consisting of drops or liquids may be used.

In another embodiment of the present invention, the pharmaceutical composition comprises suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostats, commonly used in the preparation of pharmaceutical compositions, It may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered intravenously, intraarterially, intraperitoneally, intramuscularly, intraarterially, intraperitoneally, intrasternally, transdermally, intranasally, inhalation, topical, rectal, oral, intraocular or Administration can be to a subject in a conventional manner via the intradermal route.

The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and period of administration, and may be appropriately selected by those skilled in the art, and the daily dosage is 0.01 mg /kg to 200 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, more preferably 0.1 mg/kg to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In addition, the present invention provides a coating composition for a medical device comprising the peptide as an active ingredient.

In addition, the present invention provides a cosmetic composition comprising the peptide as an active ingredient.

The cosmetic composition of the present invention is not particularly limited in its formulation, for example, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing It may be formulated as cosmetics such as water, pack, powder, body lotion, body cream, body oil and body essence, and may be applied in a form that is applied to the skin or is absorbed into the skin using a micro-needle.

In the cosmetic composition of the present invention, pharmaceutically or cosmetically acceptable carriers vary depending on the formulation thereof, but include hydrocarbons such as petrolatum, liquid paraffin, gelled hydrocarbons (alias: Plastibase); Animal and vegetable oils, such as medium-chain fatty acid triglyceride, pork fat, hard fat, and cacao fat; higher fatty acid alcohols such as cetanol, stearyl alcohol, stearic acid, and isopropyl palmitate; fatty acids and esters thereof; water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, sucrose, and sugar alcohol; Emulsifiers, such as glycerol fatty acid ester, polyoxyl stearate, and polyoxyethylene hydrogenated castor oil; Adhesives, such as acrylic acid ester and sodium alginate; propellants such as liquefied petroleum gas and carbon dioxide; and preservatives such as paraoxybenzoic acid esters, and the external preparation of the present invention can be prepared by using them according to a conventional method. In addition to these, a stabilizer, a fragrance, a colorant, a pH adjuster, a diluent, a surfactant, a preservative, an antioxidant, and the like may be blended as needed. The use of the external preparation of the present invention can be applied to a local wound by a conventional method.

In addition, the present invention provides a feed additive comprising the peptide as an active ingredient.

In addition, the present invention provides a hygiene product comprising the peptide as an active ingredient.

In addition, the present invention provides an antibacterial method in a non-human subject, comprising administering a veterinarily effective amount of the peptide to a non-human subject.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the following examples and the like are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples and the like. Examples and the like of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

[Preparation Example] Peptide

In the peptide consisting of SEQ ID NO: 1 (WRWPWWWRR; hereinafter referred to as Example 1), in the peptide consisting of SEQ ID NO: 3 (FRFPFFFRR), all phenylalanine (Phe) carbon 2 is methylated ((2-Me)FR(2) -Me)FP(2-Me)F(2-Me)F(2-Me)FRR; hereinafter referred to as Example 2), all phenylalanine (Phe) in the peptide consisting of SEQ ID NO: 3 (FRFPFFFRR) is D-type (form) of a peptide (fRfPfffRR; hereinafter referred to as Example 3), a peptide consisting of SEQ ID NO: 4 (FRWPWWWRR) in which butyramide is bonded to the 4th carbon of the N-terminal phenylalanine (Phe) ( F(4-NHCO(CH ₂ ) ₃ CH ₃ )RWPWWWRR; hereinafter referred to as Example 4) and the peptide consisting of SEQ ID NO: 2 (FRFPFKFRR; hereinafter referred to as Example 5) are powders from Peptron (Peptron, Korea), respectively. provided in the form (98% purity).

[Experimental Example 1] Confirmation of antibacterial effect

In order to confirm the antimicrobial activity of Examples 1 to 5, the most representative method, antimicrobial activity evaluation (MIC test) was performed.

1) Gram-negative bacteria Escherichia coli ) and Gram-positive bacteria Bacillus subtilis ( Bacillus subtilis ) The strain was spread on a nutrient medium (Nutrient agar plate) with 1 μl loop & needle, and cultured in an incubator at 36 ° C. for 24 hours. Put 10 ml of 1% peptone medium in a 50 ml conical tube, pick the colonies formed in the nutrient medium, put them in a conical tube, and incubate for about 18 hours while shaking at 220 rpm in an incubator at 36 ° C. . After incubation, the concentration in the culture medium was adjusted to 0.5 McFarland Standard. 2) Examples 1 to 5 and indolicidin in powder form were each mixed with distilled water to prepare a peptide solution at 200 μM.

Prepare a 96-well plate, and each well contains 50 μl of the peptide solution of 2) and 50 μl of the culture medium of 1), and the final concentration of the peptide solution is 8 wells was adjusted to be 0.78, 1.56, 3.12, 6.24, 12.48, 24.96, 50, and 100 μM, respectively. After sealing the 96-well plate with a sealing paper, it was incubated in an incubator at 36° C. for 24 hours. After 24 hours, the absorbance at O.D.600 was measured with a microplate reader.

As a result, a value corresponding to the MIC50 was confirmed, and according to Table 1, the values of Examples 1 to 5 were the same as or similar to those of indolicidin. It could be determined that Examples 1 to 5 showed an antibacterial activity similar to that of indolicidin, and showed an antibacterial effect regardless of gram-negative and gram-positive bacteria.

B. subtilis E. coli Indolicidin (control) 3.12 μM 3.12 μM Example 1 1.56 μM 1.56 μM Example 2 0.78 μM 1.56 μM Example 3 1.56 μM 3.12 μM Example 4 0.78 μM 3.12 μM Example 5 1.56 μM 1.56 μM

[Experimental Example 2] Toxicity evaluation

1. Hemolysis test by red blood cells

a) Sheep blood was mixed with 5 times the volume of PBS, centrifuged at 2000 rpm for 5 minutes to wash red blood cells of the sheep, and then PBS was further added to make a 4% RBC solution. b) Examples 1 to 5 and indolicidin in powder form were mixed with PBS, respectively, to prepare a 200 μM peptide solution, and the concentration of the peptide solution was 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 μM. was adjusted to be Then, 100 μl of each of the peptide solutions adjusted to the concentration and 100 μl of the RBC solution of a) were mixed. After 1 hour of incubation in an incubator at 36 °C, centrifugation was performed at 10000 rpm for 10 minutes, and 100 μl of the centrifuged supernatant was added to each well of a 96-well plate. Absorbance was measured at O.D.414 with a microplate reader.

As a result, according to Table 2 and FIG. 1, in Examples 1 to 5, when used at 100 μM or less, cytotoxicity was within 22%, and in particular, in Examples 3 and 5, 2% It showed cytotoxicity only to a certain extent, so it was possible to determine that it was harmless to the human body by solving the cytotoxicity that was the limit of indolicidin.

1.5625 μM 3.125 μM 6.25 μM 12.5 μM 25 μM 50 μM 100 μM indolicidin
(control group) 2 % 2 % 3% 7% 11% 23% 65% Example 1 3% 4 % 6% 7% 9% 10% 22% Example 2 2 % 3% 5% 8 % 9% 11% 16% Example 3 0 % One % One % One % 2 % 2 % 2 % Example 4 2 % 3% 3% 6% 7% 9% 12% Example 5 0 % 0 % 0 % 0 % 2 % 2 % 2 %

2. human body Cytotoxicity analysis for fibroblasts

Using human fibroblast cells (CCD-986 sk), a cytotoxicity assay (mtt assay; protocol Cat#. Ab211091) was performed. For the peptide, Example 1 was used.

As a result, the O.D.590 absorbance value was measured. According to Table 3, the viability ratio was measured to be 92.99% at 96 μM, which is a high concentration as well as a low concentration. Based on the fact that it can be determined that there is no cytotoxicity even when the viability ratio is 70% or more, it was confirmed that Example 1 had no cytotoxicity at high concentrations as well as low concentrations.

Peptide Concentration Absorbance measurements viability rate (%) Is it cytotoxic? 0 μM 0.36 100 doesn't exist 1 μM 0.36 100 doesn't exist 6 μM 0.37 100 doesn't exist 12 μM 0.37 100 doesn't exist 96 μM 0.33 92.99 doesn't exist

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

<110> REPILL Co., Ltd. <120> Antimicrobial peptide derived from indolicidin and uses thereof <130> DP-2021-0210 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> It was provided in powder form (purity 98%) from Peptron(Korea). <400> 1 Trp Arg Trp Pro Trp Trp Trp Arg Arg 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> It was provided in powder form (purity 98%) from Peptron(Korea). <400> 2 Phe Arg Phe Pro Phe Lys Phe Arg Arg 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Example 2 <400> 3 Phe Arg Phe Pro Phe Phe Phe Arg Arg 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Example 4 <400> 4 Phe Arg Trp Pro Trp Trp Trp Arg Arg 1 5

Claims (13)

i) a peptide consisting of SEQ ID NO: 1;
ii) a peptide in which all phenylalanine (Phe) carbon 2 is methylated in the peptide consisting of SEQ ID NO: 3;
iii) a peptide in which all phenylalanine (Phe) is D-form in the peptide consisting of SEQ ID NO: 3;
iv) a peptide in which valeric acid or butyramide is bonded to the 4th carbon of the N-terminal phenylalanine (Phe) in the peptide consisting of SEQ ID NO: 4; and
v) a peptide consisting of SEQ ID NO: 2; a peptide selected from the group consisting of. The peptide according to claim 1, wherein the peptide is amidated at the C-terminus. The peptide according to claim 1, wherein the peptide is an antibacterial peptide. The peptide according to claim 1, wherein the peptide is an antibacterial peptide having antibacterial activity against both Gram-positive and Gram-negative bacteria. The method according to claim 4, wherein the Gram-positive bacteria Bacillus subtilis ( Bacillus subtilis ), Enterococcus faecalis ( Enterococcus ) faecalis ), any one selected from the group consisting of Staphylococcus aureus ; And the gram-negative bacteria is Escherichia coli ), Cutibacterium acnes , Staphylococcus epidermidis and Proteus mirabilis ( Proteus ) mirabilis ) any one selected from the group consisting of; Peptide, characterized in that An antibacterial pharmaceutical composition comprising the peptide of claim 1 as an active ingredient. The pharmaceutical composition according to claim 6, wherein the concentration of the peptide in the pharmaceutical composition is 3 to 150 μM. The pharmaceutical composition according to claim 6, wherein the composition does not have toxicity to any cell line selected from the group consisting of human red blood cells and epithelial cells. A coating composition for medical devices comprising the peptide of claim 1 as an active ingredient. A cosmetic composition comprising the peptide of claim 1 as an active ingredient. A feed additive comprising the peptide of claim 1 as an active ingredient. A hygiene product comprising the peptide of claim 1 as an active ingredient. An antibacterial method in a non-human subject, comprising administering a veterinary effective amount of the peptide of claim 1 to a non-human subject.

Images