KR101930125B1 - Stapled heptapeptide and use thereof - Google Patents

Abstract

The present invention relates to a stapled novel heptapeptide, and more particularly to a stapled heptapeptide and an antimicrobial or anticancer composition comprising the stapled heptapeptide.
The stapled heptapeptide according to the present invention can effectively stabilize a short peptide having 7 amino acid residues into a spiral secondary structure by introducing staples. In addition, since proteases do not recognize helical peptides as substrates, peptides stabilized in a helical form by a secondary staple can have high stability against proteolytic enzymes. Furthermore, the staple introduced to stabilize the secondary structure of the peptide in a helical manner is hydrophobic and the positively charged amino acid is formed in the opposite direction to the staple to form a bipolar structure. As with many antimicrobial peptides present in nature, It can exhibit antibacterial action by destruction. Thus, the stapled heptapeptide according to the present invention can be used for various purposes and applications requiring antimicrobial activity, and is expected to be usefully used as an anti-cancer composition.

Description

Stapled heptapeptides and their uses {Stapled heptapeptide and use thereof}

The present invention relates to a stapled novel heptapeptide, and more particularly to a stapled heptapeptide and an antimicrobial or anticancer composition comprising the stapled heptapeptide.

Since the discovery of penicillin, a number of antibiotics have been developed and contributed greatly to the health of humankind. However, the emergence of resistant strains to antibiotics has made it difficult to easily disinfect existing antibiotics, and it is constantly demanding the development of antibiotics that exhibit activity as a new mechanism. Efforts to develop new antibiotics were not enough to keep pace with the rate of resistance to antibiotics, and resistance to antibiotics is now becoming a more serious social problem as more pharmaceutical companies abandon the study of new antibiotic development.

On the other hand, antimicrobial peptides are the primary protective agents against external infectious agents. They are found in almost all living organisms including humans, and exhibit various physiological activities such as anti-cancer activity and anti-inflammatory action . The majority of the antimicrobial peptides that are active by inducing rupture of the biological membrane are known to act on the biomembrane in the form of a helical secondary structure and for the most effective interaction with bacterial biomembranes, The amino acid residue has an amphipathic helix with a predominantly amino acid residue on the other side.

While the existing antibiotics target molecules in vivo of bacteria to easily produce resistance, the majority of these antimicrobial peptides act on the biofilm and induce rupture of the biomembrane, indicating that the activity is very small Antimicrobial peptides are a differentiated antibiotic that can cope with antibiotic resistance, and its potential is very high.

However, most of these antimicrobial peptides present in nature have a long sequence of more than 20 amino acids, and most of the derivatives that shorten this sequence lose activity, which makes it difficult to maintain a helical structure effectively in short sequence peptides . In rare cases, it has been reported that derivatives having short sequences maintain activity, but most of them are easily destroyed by protease in the body and bioavailability is very low.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems of the prior art, and the present inventors have made efforts to study antibiotics or anticancer substances capable of coping with tolerance by a unique mechanism of proteolytic enzyme and biodegradation, The peptide is stapled with a short amino acid sequence to prepare a stable peptide having an excellent antimicrobial or anticancer activity. Based on this finding, the present invention has been completed.

Accordingly, an object of the present invention is to provide a stapled heptapeptide.

It is still another object of the present invention to provide an antimicrobial or anticancer composition comprising the stapled heptapeptide.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention is described by the following formula 1,

[Formula 1]

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇

X ₁ , X _4, and X ₇ are positively charged amino acids, and X ₂ and X ₆ are stapled with hydrocarbons to provide a heptapeptide.

In one embodiment of the present invention, the hydrocarbon may be an oct-4-enyl group.

In another embodiment of the present invention, the stapled heptapeptide may be linked to a compound by a linker at X < ₁ >.

In another embodiment of the present invention, the stapled heptapeptide may be composed of any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

The present invention provides an antimicrobial composition comprising the stapled heptapeptide.

In one embodiment of the invention, the composition may exhibit an antibiotic effect against pathogenic bacteria, viruses, pathogenic yeast or fungi.

In another embodiment of the present invention, the pathogenic bacterium may be gram positive or gram negative.

In another embodiment of the present invention, the Gram positive bacteria may be Bacillus subtilis, Staphylococus aureus or Staphylococcus epidermis.

In another embodiment of the present invention, the Gram-negative bacterium is selected from the group consisting of Escherichia coli, Sigella dysentariae, Salmonella typhimurium, Klebsiella pneumoniae or pseudomonas aeruginosa, Lt; / RTI >

In another embodiment of the present invention, the pathogenic yeast or fungus is selected from the group consisting of Candida albicans, Aspergillus humigatus, Saccharomyces cerevisiae or Cryptococcus neoformans (Cryptococcus neoformans).

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the stapled heptapeptide.

The present invention provides a method for treating an infectious disease or cancer caused by a pathogenic bacterium, a virus, a yeast or a fungus comprising the step of administering the stapled heptapeptide to a subject.

The present invention provides an infectious disease caused by a pathogenic bacterium, a virus, a yeast or a fungus, or cancer treatment, comprising the step of administering the stapled heptapeptide to a subject.

The stapled heptapeptide according to the present invention can effectively stabilize a short peptide having 7 amino acid residues into a spiral secondary structure by introducing staples. In addition, proteases do not recognize helical peptides as substrates, and peptides stabilized in a helical form by a staple can have high stability against proteases. Furthermore, the staple introduced to stabilize the secondary structure of the peptide in a helical manner is hydrophobic and the positively charged amino acid is formed in the opposite direction to the staple to form a bipolar structure. As with many antimicrobial peptides present in nature, It can exhibit antibacterial action by destruction. Thus, the stapled heptapeptide according to the present invention can be used for various purposes and applications requiring antimicrobial activity, and is also expected to be useful as an anti-cancer composition.

Fig. 1 schematically shows the staple process of a peptide.
Figure 2 shows the structure of a stapled heptapeptide with a bipolar structure.
Fig. 3 schematically shows a stapling process of the heptapeptide according to the present invention.
Figures 4 and 5 show the sequences and stapled positions of the stapled heptapeptides according to the present invention.
FIGS. 6 and 7 show the results of the circular polarization duality of the stapled heptapeptide according to the present invention.

As a result of efforts to study an antibiotic or an anticancer substance capable of coping with tolerance by a unique mechanism of proteolytic enzyme and stable biodegradation, the present inventors have found that when a peptide is stapled with a short amino acid sequence of less than 10, the bipolar structure amphipathic helix), it was confirmed that it had a stable and excellent antimicrobial or anticancer activity. Based on this finding, the present invention was completed.

Hereinafter, the present invention will be described in detail.

The stapled heptapeptide of the present invention is represented by the following formula 1, wherein X ₁ , X _4, and X ₇ are positive amino acids and X ₂ and X ₆ are stapled with hydrocarbons. to provide.

[Formula 1]

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇

In the present invention, "stapling" is a technique capable of stabilizing the secondary structure of the? Helical of a peptide by introducing cross-linking of hydrocarbons to the peptide, and the stapling of the peptide is shown in the schematic diagram of FIG.

The staple of the peptide of the present invention can be applied to hydrocarbons. The hydrocarbon may be saturated or unsaturated, preferably of 2 to 40 carbon atoms, and each single, double or triple bond may be independently from 1 to 40 carbon atoms. Preferably, when the carbon number of the hydrocarbon is 8, the double bond may be located at the 4-carbon position (oct-4-enyl); When the number of carbon atoms is 10, the double bond may be located at the 5-carbon position; When the number of carbon atoms is 12, the double bond is at the 6-position carbon; When the number of carbon atoms is 14, the double bond is at the 7-position carbon; When the number of carbon atoms is 16, the double bond is at the 8-position carbon; When the number of carbon atoms is 18, the double bond is at the 9-position carbon; When the number of carbon atoms is 20, the double competition may be located at the 10 carbon position. Most preferably, the hydrocarbon is an oct-4-enyl group.

(S) -α-methyl and α-petenylglycine (S5) amino acids at any position (i) in the peptide sequence and at the 4th position (i + 4) toward the C terminal thereof from the peptide sequence And the residues of these two amino acids are cross-linked to a metathesis reaction using the Grubbs 1 catalyst.

In the present invention, the positions at which the cross-linked staple is introduced may be stapled at any one of the amino acid positions and the amino acid positions after the fourth amino acid position, more preferably the second amino acid position and the sixth amino acid position, The location may be stapled. Accordingly, the stapled peptide according to the present invention may comprise any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6.

The stapled heptapeptides according to the present invention effectively stabilize short peptides in a spiral secondary structure by introducing staples and at the same time are able to stabilize the amino acids which are positively charged to opposite positions (X ₁ , X ₄ and X ₇ ) of the hydrophobic staples To form a bipolar spiral structure (see Fig. 2). At this time, the amino acid which can be arranged is preferably lysine (Lys) or arginine (Arg), but it is not limited thereto, and in some cases alanine (Ala) or tryptophan (Trp) may be substituted.

In one embodiment of the present invention, the antibacterial activity of the stapled heptapeptide according to the present invention was found to be significantly increased as compared with the stapled control group in both Gram positive and Gram negative (See Example 4).

Therefore, the stapled heptapeptide according to the present invention can be used for various purposes and applications requiring antimicrobial activity, for example, a composition for preventing, inhibiting, improving or treating infectious diseases caused by pathogenic bacteria, viruses, yeast or fungi, But are not limited to, compositions for animal feed, animal feed additives, functional food compositions, food additives, food preservatives, disinfectants, sterilizing detergents and the like.

Accordingly, the present invention provides an antimicrobial composition comprising the stapled heptapeptide.

In the present invention, "antibacterial" means an activity capable of inhibiting the growth of pathogenic bacteria, viruses, pathogenic yeast or fungi. The pathogenic bacteria are all microorganisms which invade the living body of animals and plants to cause parasitic diseases or give harm, and may include Gram-positive bacteria and Gram-negative bacteria. For example, Gram-positive bacteria include Bacillus subtilis, Staphylococus aureus, Staphylococcus epidermis and the like. Gram-negative bacteria include Escherichia coli, But are not limited to, Salmonella typhimurium, Klebsiella pneumoniae or pseudomonas aeruginosa, and the like, such as, for example, Sigella dysentariae, Salmonella typhimurium, Pseudomonas aeruginosa, and the like. In addition, the bacterium may be an antibiotic-resistant bacterium. In addition, the pathogenic yeast and fungi are pathogenic yeasts and fungi, including, but not limited to, Candida albicans, Aspergillus humigatus, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Cryptococcus neoformans, and the like.

In one embodiment, the antimicrobial composition according to the present invention can be used in the form of a pharmaceutical composition for the prevention or treatment of infectious diseases caused by pathogenic bacteria, viruses, yeast or fungi.

In the present invention, the infectious diseases caused by the pathogenic bacteria are cholera caused by cholera; Bacterial Fertility by Agrobacterium; Pertussis by pertussis; Typhoid fever caused by typhoid fever; Laryngeal diphtheria by diphtheria, non-nasal diphtheria; Glandular fist and lung fist by pest bacterium; Scarlet fever due to hemolytic streptococci, erysipelas, sepsis, skin pyoderma; Tuberculosis caused by tubercle bacillus, joint tuberculosis, renal tuberculosis, tuberculous meningitis; Salmonella and enteritis Vibrio and the like may be bacterial food poisoning. In addition, the infectious diseases caused by the pathogenic yeast and fungi include, but are not limited to, cryptococcosis, candidasis, dermatophytosis, superficial mycoses, meningitis, brain abscess, brain tumor, histoplasmosis Histoplasmosis), neuromusstic pneumonia or aspergillosis.

In another embodiment, the antimicrobial composition according to the present invention may be used in the form of a composition for animal feed, wherein the animal is a biological group corresponding to a plant, which mainly consumes organic matter as nutrients, and digestion, And may be echinoderms, crustaceans, mollusks, fishes, amphibians, reptiles, birds, mammals, and more preferably echinoderms such as sea urchins or sea cucumbers, crabs, shrimps, Mites such as arthropods including arthropods, arthropods such as arthropods, cephalopods, gastropods or bivalves, birds including poultry such as red sea breams, sea breams, codfishes, flounder, etc., poultry or chickens or mammals such as pigs, Lt; / RTI >

The composition for animal feed of the present invention may further comprise cereals, vegetable protein feeds, animal protein feeds, sugar or dairy products, etc. in addition to the antimicrobial composition comprising the stapled heptapeptide as an active ingredient, further comprising nutritional supplements, digestion and absorption An agent such as an enhancer, growth promoter or disease preventive agent may be used together.

The antimicrobial composition contained in the composition for animal feed of the present invention may vary depending on the purpose of use and the conditions of use of the feed, and may be appropriately selected by those skilled in the art.

In another embodiment, the antimicrobial composition according to the present invention may be used in the form of a functional food composition. In order to prevent or improve diseases such as food poisoning, the functional food composition may be administered before or after the onset of the disease, Can be used simultaneously or separately as a medicament for treatment.

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

In the functional food composition of the present invention, the active ingredient may be directly added to the food or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The functional food composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned active ingredients as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients 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 above-mentioned natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the functional food composition of the present invention may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

In another embodiment, the antimicrobial composition according to the present invention may be used in the form of a food preservative composition. In this case, the food preservative may be added to the stapled heptapeptide according to the present invention Can be prepared by further adding an acceptable solvent or diluent. The food preservative composition of the present invention may be formulated together with the raw material in the process of producing food or may be added as a separate aqueous suspension. In addition, the food preservative agent of the present invention can be used to immerse the target food or spray the food preservative agent of the present invention to the target food.

In another embodiment, the antimicrobial composition according to the present invention can be used in the form of a sterilizing detergent composition, wherein the sterilizing detergent can be used for kitchen or food, but is not limited thereto. The sterilizing detergent of the present invention may further contain adversely selected additives depending on the purpose and use of the sterilizing detergent of the present invention and may be mixed with other active ingredients such as a sterilizing detergent generally used or additives such as coloring matters, surfactants and preservatives . The sterilizing detergent of the present invention may be prepared by pulverizing, granulating, tableting or liquefying according to the purpose and use of the sterilization cleaner of the present invention. For commercialization, a conventional method may be used for packaging.

In another embodiment, the antimicrobial composition according to the present invention may be used in the form of a disinfectant. In this case, the disinfectant may be prepared by using the stapled heptapeptide as an active ingredient as it is, or by using a diluent which is dermatologically and pharmacologically acceptable Or diluted with a solvent. The disinfecting agent of the present invention can be used on the surface of an organism, preferably on the skin of mammals, most preferably on human skin. The disinfectant of the present invention can also be used on surfaces of non-living organisms such as wood, metal, glass, ceramic, plastic, paper and cloth. The disinfectant of the present invention may be applied to the surface of the organism or organism using methods including immersion, swab, spray and brushing. Preferably, the disinfectant is selected from the group consisting of wound surface, pre-operative skin disinfection, It can be used in hospitals and general households for disinfection and duct sterilization.

In another embodiment, the antimicrobial composition according to the present invention can be used in the form of a cosmetic composition, wherein the cosmetic composition can be prepared in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. The cosmetic composition may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, surfactants, water, ionic or nonionic emulsifiers, Auxiliaries such as ionic sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetic compositions ≪ / RTI >

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the stapled heptapeptide. The stapled heptapeptide according to the present invention can stably maintain the secondary structure of the alpha helical peptide, thereby achieving an effective anticancer effect in a biologically stabilized form.

As used herein, the term "prophylactic " means any act that inhibits cancer or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of cancer by administration of the pharmaceutical composition of the present invention.

"Cancer" as a disease to be improved, prevented or treated by the composition of the present invention is an aggressive characteristic that cells divide and grow by ignoring the normal growth limit, invasive characteristic penetrating into surrounding tissues And diseases caused by cells having metastatic characteristics spreading to other parts of the body. Examples of cancer in the present invention include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, Cancer of the liver, cancer of the liver, cancer of the breast, cancer of the endometrium, pituitary adenocarcinoma, adrenal cancer, soft tissue sarcoma, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, But are not limited to, colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, liver cancer, prostate cancer, mucin cancer, thyroid cancer, liver cancer and head and neck cancer.

The pharmaceutical composition of the present invention may further comprise a pharmacologically contained carrier comprising stapled heptapeptide as an active ingredient. Herein, pharmaceutically acceptable carriers are those conventionally used at the time of formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, May be administered at a daily dose of 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental preparation

Commercially available solvents and reagents were used according to the manual. All of the Fmoc-protected α-amino acids (except for Fmoc- (S) -methyl and α-pentylglycine purchased from Okeanos Tech Co., Ltd.), 1 - [(1- (cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylamino-morpholino)] - uranium hexafluorophosphate (COMU), and Rink Amide MBHA resin were purchased from NovaBiochem. N-diisopropylethylamine (DIEA), Grubbs 1st generation catalyst (bis (tricyclohexylphosphine) benzylidine ruthenium (IV) dichloride), 1, 2-pyrrolidine , 2-dichloroethane (DCE), triisopropylsilane (TIS), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich.

Example 2. Peptide design and synthesis

In order to design a peptide consisting of fewer than 10 short sequences, stapled peptides were devised as shown in Equation 1 below.

[Formula 1]

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇

In order to obtain effective stabilization with a helical secondary structure, peptides having (S) -α-methyl and α-petenylglycine amino acids were synthesized on X ₂ and X ₆ , and the residues of these two amino acids were then purified using Grubbs 1 catalyst Metathesis reaction to cross-link the amino acid at that position to the oct-4-enyl staple (see FIG. 3). Since the oct-4-enyl staple introduced to stabilize the secondary structure in a helical form is hydrophobic, a lysine group with positive charge opposite to the staple was introduced to form a bipolar spiral structure, (Ala) group or tryptophan (Trp). In addition, acetyl groups were introduced at X ₁ , that is, at the N-terminus, and those without stapling were used as a control group. The specific sequence of the designed peptides is shown in FIGS. 4 and 5. FIG. Meanwhile, in FIG. 4 and FIG. 5, * and * indicate positions stapled with hydrocarbons.

For the experiment, the peptides of FIGS. 4 and 5 were synthesized. All peptides were prepared using Fmoc chemistry on Rink amide MBHA resin with a loading capacity of 0.6 mmol / g. Before use, the dried resin (50 mg, 30 μmol) was swollen in NMP for 10 minutes. After removal of the Fmoc protecting group (2 x 10 min) by treatment with 25% piperidine in NMP, 5 equiv. Of Fmoc-protected amino acid in NMP, 10 equivalents of DIEA and activating agent COMU for 30 min The amino acid was couiled (4.75 eq.). Coupling of Fmoc- (S) -a-methyl and alpha -pentylglycine was carried out for 2 hours using amino acid (3 equivalents), COMU (2.85 equivalents), and DIEA (6 equivalents). After each coupling or deprotecting reaction, the resin was dissolved in dichloromethane (DCM) (1 x 2 min), NMP (1 x 2 min), DCM (1 x 2 min), and NMP ).

Example 3. Peptide Metathesis and Purification

Ring-closing metathesis of resin-bound N-Fmoc, a side chain protected peptide, was carried out with 20 mol% of Grubbs 1 catalyst in degassed DCE at room temperature for 2 hours . The reaction was monitored using a liquid chromatography-mass spectrometry (LC / MS) after decomposition of the peptide obtained from the resin aliquot. After draining the reaction solution, the resin was washed with DCE (dichloromethane) (3 x 2 min) and again with DCM (3 x 2 min).

After removal of the Fmoc protecting group by treatment with 25% piperidine in NMP (2 x 10 min) or after the final deprotection reaction, the N-terminal amino group is reacted with 30 equivalents of acetic anhydride and 60 equivalents of DIEA .

The resin was washed with DCM (3 x 2 min) and DMF (3 x 2 min) and dried under vacuum overnight. The peptides were deprotected and the resin was cleaved from the resin for 2 hours by addition of a mixture of TFA / TIS / water (95 / 2.5 / 2.5) and a 1: 1 mixture of n-pentane and diethyl ether To precipitate. The impurities were collected on a centrifuge, dissolved in a 1: 1 mixture of acetonitrile and water, and the resin was removed by filtration. The product was purified by high-performance liquid chromatography using a Zorbax C18 column (Agilent, 5 [mu] m, 9.4 x 250 mm) and then purified by LC / MS (Liquid chromatography / mass spectrometry) (Agilent, API4000) .

S1. ESIMS m / z calculated for C ₄₈ H ₇₈ N ₁₂ O ₈ [M + 2H] ^{2 +} / 2 476.31, found 476.45.

S2. ESIMS m / z calculated for C ₄₈ H ₇₈ N ₁₂ O ₈ [M + 2H] ^{2 +} / 2 476.31, found 476.45.

S3. ESIMS m / z calcd for C ₅₁ H ₈₁ N ₁₂ O ₈ [M + 2H] ^{2 +} / 2 497.33, found 497.45.

S4. ESIMS m / z calcd for C ₅₁ H ₈₁ N ₁₂ O ₈ [M + 2H] ^{2 +} / 2 497.33, found 497.45.

S5. ESIMS m / z calculated for C ₄₅ H ₆₈ N ₁₁ O ₈ [M + 2H] ^{2 +} / 447.78, found 447.80.

S6. ESIMS m / z calculated for C ₄₅ H ₆₈ N ₁₁ O ₈ [M + 2H] ^{2 +} / 447.78, found 447.80.

U1. _{C 50 H 82 N 12 O 8} [M + 2H] 2+ / 2 ESIMS m / z 490.33 for the calculated values, measured values 490.45.

U2. _{C 50 H 82 N 12 O 8} [M + 2H] 2+ / 2 ESIMS m / z 490.33 for the calculated values, measured values 490.45.

U3. ESIMS m / z calculated for C ₅₃ H ₈₅ N ₁₂ O ₈ [M + 2H] ²⁺ / 5 511.35, measured 511.50.

U4. ESIMS m / z calculated for C ₅₃ H ₈₅ N ₁₂ O ₈ [M + 2H] ²⁺ / 5 511.35, measured 511.50.

U5. ESIMS m / z calculated for C ₄₇ H ₇₂ N ₁₁ O ₈ [M + 2H] ^{2 +} / 461.80, found 461.85.

U6. ESIMS m / z calculated for C ₄₇ H ₇₂ N ₁₁ O ₈ [M + 2H] ^{2 +} / 461.80, found 461.85.

Ac-S1. ESIMS m / z calculated for C ₄₈ H ₇₉ N ₁₂ O ₈ [M + H] ⁺ 951.61, measured 951.50.

Ac-S2. ESIMS m / z calculated for C ₄₈ H ₇₉ N ₁₂ O ₈ [M + H] ⁺ 951.61, measured 951.35.

Ac-S3. ESIMS m / z calculated for C ₅₁ H ₈₅ N ₁₂ O ₈ [M + H] ⁺ 993.66, found 993.50.

Ac-S4. ESIMS m / z calculated for C ₅₁ H ₈₅ N ₁₂ O ₈ [M + H] ⁺ 993.66, found 993.50.

H-S1. ESIMS m / z calculated for C ₄₆ H ₇₆ N ₁₂ NaO ₇ [M + Na] ⁺ 932.60, measured 931.60.

H-S2. ESIMS m / z calculated for C ₄₆ H ₇₆ N ₁₂ NaO ₇ [M + Na] ⁺ 932.60, measured 931.65.

H-S3. C ₄₉ H ₈₃ N ₁₂ O ₇ ESIMS m / z calculated for [M + H] ⁺ 951.65, found 951.60.

H-S4. C ₄₉ H ₈₃ N ₁₂ O ₇ ESIMS m / z calculated for [M + H] ⁺ 951.65, measured 951.65.

Example 4. Confirmation of antimicrobial activity of stapled peptide

In order to measure the antimicrobial activity of the stapled peptides prepared in Example 2 and Example 3, the following experiment was conducted.

That is, the antibacterial analysis was carried out by measuring the minimal inhibitory concentration (MIC) value using a standard broth microdilution method. Escherichia coli ATCC 25922, Shigella dysentariae ATCC 9752, Salmonella typhimurium (ATCC 9752), and Gram-negative strains (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538p, and Staphylococcus epidermis ATCC 12228) Protein mirabilis ATCC 25933, Pseudomonas aeruginosa ATCC 27853) were used. The strains were inoculated in 2 ml LB (Luria-Bertani) broth and cultured at 37 ° C overnight. Each peptide was dissolved in PBS (phosphate-buffered saline) and the peptide solution was prepared by diluting the peptide solution two-fold with 0.2 μg / mL to 200 μg / mL in 96-well round bottom microtitre plates. Peptide solution and bacterial inoculum were diluted using LB broth. Bacterial suspensions of 10 ⁶ to 10 ⁸ colony-forming units (CFU) / ml were added to the peptide-containing wells and cultured. After incubation at 37 ° C for 24 hours, the minimum inhibitory concentration (MIC) of the wells was analyzed, where the MIC was defined as the lowest peptide concentration that completely inhibited cell growth. The experiment was carried out twice. On the other hand, all bacterial strains were distributed from KRIBB (Korea Research Institute of Bioscience and Biotechnology, Korea) of KCTC (Korean Collection of Type Culture).

As a result, as shown in Tables 1 and 2, it was confirmed that the stapled peptide according to the present invention exhibited significantly increased antimicrobial activity against both Gram-positive and Gram-negative.

Minimum Inhibitory Concentration (μM) ^a Bacteria line U1 S1 U2 S2 U3 S3 U4 S4 U5 S5 U6 S6 C ^b Gram (+) Bacillus subtilis > 100 25 > 100 50 25 12.5 25 12.5 > 100 50 > 100 > 100 3.1 Staphylococcus aureus > 100 25 > 100 100 25 25 25 12.5 > 100 50 > 100 > 100 6.3 Staphylococcus epidermis > 100 > 100 > 100 > 100 100 100 100 100 > 100 > 100 > 100 > 100 6.3 Gram (-) Escherichia coli 100 25 > 100 50 25 12.5 25 25 > 100 50 > 100 100 12.5 Shigella  dysentariae > 100 100 > 100 > 100 100 25 > 100 25 > 100 > 100 > 100 > 100 25 Salmonella typhimurium > 100 > 100 > 100 > 100 > 100 50 > 100 50 > 100 > 100 > 100 > 100 50 Klebsiella  pneumonia > 100 50 100 100 50 25 50 25 > 100 100 > 100 > 100 12.5 Pseudomonas  aeuginose > 100 50 > 100 > 100 100 25 100 25 > 100 > 100 > 100 > 100 12.5

Minimum Inhibitory Concentration (μM) ^a Bacteria line Ac-S1 H-S1 Ac-S2 H-S2 Ac-S3 H-S3 Ac-S4 H-S4 C ^b Gram (+) Bacillus subtilis 25 25 25 37.5 18.8 12.5 12.5 25 3.1 Staphylococcus aureus 25 37.5 37.5 37.5 25 12.5 25 50 6.3 Staphylococcus
epidermis > 100 > 100 > 100 > 100 100 25 100 100 6.3 Gram (-) Escherichia coli 37.5 25 25 50 25 12.5 25 25 12.5 Shigella dysentariae 100 100 100 > 100 50 50 50 75 25 Salmonella typhimurium > 100 100 > 100 > 100 75 50 100 100 25 Klebsiella  pneumonia 50 25 37.5 50 37.5 25 37.5 37.5 12.5 Pseudomonas  aeruginose 50 25 50 37.5 50 25 50 25 12.5

Example 5. Identification of erythrocyte hemolytic activity of stapled peptide

The peptides were dissolved in PBS. 10 μl of the serially diluted peptide (final concentration 0.8-100 μM) was added to a suspension of 190 μl of human red blood cells (10% v / v in PBS) and incubated at 37 ° C for 30 minutes. After centrifugation, the supernatant was diluted 10-fold with PBS and the absorbance for each solution was measured at 405 nm. Blood suspension treated with 0.2% Triton X-100 was used as a control for 100% hemolysis. The percentage of hemolysis was determined using the following equation.

As a result, as shown in Tables 3 and 4, it was confirmed that the stapled peptide according to the present invention exhibited a low hemolytic activity against human erythrocytes.

Concentration (μM) U1 S1 U2 S2 U3 S3 U4 S4 U5 S5 U6 S6 C ^b 100.0 0.99 1.21 0.88 1.24 1.12 7.75 1.22 4.50 1.10 1.04 1.23 0.84 75.75 50.0 0.75 1.17 0.96 1.75 1.03 3.81 1.19 2.46 1.03 0.88 0.91 0.61 67.16 25.0 0.90 1.36 0.93 0.82 0.98 2.52 1.19 1.87 0.87 0.76 1.01 0.70 58.21 12.5 0.88 0.83 0.97 0.63 0.75 2.13 0.97 1.08 0.94 0.83 0.72 0.64 34.89 6.3 0.91 0.66 1.05 0.99 1.25 0.94 0.81 0.68 0.82 0.76 0.70 0.87 7.84 3.1 0.70 0.67 0.84 0.66 0.90 1.02 1.28 0.71 0.75 0.59 0.73 0.75 1.68 1.6 0.76 0.53 0.86 1.01 0.72 0.65 1.98 0.90 0.78 0.60 0.67 0.66 0.59 0.8 0.91 0.53 0.76 0.91 0.88 0.87 0.80 0.71 0.86 0.65 0.60 0.65 0.51

Concentration
(μM) Ac-S1 H-S1 Ac-S2 H-S2 Ac-S3 H-S3 Ac-S4 H-S4 C ^b 100.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 5.53 2.05 5.74 ≪ 1.0 50.8 50.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 1.59 ≪ 1.0 2.84 ≪ 1.0 36.6 25.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 1.29 ≪ 1.0 11.3 12.5 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 6.9 6.3 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 3.0 3.1 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 1.6 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 0.8 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0 ≪ 1.0

Example 6. Confirmation of conformational preferences of stapled peptides through circular dichroism

To determine whether the increased antimicrobial activity of the stapled analogs according to the present invention is related to their spiral stability, morphological preference was tested using a far-UV circular dichroism spectrum.

More specifically, each peptide was dissolved in 25 mM potassium phosphate buffer (pH 6.5) and the concentration was measured by absorbance spectroscopy at ₂₈₀ nm (extinction coefficient for tryptophan,? ₂₈₀ = 5690 cm ^-1 ). Circular polarization dichroism spectra were collected on a Chirascan HP dipolar circular dichroism spectrum with temperature controller using the following standard measurement parameters: 1 nm step resolution, 3 accumulations, 0.5 second reaction, 1 nm bandwidth, and 0.1 cm path Length. All spectra were converted to a uniform scale of molar ellipticity after excluding the background. The curves were flattened to standard parameters and the results are shown in FIG. 6 and FIG.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> Stapled heptapeptide and use thereof <130> MP15-088 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> S1-stp <220> <221> BINDING <222> (2) <223> hydrocarbon stapling cross linking with site 6 <220> <221> BINDING <222> (6) <400> 1 Lys Xaa Trp Lys Ala Xaa Lys   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> S2-stp <220> <221> BINDING <222> (2) <223> hydrocarbon stapling cross linking with site 6 <220> <221> BINDING <222> (6) <400> 2 Lys Xaa Ala Lys Trp Xaa Lys   1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> S3-stp <220> <221> BINDING <222> (2) <223> hydrocarbon stapling cross linking with site 6 <220> <221> BINDING <222> (6) <400> 3 Lys Xaa Trp Lys Leu Xaa Lys   1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> S4-stp <220> <221> BINDING <222> (2) <223> hydrocarbon stapling cross linking with site 6 <220> <221> BINDING <222> (6) <400> 4 Lys Xaa Leu Lys Trp Xaa Lys   1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> S5-stp <220> <221> BINDING <222> (2) <223> hydrocarbon stapling cross linking with site 6 <220> <221> BINDING <222> (6) <400> 5 Lys Xaa Trp Ala Lys Xaa Ala   1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> S6-stp <220> <221> BINDING <222> (2) <223> hydrocarbon stapling cross linking with site 6 <220> <221> BINDING <222> (6) <400> 6 Lys Xaa Ala Trp Lys Xaa Ala   1 5

Claims (11)

A stapled heptapeptide is described by the following formula 1,
[Formula 1]
X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇
X ₁ , X ₄ and X ₇ are positively charged amino acids, and X ₂ and X ₆ are stapled with an oct-4-enyl group.
delete delete The stapled heptapeptide according to claim 1, wherein the stapled heptapeptide comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6.
delete delete delete delete delete delete delete

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