Antimicrobial polypeptides and uses thereof

Abstract

The application belongs to the field of polypeptide drugs, and particularly relates to an antibacterial polypeptide and application thereof. The application uses Rink amide MBHA amino resin as a solid phase carrier, and according to the modification and transformation of a template polypeptide GCIFN-20-0: Ac-SYEKKINRHFKILKKNLKKK-NH2 amino acid sequence, the original amino acids are replaced by S5 or S5, R8 at i, i+4 and i, i+7 amino acid positions respectively on the basis of retaining key amino acid residues to obtain a target stapled peptide. The stapled peptide obtained relative to the template polypeptide GCIFN-20-0 can significantly improve the bacteriostatic activity on Staphylococcus epidermidis and enhance the enzyme stability.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drugs, specifically relating to an antibacterial polypeptide and its applications. Background Technology

[0002] Antibacterial drugs are a class of drugs with bactericidal or bacteriostatic activity, including antibiotics and chemically synthesized drugs. Since the discovery of penicillin, antibiotics have been widely used to treat bacterial infections. However, in recent years, the overuse of antibiotics has led to increasingly serious problems of bacterial resistance, thus urgently requiring the development of new antibacterial drugs. Staphylococcus epidermidis is a Gram-positive coccus that grows on the epidermis of organisms, found in the skin, nasal cavity, intestines, and genitals of humans. It is named Staphylococcus epidermidis because it often clusters in grape-like patterns. Staphylococcus epidermidis can cause purulent infections and even critical conditions such as sepsis. It is commonly seen in newborns, premature infants, infants, the elderly, and patients with certain chronic diseases, malnutrition, weakened immune function, developmental delays, or those with severe trauma, burns, major surgery, prolonged bed rest, or coma. Like Staphylococcus aureus, Staphylococcus epidermidis easily develops resistance to commonly used antibiotics, leading to an increased incidence of Staphylococcus epidermidis infections and sepsis, which is currently a challenge in clinical treatment. There is an urgent need to develop new therapeutic drugs to address septicemia caused by Staphylococcus epidermidis.

[0003] Antimicrobial peptides (AMPs) are a class of host defense peptides with unique mechanisms of action, such as disrupting cell membrane structure and inhibiting the synthesis of intracellular biomolecules. They do not involve binding to specific proteins, are less prone to inducing drug resistance, and can combat bacterial infections unresponsive to traditional antibiotics. Therefore, AMPs have the potential to be a next-generation antibiotic for treating drug-resistant bacterial infections. Recently, antimicrobial peptides have been widely reported. For example, patent CN116217669A discloses a staple peptide that can enhance broad-spectrum antimicrobial activity, its preparation method, and its applications. This staple peptide exhibits broad-spectrum antimicrobial activity, including activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Patent CN117756905A discloses a staple peptide and its pharmaceutical uses; the staple peptide in this invention exhibits inhibitory activity against Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Patent CN116655766A discloses a staple peptide, its preparation method, and its applications. The staple peptide enhances the inhibitory activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. While the aforementioned study showed that the staple peptide can improve the antibacterial activity against some drug-resistant bacteria, there are no reports on its activity against Staphylococcus epidermidis. Solving the problem of drug resistance in Staphylococcus epidermidis remains an urgent issue.

[0004] Patent CN108752457A discloses a derivative polypeptide derived from grass carp interferon, with the amino acid sequence SYEKKINRHFKILKKNLKKK (i.e., GCIFN-20-0 of this application). This interferon-derived polypeptide exhibits highly effective antibacterial activity, functioning against drug-resistant Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Vibrio fluvialis, and Aeromonas hydrophila. However, as a linear polypeptide, this interferon-derived polypeptide is structurally unstable and easily degraded by proteases in vivo, greatly limiting its drug-like properties. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an antimicrobial peptide, specifically a staple peptide. Compared to template peptides, this staple peptide exhibits enhanced activity against drug-resistant bacteria and improved enzyme stability, resulting in superior overall performance and making it more suitable for drug development.

[0006] Another object of the present invention is to provide pharmaceutical use of the said antimicrobial polypeptide.

[0007] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows: An antimicrobial polypeptide, wherein the antimicrobial polypeptide is a staple peptide, and the staple peptide is: GCIFN-20-2: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 7N are replaced by S5; GCIFN-20-4: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 12I and 16N are replaced by S5; GCIFN-20-5: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 2Y and 9H are replaced by R8 and S5; GCIFN-20-6: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 10F are replaced by R8 and S5; GCIFN-20-7: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 9H and 16N are replaced by R8 and S5.

[0008] Preferably, in the above-mentioned antimicrobial polypeptide, the stapler peptide is: GCIFN-20-2: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 7N are replaced by S5; GCIFN-20-5: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 2Y and 9H are replaced by R8 and S5; GCIFN-20-6: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 10F are replaced by R8 and S5; GCIFN-20-7: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 9H and 16N are replaced by R8 and S5.

[0009] More preferably, in the above-mentioned antimicrobial polypeptide, the stapler peptide is: GCIFN-20-2: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 7N are replaced by S5; GCIFN-20-7: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 9H and 16N are replaced by R8 and S5.

[0010] More preferably, in the above-mentioned antimicrobial polypeptide, the stapler peptide is: GCIFN-20-2: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 3E and 7N are replaced by S5.

[0011] Alternatively, preferably, the above-mentioned antimicrobial polypeptide, wherein the stapler peptide is: GCIFN-20-6: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 3E and 10F are replaced by R8 and S5.

[0012] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows: The above-mentioned antimicrobial peptides are used in the preparation of drugs against Staphylococcus epidermidis.

[0013] The above-mentioned antimicrobial peptides are used in the preparation of drugs against Pseudomonas aeruginosa.

[0014] The above-mentioned antimicrobial peptides are used in the preparation of drugs against Klebsiella pneumoniae.

[0015] In this invention, the abbreviations are explained as follows: Fmoc: fluorenemethyloxycarbonyl; DCE: 1,2-dichloroethane; Oxyme: ethyl 2-oxime cyanoacetate; DCM: dichloromethane; DMF: N,N-dimethylformamide; DIC: N,N-diisopropylcarbodiimide; S5: 2-amino-2-methyl-9-heptenoic acid; R8: 2-amino-2-methyl-9-decenoic acid; TFA: trifluoroacetic acid; EDT: 1,2-ethylenedithiol; Grubbs I: phenylmethylenebis(tricyclohexylphosphine)ruthenium dichloride; MS: mass spectrometry; HR-Q-TOF-MS: high-resolution matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0016] The advantages of this invention are: 1. In this invention, Rink amide MBHA amino resin is used as a solid-phase support. The amino acid sequence of the template polypeptide GCIFN-20-0: Ac-SYEKKINRHFKILKKNLKKK-NH2 is modified. While retaining the key amino acid residues, the original amino acids at the i, i+4 and i, i+7 positions are replaced by S5 or S5, R8, respectively, to obtain the target staple peptide.

[0017] 2. The seven staple peptides obtained in this invention have significantly improved antibacterial properties compared to the template peptide. Among them, GCIFN-20-2, GCIFN-20-4, GCIFN-20-5, GCIFN-20-6, and GCIFN-20-7 showed good in vitro antibacterial properties against Staphylococcus epidermidis ATCC12228. Among them, the antibacterial activity of staple peptides GCIFN-20-4 and GCIFN-20-6 against Staphylococcus epidermidis ATCC12228 was 1 times higher than that of the template peptide GCIFN-20-0; GCIFN-20-5 showed a 3-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to GCIFN-20-0; GCIFN-20-7 showed a 7-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to GCIFN-20-0; and GCIFN-20-2 showed a 31-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to GCIFN-20-0. This invention successfully prepared modified staple peptides based on GCIFN-20-0. In vitro experiments demonstrated that the synthesized staple peptides can significantly inhibit the growth and reproduction of pathogenic bacteria, showing promise for development into novel antibacterial drugs.

[0018] 3. The results of the enzymatic stability test showed that the staple peptide GCIFN-20-2 of the present invention has improved stability against chymotrypsin hydrolysis and trypsin hydrolysis compared with the template peptide GCIFN-20-0. Attached Figure Description

[0019] Figure 1The diagram shows the amino acid sequence of GCIFN-20-0 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-0, B is the HPLC chromatogram of GCIFN-20-0, and C is the mass spectrum of GCIFN-20-0.

[0020] Figure 2 The diagram shows the amino acid sequence of GCIFN-20-1 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-1, B is the HPLC chromatogram of GCIFN-20-1, and C is the mass spectrum of GCIFN-20-1.

[0021] Figure 3 The diagram shows the amino acid sequence of GCIFN-20-2 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-2, B is the HPLC chromatogram of GCIFN-20-2, and C is the mass spectrum of GCIFN-20-2.

[0022] Figure 4 The diagram shows the amino acid sequence of GCIFN-20-3 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-3, B is the HPLC chromatogram of GCIFN-20-3, and C is the mass spectrum of GCIFN-20-3.

[0023] Figure 5 The diagram shows the amino acid sequence of GCIFN-20-4 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-4, B is the HPLC chromatogram of GCIFN-20-4, and C is the mass spectrum of GCIFN-20-4.

[0024] Figure 6 The diagram shows the amino acid sequence of GCIFN-20-5 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-5, B is the HPLC chromatogram of GCIFN-20-5, and C is the mass spectrum of GCIFN-20-5.

[0025] Figure 7 The diagram shows the amino acid sequence of GCIFN-20-6 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-6, B is the HPLC chromatogram of GCIFN-20-6, and C is the mass spectrum of GCIFN-20-6.

[0026] Figure 8 The diagram shows the amino acid sequence of GCIFN-20-7 and its characterization spectrum. In the diagram, A is the amino acid sequence of GCIFN-20-7, B is the HPLC chromatogram of GCIFN-20-7, and C is the mass spectrum of GCIFN-20-7.

[0027] Figure 9The degradation kinetic curves of GCIFN-20-0 and GCIFN-20-2 trypsin are shown.

[0028] Figure 10 The degradation kinetic curves of GCIFN-20-0 and GCIFN-20-2 chymotrypsin. Detailed Implementation

[0029] 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.

[0030] Unless otherwise specified, the experimental materials and reagents used in the embodiments of this invention are all consumables and reagents that are conventionally available from commercial sources.

[0031] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Percentages and parts are by weight.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0033] This invention designs and synthesizes seven staple peptides based on the amino acid sequence of the template peptide GCIFN-20-0. A schematic diagram, HPLC chromatogram, and mass spectrum of the template peptide GCIFN-20-0 are shown below. Figure 1 .

[0034] Example 1: Preparation of staple peptide based on GCIFN-20-0 1. General Synthesis Process All stapled peptides (GCIFN-20-1 to GCIFN-20-7) were prepared using a solid-phase peptide synthesis method based on the Fmoc (fluorenemethyloxycarbonyl) protection strategy. This method used Rink amide MBHA resin as the solid-phase support at a loading capacity of 0.35 mmol / g. Synthesis was carried out in solid-phase synthesis reaction tubes, and the entire process included resin swelling, Fmoc protecting group removal, amino acid condensation, N-terminal acetylation, olefin metathesis cyclization, and final peptide cleavage.

[0035] (1) Resin pretreatment and activation: Take 400 mg of amino resin and soak it in dichloromethane solvent for 30 minutes to allow it to swell fully. Then, treat it twice with 7 mL of 20% piperidine N,N-dimethylformamide solution at 35°C, shaking for 5 minutes each time, to remove the Fmoc protecting groups on the resin surface. Finally, wash the resin three times each with N,N-dimethylformamide, dichloromethane and N,N-dimethylformamide in sequence.

[0036] (2) Amino acid condensation: Following the sequence of the target polypeptide GCIFN-20-0 (Ac-SYEKKINRHFKILKKNLKKK-NH2), amino acids were sequentially linked from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus). For common amino acids, 1 mmol of Fmoc-protected amino acid, 142 mg of ethyl 2-oxime cyanoacetate, and 200 μL of N,N-diisopropylcarbodiimide were dissolved in 7 mL of N,N-dimethylformamide, activated at 37°C for 15 minutes, and then added to a reaction tube for coupling with resin. The reaction was carried out at 60°C for 20 minutes. For non-natural amino acids S5 or R8, the amount used was 0.2 mmol, and the condensing agent was 43 mg of ethyl 2-oxime cyanoacetate and 60 μL of N,N-diisopropylcarbodiimide. After activation in the same solvent, the reaction was carried out at 60°C for 3 hours or at 37°C overnight to ensure complete coupling. After each amino acid is attached, the Fmoc protecting group must be removed with a 20% piperidine N,N-dimethylformamide solution, followed by washing.

[0037] (3) N-terminal acetylation: After the sequence synthesis was completed, the Fmoc protecting group of the terminal amino acid was removed with a 20% piperidine N,N-dimethylformamide solution. Then, 10 mL of acetylation reagent (diisopropylethylamine: acetic anhydride: N,N-dimethylformamide = 1:1:8, V / V / V) was added, and the reaction was carried out at 37℃ for 5 min to acetylate the N-terminus of the peptide. After the reaction was completed, the resin was dried and washed.

[0038] (4) Olefin metathesis reaction (cyclization): The resin was rinsed three times with 1,2-dichloroethane. A solution of 56 mg phenylmethylene bis(tricyclohexylphosphine) ruthenium dichloride dissolved in 6 mL of 1,2-dichloroethane was added, and the reaction was carried out at room temperature for 8 hours. This caused a ring-closing metathesis reaction between the introduced non-natural amino acid side chain olefins, forming a full-carbon scaffold, thereby stabilizing the α-helical conformation of the polypeptide. The resin was thoroughly washed after the reaction was completed.

[0039] (5) Peptide cleavage and purification: The resin was placed in a 50 mL centrifuge tube, and 20 mL of cleavage reagent K (trifluoroacetic acid: water: 1,2-ethylenedithiol: benzyl sulfide: phenol = 82.5: 5: 2.5: 5: 5, V / V / V / V / V) was added. The mixture was shaken at 37 °C for 3 hours. After the reaction was completed, the cleavage solution was collected, dried and concentrated under nitrogen, and the crude peptide was precipitated with pre-cooled ice-cold ether. After centrifugation at 3500 r / min for 3 min, the supernatant was discarded, and the precipitate was air-dried to obtain the target crude peptide. The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and its structure was identified by mass spectrometry.

[0040] 2. Specific synthesis of each binding peptide The general synthetic procedure described above applies to all staple peptides from GCIFN-20-1 to GCIFN-20-7. The difference between each staple peptide lies in the substitution of a specific position in its template sequence by a non-natural amino acid S5 or a combination of S5 and R8, as shown in Table 1 below. These substitutions are the basis for the olefin metathesis reaction to proceed and form the specific "staple" structure.

[0041] Table 1. Specific amino acid substitution sites for each binding peptide Note: S5 and R8 are specific non-natural amino acids that are introduced for subsequent olefin metathesis reactions.

[0042] 3. Purification of peptide samples The crude peptide was dissolved in a mixed solvent of acetonitrile and water, and purified by reversed-phase preparative high-performance liquid chromatography (RP-HPLC) to obtain the purified peptide product. The separation conditions were as follows: Instrument: Shimadzu LC-20A reversed-phase high-performance liquid chromatograph; Column: UltimateXB-C18, 21.2 × 250 mm, 5 μm; Mobile phase: Mobile phase A is an acetonitrile solution of 0.1% trifluoroacetic acid by volume, and mobile phase B is an aqueous solution of 0.1% trifluoroacetic acid by volume; Procedure and parameters: Elute with 90% B for 3 min, then elute with 90% B to 45% B for 40 min; flow rate is 10 mL / min, injection volume is 5 mL, and detection wavelengths are 214 nm and 254 nm.

[0043] Each peptide was purified individually.

[0044] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 45%, compound GCIFN-20-0 was eluted to obtain GCIFN-20-0, with a separation rate of 45.79%.

[0045] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 48%, compound GCIFN-20-1 was eluted to obtain GCIFN-20-1, with a separation rate of 6.83%.

[0046] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 51%, compound GCIFN-20-2 was eluted to obtain GCIFN-20-2, with a separation rate of 12.85%.

[0047] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 47%, compound GCIFN-20-3 was eluted to obtain GCIFN-20-3, with a separation rate of 8.52%.

[0048] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 52%, compound GCIFN-20-4 was eluted to obtain GCIFN-20-4, with a separation rate of 39.60%.

[0049] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 55%, compound GCIFN-20-5 was eluted to obtain GCIFN-20-5, with a separation rate of 6.67%.

[0050] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 49%, compound GCIFN-20-6 was eluted to obtain GCIFN-20-6, with a separation rate of 8.62%.

[0051] During the gradient elution process, when the volume fraction of mobile phase A increased to about 50%, compound GCIFN-20-7 was eluted to obtain GCIFN-20-7, with a separation rate of 25.01%.

[0052] Identification and structural analysis of the product in Example 2 The product obtained in step 2 of Example 1 was identified by reversed-phase HPLC. The analytical column was Welch C18; mobile phase A was an acetonitrile solution of 0.1% trifluoroacetic acid (v / v), and mobile phase B was an aqueous solution of 0.1% trifluoroacetic acid (v / v). Gradient elution was used (0–2 min, mobile phase B 90%; 3–25 min, mobile phase B 90%–10%); flow rate was 1.0 mL / min; 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%. The HPLC chromatograms of the staple peptide GCIFN-20-1 and GCIFN-20-7 are shown below. Figures 2-8 Structural analysis was performed by HR-Q-TOF-MS, and the mass spectrometry results of GCIFN-20-1 and GCIFN-20-7 are shown below. Figures 2-8The structure of the binding peptide is shown in Table 2.

[0053] Table 2. Sequences of the template peptide and the modified binding peptide used in this invention. The template peptides and the amino acid sequences of the obtained binding peptides mentioned in Tables 1 and 2 of this invention are shown below. Figure 1-8 The amino acid sequence is shown in SEQ ID NO:1.

[0054] Example 3: Antibacterial activity experiment of the polypeptide of the present invention In vitro anti-drug resistant bacteria test: Prepare solid LB medium, autoclave, plate, and prepare liquid LB medium, store at 4℃. Spread the bacterial suspension onto solid LB medium and incubate overnight in an inverted incubator at 37℃. Take a single colony, add 3 mL of liquid LB medium, and incubate at 37℃ and 220 rpm for 6 h in a constant temperature shaker to allow the bacteria to grow to the logarithmic phase. Take 1 mL of bacterial suspension, centrifuge at 4000 rpm for 5 min, discard the supernatant, add PBS, and adjust the bacterial concentration to 2 × 10⁻⁶ based on the OD value. 6 CFU / mL. Different concentrations of antimicrobial peptides were added to 96-well plates, along with bacterial culture. The plates were incubated at 37°C for 8 h, and detected 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.

[0055] Table 3. Antibacterial experimental results of the binding peptide of this invention. The results in Table 3 show that the seven staple peptides obtained in this invention have significantly improved antibacterial properties compared with the template peptide. Among them, GCIFN-20-2, GCIFN-20-4, GCIFN-20-5, GCIFN-20-6, and GCIFN-20-7 showed good in vitro antibacterial properties against Staphylococcus epidermidis ATCC12228. Among them, the antibacterial properties of the staple peptides GCIFN-20-4 and GCIFN-20-6 against Staphylococcus epidermidis ATCC12228 were 1 times higher than those of the template peptide GCIFN-20-0; the antibacterial properties of GCIFN-20-5 against Staphylococcus epidermidis ATCC12228 were 3 times higher than those of the template peptide GCIFN-20-0; the antibacterial properties of GCIFN-20-7 against Staphylococcus epidermidis ATCC12228 were 7 times higher than those of the template peptide GCIFN-20-0; the antibacterial properties of GCIFN-20-2 against Staphylococcus epidermidis ATCC12228 were 31 times higher than those of the template peptide GCIFN-20-0; and the antibacterial properties of GCIFN-20-2 against Pseudomonas aeruginosa ATCC27853 were 1 time higher than those of the template peptide GCIFN-20-0.

[0056] GCIFN-20-6 showed twice the antibacterial activity against Pseudomonas aeruginosa ATCC27853 and Klebsiella pneumoniae CMCC46117 compared to the template peptide GCIFN-20-0.

[0057] The above embodiments demonstrate that the present invention successfully prepared a modified staple peptide based on GCIFN-20-0. In vitro experiments have shown that the synthesized staple peptide can significantly inhibit the growth and reproduction of pathogenic bacteria, and has the potential to be developed into a novel antibacterial drug.

[0058] Example 4: Helicity test of the polypeptide of the present invention Linear peptides GCIFN-20-0 and stapled peptides GCIFN-20-1 to GCIFN-20-7 were accurately weighed at 1 mg and dissolved, respectively, in water and trifluoroethanol (V / V = 1:1) to a final concentration of 50 mM. The concentration was measured at room temperature using circular dichroism JASCO. J Characterization was performed using -1500 and 1 mm quartz cuvettes. The following experimental parameters were measured: wavelength, 190-260 nm; velocity, 20 nm / min. -1 Bandwidth, 1 nm. For the α-helical structure, there is a positive band near 192 nm and two negative bands at 222 nm and 208 nm. The helicity of each peptide was calculated using the equation helicity (%) = [θ]222 / (-39500(1-2.57 / n)) × 100, based on the ellipticity of the peptide spectrum at 222 nm and the number of amino acids in the peptide sequence. The results are shown in Table 4.

[0059] Table 4 Helicity of Peptides Table 4 shows that the staple peptide of the present invention can improve the α-helix degree of the template peptide. The above examples demonstrate that the modified staple peptide based on GCIFN-20-0 prepared by the present invention, through circular dichroism spectroscopy analysis, revealed that, except for GCIFN-20-7, the helix degree of the other staple peptides was greater than that of the template peptide GCIFN-20-0. This indicates that the staple locking effect after modification by the staple peptide strategy plays a certain role in reinforcing the peptide chain and improving the conformational stability of the peptide.

[0060] Example 5: Stability test of the polypeptide enzymatic hydrolysis of the present invention 1. Methods and results for determining the stability of trypsin digestion (1) Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH=7.4) until it is fully dissolved, so that the final concentration is 2 mM.

[0061] (2) Preparation of trypsin solution: Weigh a small amount of trypsin and dissolve it in the prepared buffer solution to make the final solubility 0.01 ng / μL.

[0062] (3) Preparation of peptide solution: Weigh 1 mg of template peptide GCIFN-20-0 and GCIFN-20-2, the optimal active derivative of the staple peptide after staple peptide strategy modification, and add them to the buffer solution to make the final solubility 1 mM.

[0063] (4) Reaction and Sampling: 1950 μL of trypsin solution was placed in a 5 mL centrifuge tube, and 50 μL of the prepared 1 mM polypeptide solution was added to the centrifuge tube containing the trypsin solution to carry out the trypsin degradation experiment. At time points of 0 min, 30 min, 60 min, 90 min, 120 min, 150 min and 180 min during the experiment, 60 μL of the reaction mixture was taken and 20 μL of concentrated hydrochloric acid (1M) was added to quench and terminate the enzymatic hydrolysis reaction.

[0064] (5) Results analysis: Take 50 μL for HPLC analysis, determine the residual amount of peptide by integrating the peak area of ​​peptide, calculate the percentage of peak area at each time point to peak area at time 0 and draw a curve to obtain the analysis chart of peptide antitrypsin hydrolysis stability.

[0065] The principle behind trypsin degradation of peptides is that trypsin primarily cleaves peptide bonds at the carboxyl terminus of arginine or lysine, specifically cutting these bonds. The results are shown in Tables 5 and 6.

[0066] Table 5. Stability data of GCIFN-20-0 trypsin Table 6. Stability data of GCIFN-20-2 trypsin The results in Tables 5 and 6 show that the staple peptide GCIFN-20-2, which exhibits the best inhibitory activity against Staphylococcus epidermidis in this invention, still retains 72.60% of its staple peptide intact after 3 hours of trypsin digestion. The trypsin hydrolysis stability test demonstrates that the staple peptide GCIFN-20-2 exhibits significantly improved stability against trypsin hydrolysis compared to the template peptide GCIFN-20-0.

[0067] 2. Methods and results for determining the stability of chymotrypsin hydrolysis (1) Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH=7.4) until it is fully dissolved, so that the final concentration is 2 mM.

[0068] (2) Preparation of chymotrypsin solution: Weigh a small amount of chymotrypsin and dissolve it in the prepared buffer solution to make the final solubility 0.01 ng / μL.

[0069] (3) Preparation of peptide solution: Weigh 1 mg of template peptide GCIFN-20-0 and GCIFN-20-2, the optimal active derivative of the staple peptide after staple peptide strategy modification, and add them to the buffer solution to make the final solubility 1 mM.

[0070] (4) Reaction and Sampling: 1950 μL of chymotrypsin solution was placed in a 5 mL centrifuge tube, and 50 μL of the prepared 1 mM polypeptide solution was added to the centrifuge tube containing the chymotrypsin solution to carry out the chymotrypsin degradation experiment. At time points of 0 min, 30 min, 60 min, 90 min, 120 min, 150 min and 180 min during the experiment, 60 μL of the reaction mixture was taken and 20 μL of concentrated hydrochloric acid (1M) was added to quench and terminate the enzymatic hydrolysis reaction.

[0071] (5) Results analysis: Take 50 μL for HPLC analysis, determine the residual amount of peptide by integrating the peak area of ​​peptide, calculate the percentage of peak area at each time point to peak area at time 0 and draw a curve to obtain the analysis chart of peptide antichymotrypsin hydrolysis stability.

[0072] The principle behind chymotrypsin's degradation of peptides is that chymotrypsin's main cleavage sites are tryptophan, tyrosine, phenylalanine, and threonine, allowing it to specifically cleave the peptide bonds formed at the carboxyl ends of tryptophan, tyrosine, phenylalanine, and threonine in the peptide chain. The results are shown in Tables 7 and 8.

[0073] Table 7. GCIFN-20-0 chymotrypsin hydrolysis data Table 8. Hydrolysis data of GCIFN-20-2 chymotrypsin The results in Tables 7 and 8 show that the staple peptide GCIFN-20-2, which exhibits the best inhibitory activity against Staphylococcus epidermidis in this invention, was not completely degraded after 3 hours of exposure to chymotrypsin, retaining 20.23% of its original content. The chymotrypsin hydrolysis stability test indicates that the staple peptide GCIFN-20-2 demonstrates significantly improved stability against chymotrypsin hydrolysis compared to the template peptide GCIFN-20-0.

Claims

1. An antimicrobial polypeptide, characterized in that, The antibacterial polypeptide is a stapler peptide, and the stapler peptide is: GCIFN-20-2: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 7N are replaced by S5; GCIFN-20-4: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 12I and 16N are replaced by S5; GCIFN-20-5: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 2Y and 9H are replaced by R8 and S5; GCIFN-20-6: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 10F are replaced by R8 and S5; GCIFN-20-7: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 9H and 16N are replaced by R8 and S5.

2. The antimicrobial polypeptide according to claim 1, characterized in that, The stapler peptide is: GCIFN-20-2: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 7N are replaced by S5; GCIFN-20-5: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 2Y and 9H are replaced by R8 and S5; GCIFN-20-6: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 10F are replaced by R8 and S5; GCIFN-20-7: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 9H and 16N are replaced by R8 and S5.

3. The antimicrobial polypeptide according to claim 2, characterized in that, The stapler peptide is: GCIFN-20-2: Using Ac-SYEKKINRHFKILKKNLKKK-NH2 as a peptide template, amino acid residues 3E and 7N are replaced by S5; GCIFN-20-7: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 9H and 16N are replaced by R8 and S5.

4. The antimicrobial polypeptide according to claim 3, characterized in that, The stapler peptide is: GCIFN-20-2: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 3E and 7N are replaced by S5.

5. The use of the antimicrobial polypeptide according to claim 1, 2, 3 or 4 in the preparation of an anti-Staphylococcus epidermidis drug.

6. The antimicrobial polypeptide according to claim 1, characterized in that, The stapler peptide is: GCIFN-20-6: The peptide template is Ac-SYEKKINRHFKILKKNLKKK-NH2, in which amino acid residues 3E and 10F are replaced by R8 and S5.

7. The use of the antimicrobial polypeptide according to claim 4 or 6 in the preparation of an anti-Pseudomonas aeruginosa drug.

8. The use of the antimicrobial polypeptide according to claim 6 in the preparation of an anti-Klebsiella pneumoniae drug.

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