KR20140140191A - Stapled gaegurin 4 derived peptide and composition for antibiotic or anticancer comprising the same - Google Patents

Abstract

The present invention relates to a stapled gaegurin 4-derived peptide or derivatives of the same. Moreover, the present invention relates to an antibiotic or anticancer composition including the stapled peptide. The stapled gaegurin 4-derived peptide of the present invention stably maintains a secondary structure of α spiral peptide, so that high antibiotic properties and high resistance against protease are exhibited. Accordingly, high antibiotic or anticancer effects can be expected.

Description

[0001] Stapled gagurin 4 derived peptides and antimicrobial or anticancer compositions comprising the same [0001] The present invention relates to a stapled gagulin-

The present invention relates to stapled gagurin 4-derived peptides or derivatives thereof, and antimicrobial or anti-cancer compositions comprising the stapled peptides.

Geagurin 4 is a natural peptide showing antibiotic and anticancer activities isolated from domestic rats (Rana rugosa). Gagurin 4 is known to exhibit antibiotic action by acting on the cell membrane of bacteria in the form of α-helices like other gagurin peptide derivatives, destroying the cell membrane. However, since the peptide has a long sequence containing 37 amino acids, there is a limit to the actual use of the peptide. Recently, Professor Lee Bong-jin of the College of Pharmacy at Seoul National University has shown that the introduction of tryptophan in place of the aspartic acid at position 16 of the peptide results in a high antibiotic activity of doggurin 4 derivatives consisting of 23 amino acids. However, 23 peptides still require high cost and manpower to synthesize and manufacture, and they are still difficult to use as medicines since they are easily degraded by various proteases when administered into the body, like other peptides.

One aspect of the present invention is to provide a stapled doggulin-derived peptide or derivative thereof.

In addition, one aspect of the present invention is to provide an antibacterial or anticancer composition comprising the stapled peptide.

One aspect of the invention provides a stapled doggulin-derived peptide or derivative thereof.

Hereinafter, the present invention will be described in detail.

Esculentin-2EM (Gagrin 4, geagurin 4) is the longest peptide of the open-chain peptide containing 37 amino acids. Like other open-chain peptides, escululin-2EM is a cationic, bipolar, α-helical peptide with a disulfide bond at the C-terminus and is known to have an antibacterial action through the destruction of the bacterial membrane. The helical form and bipolar properties of escululin-2EM are essential for interaction with the bacterial membrane and membrane lytic activity. Previous studies have focused on the formation of a short sequence of escululantin-2EM analogs. However, cleavage of the residue in the esculentin-2EM sequence has always accompanied a decrease in the antimicrobial activity. See, for example, 1999, P.D. Ryu et al. Have shown that the N-terminal 23-residue fragment of escululin-2 EM does not exhibit any antimicrobial activity at all. This is because this truncated derivative is not capable of forming a biologically active alpha helical form. In a recent study, B.-J. Lee et al. Have shown that the substitution of the N-terminal 23-residue fragment of escululenthin-2EM with the N-terminal 16-position tryptophan (E2EM23W) can restore spiral and antimicrobial activity (Eur JBiochem 2002 4367) . The 16th tryptophan residue is located between the hydrophilic and hydrophobic surfaces of the bipolar E2EM23W, allowing bipolar interactions between the E2EM23W and the bacterial membrane surface. This revealed the shortest analog of escululin-2EM with antimicrobial activity.

The inventors of the present invention have invented a new peptide which has a shorter and higher antimicrobial activity than conventional escululin-2-EM and is highly resistant to proteolytic enzymes. Specifically, in E2EM15W consisting of 5-19 residues of E2EM23W, oct-4-enyl staple cross-linked staple fused at positions 10 and 14 of the amino acid sequence of escululin- E2EM15W-S1 and E2EM15W-S2 and E2EM15W-S3 with tryptophan substitutions at different positions were designed.

Thus, the present invention provides a peptide comprising an amino acid sequence of SEQ ID NO: 3 (E2EM15W); Or a peptide derivative in which at least one of amino acids 1 to 15 in the amino acid sequence of SEQ ID NO: 3 is substituted with tryptophan, the amino acid position of any one of the first to eleventh amino acids and the amino acid position after the fourth amino acid residue thereof are stapled with hydrocarbons stapling peptide. The substituted peptide may preferably be one in which any one of the 5th, 9th, or 12th amino acid positions is substituted with tryptophan.

In the present invention, stapling is a new technique ( J. Am . Chem. Soc . 2000, 122 , 5891; and US 7192713, which can stabilize the secondary structure of the? Helical of a peptide by introducing cross- )to be. The staple 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.

The position i introducing the cross-linked staple may be any amino acid position in the peptide sequence and i + 4 is the fourth position from it to the C terminus. Wherein the amino acid position is selected from the group consisting of the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of any one of the first to eleventh amino acids and the amino acid position after the fourth amino acid residue thereof stapled with hydrocarbons stapling). More preferably, the 6th amino acid position and the 10th amino acid position may be stapled. Most preferably, the stapled peptide may be composed of an amino acid sequence of any one of SEQ ID NOS: 4 to 6.

Another aspect of the present invention provides a pharmaceutical composition for antimicrobial use comprising the stapled peptide.

The open-loop staple-derived peptide of the present invention stably maintains the secondary structure of the alpha helical peptide, thereby exhibiting high antimicrobial activity.

The antimicrobial activity of the present invention means an activity capable of inhibiting the growth of pathogenic bacteria, viruses, pathogenic yeast or fungi.

The pathogenic bacteria include all gram-positive bacteria and gram-negative bacteria that invade the living body of a plant or animal to cause parasitic disease or cause harm or damage. For example, Gram-positive bacteria include Bacillus subtilis subtilis), Staphylococcus aureus (Staphylococus aureus and Staphylococcus epidermis , and the gram-negative bacteria include Escherichia coli coli , Sigella < RTI ID = 0.0 > dysenteriae ), pneumococcus ( Klebsiella pneumoniae ), 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 ( Candida albicans), Aspergillus Hugh migeo tooth (Aspergillus humic acid , < RTI ID = 0.0 > Humigatus , < / RTI > Saccharomyces cerevisiae and Cryptococcus neoformans .

The composition of the present invention may be a pharmaceutical composition for preventing or treating 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 may be caused by cryptococcosis, candidasis, dermatophytosis, superficial mycoses, meningitis, brain abscess, brain tumor, histoplasmosis Histoplasmosis), neuromusstic pneumonia or aspergillosis.

In addition, the composition of the present invention can be used in quasi-drugs for antimicrobial use.

"Quasi-drugs" means any substance which is not intended to be used in the treatment of human or animal diseases, such as fibers, rubber products or the like, used for the purpose of treatment, alleviation, treatment or prevention, Similar to that used for sterilization, insecticide and similar uses for the prevention of infectious diseases, which is used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals, Machinery, or apparatus, and that is not an apparatus, machine or apparatus of an article used for the purpose of giving pharmacological effects to the structure or function of a person or animal.

When the antimicrobial agent of the present invention is used as a quasi-drug additive, the antimicrobial agent may be added as it is, or may be used together with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention may be, but is not limited to, disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

In addition, the antimicrobial composition of the present invention provides a food additive for preventing or ameliorating a disease caused by a pathogenic bacterium, a virus, a yeast or a fungus.

The foodstuff to which the food additive can be used is not particularly limited, and the content of the stapled peptide contained in the food additive is also not particularly limited, and it is preferable that the content of the stapled peptide contained in the food additive And can be easily determined by those skilled in the art as needed.

Examples of foods to which the antimicrobial agent of the present invention can be added include meat products, sausages, breads, chocolates, candies, snacks, confectionery products, pizza, ramen noodles, other noodles, gums, dairy products including ice cream, Alcoholic beverages, and vitamin complexes, and may include all the health functional foods in the conventional sense, and foods used as feeds for animals.

In addition, the antimicrobial composition of the present invention can be used as a feed additive for preventing or ameliorating diseases caused by pathogenic bacteria, viruses, yeast or fungi.

The content of the staple peptide added to the feed is not limited and can be easily determined by a person skilled in the art depending on the kind of animal to which the feed is applied, age, sex, pregnancy, ease of infection or degree of infection.

In another embodiment, the stapled peptide of the present invention can be applied to cosmetics.

The cosmetic of the present invention can be produced 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 comprise one or more additives selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, Adjuvants such as chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetic compositions ≪ / RTI >

Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the stapled peptide described above.

The archival 4-derived staple-derived peptide of the present invention stably maintains the secondary structure of the α-helical peptide, thereby achieving an effective anticancer effect in a biologically stabilized form.

The cancer is selected from the group consisting of 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, Pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon cancer, colon cancer, colon cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, Endometrial or uterine cancer, salivary gland cancer, renal cancer, liver cancer, prostate cancer, mucin cancer, thyroid cancer, liver cancer, and head and neck cancer.

When the composition is prepared with a pharmaceutical composition for the prevention or treatment of cancer, the composition may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the composition include those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition for preventing or treating cancer may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The appropriate dosage of the pharmaceutical composition for prevention or treatment of cancer may be determined by factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, Can be prescribed in various ways. The preferred dose of the composition is in the range of 0.001-100 mg / kg on an adult basis. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer, or to prevent or treat a disease caused by angiogenesis.

The composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily practiced by those skilled in the art, or may be manufactured by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

The inventive staple-derived 4-staple derived peptide stably maintains the secondary structure of the α-helical peptide and shows high antimicrobial activity and high resistance to proteolytic enzymes. .

FIG. 1 schematically shows the staple process of a peptide. FIG.
FIG. 2 is a diagram showing sequence information and stapled positions of a stapled dogleurin 4-derived or tryptophan-substituted peptide derivative. FIG.
3 is a diagram showing the circular polarization duality of each peptide.
Figure 4 shows the effect of each peptide on trypsin digestion.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Preparation of reagents

Commercially available solvents and reagents were used according to the manual. All Fmoc-? -Amino acids (Fmoc- (S) -methyl,? -Pentylglycine-OH) purchased from Okeanos Tech Co. Ltd., 2- (6-chloro-1- H- benzotriazole- Tetramethylammonium hexafluorophosphate (HCTU), 6-chloro-benzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (Chloro-benzotriazole -1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock) and Rink amide MBHA resin were purchased from NovaBiochem.

Example  2: Peptides  Design and synthesis

(E2EM23W) in which the 16th position of the N-terminus was substituted with tryptophan in the N-terminal 23-residue fragment of escululatin-2EM and its truncated analogue E2EM15W was prepared as a control. E2EM15W consists of 5-19 residues of E2EM23W containing the core spiral section of E2EM23W in SDS micelles. In order to design stapled analogues of E2EM15W, oct-4-enyl staple cross-linked stapled analogs at positions 10 and 14 of the amino acid sequence of escululin- E2EM15W-S1 was prepared. A schematic diagram of the staple of the peptide is shown in Fig. These two locations were identified as hydrocarbon knots that can minimize interference to bipolar properties; These two residues are located at the interface between hydrophilic and hydrophobic surfaces. In addition, E2EM15W-S2 and E2EM15W-S3 substituted with tryptophan at different positions were prepared. The two stapled peptides were designed to observe the effects of substitution of tryptophan at different positions. Specific sequence information of the control and experimental group peptides is shown in Fig. In Fig. 2, * and * indicate staples stapled with hydrocarbons.

The ESI-MS information of the control and experimental peptides is shown in Table 1 below.

Peptides Molecular formula Calculated mass (M + 3H) ³⁺ / 3 Check mass (M + 3H) ³⁺ / 3 Ac-GGN4-23W-NH ₂ C ₁₁₅ H ₁₈₉ O ₂₉ N ₃₁ 823.8 824 Ac-GGN4-15W1-NH ₂ C ₈₄ H ₁₃₈ O ₁₈ N ₂₂ 582.0 582 Ac-GGN4-15W1-Stp-NH ₂ C ₉₃ H ₁₅₂ O ₁₈ N ₂₂ 622.7 623 Ac- GGN4-15W2-Stp -NH ₂ C ₉₂ H ₁₄₈ O ₂₀ N ₂₂ 628.0 628 Ac- GGN4-15W3-Stp -NH ₂ C ₈₈ H ₁₄₈ O ₂₀ N ₂₂ 612.0 612

The peptides of Table 1 were synthesized for the experiment. Specifically, peptides were treated with 0.6 mmol / g Fmoc chemistry technique. Lt; RTI ID = 0.0 > ^5A < / RTI > The dried resin (50 mg, 30 μmol) was called for 10 minutes in NMP before use. The Fmoc-protecting group was removed by treatment with 25% piperidine in NMP (2 x 10 min). Naturally occurring amino acids were coupled for 30 minutes using HCTU (4.75 equivalents) as activator, 5 equivalents of Fmoc-amino acid and 10 equivalents of DIEA in NMP. Coupling of 5 equivalents of olefinic amino acid was carried out for 2 hours using Fmoc-amino acid (3 equivalents), PyClock (3 equivalents), 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 (1 x 2 minutes).

Example  3: Metathesis reaction ( Metathesis ) And tablets

A ring-closing metathesis of resin-bound N-Fmoc, a side-chain protected peptide, was performed using a 20 mol% Grubbs I catalyst. The reaction was monitored by dissolution of the peptide obtained from the resin aliquot by liquid chromatography-mass spectrometry (LC / MS). After draining the reaction solution, the resin was washed with DCE (dichloromethane) (3 x 2 min) and then with DCM (3 x 2 min). After the final deprotection reaction, the N-terminal amino group was treated with 30 equivalents of acetic anhydride and 60 equivalents of DIEA in NMP for 45 min. The resin was washed with DCM and DMF and dried in 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 supernatant was collected on a centrifuge and dissolved in a 1: 1 mixture of acetonitrile and water. The product was purified by high-performance liquid chromatography using a Zorbax C18 column (Agilent, 5 탆, 9.4 x 250 mm) and then purified by LC / MS (Agilent, API 4000) Respectively.

E2EM23W. ESIMS m / z calculated for C ₁₁₅ H ₁₈₉ N ₂₉ O ₃₁ [M + 3H] 3 ⁺ / 3 823.82, found 824.15.

E2EM15W. ESIMS m / z calcd for C ₈₄ H ₁₃₈ N ₁₈ O ₂₂ [M + 3H] 3 ⁺ / 3 582.03, found 582.40.

E2EM15W-S1. ESIMS m / z calculated for C ₉₃ H ₁₅₂ N ₁₈ O ₂₂ [M + 3H] 3 ⁺ / 3 622.73, measured 623.10.

E2EM15W-S2. ESIMS m / z calculated for C ₉₂ H ₁₄₈ N ₂₀ O ₂₂ [M + 3H] 3 ⁺ / 3 628.05, found 628.40.

E2EM15W-S3. ESIMS m / z calculated for C ₈₈ H ₁₄₈ N ₂₀ O ₂₂ [M + 3H] 3 ⁺ / 3 612.05, measured .612.35.

Example  3: Antibacterial for confirming antimicrobial activity Assay

The above line of E2EM peptide was used to measure the antimicrobial activity against selected gram-positive and gram-negative bacteria.

Specifically, the antimicrobial assay was performed by measuring the minimal inhibitory concentration (MIC) using a standard broth microdilution method. The antimicrobial activity of each peptide was determined using three strains of Gram-positive bacteria ( Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538p, Staphylococcus epidermis ATCC 12228) and six strains of Gram-negative bacteria ( Escherichia E. coli ATCC 25922, Shigella dysentariae ATCC 9752, Salmonella typhimurium ATCC 14028, Klebsiella pneumonia ATCC 10031, Proteus mirabilis ATCC 25933, Pseudomonas aeruginosa ATCC 27853). These strains were inoculated into 2 ml LB (Luria-Bertani) broth and incubated overnight at 37 ° C.

The peptide was dissolved in distilled water. The peptide solution was prepared by two-fold dilution in a 96 well round bottom microtitre plate at 0.2 / / mL to 200 / / mL. The wells containing the peptides were incubated in each well with a bacterial suspension of 10 ⁶ to 10 ⁸ colony-forming units / ml. LB broth was used to dilute the peptide solution and to dilute the bacterial inoculum. After incubation at 37 占 폚 for 24 hours, the minimum inhibitory concentration (MIC) of the wells was analyzed. The MIC was defined as the lowest peptide concentration that completely inhibited cell growth. The experiment was carried out twice. All bacterial strains were distributed from KRIBB (Korea Research Institute of Bioscience and Biotechnology, Korea) of KCTC (Korean Collection of Type Culture).

The results of the antimicrobial assay are shown in Table 2 below.

Bacterial strain E2EM23W E2EM15W E2EM15W-S1 E2EM15W-S2 E2EM15W-S3 Gram (+) Bacillus subtilis ATCC6633 50 200 3.13 6.25 6.25 Staphylococcus aureus ATCC6538p 100 100 3.13 6.25 6.25 Staphylococcus epidermis ATCC12228 > 200 > 200 > 200 > 200 100 Gram  (-) Escherichia coli ATCC 25922 > 200 200 > 200 100 100 Shigella dysentariae ATCC9752 > 200 > 200 > 200 50 50 Salmonella typhimurium ATCC14028 > 200 > 200 > 200 > 200 > 200 Klebsiella pneumoniae ATCC10031 > 200 > 200 > 200 50 50 Proteus mirabilis ATCC 25933 > 200 > 200 > 200 > 200 > 200 Pseudomonas aeuginose ATCC 27853 > 200 > 200 > 200 200 > 200 Minimum inhibitory concentration ( Minimum Inhibitory Concentrations ) (/ / mL )

As shown in Table 2, under the assay conditions used in this experiment, the 23-residue non-stapled control E2EM23W showed a minimum inhibitory concentration (MIC, / / ml) of 50 and 100 for Bacillus subtilis and Staphylococcus aureus But did not exhibit detectable antimicrobial activity against other bacteria including all types of Gram-positive bacteria. In addition, E2EM15W, a 15-residue non-stapled control, showed no activity in this assay. In contrast, all of the stapled peptides exhibited significantly increased antimicrobial activity against both species of bacteria. In particular, E2EM15W-S1 showed the highest antimicrobial activity compared to other peptides: the minimum inhibitory concentration (MICs, / / mL) of this peptide was lower than that of Bacillus subtilis and Staphylococcus aureus, respectively. E2EM15W-S1 is a stapled derivative of E2EM15W that is inactive, and these results demonstrate that the hydrocarbon staple system can enhance the antimicrobial activity of the peptide. However, E2EM15W-S1 did not exhibit antimicrobial activity against Staphylococcus epidermis, another Gram-positive strain, and all Gram-negative bacteria.

Other stapled analogues E2EM15W-S2 and E2EM15W-S3 show slightly less effect than E2EM15W-S1, but still Bacillus subtilis and Staphylococcus About aureus And exhibited significantly increased antimicrobial activity. However, unlike E2EM15W-S1, E2EM15W-S2 and E2EM15W-S3 is Shigella dysenteriae, and Klebsiella pneumonia. < / RTI >

Example  4: morphological preference ( Conformational preferences ) For confirmation Circular polarization  Dichroism ( Circular dichroism )

To determine whether the increased antimicrobial activity of the stapled analogs correlates with their spiral stability, morphological preference was tested using a far-UV circular dichroism spectrum.

Specifically, the peptide was dissolved in 25 mM potassium phosphate buffer (pH 6.5) and the concentration was measured by absorption spectroscopy at ₂₈₀ nm (extinction coefficient against tryptophan,? ₂₈₀ = 5690 cm ^-1 ). The circular polarization dichroism spectrum was collected on a Chirascan HP dipolar circular dichroism spectrum with a temperature controller using the following standard measurement parameters: 1 nm bandwidth, and 0.1 cm path length. All spectra were converted to equilibrium scales of molar ellipticity after excluding the background. Curves were flattened to standard parameters.

In a previous study, E2EM23W was found to be an irregular coil in aqueous solution, but it was found to be in an α-helical form in membrane-like environments. Hydrocarbon staple systems are an effective chemical means of transforming peptides into an a-helical form through covalent bonds, the helicity of stapled peptides being relatively independent of the environment. Thus, the present inventors analyzed peptide samples in aqueous solutions. The helicity of each peptide was measured based on the ellipticity of the average residues, which is used extensively for the quantitative measurement of the peptide spiral. The results are shown in Fig.

As shown in Figure 3, E2EM23W exhibited typical circular dichroism spectrum of random coils with only a spiral content of 14%. The circular dichroism showed that E2EM15W had a lower spiral under the same conditions, with a spiral content of only 4%; This indicates that the shorter the peptide, the more difficult it is to form an a-helical. In contrast, the stapled three analogs of E2EM15W exhibited significantly increased helix content, demonstrating the high efficiency of helix-stabilization by all hydrocarbon staple techniques. E2EM15W-S1, the most active analogue in the antimicrobial assay, exhibited the highest helicity (47%), indicating a close relationship between spiral and antibacterial activity. Other analogues E2EM15W-S2 and E2EM15W-S3 with oct-4-enyl staple in the same position as E2EM15W-S1 showed lesser helix content, 27% and 37%, respectively. This result implies that the substitution position of tryptophan in the stapled peptide scaffold also affects the helix-stabilization.

Example  5: Protease resistance ( Protease resistance ) Trypsin digestion for confirmation Assay

A significant feature of the stapled peptide is its increased resistance to protease digestion. The increased stability of the stapled peptide to protease digestion due to the extended form of the protease in the recognition of the peptide material is due to its morphological strength. A trypsin digestion assay was performed to determine if stapled analogs retained their important specificity.

Trypsin generally cleaves positively charged carboxyl groups such as lysine and arginine. Escululentin-2EM analogs contain a large number of lysine residues within the sequence, which are fundamentally vulnerable to trypsin protein degradation.

Specifically, 25 μl of trypsin solution (1 μm, Sigma) in digestion buffer (0.1 M NH ₄ HCO ₃ buffer, pH 8.0) was added to 250 μl of peptide solution (80 μM) in digestion buffer (substrate / enzyme = 1,600 / 1) and the resulting mixture was incubated at room temperature under rapid agitation (600 rpm). 50 μl of the digestion mixture was taken at 0, 15, 30, 45 and 60 minutes and quenched with 2 μl of trifluoroacetic acid. The residual substrate and the hydrolyzed product were quantified by LC-based pit detection at 280 nm. Each experiment was performed twice. Half-life (t _1/2) to lnS (S is a matter% is not cut) was determined by linear regression analysis from the plot of versus time (min), was used for Kaleida Graph (Synergy Software) (t 1/2 = ln2 / slope). Each peptide was treated with trypsin at an enzyme / substrate ratio of 1/800 at 25 ° C. The results are shown in FIG.

As shown in Fig. 4, at the first time point (15 min), the unstapled analogues E2EM23W and E2EM15W were completely degraded, while 90% of the E2EM15W-1 and 3 and E2EM15W-2 75% were found to be intact. More than half of E2EM15W-1 and 3 were not cleaved at 60 min after trypsin treatment. Despite the differences in their helix gender (47% vs 37%) and, E2EM15W-1 and 3 showed a similar protein hydrolytic stability, half-life (t _1/2) was 72 and 71 minutes respectively. In contrast was the most susceptible, compared with differently, for E2EM15W-2 shows the lowest spiral Castle, trypsin digestion ever other staple screen peptides according to (t _1/2 = 37 min). Taken together, the results show that the staple stability induced by hydrocarbon staple increases the resistance to protease digestion.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> Stapled gaegurin 4 derived peptide and composition for antibiotic          or anticancer comprising the same <130> 1-116 p <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 37 <212> PRT <213> Gaegurin 4 peptide <400> 1 Gly Ile Leu Asp Thr Leu Lys Gln Phe Ala Lys Gly Val Gly Lys Asp   1 5 10 15 Leu Val Lys Gly Ala Gln Gly Val Leu Ser Thr Val Ser Cys Lys              20 25 30 Leu Ala Lys Thr Cys          35 <210> 2 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> GGN4-23W <400> 2 Gly Ile Leu Asp Thr Leu Lys Gln Phe Ala Lys Gly Val Gly Lys Trp   1 5 10 15 Leu Val Lys Gly Ala Ala Gln              20 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> GGN4-15W1 <400> 3 Thr Leu Lys Gln Phe Ala Lys Gly Val Gly Lys Trp Leu Val Lys   1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> GGN4-15W1-Stp <220> <221> BINDING <222> (6) <223> hydrocarbon stapling cross linking with site 10 <220> <221> BINDING <10> <400> 4 Thr Leu Lys Gln Phe Xaa Lys Gly Val Xaa Lys Trp Leu Val Lys   1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> GGN4-15W2-Stp <220> <221> BINDING <222> (6) <223> hydrocarbon stapling cross linking with site 10 <220> <221> BINDING <10> <400> 5 Thr Leu Lys Gln Phe Xaa Lys Gly Trp Xaa Lys Asp Leu Val Lys   1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> GGN4-15W3-Stp <220> <221> BINDING <222> (6) <223> hydrocarbon stapling cross linking with site 10 <220> <221> BINDING <10> <400> 6 Thr Leu Lys Gln Trp Xaa Lys Gly Val Xaa Lys Asp Leu Val Lys   1 5 10 15

Claims (14)

A doggulin-derived peptide consisting of the amino acid sequence of SEQ ID NO: 3; Or a peptide derivative in which at least one of amino acids 1 to 15 in the amino acid sequence of SEQ ID NO: 3 is substituted with tryptophan, the amino acid position of any one of the first to eleventh amino acids and the amino acid position after the fourth amino acid residue thereof are stapled with hydrocarbons stapling peptide. 3. The staple peptide of claim 1, wherein the hydrocarbon is an oct-4-enyl group. The stapled peptide of claim 1, wherein the substituted peptide is one wherein the 5, 9, or 12 th amino acid position is replaced with tryptophan. The stapled peptide according to claim 1, wherein the staple is stapled at a 6th amino acid position and a 10th amino acid position. 5. The stapled peptide of claim 4, wherein said stapled peptide comprises an amino acid sequence of any one of SEQ ID NOS: 4 to 6. A pharmaceutical composition for antimicrobial use comprising the stapled peptide according to any one of claims 1 to 5. The antimicrobial pharmaceutical composition according to claim 6, wherein the composition exhibits an antibiotic effect against pathogenic bacteria, viruses, pathogenic yeast or fungi. [Claim 7] The pharmaceutical composition for antimicrobial use according to claim 7, wherein the pathogenic bacterium is Gram positive bacteria or Gram negative bacteria. The method according to claim 8, wherein the Gram positive bacteria is Bacillus sub-blocks bus (Bacillus subtilis), Staphylococcus aureus (Staphylococus aureus ) or Staphylococcus ( Staphylococcus epidermis) epidermis ). &lt; / RTI &gt; The method according to claim 8, wherein the Gram-negative bacteria are E. coli (Escherichia coli , Sigella &lt; RTI ID = 0.0 &gt; dysentariae ) or Pneumoniae ( Klebsiella pneumoniae ). &lt; / RTI &gt; The system according to claim 7, wherein the pathogenic yeast or fungi Candida albicans (Candida albicans), Aspergillus Hugh migeo tooth (Aspergillus humigatus), in my process serie busy as Saccharomyces (Saccharomyces cerevisiae , or Cryptococcus neoformans . An antimicrobial food additive comprising the stapled peptide of any one of claims 1 to 5. An antimicrobial feed additive comprising the stapled peptide of any one of claims 1 to 5. A pharmaceutical composition for preventing or treating cancer comprising the stapled peptide according to any one of claims 1 to 5.

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