Antibacterial stapling peptide and use 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 8V is replaced by K to obtain a target stapling peptide SLP-4. Hemolysis of the obtained stapling peptide SLP-4 is significantly improved relative to the template polypeptide.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drugs, specifically relating to an antibacterial staple peptide and its application, which has improved hemolytic properties compared to the template polypeptide. Background Technology

[0002] Antimicrobial peptides are a class of polypeptides with antibacterial activity, possessing numerous advantages, including safety and non-toxicity, broad antibacterial spectrum, good stability, low bactericidal concentration, small molecular weight, and weak sensitization. They have become a research hotspot in the biological field. In recent years, with the overuse of antibiotics, problems such as bacterial resistance, drug residues, and environmental pollution have become increasingly serious. Given the growing severity of antibiotic residues and bacterial resistance, antimicrobial peptides, due to their low sensitization, lack of residue, low bactericidal concentration, and low likelihood of inducing resistance, are highly anticipated as a potential alternative to antibiotics.

[0003] Antimicrobial peptides (AMPs) are a class of host defense peptides that can kill bacteria through various pathways, such as biofilm lysis, oxidative damage, and inhibition of biofilm formation. They do not involve binding to specific proteins, are less prone to developing resistance, and can combat bacterial infections unresponsive to traditional antibiotics. AMPs exhibit high binding and selectivity to microbial membranes. The amino acid composition of AMPs is a key factor in their mechanism of action and selectivity, determining their physicochemical properties, such as charge and amphiphilicity. Upon contact with the plasma membrane, AMPs typically undergo structural changes, adopting a defined secondary structure or oligomerizing into aggregates. During the formation of the AMP secondary structure, the separation of hydrophilic and hydrophobic residues leads to peptide penetration into the plasma membrane; therefore, the presence of hydrophobic amino acids is essential.

[0004] Patent CN117486993A discloses a template peptide SLP-0: Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2. This template peptide exhibits good antibacterial effects against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Further research by the inventors revealed that SLP-0 possesses 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 this invention is to address the shortcomings of existing technologies by providing a staple peptide. This peptide exhibits improved hemolytic activity compared to the template peptide and demonstrates significant antibacterial activity, showing promise for development into a novel antibacterial drug.

[0006] Another object of the present invention is to provide the use of the staple peptide.

[0007] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows:

[0008] Stapling peptide, wherein the stapling peptide is selected from one of the following:

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

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

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

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

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

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

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

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

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

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

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

[0020] l) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, amino acid residue 23V is replaced by K;

[0021] The sequences of the template peptide and the modified staple peptide in this invention are shown in Table 1.

[0022]

[0023] Preferably, the above-mentioned stapler peptide is selected from one of the following:

[0024] a) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 1L is replaced by K;

[0025] c) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 5W is replaced by K;

[0026] d) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 8V is replaced by K;

[0027] e) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 9F is replaced by K;

[0028] f) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 11L is replaced by K;

[0029] g) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 12L is replaced by K;

[0030] h) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 15Y is replaced by K;

[0031] i) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 16W is replaced by K.

[0032] More preferably, the above-mentioned stapler peptide is selected from one of the following:

[0033] d) Using Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 as a peptide template, where amino acid residue 8V is replaced by K;

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

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

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

[0037] The above-mentioned staple peptides are used in the preparation of antibacterial drugs.

[0038] In the application of the above-mentioned staple peptide in the preparation of antibacterial drugs, the bacteria is Streptococcus pneumoniae (SP).

[0039] In the application of the above-mentioned staple peptide in the preparation of antibacterial drugs, the bacteria is methicillin-resistant Staphylococcus aureus (MRSA).

[0040] In the application of the above-mentioned staple peptide in the preparation of antibacterial drugs, the bacteria is Pseudomonas aeruginosa (PA).

[0041] In the application of the above-mentioned staple peptide in the preparation of antibacterial drugs, the bacteria is Klebsiella pneumoniae (KP).

[0042] The advantages of this invention are:

[0043] 1. This invention provides a series of novel staple peptides. New staple peptides are obtained by structurally modifying SLP-0 (Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2). While retaining key residues, lysine residues are replaced with lysine residues at non-critical residue positions in the peptide chain, followed by cyclization to obtain structurally stable staple peptides. Hemolysis experiments show that, compared to the template peptides, the hemolytic properties of the staple peptides of this invention (SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8, and SLP-9) are significantly improved. Among these, the hemolytic properties of SLP-4, SLP-6, SLP-7, and SLP-8 are particularly significantly improved.

[0044] 2. In vitro antibacterial experiments showed that the antibacterial activity of the stapling peptide of this invention was basically equivalent to that of the template peptide, and it could significantly inhibit the growth and reproduction of SP (Streptococcus pneumoniae), MRSA (methicillin-resistant Staphylococcus aureus), PA (Pseudomonas aeruginosa), and KP (Klebsiella pneumoniae), demonstrating its potential as a novel antibacterial drug. Among these, SLP-1 showed superior antibacterial activity against Pseudomonas aeruginosa compared to the template peptide.

[0045] 3. The results of the peptide anti-enzymatic hydrolysis experiment show that the binding peptide SLP-1 of the present invention has the same anti-enzymatic hydrolysis ability as the template peptide. Attached Figure Description

[0046] Figure 1 This is a synthetic route diagram for the staple peptide of this invention.

[0047] Figure 2 The diagram shows the amino acid sequence of SLP-0 and its characterization chromatogram. 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), yielding SLP-0 with a separation rate of 15.1%. In the diagram, A represents the amino acid sequence of SLP-0, B represents the HPLC chromatogram of SLP-0 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-0.

[0048] Figure 3 The diagram shows the amino acid sequence of SLP-1 and its characterization chromatogram. 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), yielding SLP-1 with a separation rate of 26.1%. In the diagram, A represents the amino acid sequence of SLP-1, B represents the HPLC chromatogram of SLP-1 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-1.

[0049] Figure 4The diagram shows the amino acid sequence of SLP-2 and its characterization chromatogram. 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), yielding SLP-2 with a separation rate of 25.18%. In the diagram, A represents the amino acid sequence of SLP-2, B represents the HPLC chromatogram of SLP-2 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-2.

[0050] Figure 5 The diagram shows the amino acid sequence of SLP-3 and its characterization chromatogram. 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), yielding SLP-3 with a separation rate of 19.39%. In the diagram, A represents the amino acid sequence of SLP-3, B represents the HPLC chromatogram of SLP-3 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-3.

[0051] Figure 6 The diagram shows the amino acid sequence of SLP-4 and its characterization chromatogram. 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%. In the diagram, A is the amino acid sequence of SLP-4, B is the HPLC chromatogram of SLP-4 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C is the mass spectrum of SLP-4.

[0052] Figure 7The diagram shows the amino acid sequence of SLP-5 and its characterization chromatogram. 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), yielding SLP-5 with a separation rate of 21.56%. In the diagram, A represents the amino acid sequence of SLP-5, B represents the HPLC chromatogram of SLP-5 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-5.

[0053] Figure 8 The diagram shows the amino acid sequence of SLP-6 and its characterization chromatogram. 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), yielding SLP-6 with a separation rate of 17.41%. In the diagram, A represents the amino acid sequence of SLP-6, B represents the HPLC chromatogram of SLP-6 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-6.

[0054] Figure 9 The diagram shows the amino acid sequence of SLP-7 and its characterization chromatogram. 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), yielding SLP-7 with a separation rate of 18.71%. In the diagram, A represents the amino acid sequence of SLP-7, B represents the HPLC chromatogram of SLP-7 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-7.

[0055] Figure 10The diagram shows the amino acid sequence of SLP-8 and its characterization chromatogram. 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), yielding SLP-8 with a separation rate of 12.34%. In the diagram, A represents the amino acid sequence of SLP-8, B represents the HPLC chromatogram of SLP-8 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-8.

[0056] Figure 11 The diagram shows the amino acid sequence of SLP-9 and its characterization chromatogram. 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), yielding SLP-9 with a separation rate of 25.12%. In the diagram, A represents the amino acid sequence of SLP-9, B represents the HPLC chromatogram of SLP-9 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-9.

[0057] Figure 12 The diagram shows the amino acid sequence of SLP-10 and its characterization chromatogram. 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), yielding SLP-10 with a separation rate of 18.52%. In the diagram, A represents the amino acid sequence of SLP-10, B represents the HPLC chromatogram of SLP-10 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-10.

[0058] Figure 13The diagram shows the amino acid sequence of SLP-11 and its characterization chromatogram. 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), yielding SLP-11 with a separation rate of 19.76%. In the diagram, A represents the amino acid sequence of SLP-11, B represents the HPLC chromatogram of SLP-11 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-11.

[0059] Figure 14 The diagram shows the amino acid sequence of SLP-12 and its characterization chromatogram. 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), yielding SLP-12 with a separation rate of 21.38%. In the diagram, A represents the amino acid sequence of SLP-12, B represents the HPLC chromatogram of SLP-12 (analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm), and C represents the mass spectrum of SLP-12.

[0060] Figure 15 The results of the peptide hemolysis experiment of this invention are shown.

[0061] Figure 16 The results of the degradation experiments of the stapling peptide trypsin (left) and chymotrypsin (right) of this invention are shown. Detailed Implementation

[0062] The present application will be further described below with reference to the accompanying drawings and specific embodiments, 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 intended to limit the scope of the present invention. That is, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0063] Therefore, the following detailed description of a portion of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but is merely a selection of embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0064] This invention designs and synthesizes 12 staple peptides based on the amino acid sequence of template polypeptide SLP-0: Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2. The peptide structures and molecular weights are as follows: Figure 1 As shown.

[0065] The experimental materials involved in the embodiments of this invention are sourced from the following sources:

[0066] Fmoc-amino acids and RinkamideMBHA amino resin were purchased from Nankai Synthetic Co., Ltd.; NMP, DIC, Oxyme, TFA, and acetonitrile (chromatographic grade) were purchased from the Exploration Platform; DMF, anhydrous diethyl ether, DCM, DCE, piperidine, and phenol were all analytical grade and purchased from Sinopharm Chemical Reagents Shanghai Co., Ltd.; S5 was purchased from Beijing Oukenas Biochemical Technology Co., Ltd.

[0067] In this invention, the abbreviations are explained as follows:

[0068] Fmoc: fluorenemethyloxycarbonyl; DCM: dichloromethane;

[0069] DCE: 1,2-Dichloroethane;

[0070] DMF: N,N-dimethylformamide;

[0071] Oxyme: EthylCyanoglyoxylate-2-Oxime;

[0072] DIC: N,N-diisopropylcarbodiimide;

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

[0074] EDT: 1,2-Ethylenedithiol;

[0075] GrubbsⅠ: Phenylenemethylene bis(tricyclohexylphosphine)ruthenium dichloride;

[0076] MS: Mass Spectrometry;

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

[0078] Example 1: Preparation of SLP-0-based staple peptide

[0079] 1. Synthesis of staple peptide

[0080] Synthetic routes such as Figure 1 As shown:

[0081] (1) Preparation of compound 1

[0082] Take 400 mg of amino resin (sample loading capacity is 0.30 mmol·g). -1 Add the resin to the solid-phase synthesis reaction tube, soak it in DCM for 30 minutes to allow the resin to fully swell, and then dry it for later use.

[0083] 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 in sequence.

[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 DMF and added to the resin. The mixture was shaken at 60 °C for 20 min (the amino acid after S5 reacted for 1 h and the reaction was repeated once). The resin was washed three times each with DMF, DCM and DMF.

[0086] (3) Preparation of compound 3

[0087] Repeat steps (1) and (2). According to the polypeptide sequence, dissolve Fmoc amino acids (1 mmol), Oxyme (142 mg), and DIC (200 μl) in 7 ml of DMF, then add them to the resin. Shake and react at 60 °C for 30 min. Repeat the process of removing Fmoc protection → condensation → removing Fmoc protection until all amino acids have condensed. After removing the Fmoc protecting group from the last amino acid, add 7 ml of acetic anhydride:DIEA:DMF (1:1:8) mixture and shake at 37 °C for 15 min. Dry under vacuum, add acetylation reagent again, react for 15 min, wash the resin three times each with DMF, DCM, and DMF, and then dry the resin under vacuum using an oil pump.

[0088] (4) Preparation of compound 4

[0089] After the resin is completely dry, add 7 ml of 1,2-dichloroethane solution of Grubbs I (58 mg) reagent, and shake the reaction twice at 37°C for 2 hours each time. After the reaction is completed, wash the resin three times each with DMF, DCM and DMF in sequence, and then dry the resin by vacuum pump.

[0090] (5) Preparation of the target compound

[0091] First, wash and dry the resin. Add 20 mL of a mixture of TFA:phenol:H2O:anisole: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 small amount of TFA, and collect the filtrate. Blow away excess TFA with argon gas, pour in ice-cold diethyl ether to precipitate, centrifuge, discard the supernatant, and repeat the above steps. Wash and centrifuge the ice-cold diethyl ether precipitate three times, and allow it to air dry in a fume hood to obtain the crude peptide sample.

[0092] 2. Purification of staple peptide samples

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

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

[0095] Column: UltimateXB-C18, 21.2×250mm, 5μm;

[0096] Mobile phase: Mobile phase A is an 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;

[0097] Procedure and parameters: Elute with 90% B for 3 min, then elute with 90% B to 50% B for 40 min; flow rate is 8 ml / min, injection volume is 3 ml, and detection wavelengths are 214 nm and 254 nm.

[0098] Identification and structural analysis of the product in Example 2

[0099] The product obtained in step 2 of Example 1 was identified by reversed-phase HPLC and its structure was analyzed by HR-Q-TOF-MS. The mobile phase was 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. Gradient elution was used (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. The peak elution time was consistent with that of the crude product, and the purity of the staple peptide prepared by this method was >95%. Mass spectrometry analysis results are as follows: Figures 2-14 As shown in the figure. The structure of the staple peptide obtained after analysis is shown in Table 1.

[0100]

[0101] Example 3: Hemolysis experiment of the staple peptide of the present invention

[0102] After obtaining high-purity, structurally homogeneous peptides, 2% mouse erythrocyte suspension was treated in Tris-buffered saline (v / v) 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% 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 % = (OD sample - ODPBS) / (ODTriton - ODPBS) × 100%.

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

[0104]

[0105] The hemolysis test results showed that, compared with the template peptide, the hemolytic activity of the stapled peptides of the present invention, namely SLP-1, SLP-3, SLP-4, SLP-5, SLP-6, SLP-7, SLP-8, and SLP-9, was significantly improved. Among them, the hemolytic activity of SLP-4, SLP-6, SLP-7, and SLP-8 was significantly improved.

[0106] Data analysis shows that, except for a few data groups (such as SLP-1 and SLP-5, SLP-2 and SLP-11, etc.) where the differences are not significant, there are significant differences between the SLP-2 and SLP-3 and SLP-2 and SLP-9 data groups, and all other pairs of data show extremely significant differences.

[0107] Example 4: Experiment on the inhibition of Gram-positive and Gram-negative bacteria by the staple peptide of the present invention.

[0108] In vitro antimicrobial resistance assay: Solid LB medium was prepared, autoclaved, plated, and liquid LB medium was prepared and stored at 4°C. Bacterial suspension was spread onto solid LB medium and incubated overnight at 37°C. Single colonies were added to 3 mL of liquid LB medium and incubated at 37°C, 220 rpm for 6 h in a shaker to allow the bacteria to reach the logarithmic growth phase. 1 mL of bacterial suspension was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and PBS was added. The bacterial concentration was adjusted to 2 × 10⁶ CFU / mL based on the OD value. Different concentrations of antimicrobial peptides and bacterial suspensions were added to 96-well plates and incubated at 37°C for 8 h. Detection was performed using a microplate reader at 595 nm. The assay was repeated three times, and the MIC values ​​were statistically analyzed. The results are shown in Table 3.

[0109]

[0110] Table 3 shows that the antibacterial activity of the staple peptide and the template peptide of this invention is basically the same, and they can significantly inhibit the growth and reproduction of SP (Streptococcus pneumoniae), MRSA (methicillin-resistant Staphylococcus aureus), PA (Pseudomonas aeruginosa), and KP (Klebsiella pneumoniae), showing promise for development into novel antibacterial drugs. Among them, SLP-1 exhibits superior antibacterial activity against Pseudomonas aeruginosa compared to the template peptide.

[0111] Example 5: Experiment on the resistance of the binding peptide of the present invention to enzymatic hydrolysis

[0112] The peptides were dissolved in PBS buffer (50 mM, pH 7.4) to a final concentration of 1 mM. Since trypsin degradation of peptides primarily involves the enzyme acting on the peptide bonds at the carboxyl terminus of arginine or lysine residues, specifically cleaving the peptide, trypsin was dissolved in PBS buffer (50 mM, containing 2 mM CaCl2, pH 8) to a final concentration of 5 ng / mL. The peptide solution (100 mL) was then incubated with the trypsin solution (1 mL) at room temperature. Labels were applied at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 h, and 100 mL of the digest mixture was taken and quenched with 20 mL of hydrochloric acid (1 M). The solution of trypsin peptide fragments was analyzed by HPLC at different time points to determine the protease degradation rate at 214 nm. Results are shown below. Figure 16 .

[0113] Figure 16 The results showed that the binding peptide SLP-1 of the present invention had the same anti-protease digestion ability as the template peptide.

Claims

1. A stapling peptide, characterized in that, The stapling peptide is: Ac-LKRVWKRVFKLLKS5YWRS5LKKPVR-NH2 is used as a peptide chain template, wherein the amino acid residue 8V is replaced by K.

2. Use of the stapling peptide of claim 1 for the preparation of an antibacterial medicament, characterized in that, The bacteria are Streptococcus pneumoniae.

3. Use of the stapling peptide of claim 1 for the preparation of an antibacterial medicament, characterized in that, The bacteria are methicillin-resistant Staphylococcus aureus.

4. Use of the stapling peptide of claim 1 for the preparation of an antibacterial medicament, characterized in that, The bacteria are Pseudomonas aeruginosa.

5. Use of the stapling peptide of claim 1 for the preparation of an antibacterial medicament, characterized in that, The bacteria are Klebsiella pneumoniae.

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