阅读说明：本技术 新型多功能肽及其用途 (Novel multifunctional peptide and use thereof ) 是由 李泰勋 金载允 李泫珠 于 2020-03-19 设计创作，主要内容包括：本发明提供了一种新型多功能肽,能够有效调节免疫细胞的活性,同时还对各种细菌如革兰氏阴性菌和革兰氏阳性菌表现出优异的抗菌活性,因此可以用作一种抗菌剂,一种用于免疫疾病的治疗剂,以及一种用于改善皮肤免疫疾病的化妆品组合物。(The present invention provides a novel multifunctional peptide which is capable of effectively modulating the activity of immune cells while also exhibiting excellent antibacterial activity against various bacteria such as gram-negative bacteria and gram-positive bacteria, and thus can be used as an antibacterial agent, a therapeutic agent for immune diseases, and a cosmetic composition for improving skin immune diseases.)

1. A peptide having antibacterial and immunomodulatory activity, consisting of 6 to 12 amino acids comprising an amino acid sequence selected from the group consisting of:

i)BOBWX₁OU; and

ii)X₁RWWUX₁X₂m;

wherein B is a basic amino acid independently selected from the group consisting of lysine (K) and arginine (R), O is an aromatic amino acid independently selected from the group consisting of phenylalanine (F), tyrosine (Y) and tryptophan (W), and X is₁Independently selected from the group consisting of arginine (R), norleucine (Nle) or 2-naphthyl-L-alanine (Z), said U being absent or selected from m, X₂WW、RX₂The group consisting of WW and MVm,

wherein said X₂Is norleucine (Nle), tryptophan (W) or valine (V), and said m is D-methionine, and KFKWRYm is excluded from i).

2. The peptide according to claim 1, wherein said i) comprises an amino acid sequence selected from the group consisting of BOBW-Nle-Om, BOBWBO, BOBWZO, BOBWZOm and BOBWBOMVm.

3. The peptide of claim 1, wherein the ii) comprises ZRWWX₁X₂m，RRWWX₁X₂m and ZRWRX₂WWRWm。

4. The peptide according to any one of claims 1 to 3, wherein an octanoyl group or an acetyl group is added to the N-terminus or C-terminus of the peptide.

5. The peptide of any one of claims 1 to 3, wherein the C-terminus of the peptide is amidated.

6. The peptide according to any one of claims 1 to 3, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos 1 to 17.

7. An antibacterial agent comprising the peptide according to any one of claims 1 to 3 as an active ingredient.

8. The antibacterial agent of claim 7, wherein the peptide has antibacterial activity against Pseudomonas aeruginosa (P.aeruginosa) or Staphylococcus aureus (S.aureus).

9. A pharmaceutical composition for treating immune-related diseases, comprising the peptide of any one of claims 1 to 3 as an active ingredient.

10. The pharmaceutical composition of claim 9, wherein the immune related disease is atopic dermatitis, psoriasis, conjunctivitis, keratitis, dry eye, pneumonia, asthma, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, or crohn's disease.

11. A cosmetic composition for improving the symptoms of skin autoimmune diseases selected from the group consisting of atopic dermatitis, lupus and psoriasis, comprising the peptide as an active ingredient.

Technical Field

The present invention relates to a novel multifunctional peptide and use thereof, and more particularly, to a novel multifunctional peptide having antibacterial effect and modulating immune cell activity and use thereof.

Background

Peptides with antibacterial action may sometimes be found in nature, and some synthetic peptides have also been found to have antibacterial activity. These antibacterial peptides consist of a relatively short amino acid sequence (10 to 100aa) compared to normal proteins, and when mainly bound to cell membranes 1) form ion channels in the cell membrane, thereby inhibiting the generation of energy by microorganisms, or 2) create large pores on the cell membrane, resulting in cell death. Unlike conventional antibiotics that physically destroy microorganisms by inhibiting synthesis of polymers in the cell walls or cells of the microorganisms, according to reports so far, the microorganisms have difficulty in developing resistance to antibacterial peptides. Although many of the antibacterial peptides known to date have little similarity in amino acid sequence, they show some general trends in structure or activity. For example, antimicrobial peptides have positively charged amino acids such as lysine, arginine, and histidine, and hydrophobic regions. According to the most feasible hypothesis currently associated with the mechanism of action of antimicrobial peptides, the Shai-Matsuzaki-Huang (SMH) model explains the amino acid sequence features and mechanism of action of antimicrobial peptides as follows: the positively charged hydrophilic regions bind to the membrane of negatively charged bacteria and then bind to the hydrophobic regions of peptides of the bacterial membrane, interact with the hydrophobic regions of the membrane phospholipids and form pores in the membrane, and ultimately kill the bacteria by altering the permeability of the membrane.

Formyl peptide receptor 1(FPR1) and formyl peptide receptor 2(FPR2) expressed in phagocytes such as neutrophils and monocytes play an important role in the defense and elimination of inflammation in hosts against pathogen infection (Mangmool, S.et al, Toxins,3: 884-. The receptors are known to bind to Gi proteins that are sensitive to pertussis toxin (Nakashima, k.et al., j.biol.chem.,290(22): 13678-. Among them, FPR2 plays an important role in inflammatory diseases. FPR2 induces separation of the G.beta.gamma subunit from the G.alpha.i subunit, and the G.beta.gamma subunit induces activation of phospholipase C.beta.or phosphoinositide 3-kinase (Duru, E.A. et al, J.Surg. Res.,195(2):396-405, 2015). Activation of these molecules mediates various cellular responses such as chemotactic migration, degranulation, and superoxide generation, thereby modulating the host's immune response and ultimately mediating the host's defense against pathogen infection and eliminating inflammation by inducing complex downstream signaling.

Disclosed is a

Technical problem

However, the present invention has been made to solve various problems including the above problems, and an object thereof is to provide a novel multifunctional peptide capable of modulating the activity of immune cells and exhibiting excellent antibacterial activity against various bacteria. However, these issues are exemplary, and the scope of the present invention is not limited thereto.

Technical scheme

In one aspect of the present invention, there is provided a peptide having antibacterial and immunomodulatory activity, consisting of 6 to 12 amino acids comprising an amino acid sequence selected from the group consisting of:

i)BOBWX₁OU; and

ii)X₁RWWUX₁X₂m;

wherein B is a basic amino acid independently selected from the group consisting of lysine (K) and arginine (R), and O is independently selected from the group consisting of phenylalanine (F), tyrosine (Y), and tryptophan (W)The aromatic amino acid of (1), the X₁Independently selected from the group consisting of arginine (R), norleucine (Nle) or 2-naphthyl-L-alanine (Z), said U being absent or selected from the group consisting of m, X₂WW、RX₂The group consisting of WW and MVm,

wherein said X₂Is norleucine (Nle), tryptophan (W) or valine (V), and said m is D-methionine, and KFKWRYm is excluded from i).

In another aspect of the present invention, there is provided an antibacterial agent comprising the above peptide as an active ingredient.

In another aspect of the present invention, there is provided an agent for treating an immune-related disease, comprising the peptide as an active ingredient.

In another aspect of the present invention, there is provided a cosmetic composition for improving a symptom of an autoimmune disease of the skin selected from the group consisting of atopic dermatitis, lupus and psoriasis, containing the peptide as an active ingredient.

Effects of the invention

As described above, according to the embodiments of the present invention, it is possible to prepare a novel multifunctional peptide having not only high antibacterial activity against various bacteria (e.g., gram-negative bacteria and gram-positive bacteria) but also simultaneously modulating activity of immune cells. However, the scope of the present invention is not limited thereto.

Drawings

FIG. 1 is a graph showing the effect of the novel multifunctional peptide of the present invention on FPR2 activation by increasing FPR 2-dependent intracellular calcium ion activity.

Fig. 2 is a graph showing antibacterial activity of the novel multifunctional peptide of the present invention against pseudomonas aeruginosa (p.

Fig. 3 is a graph showing antibacterial activity of the novel multifunctional peptide of the present invention against staphylococcus aureus (s.

Detailed Description

Definition of terms:

the term "immunomodulatory peptide" as used herein refers to a peptide having direct or indirect antimicrobial and/or antiviral activity and immunomodulatory activity, such as inflammatory response control of immune cells, immune cell migration, and inflammatory response control of epidermal or endothelial cells. In other words, the peptide plays an important role in host resistance to pathogen infection through formyl peptide receptor 1(FPR1) and formyl peptide receptor 2(FPR2) expressed in phagocytes such as neutrophils and monocytes, and epidermal cells, etc. The above receptors are known to bind to Gi proteins that are sensitive to pertussis toxin. Activation of FPR1 and FPR2 induces separation of the G β γ subunit from the G α i subunit, which induces activation of phospholipase C β or phosphoinositide 3-kinase. Activation of these molecules induces complex downstream intracellular signaling to mediate cellular responses such as chemotactic migration, degranulation, and superoxide generation.

The term "antimicrobial peptide" as used herein is a cationic peptidic compound, generally consisting of a relatively simple structure, capable of combating a broad spectrum of microorganisms including gram-positive bacteria, gram-negative bacteria, fungi, viruses, and the like. Although the mechanism of action is not completely understood, it is thought that the mechanism of action is to exhibit antibacterial activity by disrupting the cell membrane of a microorganism.

Best mode:

in one aspect of the present invention, there is provided a peptide having antibacterial and immunomodulatory activity, consisting of 6 to 12 amino acids comprising an amino acid sequence selected from the group consisting of:

i)BOBWX₁OU; and

ii)X₁RWWUX₁X₂m;

wherein B is a basic amino acid independently selected from the group consisting of lysine (K) and arginine (R), O is an aromatic amino acid independently selected from the group consisting of phenylalanine (F), tyrosine (Y) and tryptophan (W), and X is₁Independently selected from the group consisting of arginine (R), norleucine (Nle) or 2-naphthyl-L-alanine (Z), said U being absent or selected from m, X₂WW、RX₂The group consisting of WW and MVm,

wherein said X₂Is norleucine (Nle), tryptophan (W) or valine (V), and said m is D-methionine, and KFKWRYm is excluded from i).

According to the above peptide, said i) is a peptide comprising an amino acid sequence selected from the group consisting of BOBW-Nle-Om, BOBWBO, BOBWZO, BOBWZOm and BOBWBOMVm.

The peptide of above, wherein ii) is a peptide comprising a peptide selected from the group consisting of ZRWWX₁X₂m、RRWWX₁X₂m and ZRWRX₂A peptide of an amino acid sequence in the group consisting of WWRWm.

According to the above-mentioned peptide, an octanoyl group or an acetyl group may be added to the N-terminus or C-terminus of the above-mentioned peptide, or the C-terminus of the above-mentioned peptide may be amidated.

The above peptide may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 17.

In another aspect of the present invention, there is provided an antibacterial agent comprising the above peptide as an active ingredient.

The antimicrobial agent may have antimicrobial activity against pseudomonas aeruginosa or staphylococcus aureus.

In another aspect of the present invention, there is provided a pharmaceutical composition for treating immune-related diseases, comprising the peptide as an active ingredient.

According to the pharmaceutical composition, the immune-related disease may be atopic dermatitis, psoriasis, conjunctivitis, keratitis, dry eye, pneumonia, asthma, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, or crohn's disease.

In another aspect of the present invention, there is provided a cosmetic composition for improving a symptom of an autoimmune disease of the skin selected from the group consisting of atopic dermatitis, lupus and psoriasis, containing the peptide as an active ingredient.

The pharmaceutical composition comprising the peptide as an active ingredient may include at least one of pharmaceutical diluents selected from the group consisting of salts, buffer salts, glucose, water, glycerol and ethanol, but the diluent is not limited thereto. The pharmaceutical composition may be variously applied according to the administration purpose and the disease state. The amount of the active ingredient actually administered will take into account a variety of relevant factors such as the disease to be treated, the severity of the patient's condition, the co-administration with other drugs (e.g., chemotherapeutic agents), the age, sex, body weight, diet, time of administration, route of administration, and the dosage ratio of the composition. The composition can be administered once or 1-3 times per day, but the dosage and route of administration can be adjusted depending on the type and severity of the disease.

The Pharmaceutical composition comprising the peptide as an active ingredient may be prepared using any formulation conventionally used in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), and the form of the formulation may preferably be an external preparation, but is not limited thereto. The external preparation of the present invention includes conventional external preparations such as tablets, liquid coatings, sprays, emulsions, creams, plasters, powders, osmotic pads, sprays, gels (including hydrogels), pastes, liniments, ointments, aerosols, suspensions, and transdermal absorbents. These formulations are described in Remington's Pharmaceutical Science, 15 th edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), which is well known in all fields of Pharmaceutical chemistry.

The pharmaceutical compositions comprising the peptides or substances of the invention can be administered orally or parenterally. Parenteral administration means administration by non-oral routes, i.e., intrarectal, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, inhalation, intraocular, and subcutaneous. The pharmaceutical composition comprising the peptide or substance may be formulated in any form, such as oral dosage forms, injections or topical formulations. It is preferably prepared in formulations for oral and injectable administration (true solutions, suspensions or emulsions), and most preferably in oral form, such as tablets, capsules, soft capsules, aqueous pharmaceuticals, pills, granules, and the like as desired. In the above preparations, the peptide of the present invention is filled in soft capsules without an adjuvant, mixed with a carrier or diluted to prepare a suitable preparation. Examples of suitable carriers include starch, water, saline, ringer's solution and dextrose.

The cosmetic composition comprising the peptide of the present invention as an active ingredient may comprise one or more additives for the formulation of the cosmetic composition. For example, such additives include 1, 3-butylene glycol, soybean phospholipid choline, sphingosine, cholesterol, tween 80, phytosphingosine, salicylic acid, skin moisturizers (moisturizers), emollients, natural oils, natural extracts, keratin, lipid-like substances, water-absorbing water-soluble substances, stratum corneum ceramides, fatty acids of the epidermal lipid membrane, cholesterol esters, ethanol, distilled water, and the like.

The pharmaceutical composition comprising the peptide of the present invention as an active ingredient may be administered by oral administration or parenteral route, more preferably oral administration, but is not limited thereto. When administered parenterally, the administration may be carried out by various routes such as intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, transdermal absorption, and the like.

Furthermore, a pharmaceutical composition comprising the peptide of the present invention as an active ingredient may be administered at a dose of 0.1mg/kg to 1g/kg, more preferably at a dose of 1mg/kg to 600 mg/kg. Meanwhile, the dosage can be properly adjusted according to the age, sex and condition of the patient.

Modes of the invention:

hereinafter, the present invention will be described in more detail by examples and experimental examples. However, the present invention is not limited to the examples and experimental examples disclosed below, which make the disclosure of the present invention complete and fully inform the scope of those skilled in the art, and can be implemented in various different ways.

Example 1: preparation of antibacterial peptides

The present inventors developed a peptide (KFKWRYm) having antibacterial activity through conventional basic screening studies and obtained a patent for the peptide (Korean patent No. 10-1855170). Subsequently, in order to develop an improved peptide having better antibacterial and immunoregulatory functions than the peptide, the present inventors designed various variants different from the amino acid sequences of the patent peptides and synthesized various antibacterial candidate peptides using a general amino acid synthesis method (Umbarger, HE, Ann. Rev. biochem.,47: 533. 606, 1978).

The present inventors further added modifications such as addition of octanoyl or acetyl groups to the N-terminus of the synthesized peptide and/or substitution of the C-terminal carboxyl group with an amine group.

Experimental example 1: FPR2 activation effect analysis of novel peptides

The present inventors observed changes in calcium ion permeability of candidate peptides according to embodiments of the present invention to determine whether they can activate immunomodulatory functions in vivo. In particular, intracellular calcium ion concentrations were measured to confirm that the peptides were able to activate FPR 2. RBL cells expressing neither FPR1 nor FPR2, RBL cells overexpressing FPR 1(FPR 1-RBL), and RBL cells overexpressing FPR 2(FPR 2-RBL) were used and intracellular calcium ion levels were sensitively detected using Fura-2/AM, a fluorophore with high binding affinity for calcium. For this, cells were cultured in RPMI medium supplemented with 10% fetal bovine serum and in the log phase (mid-log phase, 1-3X 10)⁷Individual cells/ml) were harvested by centrifugation, then washed several times with RPMI medium without addition of fetal bovine serum, and then the cells were resuspended to 1 × 10 in RPMI medium⁷Individual cells/ml. Subsequently, Fura-2/AM was added to a final concentration of 3. mu.M, and the cells were incubated at 37 ℃ with 5% CO₂Was cultured in the incubator of (1) for 45 minutes. After an appropriate time, the cells were harvested, washed again with RPMI medium, and then suspended in an appropriate amount of RPMI medium supplemented with 250 μ M sulpirenone (sulfipyrazone) to prevent Fura-2 from entering the cells and being lost. Each time taking about 2 × 10⁶The cells were harvested by rapid centrifugation, resuspended in 1ml of Locke solution without calcium ions and EGTA, treated every about 1 minute with different concentrations (1. mu.M, 0.1. mu.M and 0.01. mu.M) of the peptide according to an embodiment of the invention, the ratio of the absorbance at both wavelengths, 340nm and 380nm, was monitored on a spectrophotometer, and then the difference in absorbance at both wavelengths was investigated. It is converted to a concentration of free calcium ions entering the cell according to the method of Grynkiewicz.

The results showed that the effect of activating FPR2 was shown in most of the peptides, which indicates that the peptides according to the present embodiment can modulate immune function of individuals by binding to and activating FPR2 (fig. 1). The results of analyzing the effect of the peptides on the activation of FPR2 are summarized in table 1 below.

TABLE 1

Analysis of FPR2 Activity of peptides tested

Peptides	FPR2 Activity (EC)50,nm)	SEQ ID Nos
			V3	106.9	1
V4	64.0	2
			V5	41.5	3
V6	4.2	4
			V7	3.2	5
V8	6.4	6
			V9	4.7	7
V16	-	8
			V16_0	-	9
V18	62.8	10
			V18_0	10.0	11
V19_Ac	69.4	12
			V20_Ac	45.3	13
V21_Ac	92.3	14
			V22_Ac	16.2	15
V23_Ac	61.3	16
			V24_Ac	58.6	17

Experimental example 2: novel peptide antibacterial activity assay

The present inventors evaluated their ability to activate FPR2 in order to select a peptide excellent in performance among the various peptides synthesized in example 1. Specifically, in order to examine the antimicrobial activity of the peptide, gram-positive bacteria Staphylococcus aureus (Staphylococcus aureus) and gram-negative bacteria Pseudomonas aeruginosa (Pseudomonas aeruginosa) were inoculated on an agar plate medium by four-zone streaking and cultured overnight in an incubator at 36 ℃. The next day, colonies of the strain produced on agar plate medium were inoculated into 3ml of nutrient medium and cultured overnight at 36 ℃ and in a shaker at 220 rpm. The next day, the bacteria were diluted to measure absorbance at 600nm, adjusted to OD₆₀₀0.5 and diluted 1:100 in nutrient medium. Then, the peptides prepared in the above examples were diluted in the nutrient medium to concentrations of 0, 1.25, 2.5, 5, 10, 20. mu.M in order to prepare 1ml each, and then 1ml of the diluted bacteria were inoculated. Subsequently, the mixture was shaken at 36 ℃ and 220rpm, incubated for 18 hours, and the absorbance at 600nm was measured.

The results showed that 20 peptides showing high antibacterial activity against pseudomonas aeruginosa and staphylococcus aureus were selected (fig. 2 and 3). The sequence information and antimicrobial activity of these peptides are summarized in the table.

TABLE 2

Amino acid sequence information and antibacterial activity of the antibacterial peptide

The invention has been described with reference to the foregoing embodiments and experimental examples, but it is understood that these are exemplary only and that various modifications and equivalent other embodiments and experimental examples may be made by those skilled in the art. Therefore, the true scope of the present invention should be determined by the technical spirit of the appended claims.

SEQUENCE LISTING

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<120> novel multifunctional peptide and use thereof

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<151> 2019-03-19

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