Antibacterial stapling peptide and application thereof

Abstract

The invention belongs to the field of polypeptide drugs, and particularly relates to an antibacterial stapling peptide and application thereof. According to the present invention, Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 is adopted as a peptide chain template, and an amino acid residue 12L is replaced by K so as to obtain the target stapled peptide SLP-7; compared with the template polypeptide, the hemolytic activity of the obtained stapled peptide SLP-7 is obviously improved.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide drugs, and in particular relates to an antibacterial stapling peptide and application thereof. Compared with a template polypeptide, the hemolytic property of the polypeptide is improved. Background Art

[0002] Antimicrobial peptides are a class of polypeptide substances with antibacterial activity. They have many advantages, including safety and non-toxicity, broad antibacterial spectrum, good stability, low bactericidal concentration, small molecular weight, weak sensitization, etc., and have become one of the research hotspots in the biological field. In recent years, with the abuse of antibiotics, bacterial resistance, drug residues and environmental pollution have become increasingly serious. In the situation where antibiotic residues and bacterial resistance are becoming more and more serious, antimicrobial peptides are expected to be a substitute for antibiotics because of their advantages such as low sensitization, no residue, low bactericidal concentration, and low resistance.

[0003] Antimicrobial peptides (AMPs) are a class of host defense peptides that can kill bacteria through a variety of pathways, such as biofilm lysis, oxidative damage, and inhibition of biofilm formation. They do not involve binding to specific proteins, are not prone to drug resistance, and can fight bacterial infections that are ineffective against traditional antibiotics. Antimicrobial peptides (AMPs) have high binding 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 and amphipathicity. After contact with the plasma membrane, AMPs usually undergo structural changes, adopting a certain secondary structure or oligomerizing into aggregates. When the secondary structure of AMP is formed, the separation of hydrophilic and hydrophobic residues causes the peptide to penetrate 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 a good antibacterial effect on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii. The inventors found in further research that SLP-0 has strong hemolytic activity. Hemolytic activity has become one of the key factors restricting the further development and application of this antimicrobial peptide. Summary of the invention

[0005] The purpose of the present invention is to provide a stapling peptide in view of the deficiencies in the prior art. Compared with the template polypeptide, the hemolytic property of the polypeptide is improved, and the polypeptide has significant antibacterial activity, and has the application prospect of being developed into a new type of antibacterial drug.

[0006] Another object of the present invention is to provide uses of the stapled peptide.

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

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

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

[0010] The use of the above-mentioned stapled peptide in the preparation of antibacterial drugs.

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

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

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

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

[0015] The advantages of the present invention are: 1. The present invention provides a series of new stapled peptides. The new stapled peptides are obtained by structurally modifying SLP-0 (Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2). On the basis of retaining the key residues, Lys (lysine) is used to replace the original amino acid at the non-key residue position of the peptide chain, and the structure-stable stapled peptide is obtained after cyclization. The results of the hemolytic experiment show that compared with the template peptide, the hemolytic properties of the stapled peptides of the present invention, SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8, and SLP-9 polypeptides 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 results of in vitro antibacterial experiments show that the antibacterial activity of the stapled peptides of the present invention is basically the same as that of the template polypeptide, and 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 being developed 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 results of the peptide anti-enzyme hydrolysis experiment show that the stapled peptide SLP-1 of the present invention has the same anti-proteolytic ability as the template polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The synthetic route of the stapled peptide of the present invention is shown in FIG.

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

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

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

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

[0023] Figure 6 The diagram is a schematic diagram of the amino acid sequence of SLP-4 and its characterization spectrum. SLP-4 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), 55 minutes on a Welch C18 column). 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 spectrum of SLP-4, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214nm). C is the mass spectrum of SLP-4.

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

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

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

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

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

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

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

[0031] Fig.14The following is a schematic diagram of the amino acid sequence of SLP-12 and its characterization spectrum. SLP-12 was purified by HPLC (purification conditions: 10%-65% CH3CN (0.1% TFA) in H2O (0.1% TFA), 55 minutes on a Welch C18 column) 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 spectrum of SLP-12, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214nm). C is the mass spectrum of SLP-12.

[0032] Fig.15 This is the hemolytic experiment result of the stapled peptide of the present invention.

[0033] Fig.16 These are the results of the degradation experiments of the stapled peptide of the present invention using trypsin (left) and chymotrypsin (right). DETAILED DESCRIPTION

[0034] The present application will be further described below in conjunction with the accompanying drawings and specific implementation methods so that those skilled in the art can better understand the present application. However, these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. That is, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0035] Therefore, the following detailed description of some embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but is only a selected embodiment of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0036] The present invention designs and synthesizes 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 shown in Figure 1 shown.

[0037] The sources of the experimental materials involved in the embodiments of the present invention are as follows: Fmoc-amino acid and RinkamideMBHA 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 analytical grade and purchased from Sinopharm Chemical Reagent Shanghai Co., Ltd.; S5 was purchased from Beijing Okainas Biochemical Technology Co., Ltd.

[0038] In the present invention, the abbreviations involved are explained as follows: Fmoc: fluorenylmethoxycarbonyl; 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-pentanoic acid TFA: trifluoroacetic acid; EDT: 1,2-ethanedithiol; GrubbsⅠ: phenylmethylenebis(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 SLP-0-based stapled peptides 1. Synthesis of stapled peptides Synthesis route such as Figure 1 As shown: (1) Preparation of compound 1 Take 400 mg of amino resin (loaded with 0.30 mmol g -1 ) was added into the solid phase synthesis reaction tube, soaked in DCM for 30 min to allow the resin to fully swell, and then drained 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 The first amino acid in the sequence (1 mmol), Oxyme (142 mg, 1 mmol) and DIC (200 μL) were mixed in 7 ml DMF, added to the resin and shaken at 60 °C for 20 min (one amino acid after S5 was reacted for 1 h, and the reaction was repeated once), and the resin was washed 3 times with DMF, DCM and DMF respectively.

[0042] (3) Preparation of compound 3 Repeat steps (1) and (2), dissolve Fmoc amino acid (1mmol), Oxyme (142mg) and DIC (200μl) in 7ml DMF according to the peptide sequence, then add to the resin, shake and react at 60℃ for 30min, repeat the process of removing Fmoc protection → condensation → removing Fmoc protection until all amino acids are condensed. After the last amino acid is freed from the Fmoc protecting group, add 7ml of acetic anhydride: DIEA: DMF (1:1:8) mixture, shake at 37℃ for 15min, drain, add acetylation reagent again, react for 15min, wash the resin with DMF, DCM and DMF three times each, and dry the resin by vacuum pump.

[0043] (4) Preparation of compound 4 After the resin is completely dried, add GrubbsⅠ (58 mg) reagent in 1,2-dichloroethane solution (7 ml) and shake the reaction twice at 37°C, each time for 2 h. After the reaction is completed, wash the resin with DMF, DCM, and DMF three times each, and dry the resin with an oil pump.

[0044] (5) Preparation of target compound First, wash and dry the resin, add 20 mL of TFA:phenol:H2O:thioanisole:EDT=82.5:5:5:5:2.5 (V / V / V / V), shake at 37°C for 3 hours, filter, wash the resin with a little TFA, and collect the filtrate. Use argon bubbling to blow away the excess TFA, pour in ice ether precipitation and centrifuge, discard the supernatant, repeat the above steps, wash the ice ether precipitation and centrifuge three times, place it in a fume hood and evaporate it naturally to obtain a crude polypeptide sample.

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

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

[0047] Example 3 Hemolytic experiment of the stapled peptide of the present invention After obtaining high-purity and uniform peptides, 2% mouse erythrocyte 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 96-well plates at room temperature for 2 h. Then 1% TritonX-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 % = (OD sample-ODPBS) / (ODTriton-ODPBS) × 100%.

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

[0049] The results of the hemolytic experiment showed that the hemolytic activity of the stapled peptides of the present invention was significantly improved compared with the template peptides, including SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8, and SLP-9. Among them, the hemolytic activity of SLP-4, SLP-6, SLP-7, and SLP-8 was significantly improved.

[0050] Data analysis shows that, except for some individual data groups (such as SLP-1 and SLP-5, SLP-2 and SLP-11, etc.), there are significant differences between SLP-2 and SLP-3, and SLP-2 and SLP-9, and any other two groups show extremely significant differences.

[0051] Example 4 Experiment on the inhibition of Gram-positive bacteria and Gram-negative bacteria by the stapled peptide of the present invention In vitro anti-resistant bacteria test: Prepare solid LB medium, sterilize by high pressure, plate and prepare LB liquid medium, and keep in a refrigerator at 4℃ for use. Spread the bacterial solution on solid LB medium and invert and culture overnight in a 37℃ incubator; take a single clone, add it to 3mL liquid LB medium, and culture it in a constant temperature shaker at 37℃, 220rpm for 6h to grow the bacteria to the logarithmic phase; take 1mL of bacterial solution, centrifuge at 4000rpm for 5min, discard the supernatant, add PBS, and adjust the bacterial solution concentration to 2×106CFU / mL by OD value. Add different concentrations of antimicrobial peptides to a 96-well plate, and add the bacterial solution to a 96-well plate at the same time, culture at 37℃ for 8h, and use a microplate reader to detect at 595nm, repeat three times, and statistically analyze the MIC value. The results are shown in Table 3.

[0052] The results in Table 3 show that the antibacterial activity of the stapled peptides of the present invention is basically the same as that of the template polypeptide, and 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 being developed 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.

[0053] Example 5 Anti-enzymatic Experiment of Staple Peptide of the Present Invention The polypeptides were dissolved in PBS buffer solution (50mM, pH=7.4) to a final concentration of 1mM. The principle of trypsin degradation of polypeptides is that trypsin mainly acts on the peptide bond at the carboxyl end of arginine or lysine, and then specifically cuts the polypeptide. Therefore, trypsin was dissolved in PBS buffer (50mM, containing 2mMCaCl2, pH=8) to a final concentration of 5ng / mL. The peptide solution (100mL) was then incubated with the trypsin solution (1mL) at room temperature. At 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5h, 100mL of the digestion mixture was taken and then quenched with 20mL of hydrochloric acid (1M). The solution of tryptic peptide fragments was detected by HPLC at different times to determine the protease degradation rate at 214nm. The results are shown in. Fig.16 .

[0054] Fig.16 The results showed that the stapled peptide SLP-1 of the present invention had the same ability to resist proteolysis as the template polypeptide.

Claims

1. A stapler peptide, characterized in that The stapled peptide is: Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 was used as the peptide chain template, in which amino acid residue 12L was replaced by K.

2. The use of the stapled peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that: The bacteria is Streptococcus pneumoniae.

3. The use of the stapled peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that: The bacteria is methicillin-resistant Staphylococcus aureus.

4. Use of the stapled peptide according to claim 1 in the preparation of antibacterial drugs, 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 bacteria is Klebsiella pneumoniae.

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