Antimicrobial stapling peptides and uses thereof

Abstract

The application belongs to the field of polypeptide drugs, and particularly relates to an antibacterial stapling peptide and application thereof. The application takes Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein L of the amino acid residue 12 is replaced by K to obtain a target stapling peptide SLP-7. Hemolysis of the obtained stapling peptide SLP-7 is significantly improved relative to the template polypeptide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptide drugs, and particularly relates to an antibacterial stapling peptide and application thereof, wherein the polypeptide has improved hemolytic property relative to a template polypeptide. BACKGROUND

[0002] Antibacterial peptides are a kind of polypeptide substances with antibacterial activity, and have many advantages, including safety, non-toxicity, wide antibacterial spectrum, good stability, low bactericidal concentration, small molecular weight, weak allergenicity, etc., and have become one of the research hotspots in the biological field. In recent years, with the abuse of antibiotics, the problems of bacterial drug resistance, drug residues and environmental pollution are becoming increasingly serious. Under the situation that the problems of antibiotic residues and bacterial drug resistance are becoming increasingly serious, antibacterial peptides are highly expected as a substitute for antibiotics due to their low allergenicity, no residues, low bactericidal concentration, and difficulty in developing drug resistance.

[0003] Antibacterial peptides (AMPs) are a kind of host defense peptides, which can kill bacteria through various ways, such as bacterial membrane lysis, oxidative damage, inhibition of biofilm formation, etc., without involving the binding of specific proteins, and are not easy to develop drug resistance, and can resist bacterial infections that are ineffective to traditional antibiotics. AMPs have high binding and selectivity to microbial membranes, and the amino acid composition of AMPs is a key factor for their action mechanism and selectivity, which determines the physical and chemical properties of the peptides, such as charge and amphiphilicity; after contacting with the plasma membrane, AMPs usually undergo structural changes, adopting a certain secondary structure or oligomerizing into aggregates. When the secondary structure of AMPs is formed, the separation of hydrophilic and hydrophobic residues leads to the penetration of the peptide into the plasma membrane, and therefore the presence of hydrophobic amino acids is necessary.

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

[0005] The present application aims at the deficiencies in the prior art, and provides a stapling peptide. The polypeptide has improved hemolytic property relative to a template polypeptide, and has significant antibacterial activity, and has application prospect for developing new antibacterial drugs.

[0006] Another object of the present application is to provide the use of the stapling peptide.

[0007] To achieve the first object, the technical solution adopted by the present application is as follows:

[0008] a stapling peptide selected from one of the following:

[0009] a) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 1 L is replaced by K;

[0010] b) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 4 V is replaced by K;

[0011] c) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 5 W is replaced by K;

[0012] d) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 8 V is replaced by K;

[0013] e) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 9 F is replaced by K;

[0014] f) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 11 L is replaced by K;

[0015] g) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 12 L is replaced by K;

[0016] h) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 15 Y is replaced by K;

[0017] i) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 16 W is replaced by K;

[0018] j) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 19 L is replaced by K;

[0019] k) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein amino acid residue 22 P is replaced by K;

[0020] l) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 23V is replaced by K;

[0021] The sequences of the template polypeptides and engineered stapled peptides of the present application are shown in Table 1.

[0022]

[0023] Preferably, the stapled peptide described above is selected from one of the following:

[0024] a) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 1L is replaced by K;

[0025] c) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 5W is replaced by K;

[0026] d) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 8V is replaced by K;

[0027] e) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 9F is replaced by K;

[0028] f) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 11L is replaced by K;

[0029] g) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 12L is replaced by K;

[0030] h) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 15Y is replaced by K;

[0031] i) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 16W is replaced by K.

[0032] More preferably, the stapled peptide described above is selected from one of the following:

[0033] d) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2, wherein amino acid residue 8V is replaced by K;

[0034] f) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein the amino acid residue 11L is replaced by K;

[0035] g) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein the amino acid residue 12L is replaced by K;

[0036] h) Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide chain template, wherein the amino acid residue 15Y is replaced by K.

[0037] The stapling peptide is used for preparing an antibacterial drug.

[0038] The stapling peptide is used for preparing an antibacterial drug, and the bacteria are Streptococcus pneumoniae (SP).

[0039] The stapling peptide is used for preparing an antibacterial drug, and the bacteria are Methicillin-resistant Staphylococcus aureus (MRSA).

[0040] The stapling peptide is used for preparing an antibacterial drug, and the bacteria are Pseudomonas aeruginosa (PA).

[0041] The stapling peptide is used for preparing an antibacterial drug, and the bacteria are Klebsiella pneumoniae (KP).

[0042] The present application has the following advantages:

[0043] 1. The present application provides a series of new stapling peptides, which are obtained by modifying the structure of SLP-0 (Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2), replacing the original amino acid with Lys (lysine) at the non-key residue position of the peptide chain on the basis of retaining the key residues, and obtaining the stapling peptide with stable structure after cyclization. The hemolysis experiment results show that the hemolysis of SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8, and SLP-9 polypeptides can be obviously improved compared with the template peptide. Among them, the hemolysis of LP-4, SLP-6, SLP-7, and SLP-8 is significantly improved.

[0044] 2、In vitro antibacterial experiment results show that the stapling peptide of the application and the template polypeptide have basically the same antibacterial activity, 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 to Pseudomonas aeruginosa is better than that of the template polypeptide.

[0045] 3、Peptide anti-enzymolysis experiment results show that the anti-protein enzymolysis ability of the stapling peptide SLP-1 of the application and the template polypeptide is the same. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 It is a synthesis route map of the stapling peptide of the application.

[0047] Figure 2 It is a schematic diagram of the amino acid sequence of SLP-0 and its characterization map. SLP-0 is purified by HPLC (purification conditions: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA) on a Welch C18 column for 55 minutes), to obtain SLP-0, and the separation rate is 15.1%. Among them, A is the amino acid sequence of SLP-0, B is the HPLC map of SLP-0, the analysis column is Welch C18, the gradient is 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214nm). C is the mass spectrum of SLP-0.

[0048] Figure 3 It is a schematic diagram of the amino acid sequence of SLP-1 and its characterization map. SLP-1 is purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA) on a Welch C18 column for 55 minutes), to obtain SLP-1, and the separation rate is 26.1%. Among them, A is the amino acid sequence of SLP-1, B is the HPLC map of SLP-1, the analysis column is Welch C18, the gradient is 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214nm). C is the mass spectrum of SLP-1.

[0049] Figure 4Figure 1 is a schematic diagram of the amino acid sequence of SLP-2 and its characterization profile. SLP-2 was purified by HPLC (purification condition: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA) on a Welch C18 column over 55 minutes) to obtain SLP-2 at a separation rate of 25.18%. Wherein, A is the amino acid sequence of SLP-2, B is the HPLC profile 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 profile of SLP-2.

[0050] Figure 5 Figure 3 is a schematic diagram of the amino acid sequence of SLP-3 and its characterization profile. SLP-3 was purified by HPLC (purification condition: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA) on a Welch C18 column over 55 minutes) to obtain SLP-3 at a separation rate of 19.39%. Wherein, A is the amino acid sequence of SLP-3, B is the HPLC profile 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 profile of SLP-3.

[0051] Figure 6 Figure 4 is a schematic diagram of the amino acid sequence of SLP-4 and its characterization profile. SLP-4 was purified by HPLC (purification condition: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA) on a Welch C18 column over 55 minutes) to obtain SLP-4 at a separation rate of 31.58%. Wherein, A is the amino acid sequence of SLP-4, B is the HPLC profile 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 profile of SLP-4.

[0052] Figure 7Figure 1 shows the amino acid sequence of SLP-5 and its characterization profile. SLP-5 was purified by HPLC (purification condition: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA) over a Welch C18 column in 55 minutes) with a separation rate of 21.56%. Wherein, A is the amino acid sequence of SLP-5, B is the HPLC profile of SLP-5, 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 profile of SLP-5.

[0053] Figure 8 Figure 2 shows the amino acid sequence of SLP-6 and its characterization profile. SLP-6 was purified by HPLC (purification condition: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA) over a Welch C18 column in 55 minutes) with a separation rate of 17.41%. Wherein, A is the amino acid sequence of SLP-6, B is the HPLC profile 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 profile of SLP-6.

[0054] Figure 9 Figure 3 shows the amino acid sequence of SLP-7 and its characterization profile. SLP-7 was purified by HPLC (purification condition: 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA) over a Welch C18 column in 55 minutes) with a separation rate of 18.71%. Wherein, A is the amino acid sequence of SLP-7, B is the HPLC profile 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 profile of SLP-7.

[0055] Figure 10The figure A is the amino acid sequence of SLP-8, the figure B is the HPLC chromatogram of SLP-8, the analysis column is Welch C18, the gradient is 10%-60% CH3CN (0.1% TFA) in H2O (0.1% TFA), 55 minutes (λ=214 nm), and the figure C is the mass spectrum of SLP-8.

[0056] Figure 11 The figure A is the amino acid sequence of SLP-9, the figure B is the HPLC chromatogram of SLP-9, the analysis column is Welch C18, the gradient is 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and the figure C is the mass spectrum of SLP-9.

[0057] Figure 12 The figure A is the amino acid sequence of SLP-10, the figure B is the HPLC chromatogram of SLP-10, the analysis column is Welch C18, the gradient is 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and the figure C is the mass spectrum of SLP-10.

[0058] Figure 13The SLP-11 amino acid sequence and its characterization map are shown in the figure, and SLP-11 is obtained by HPLC purification (purification conditions: 10%-65% CH3CN (0.1% TFA) in H2O (0.1% TFA) on a Welch C18 column for 55 minutes), and the separation rate is 19.76%. Wherein, A is the amino acid sequence of SLP-11, B is the HPLC map of SLP-11, the analysis column is Welch C18, the gradient is 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm). C is the mass spectrum of SLP-11.

[0059] Figure 14 The SLP-12 amino acid sequence and its characterization map are shown in the figure, and SLP-12 is obtained by HPLC purification (purification conditions: 10%-65% CH3CN (0.1% TFA) in H2O (0.1% TFA) on a Welch C18 column for 55 minutes), and the separation rate is 21.38%. Wherein, A is the amino acid sequence of SLP-12, B is the HPLC map of SLP-12, the analysis column is Welch C18, the gradient is 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm). C is the mass spectrum of SLP-12.

[0060] Figure 15 The hemolysis experiment results of the stapling peptide of the application are shown in the figure.

[0061] Figure 16 The trypsin (left) and chymotrypsin (right) degradation experiment results of the stapling peptide of the application are shown in the figure. DETAILED DESCRIPTION

[0062] The application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the application, but these embodiments are only used to illustrate the application and not to limit the scope of the application, that is, the described embodiments are only a part of the embodiments of the application, not all the embodiments.

[0063] Therefore, the following detailed description of a part of the embodiments of the application provided herein is not intended to limit the scope of the claimed application, but only selected embodiments of the application. Based on the embodiments of the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0064] The present application designs and synthesizes 12 stapled peptides according to the template polypeptide SLP-0: Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 amino acid sequence. The structure and molecular weight of the peptide are shown in Figure 1

[0065] The experimental materials involved in the embodiments of the present application are as follows:

[0066] Fmoc-amino acid, Rinkamide MBHA amino resin are purchased from Nankai Synthetic Co., Ltd.; NMP, DIC, Oxyme, TFA, acetonitrile (chromatographically pure) are purchased from Exploration Platform; DMF, anhydrous diethyl ether, DCM, DCE, piperidine, phenol are all analytical pure and are purchased from Shanghai Chemical Reagent Co., Ltd. of China National Pharmaceutical Group; S5 is purchased from Beijing Oukaisi Biochemical Technology Co., Ltd.

[0067] In the present application, the abbreviations involved are explained as follows:

[0068] Fmoc: fluorenylmethyloxycarbonyl; DCM: dichloromethane;

[0069] DCE: 1,2-dichloroethane;

[0070] DMF: N,N-dimethylformamide;

[0071] Oxyme: Ethyl Cyanoglyoxylate-2-Oxime;

[0072] DIC: N,N-diisopropylcarbodiimide;

[0073] S5: (S)-2-amino-2-methyl-4-pentanoic acid TFA: trifluoroacetic acid;

[0074] EDT: 1,2-ethanedithiol;

[0075] Grubbs I: phenylmethylenebis(tricyclohexylphosphine) ruthenium dichloride;

[0076] MS: mass spectrum;

[0077] HR-Q-TOF-MS: high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

[0078] Example 1 Preparation of stapled peptides based on SLP-0

[0079] 1. Synthesis of stapled peptides

[0080] The synthesis route is shown in Figure 1

[0081] (1) Preparation of compound 1

[0082] ​​Amino resin 400 mg (loading capacity 0.30 mmol.g -1 ) was added to the solid-phase synthesis reaction tube, and the resin was soaked in DCM for 30 min to fully swell the resin, and then dried for use.

[0083] 7 ml of 20% piperidine-DMF solution was added to the resin, and the resin was shaken at 35°C for 5 min x 2 to remove the Fmoc protecting group on the resin, and the resin was washed with DMF, DCM, and DMF for 3 times, respectively.

[0084] (2) Preparation of compound 2

[0085] The first amino acid in the sequence (1 mmol), Oxyme (142 mg, 1 mmol), and DIC (200 μL) were mixed in 7 ml of DMF, and added to the resin, and shaken at 60°C for 20 min (1 h for one amino acid after S5, and repeated once), and the resin was washed with DMF, DCM, and DMF for 3 times, respectively.

[0086] (3) Preparation of compound 3

[0087] The procedures of (1) and (2) were repeated, and according to the polypeptide sequence, Fmoc amino acid (1 mmol), Oxyme (142 mg), and DIC (200 μl) were dissolved in 7 ml of DMF, and then added to the resin, and shaken at 60°C for 30 min, and the process of removing Fmoc protection, condensation, and removing Fmoc protection was repeated until all the amino acids were condensed. After the last amino acid was removed from the Fmoc protecting group, 7 ml of a mixture of acetic anhydride:DIEA:DMF (1:1:8) was added, and shaken at 37°C for 15 min, and then dried, and the acetylation reagent was added again, and reacted for 15 min, and the resin was washed with DMF, DCM, and DMF for 3 times, respectively, and then dried by oil pump vacuum.

[0088] (4) Preparation of compound 4

[0089] After the resin was completely dried, 7 ml of Grubbs I (58 mg) reagent in 1,2-dichloroethane was added, and shaken at 37°C for 2 h, and then the resin was washed with DMF, DCM, and DMF for 3 times, respectively, and dried by oil pump vacuum.

[0090] (5) Preparation of target compound

[0091] Firstly, the resin was washed and dried, then 20 mL of TFA:phenol:H2O:benzyl thioether:EDT=82.5:5:5:5:2.5 (V / V / V / V) was added, and the mixture was shaken at 37°C for 3 h. The resin was filtered, washed with a small amount of TFA, and the filtrate was collected. The excess TFA was blown away by argon gas blowing, and the product was precipitated in ice ethyl ether. After centrifugation, the supernatant was discarded, and the above steps were repeated three times. The product was placed in a fume hood and naturally volatilized to obtain a crude polypeptide sample.

[0092] 2. Purification of stapled peptide sample

[0093] The crude polypeptide was dissolved in a mixed solvent of acetonitrile and water, and was purified by reverse phase preparative RP-HPLC to obtain a purified stapled peptide product. The separation conditions are as follows:

[0094] Instrument: Shimadzu LC-20A reverse phase high performance liquid chromatograph;

[0095] Chromatographic column: Ultimate XB-C18, 21.2 x 250 mm, 5 μm;

[0096] Mobile phase: mobile phase A is acetonitrile solution with a volume fraction of 0.1% TFA, and mobile phase B is water solution with a volume fraction of 0.1% TFA;

[0097] Step and parameter: 90% B elution for 3 min, 90% B to 50% B elution for 40 min; flow rate is 8 ml / min, sample injection amount is 3 ml, and detection wavelength is 214 nm and 254 nm.

[0098] Identification and structure analysis of product of Example 2

[0099] The product obtained in step 2 of Example 1 was identified by reverse phase HPLC and structure analysis by HR-Q-TOF-MS, and the chromatographic mobile phase was acetonitrile and water. Mobile phase A is acetonitrile solution with a volume fraction of 0.1% TFA, and mobile phase B is water solution with a volume fraction of 0.1% TFA, gradient elution (0-2 min, mobile phase B: 90%; 3-25 min, mobile phase B: 90% to 10%); flow rate is 1.0 mL·min -1 ; detection wavelength is 214 nm and 254 nm, and sample injection volume is 24 μl. It is determined that the peak time of the main peak of the crude product is consistent, and the purity of the stapled peptide prepared by the method is greater than 95%, and the mass spectrum analysis result is shown in Figures 2-14 . After analysis, the structure of the obtained stapled peptide is shown in Table 1.

[0100]

[0101] Example 3 Hemolysis experiment of stapled peptide of the application

[0102] After obtaining the polypeptide with high purity and uniform structure, a 2% mouse red blood cell suspension 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 in Tris-buffered saline solution (v / v) 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 by a microplate spectrophotometer. The hemolysis rate was calculated using the following formula: Hemolysis rate % = (OD sample - OD PBS) / (OD Triton - OD PBS) x 100%.

[0103] The results are shown in Table 2 and Figure 15 .

[0104]

[0105] The results of the hemolysis experiment show that the hemolytic activity of the SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8 and SLP-9 polypeptides of the stapled peptide of the application can be significantly improved compared with the template peptide. Among them, the hemolytic activity of SLP-4, SLP-6, SLP-7 and SLP-8 is significantly improved.

[0106] Through data analysis, it can be known that, except for individual data groups (such as SLP-1 and SLP-5, SLP-2 and SLP-11, etc.) which have no significant difference, there is a significant difference between SLP-2 and SLP-3, SLP-2 and SLP-9, and any two groups show extremely significant difference.

[0107] Example 4 Inhibition of Gram-positive bacteria and Gram-negative bacteria by the stapled peptide of the application

[0108] In vitro anti-drug resistant bacteria test: solid LB medium was prepared, sterilized by high pressure, and then plated and prepared LB liquid medium, which was stored in a 4°C refrigerator. The bacterial liquid was spread on the solid LB medium and cultured in an inverted incubator at 37°C overnight; a single colony was taken and added to 3 mL of liquid LB medium, which was cultured at 37°C, 220 rpm, in a constant temperature shaker for 6 h to make the bacteria grow to the logarithmic phase; 1 mL of bacterial liquid was centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. PBS was added to adjust the concentration of the bacterial liquid to 2 x 106 CFU / mL. Different concentrations of antibacterial peptides were added to the 96-well plate, and the bacterial liquid was also added to the 96-well plate. After incubation at 37°C for 8 h, the microplate reader was used to detect at 595 nm, and the MIC value was calculated by repeating three times. The results are shown in Table 3.

[0109]

[0110] The results in Table 3 show that the stapling peptide of the application is basically on a par with the template polypeptide in antibacterial activity, 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 application prospect of developing into a new type of antibacterial drug. Among them, the antibacterial activity of SLP-1 on Pseudomonas aeruginosa is better than that of the template polypeptide.

[0111] Example 5 Anti-enzymolysis experiment of stapling peptide of the application

[0112] The polypeptide was dissolved in PBS buffer solution (50 mM, pH = 7.4) to a final concentration of 1 mM. Since the principle of trypsin degrading polypeptide 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 solution (50 mM, containing 2 mM CaCl2, pH = 8) to a final concentration of 5 ng / mL. Then the peptide solution (100 mL) was incubated with the trypsin solution (1 mL) at room temperature. At 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 h, 100 mL of digestion mixture was taken, and then quenched with 20 mL of hydrochloric acid (1 M). The trypsin peptide fragment solution was detected by HPLC at different times to determine the protease degradation rate at 214 nm. The results are shown in Figure 16 .

[0113] Figure 16 The results show that the stapling peptide SLP-1 of the application is on a par with the template polypeptide in anti-protease degradation ability.

Claims

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

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

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

4. The use of the staple peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that, The bacteria in question is Pseudomonas aeruginosa.

5. The use of the staple peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that, The bacteria in question is Klebsiella pneumoniae.

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