Stapling peptide with antibacterial effect and application thereof

Abstract

The invention belongs to the field of polypeptide drugs, and particularly relates to a stapling peptide with an antibacterial effect and application thereof. According to the present invention, Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 is adopted as a peptide chain template, and an amino acid residue 11L is replaced by K so as to obtain the target stapled peptide SLP-6; compared with the template polypeptide, the hemolytic activity of the obtained stapled peptide SLP-6 is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drugs, and particularly relates to a stapled peptide with antibacterial effects and its applications. Compared with the template polypeptide, the hemolytic property of this polypeptide has been improved. Background Art

[0002] Antibacterial peptides are a class of polypeptide substances with bacteriostatic activities, having various advantages, including being safe and non-toxic, having a broad antibacterial spectrum, good stability, low bactericidal concentration, small molecular weight, weak allergenicity, etc., and becoming one of the research hotspots in the biological field. In recent years, with the abuse of antibiotics, problems such as bacterial drug resistance, drug residues, and environmental pollution have become increasingly serious. In the situation where the problems of antibiotic residues and bacterial drug resistance are becoming increasingly serious, antibacterial peptides are highly expected as alternatives to antibiotics due to their low allergenicity, no residues, low bactericidal concentration, and difficulty in generating drug resistance.

[0003] Antibacterial peptides (AMPs) are a class of host defense peptides that can kill bacteria through various pathways, such as bacterial membrane lysis, oxidative damage, inhibition of biofilm formation, etc., without involving the binding of specific proteins and being difficult to generate drug resistance, and can combat bacterial infections ineffective against traditional antibiotics. Antibacterial peptides (AMPs) have high binding affinity and selectivity for microbial membranes. The amino acid composition of AMPs is a key factor in their mechanism of action and selectivity, which determines the physicochemical properties of the peptide, such as charge, amphiphilicity, etc.; after contacting with the plasma membrane, AMPs usually undergo structural changes, adopting a defined secondary structure or oligomerizing into aggregates. When the secondary structure of AMPs forms, the separation of hydrophilic and hydrophobic residues leads to the penetration of the peptide into the plasma membrane, so the presence of hydrophobic amino acids is necessary.

[0004] Patent CN117486993A discloses a template polypeptide SLP - 0: Ac - LKRVWKRVFKLLKS5YWRS5LKKPVR - NH2, which has good bacteriostatic effects on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. The inventors found in further research that SLP - 0 has strong hemolytic property. Hemolytic property has become one of the key factors restricting the further development and application of this antibacterial peptide. Summary of the Invention

[0005] The purpose of the present invention is to provide a stapled peptide for the deficiencies in the prior art. Compared with the template polypeptide, the hemolytic property of this polypeptide has been improved, and it has significant bacteriostatic activity, having the application prospect of developing into a new antibacterial drug.

[0006] Another purpose of the present invention is to provide the uses of the above - mentioned stapled peptide.

[0007] To achieve the above - mentioned first purpose, the technical solution adopted by the present invention is: Stapled peptide, wherein the stapled peptide is selected from one of the following: a) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 1L is replaced by K; b) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 4V is replaced by K; c) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 5W is replaced by K; d) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 8V is replaced by K; e) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 9F is replaced by K; f) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 11L is replaced by K; g) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 12L is replaced by K; h) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 15Y is replaced by K; i) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 16W is replaced by K; j) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 19L is replaced by K; k) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 22P is replaced by K; l) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, wherein amino acid residue 23V is replaced by K; The sequences of the template polypeptide and the modified stapled peptide in the present invention are shown in Table 1.

[0008] Preferably, the above-mentioned stapled peptide is selected from one of the following: a) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 1L is replaced by K; c) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 5W is replaced by K; d) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 8V is replaced by K; e) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 9F is replaced by K; f) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 11L is replaced by K; g) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 12L is replaced by K; h) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 15Y is replaced by K; i) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 16W is replaced by K.

[0009] More preferably, for the above stapled peptide, the stapled peptide is selected from one of the following: d) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 8V is replaced by K; f) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 11L is replaced by K; g) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 12L is replaced by K; h) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 15Y is replaced by K.

[0010] Use of the above stapled peptide in the preparation of antibacterial drugs.

[0011] In the use of the above stapled peptide in the preparation of antibacterial drugs, the bacterium is Streptococcus pneumoniae (SP).

[0012] In the application of the above-mentioned stapled peptide in the preparation of antibacterial drugs, the bacterium is methicillin-resistant Staphylococcus aureus (MRSA).

[0013] In the application of the above-mentioned stapled peptide in the preparation of antibacterial drugs, the bacterium is Pseudomonas aeruginosa (PA).

[0014] In the application of the above-mentioned stapled peptide in the preparation of antibacterial drugs, the bacterium is Klebsiella pneumoniae (KP).

[0015] The advantages of the present invention are as follows: 1. The present invention provides a series of new stapled peptides. By structurally modifying SLP-0 (Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2) to obtain new stapled peptides, on the basis of retaining the key residues, Lys (lysine) is used to replace the original amino acids at the non-key residue positions of the peptide chain, and after cyclization, a stapled peptide with stable structure is obtained. The hemolysis experiment results show that compared with the template peptide, the hemolytic properties of SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8, and SLP-9 polypeptides of the present invention can be significantly improved. Among them, the hemolytic properties of LP-4, SLP-6, SLP-7, and SLP-8 have been significantly improved.

[0016] 2. The in vitro antibacterial experiment results show that the antibacterial activity of the stapled peptides of the present invention is basically the same as that of the template polypeptide, and it can significantly inhibit the growth and reproduction of SP (Streptococcus pneumoniae), MRSA (methicillin-resistant Staphylococcus aureus), PA (Pseudomonas aeruginosa), and KP (Klebsiella pneumoniae), and has the application prospect of developing into a new type of antibacterial drug. Among them, the antibacterial activity of SLP-1 against Pseudomonas aeruginosa is better than that of the template polypeptide.

[0017] 3. The peptide anti-enzyme digestion experiment results show that the anti-protease digestion ability of SLP-1 of the present invention is the same as that of the template polypeptide. Description of the Drawings

[0018] Figure 1 It is the synthesis route diagram of the stapled peptide of the present invention.

[0019] Figure 2Schematic diagram of the amino acid sequence of SLP-0 and its characterization map. SLP-0 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-0 was obtained with a separation rate of 15.1%. Among them, A is the amino acid sequence of SLP-0, B is the HPLC map of SLP-0, analysis column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-0.

[0020] Figure 3 Schematic diagram of the amino acid sequence of SLP-1 and its characterization map. SLP-1 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-1 was obtained with a separation rate of 26.1%. Among them, A is the amino acid sequence of SLP-1, B is the HPLC map of SLP-1, analysis column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-1.

[0021] Figure 4 Schematic diagram of the amino acid sequence of SLP-2 and its characterization map. SLP-2 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-2 was obtained with a separation rate of 25.18%. Among them, A is the amino acid sequence of SLP-2, B is the HPLC map of SLP-2, analysis column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-2.

[0022] Figure 5Schematic diagram of the SLP-3 amino acid sequence and its characterization map. SLP-3 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-3 was obtained with a separation rate of 19.39%. Among them, A is the amino acid sequence of SLP-3, B is the HPLC map of SLP-3, analysis column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-3.

[0023] Figure 6 Schematic diagram of the SLP-4 amino acid sequence and its characterization map. SLP-4 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes). SLP-4 was obtained with a separation rate of 31.58%. Among them, A is the amino acid sequence of SLP-4, B is the HPLC map of SLP-4, analysis column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-4.

[0024] Figure 7 Schematic diagram of the SLP-5 amino acid sequence and its characterization map. SLP-5 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-5 was obtained with a separation rate of 21.56%. Among them, A is the amino acid sequence of SLP-5, B is the HPLC map of SLP-5, analysis column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-5.

[0025] Figure 8Schematic diagram of the SLP-6 amino acid sequence and its characterization map. SLP-6 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-6 was obtained with a separation rate of 17.41%. Among them, A is the amino acid sequence of SLP-6, B is the HPLC map of SLP-6, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-6.

[0026] Figure 9 Schematic diagram of the SLP-7 amino acid sequence and its characterization map. SLP-7 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-7 was obtained with a separation rate of 18.71%. Among them, A is the amino acid sequence of SLP-7, B is the HPLC map of SLP-7, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-7.

[0027] Figure 10 Schematic diagram of the SLP-8 amino acid sequence and its characterization map. SLP-8 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-8 was obtained with a separation rate of 12.34%. Among them, A is the amino acid sequence of SLP-8, B is the HPLC map of SLP-8, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-8.

[0028] Figure 11Schematic diagram of the SLP-9 amino acid sequence and its characterization map. SLP-9 was purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-9 was obtained with a separation rate of 25.12%. Among them, A is the amino acid sequence of SLP-9, B is the HPLC map of SLP-9, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-9.

[0029] Figure 12 Schematic diagram of the SLP-10 amino acid sequence and its characterization map. SLP-10 was purified by HPLC (purification conditions: 10%-65% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-10 was obtained with a separation rate of 18.52%. Among them, A is the amino acid sequence of SLP-10, B is the HPLC map of SLP-10, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-10.

[0030] Figure 13 Schematic diagram of the SLP-11 amino acid sequence and its characterization map. SLP-11 was purified by HPLC (purification conditions: 10%-65% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes), and SLP-11 was obtained with a separation rate of 19.76%. Among them, A is the amino acid sequence of SLP-11, B is the HPLC map of SLP-11, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-11.

[0031] Figure 14Schematic diagram of the SLP-12 amino acid sequence and its characterization map. SLP-12 was purified by HPLC (purification conditions: 10%-65% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes) to obtain SLP-12, with a separation rate of 21.38%. Among them, A is the amino acid sequence of SLP-12, B is the HPLC map of SLP-12, analysis column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ = 214 nm). C is the mass spectrum of SLP-12.

[0032] Figure 15 Results of the hemolysis experiment of the stapled peptides of the present invention.

[0033] Figure 16 Results of the degradation experiments of the stapled peptides of the present invention by trypsin (left) and chymotrypsin (right). Detailed implementation manners

[0034] The following further illustrates the present application in conjunction with the accompanying drawings and specific implementation manners, so that those skilled in the art can better understand the present application. However, these examples are only used to illustrate the present invention and not to limit the scope of the present invention, that is, the described examples are only a part of the embodiments of the present invention, rather than all the embodiments.

[0035] Therefore, the following detailed description of some embodiments of the present invention provided is not intended to limit the scope of the claimed present invention, but is merely selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0036] The present invention designed and synthesized 12 stapled peptides according to the amino acid sequence of the template polypeptide SLP-0: Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2. The structure and molecular weight of the peptides are as Figure 1 shown.

[0037] The sources of the experimental materials involved in the embodiments of the present invention are as follows: Fmoc-amino acids and Rinkamide MBHA amino resin were purchased from Nankai Synthesis Co., Ltd.; NMP, DIC, Oxyme, TFA, and acetonitrile (chromatographic grade) were purchased from the exploration platform; DMF, anhydrous ether, DCM, DCE, piperidine, and phenol were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. Shanghai; S5 was purchased from Beijing Okinas Biochemical Technology Co., Ltd.

[0038] In the present invention, the abbreviations involved are explained as follows: Fmoc: 9-fluorenylmethyloxycarbonyl; DCM: dichloromethane; DCE: 1,2-dichloroethane; DMF: N,N-dimethylformamide; Oxyme: EthylCyanoglyoxylate-2-Oxime; DIC: N,N'-diisopropylcarbodiimide; S5: (S)-2-amino-2-methyl-4-pentenoic acid TFA: trifluoroacetic acid; EDT: 1,2-ethanedithiol; GrubbsⅠ: Benzylidene bis(tricyclohexylphosphine) ruthenium dichloride; MS: mass spectrometry; HR-Q-TOF-MS: high resolution matrix assisted laser desorption ionization time of flight mass spectrometry.

[0039] Example 1 Preparation of stapled peptide based on SLP-0 1. Synthesis of stapled peptide The synthesis route is as Figure 1 shown: (1) Preparation of Compound 1 Take 400 mg of amino resin (loading capacity is 0.30 mmol·g -1 ) and add it to the solid-phase synthesis reaction tube. Soak the resin in DCM for 30 min to fully swell the resin, and then drain it for later use.

[0040] Add 7 ml of 20% piperidine-DMF solution to the resin, shake at 35 °C for 5 min × 2 to remove the Fmoc protecting group on the resin, and wash the resin with DMF, DCM, and DMF three times each.

[0041] (2) Preparation of Compound 2 Mix the first amino acid in the sequence (1 mmol), Oxyme (142 mg, 1 mmol), and DIC (200 μL) in 7 ml of DMF, add it to the resin, and shake at 60 °C for 20 min (the amino acid after S5 reacts for 1 h and the reaction is repeated once), and wash the resin with DMF, DCM, and DMF three times each.

[0042] (3) Preparation of Compound 3 Repeat the procedures of (1) and (2). According to the polypeptide sequence, dissolve Fmoc amino acid (1 mmol), Oxyme (142 mg) and DIC (200 μl) in 7 ml of DMF successively, then add them to the resin, and react with shaking at 60 °C for 30 min. Repeat the process of removing Fmoc protection → condensation → removing Fmoc protection until all amino acids are condensed. After the last amino acid removes the Fmoc protecting group, add 7 ml of the mixture of acetic anhydride:DIEA:DMF (1:1:8), shake at 37 °C for 15 min, drain, add the acetylation reagent again, react for 15 min, wash the resin with DMF, DCM and DMF three times respectively, and then dry the resin by pumping vacuum with an oil pump.

[0043] (4) Preparation of Compound 4 After the resin is completely dried, add a 1,2-dichloroethane solution (7 ml) of Grubbs I (58 mg) reagent, react with shaking at 37 °C twice, 2 h each time. After the reaction is completed, wash the resin with DMF, DCM and DMF three times respectively, and dry the resin by pumping vacuum with an oil pump.

[0044] (5) Preparation of the target compound First, wash and drain the resin, add 20 mL of TFA:phenol:H2O:benzyl mercaptan:EDT = 82.5:5:5:5:2.5 (V / V / V / V), react with shaking at 37 °C for 3 h, filter, wash the resin with a little TFA, and collect the filtrate. Bubble with argon to blow away the excess TFA, pour it into ice ether for precipitation and centrifugation, then discard the supernatant. Repeat the above steps, wash and centrifuge with ice ether three times, and let it dry naturally under the fume hood to obtain the crude polypeptide sample.

[0045] 2. Purification of the stapled peptide sample Dissolve the crude polypeptide in a mixed solvent of acetonitrile and water, and purify it by reverse-phase preparative RP-HPLC to obtain the purified stapled peptide pure product. The separation conditions are as follows: Instrument: Shimadzu LC-20A reverse-phase high-performance liquid chromatograph; Chromatographic column: Ultimate XB-C18, 21.2×250 mm, 5 μm; Mobile phase: Mobile phase A is an acetonitrile solution with 0.1% TFA by volume, and mobile phase B is an aqueous solution with 0.1% TFA by volume; Steps and parameters: Elute with 90% B for 3 min, then elute with 90% B to 50% B for 40 min; the flow rate is 8 ml / min, the injection volume is 3 ml, and the detection wavelengths are 214 nm and 254 nm.

[0046] Identification and structural analysis of the product of Example 2 The product obtained in Step 2 of Example 1 was identified by reverse-phase HPLC and its structure was analyzed by HR-Q-TOF-MS. The chromatographic mobile phase was acetonitrile and water. Mobile phase A was an acetonitrile solution containing 0.1% TFA by volume, and mobile phase B was an aqueous solution containing 0.1% TFA by volume. Gradient elution was performed (0 - 2 min, mobile phase B: 90%; 3 - 25 min, mobile phase B: 90% - 10%); the flow rate was 1.0 mL·min -1 ; the detection wavelengths were 214 nm and 254 nm, and the injection volume was 24 μl. It was determined that the retention time was consistent with that of the main peak of the crude product, and the purity of the stapled peptide prepared by this method was >95%. The results of mass spectrometry analysis are as Figures 2 - 14 shown. After analysis, the structure of the stapled peptide obtained is shown in Table 1.

[0047] Example 3 Hemolysis experiment of the stapled peptide of the present invention After obtaining high-purity and structurally homogeneous polypeptides, 2% mouse red blood cell suspension in Tris-buffered saline solution (v / v) was treated with different concentrations of SLP-0 and its derivatives (6.25, 12.5, 25, 50, and 100 μM) in a 96-well plate at room temperature for 2 h. Then 1% Triton X-100 and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. The absorbance of the supernatant at 540 nm was measured using a microplate spectrophotometer. The hemolysis rate was calculated using the following formula: Hemolysis rate % = (ODsample - OD PBS) / (OD Triton - OD PBS) × 100%.

[0048] The results are shown in Table 2 and Figure 15 .

[0049] The results of the hemolysis experiment showed that compared with the template peptide, the hemolytic properties of the stapled peptides SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8, and SLP-9 of the present invention could be significantly improved. Among them, the hemolytic properties of SLP-4, SLP-6, SLP-7, and SLP-8 were significantly improved.

[0050] Through data analysis, it was found that except for insignificant differences between individual data groups (such as SLP-1 and SLP-5, SLP-2 and SLP-11, etc.), there were significant differences between the two data groups of SLP-2 and SLP-3, and SLP-2 and SLP-9, and extremely significant differences were shown between any other two groups.

[0051] Example 4 Experiment on the stapled peptide of the present invention inhibiting Gram-positive bacteria and Gram-negative bacteria In vitro anti-drug-resistant bacteria test: Prepare solid LB medium, plate it after autoclaving, and prepare LB liquid medium for standby in a 4°C refrigerator. Spread the bacterial solution on the solid LB medium and incubate it upside down in a 37°C incubator overnight; pick monoclonal colonies, add them to 3 mL of liquid LB medium, and culture them at 37°C and 220 rpm in a constant temperature shaker for 6 h to grow the bacteria to the logarithmic phase; take 1 mL of the bacterial solution, centrifuge it at 4000 rpm for 5 min, discard the supernatant, add PBS, and adjust the bacterial solution concentration to 2×106 CFU / mL by OD value. Add antibacterial peptides with different concentrations to a 96-well plate, and at the same time add the bacterial solution to the 96-well plate, incubate at 37°C for 8 h, detect with an enzyme-linked immunosorbent assay (ELISA) reader at 595 nm, repeat three times, and statistically analyze the MIC value. The results are shown in Table 3.

[0052] Table 3 results show that the stapled peptide of the present invention has basically the same antibacterial activity as the template polypeptide, can significantly inhibit the growth and reproduction of SP (Streptococcus pneumoniae), MRSA (methicillin-resistant Staphylococcus aureus), PA (Pseudomonas aeruginosa), and KP (Klebsiella pneumoniae), and has the application prospect of developing into a new antibacterial drug. Among them, the antibacterial activity of SLP-1 against Pseudomonas aeruginosa is better than that of the template polypeptide.

[0053] Example 5 Proteolytic resistance experiment of the stapled peptide of the present invention Dissolve the polypeptides in PBS buffer solution (50 mM, pH = 7.4) to a final concentration of 1 mM. Since the principle of trypsin degrading polypeptides is that trypsin mainly acts on the peptide bonds at the carboxyl terminus of arginine or lysine and then specifically cleaves the polypeptides. Therefore, trypsin is dissolved in PBS buffer solution (50 mM, containing 2 mM CaCl2, pH = 8) to a final concentration of 5 ng / mL. Then incubate the peptide solution (100 μL) with the trypsin solution (1 mL) at room temperature. Mark at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 h, take 100 μL of the digestion mixture, and then quench it with 20 μL of hydrochloric acid (1 M). Detect the solution of trypsin peptide fragments by HPLC at different times to determine the protease degradation rate at 214 nm. The results are shown in Figure 16 .

[0054] Figure 16 The results show that the proteolytic resistance ability of the stapled peptide SLP-1 of the present invention is comparable to that of the template polypeptide.

Claims

1. A stapled peptide, characterized in that, The stapled peptide is as follows: Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as the peptide chain template, where amino acid residue 11L is replaced by K.

2. Use of the stapled peptide according to claim 1 in the preparation of an antibacterial drug, characterized in that, The bacterium is Streptococcus pneumoniae.

3. Use of the stapled peptide according to claim 1 in the preparation of an antibacterial drug, characterized in that, The bacterium is methicillin-resistant Staphylococcus aureus.

4. Use of the stapled peptide according to claim 1 in the preparation of an antibacterial drug, characterized in that, The bacterium is Pseudomonas aeruginosa.

5. Use of the stapled peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that, The bacterium is Klebsiella pneumoniae.

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