Antibacterial peptide derivative and use thereof

Abstract

The application provides an antibacterial peptide derivative and application thereof. The antibacterial peptide derivative is any one of polypeptides in (a)-(d) below: (a) a polypeptide with an amino acid sequence as shown in general formula (1) or general formula (2): Ac-GX1 a ZOWX1 b ZOFWNOIOZ-NH2 (1), Ac-GX2 a PZOWX2 b ZOFWNOIOZ-NH2 (2). The antibacterial peptide has excellent in-vitro antibacterial activity, and has lower hemolysis and higher anti-enzymatic stability.

Description

Technical Field

[0001] This invention relates to the field of biopeptide technology, and in particular to an antimicrobial peptide derivative and its applications. Background Technology

[0002] In recent years, antimicrobial peptides have attracted widespread attention due to their broad-spectrum antimicrobial activity and low tendency to induce bacterial resistance, and are considered promising next-generation antimicrobial drugs to replace traditional antibiotics. P-α-O2-B, a derivative peptide derived from the natural antimicrobial peptide sC18, exhibits good activity against drug-resistant bacteria and has shown promising efficacy in treating bacteremia models in mice. However, like most antimicrobial peptides, P-α-O2-B suffers from poor enzymatic stability, which limits its clinical application. Therefore, improving the enzymatic stability of P-α-O2-B has become an urgent problem to be solved. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides an antimicrobial peptide derivative and its applications. It solves the problem of instability in existing antimicrobial peptides.

[0004] In a first aspect, the present invention provides an antimicrobial peptide derivative or a pharmaceutically acceptable salt thereof, said antimicrobial peptide derivative being any one of the following polypeptides (a)-(d):

[0005] (a) A polypeptide having an amino acid sequence as shown in general formula (1) or general formula (2):

[0006] Ac-GX1 a ZOWX1 b ZOFWNOIOZ-NH2(1)

[0007] Ac-GX2 a PZOWX2 b ZOFWNOIOZ-NH2(2)

[0008] X1 a X1 b Same or different, X1 a X1 b Each is independently selected from one of L-type isoleucine (I), D-type leucine (l), and D-type isoleucine (i), where O represents ornithine and Z represents arginine.

[0009] X2 a X2 b Same or different, X2 a X2 bEach of the following is independently selected from one of L-leucine (L), L-isoleucine (I), D-leucine (l), and D-isoleucine (i), where O represents ornithine and Z represents arginine.

[0010] (b) A polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues into the amino acid sequence defined in (a);

[0011] (c) A polypeptide obtained by modifying the N-terminus and / or C-terminus of the polypeptide defined in (a);

[0012] (d) is a polypeptide that has 80% or more, 85% or more, 90% or more, 95% or more or more, 99% or more homology with the amino acid sequence defined in (a).

[0013] According to an embodiment of the present invention, the antimicrobial peptide derivative comprises any of the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:13 below, or a polypeptide having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any of the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:13.

[0014]

[0015] According to an embodiment of the present invention, the antimicrobial peptide derivative is H-08, and its amino acid sequence is shown in SEQ ID NO:8.

[0016] According to an embodiment of the present invention, the antimicrobial peptide derivative has good antimicrobial activity.

[0017] According to embodiments of the present invention, the strains include, but are not limited to, drug-resistant strains of Staphylococcus aureus, Enterococcus faecalis, Bacillus mycoides, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Citrobacter freundii, and Klebsiella pneumoniae.

[0018] According to an embodiment of the present invention, the minimum inhibitory concentration of the antimicrobial peptide derivative is 1.5-25 μM.

[0019] According to an embodiment of the present invention, the antimicrobial peptide derivative H-08 exhibits good antimicrobial activity against all of the aforementioned strains. In one embodiment of the present invention, the minimum inhibitory concentrations (MICs) of the antimicrobial peptide derivative H-08 against resistant strains of Staphylococcus aureus, Enterococcus faecalis, Bacillus mycoides, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Citrobacter freundii, and Klebsiella pneumoniae are 1.56 μM, 3.13 μM, 3.13 μM, 3.13 μM, 3.13 μM, 3.13 μM, 3.13 μM, and 3.13 μM, respectively.

[0020] According to an embodiment of the present invention, the antimicrobial peptide derivative H-08 exhibits significant bactericidal activity against Staphylococcus aureus and Escherichia coli. In one embodiment of the present invention, the antimicrobial peptide derivative H-08 can effectively eliminate Staphylococcus aureus and Escherichia coli within 24 hours and 6 hours, respectively.

[0021] According to an embodiment of the present invention, the antimicrobial peptide derivative has lower hemolytic toxicity compared to P-α-O2-B.

[0022] According to an embodiment of the present invention, the antimicrobial peptide derivative exhibits good stability. In one embodiment of the present invention, the antimicrobial peptide derivative exhibits high stability and anti-degradation properties in salt ion environments and in trypsin, proteinase K, and chymotrypsin environments.

[0023] According to embodiments of the present invention, the antimicrobial peptide derivative exhibits good in vivo safety and therapeutic efficacy against bacterial infections. In one embodiment of the present invention, the dosage of the antimicrobial peptide derivative is 10 mg / kg, demonstrating good in vivo safety and efficacy.

[0024] According to embodiments of the present invention, the antimicrobial peptide derivative can be used alone, in combination, or in the form of a polymer. For example, it can be in the form of a peptide dimer, peptide trimer, etc., and the polymer can be composed of peptide derivatives having the same sequence or of several peptide derivatives of different sequences of general formula 1 or general formula 2.

[0025] According to an embodiment of the present invention, the antimicrobial peptide derivative further includes a modified antimicrobial peptide derivative.

[0026] According to embodiments of the present invention, the antimicrobial peptide derivative can be fused with other proteins to form a fusion protein, or the antimicrobial peptide derivative can be linked to a polymer or carrier. Examples include human serum albumin, humanized antibodies, liposomes, microarrays, synthetic polymers, nanoparticles, and bacteriophages.

[0027] According to embodiments of the present invention, the antimicrobial peptide derivative is obtained through chemical synthesis or recombinant technology. In one embodiment of the present invention, the peptide is synthesized by solid-phase peptide synthesis and purified by reversed-phase high-performance liquid chromatography.

[0028] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antimicrobial peptide derivative and a pharmaceutically acceptable carrier or excipient.

[0029] According to embodiments of the present invention, the pharmaceutical composition can be delivered by topical application or other forms of administration, such as oral, parenteral, subcutaneous, sublingual, intralesional, intraperitoneal, intravenous, or intramuscular administration.

[0030] According to embodiments of the present invention, the pharmaceutical composition may be in the form of capsules, tablets, pills, drops, suppositories, powders, sprays, vaccines, ointments, pastes, creams, inhalers, patches, aerosols, etc.

[0031] According to an embodiment of the present invention, the pharmaceutically acceptable carrier or excipient is compatible with any solvent, diluent or other liquid medium, dispersant or suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, capsule, solid binder or lubricant.

[0032] In a third aspect, the present invention provides a nucleic acid encoding the antimicrobial peptide derivative.

[0033] In a fourth aspect, the present invention provides a vector containing the nucleic acid.

[0034] In a fifth aspect, the present invention provides a host cell containing the nucleic acid, the vector, or expressing the antimicrobial peptide derivative.

[0035] In a sixth aspect, the present invention provides the use of an antimicrobial peptide derivative, a pharmaceutical composition, the aforementioned nucleic acid, a carrier, or a host cell in the preparation of an antimicrobial drug.

[0036] According to embodiments of the present invention, the strains include, but are not limited to, drug-resistant strains of Staphylococcus aureus, Enterococcus faecalis, Bacillus mycoides, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Citrobacter freundii, and Klebsiella pneumoniae.

[0037] According to an embodiment of the present invention, the antimicrobial peptide derivative and pharmaceutical composition can be used to treat drug-resistant bacterial infectious diseases.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention modifies the antimicrobial peptide P-α-02-B using strategies such as proline insertion, isoleucine substitution, and D-amino acid substitution, discovering a novel class of antimicrobial peptide sequences. These antimicrobial peptides exhibit excellent in vitro antimicrobial activity, low hemolytic activity, and high resistance to enzymatic degradation. In particular, H-08 showed superior in vivo therapeutic effects compared to P-α-02-B in the treatment of bacteremia in mice. Attached Figure Description

[0040] Figure 1 shows the high performance liquid chromatograms of antimicrobial peptides H-01 to H-13; Figure 1A It is an antimicrobial peptide H-01; Figure 1B It is an antimicrobial peptide H-02; Figure 1C It is an antimicrobial peptide H-03; Figure 1D It is an antimicrobial peptide H-04; Figure 1E It is an antimicrobial peptide H-05; Figure 1F It is an antimicrobial peptide H-06; Figure 1G It is an antimicrobial peptide H-07; Figure 1H It is an antimicrobial peptide H-08; Figure 1I It is an antimicrobial peptide H-09; Figure 1J It is an antimicrobial peptide H-10; Figure 1K It is an antimicrobial peptide H-11; Figure 1L It is an antimicrobial peptide H-12; Figure 1M It is an antimicrobial peptide H-13.

[0041] Figure 2 shows the mass spectra of antimicrobial peptides H-01 to H-13; Figure 2A It is an antimicrobial peptide H-01; Figure 2B It is an antimicrobial peptide H-02; Figure 2C It is an antimicrobial peptide H-03; Figure 2D It is an antimicrobial peptide H-04; Figure 2E It is an antimicrobial peptide H-05; Figure 2F It is an antimicrobial peptide H-06; Figure 2G It is an antimicrobial peptide H-07; Figure 2H It is an antimicrobial peptide H-08; Figure 2I It is an antimicrobial peptide H-09; Figure 2J It is an antimicrobial peptide H-10; Figure 2K It is an antimicrobial peptide H-11; Figure 2L It is an antimicrobial peptide H-12; Figure 2M It is an antimicrobial peptide H-13.

[0042] Figure 3 The image shows the hemolytic effects of the antimicrobial peptide P-α-O2-B and its derivative peptides.

[0043] Figure 4 Figure 1 shows the stability test results of antimicrobial peptide P-α-02-B and its derivative peptides under enzyme and salt treatment conditions. (A) Degradation of P-α-02-B and its derivative peptides in the presence of chymotrypsin; (B) Degradation of P-α-02-B and its derivative peptides in the presence of trypsin and proteinase K; Changes in MIC values ​​of P-α-02-B and H-08 against Staphylococcus aureus (C) and Escherichia coli (D) under salt treatment conditions.

[0044] Figure 5 The bactericidal rate curves of antimicrobial peptide H-08 against Staphylococcus aureus (left) and Escherichia coli (right).

[0045] Figure 6Figure 1 shows the in vivo safety results of the antimicrobial peptide H-08. (A) Survival curve of mice after H-08 treatment; (B) Survival curve of mice after polymyxin B treatment; (C) Median lethal dose of mice treated with H-08 and polymyxin B; Organ index (D) and blood biochemical indicators (E, F) of mice after H-08 treatment; (G) HE staining images of heart, liver, spleen, lung and kidney of mice after H-08 treatment.

[0046] Figure 7 The following are the results of the in vivo antimicrobial experiment of antimicrobial peptide H-08. (A) Flowchart of the in vivo antimicrobial experiment; (B) Survival curve of mice; (C) Weight change of mice; (D) Staphylococcus aureus load in the liver, spleen, lungs and kidneys of mice; (E) HE staining of the heart, liver, spleen, lungs and kidneys of mice. Detailed Implementation

[0047] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0048] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0049] "Derivatives" refer to antimicrobial peptide variants that have one or more amino acid residues replaced, deleted, or added at a specific position in the sequence of the antimicrobial peptide P-α-02-B, thereby affecting the biological activity (e.g., antimicrobial activity) of the antimicrobial peptide.

[0050] The term "modification" refers to the molecular engineering process that introduces non-amino acid-derived functional chemical groups (such as PEGylation, glycosylation, or lipidation) into a polypeptide molecule through covalent linkage or other chemical bonding, ensuring that such structural modifications do not significantly affect its biological conformation and functional potency (such as antibacterial activity). Such modifications can be natural or non-natural. In some embodiments, non-natural modifications may include the addition of protective or capping groups to the reactive moiety, the addition of detectable markers (such as antibodies and / or fluorescent markers), the addition or modification of glycosylation, or the addition of filler groups (such as PEGylation), and other variations known to those skilled in the art. In some embodiments, non-natural modifications may be capping modifications, such as N-terminal acetylation and C-terminal amidation. Protective groups that can be added include (but are not limited to) t-Boc and Fmoc. Commonly used fluorescently labeled proteins include (but are not limited to) green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and mCherry. Polyethylene glycol (PEG) conjugation to proteins has been used as a method to extend the cycling half-life of many pharmaceutical proteins. The term "modification" includes peptides that are chemically modified by covalently linking one or more PEG molecules.

[0051] "Amino acid sequence homology percentage" refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, such as an antimicrobial peptide derivative, after sequence alignment and the introduction of gaps (if necessary) to achieve the maximum sequence identity percentage, and without considering any conserved substitutions as part of sequence identity. Alignment for determining the amino acid sequence homology percentage can be performed in various ways within the art, for example, using publicly available software such as BLAST or software commercially available, for example, from DNASTAR. Two or more polypeptide sequences can be any value from 0 to 100% identity, or any integer value between them. As used herein, two amino acid sequences are "substantially homologous" when at least about 80% of the amino acid residues (e.g., at least about 85%, at least about 90%, at least about 92.5%, at least about 95%, at least about 98%, or at least about 99%) are identical or represent conserved substitutions. When one or more, for example, up to 10%, up to 15%, or up to 20%, of the amino acids of a polypeptide, such as the antimicrobial peptide derivative described herein, are substituted with similar or conserved amino acids, and the resulting peptide has at least one activity (e.g., antimicrobial activity) of a reference polypeptide, such as the antimicrobial peptide derivative described herein, the sequences of the disclosed polypeptides are substantially homologous.

[0052] A therapeutically effective dose refers to the amount of active ingredient that improves symptoms or signs of a disease. The dose should be sufficient to improve the symptoms or signs of the disease being treated without causing unacceptable toxicity to the patient. The dose can be varied by adjusting it to the specific circumstances of the invention to produce the desired therapeutic effect. Appropriate conversion of drug doses from animal studies to human studies (human equivalent dose, HED) is obtained using a body surface area (BSA) normalization method. The dose-response relationships between animals and humans (based on milligrams per square meter of body surface area) are described by Reagan-Shaw et al. (2007. FASEB J.22:659-661).

[0053] Pharmaceutically acceptable salts may be used in this article, such as inorganic acid salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids like acetate, propionate, malonate, and benzoate. A complete list of pharmaceutically acceptable salts is available at Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0054] Example 1: Design and Modification of Antimicrobial Peptides

[0055] To improve the enzymatic stability of P-α-02-B, a series of P-α-02-B-derived peptides H-01 to H-13 were designed using strategies such as proline insertion, isoleucine substitution, and D-amino acid substitution. Through structural prediction and sequence analysis, proline was inserted into the C-terminal side of the leucine residue at position 2 of the N-terminus of P-α-02-B to obtain H-01. Furthermore, L-isoleucine was used to replace the leucine residues at positions 2 and / or 6 of the N-terminus of P-α-02-B to obtain H-02, H-03, and H-04; L-isoleucine was used to replace the leucine residues at positions 2 and / or 6 of the N-terminus of H-01 to obtain H-05, H-06, and H-07; and D-isoleucine was used to replace the L-isoleucine residues in H-02 to H-07 to obtain H-08 to H-13. The synthesized P-α-02-B-derived peptides are shown in Table 1.

[0056] Table 1. Sequences and molecular weights of antimicrobial peptide P-α-O2-B and its derivative peptides

[0057]

[0058]

[0059] i represents D-type isoleucine, O represents ornithine, and Z represents high arginine.

[0060] Example 2 Synthesis and Preparation of Antimicrobial Peptides

[0061] 1. Reagent preparation

[0062] (1) Deprotection solution: 20% piperidine / N,N-dimethylformamide (DMF) solution.

[0063] (2) Ninhydrin solution: Weigh 0.5g of ninhydrin, transfer it to a 10mL volumetric flask, add anhydrous methanol to make up to the mark, and mix well to obtain ninhydrin solution.

[0064] (3) Pyridine solution: Pyridine is redistilled to remove water.

[0065] (4) Peptide lysis buffer: This reagent is prepared fresh for use. Add 45 mL of trifluoroacetic acid, 2.5 mL of m-methylphenol, 1.5 mL of anisole, and 0.5 mL of water to a 50 mL centrifuge tube and mix well to obtain the lysis buffer.

[0066] (5) Amino acid and condensation reagent solution: Weigh three times the amount of the resin (0.75 mmol) of the corresponding amino acid and 0.38 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and 0.13 g of 1-hydroxy-7-azobenzotriazole (HOAT) into a 10 mL centrifuge tube, add 5 mL of DMF and 150 μL of N,N-diisopropylethylamine (DIEA) and mix well.

[0067] 2. Synthesis process

[0068] (1) Swelling of resin: Weigh 0.47g (0.25mmol) of Rink amide resin (loading capacity of 0.53mmol / g) and put it into the reactor. Add 10mL of DMF and stir to swell for 30min. After the resin expands, turn on the vacuum pump and dry the reaction solution.

[0069] (2) Deprotection: Add 10 mL of deprotection reagent and stir to react. After 5 min, dry the mixture under vacuum. Add another 10 mL of deprotection reagent and stir to react for 25 min to remove the Fmoc protecting group on the resin, exposing the amino group. Wash the resin three times each with DMF and dichloromethane (DCM) alternately.

[0070] (3) Amino acid detection: Dip a small amount of resin into a centrifuge tube using a capillary tube, add one drop each of ninhydrin solution and pyridine solution, place in a 100℃ metal bath heating device and heat for 5 minutes, then remove and observe. If the resin turns blue, it indicates that deprotection was successful and proceed to the next step; if there is no color change, deprotection failed, and the deprotection reagent is added again to repeat the above steps.

[0071] (4) Condensation: After thoroughly mixing the amino acid with the condensation reagent solution, add it to the reactor, start stirring, and react at room temperature for 1 hour. After the reaction is complete, drain the reaction solution, clean the resin with DMF and DCM, and repeat the detection operation. If the resin shows no color reaction, the amino acid condensation is successful; if the resin shows blue, the condensation has failed or the reaction is incomplete. Add the material again and repeat the condensation operation until the condensation is successful. Then repeat the deprotection operation and couple the next cis amino acid until the last amino acid condensation is complete.

[0072] (5) Pyrolysis: Add anhydrous diethyl ether to the resin after the reaction is complete, wash three times, and dry under vacuum. Transfer the resin to a round-bottom flask, add 15 mL of lysis buffer, and start stirring. For the first 30 min of the reaction, place the flask in an ice bath, then remove the ice bath and stir at room temperature for 3 h. After lysis is complete, add anhydrous diethyl ether according to the peptide mass, and stir rapidly for 0.5-1 h. Then let stand until the peptide is completely precipitated, pour into a G4 funnel, and dry under vacuum. Wash the resin and the peptide solid repeatedly with diethyl ether to remove any residual lysis buffer. Replace with a new clean vacuum flask, and dissolve the peptide on the funnel in small amounts of double-distilled water several times until all the peptide in the funnel is dissolved and the remaining resin appears as loose sand.

[0073] (6) Freeze-drying: Transfer the crude peptide solution in the filtration flask to a centrifuge tube, freeze it with liquid nitrogen, and freeze-dry the crude peptide for 48 hours using a freeze dryer to obtain crude peptide powder of H-01 to H-13.

[0074] 3. Analysis and purification

[0075] (1) Molecular weight determination: 0.1 mg of lyophilized crude peptide powder was dissolved in 100 μL of ultrapure water and analyzed by analytical high performance liquid chromatography. The peak tip of the main peak was collected, and the molecular weight of the peptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After confirming the correct molecular weight, the peptide was purified. As shown in Figure 2.

[0076] (2) Peptide purification: Based on the physicochemical properties of each target peptide, appropriate analytical and purification conditions were selected. A preparative high-performance liquid chromatography (HPLC) system was used for gradient elution at room temperature, and the target peptides were collected. Purity analysis was performed using an analytical HPLC system, and peptide solutions with a purity greater than 95% were collected. These solutions were then lyophilized to obtain pure peptide powder, which was collected, weighed, sealed in centrifuge tubes, and stored at -40℃. (See Figure 1.)

[0077] Chromatographic column selection: analytical high performance liquid chromatography column (C18), preparative high performance liquid chromatography column (C18).

[0078] Mobile phase preparation: Mobile phase A is a 1‰ trifluoroacetic acid (TFA) / water solution. After mixing, filter through a 0.45μm aqueous phase filter membrane to remove impurities. Mobile phase B is a 1‰ TFA / (acetonitrile:water = 7:3) solution. After mixing, filter through a 0.45μm organic phase filter membrane to remove impurities.

[0079] Example 3: In vitro antibacterial activity assay

[0080] The minimum inhibitory concentration (MIC) of P-α-O2-B and its derived peptides in resistant strains of *Staphylococcus aureus*, *Enterococcus faecalis*, *Bacillus mycoides*, *Staphylococcus epidermidis*, *Escherichia coli*, *Acinetobacter baumannii*, *Citrobacter freundii*, and *Klebsiella pneumoniae* was determined using a dilution method. 50 μL of liquid culture medium was pipetted into rows 1-8 of a 96-well plate. 50 μL of each 400 μM antimicrobial peptide solution was pipetted into each well in the first row. Then, using a twofold dilution method, 50 μL of the peptide solution was added from the first row to the second row, mixed, and then 50 μL of the peptide solution from the second row was transferred to the third row. This process was repeated until the eighth row. Finally, 50 μL was aspirated from the last row and discarded. Then, take a sterile sample loading vessel, add the diluted bacterial suspension (dilution ratio of bacterial suspension to culture medium 1:1000), and use a pipette to add 50 μL sequentially to rows 1-8 of a 96-well plate. Mix the bacterial suspension and the solution thoroughly, incubate at 37℃ with shaking at 180 rpm for 18 h, and observe and determine the MIC value. The MIC value is defined as the lowest concentration of peptides at which bacterial growth is visible in the wells when the plate is placed under fluorescent light and the liquid in the wells is clear with no visible bacterial growth. The experimental results are shown in Table 2.

[0081] Table 2 MIC values ​​of antimicrobial peptide P-α-O2-B and its derivative peptides

[0082]

[0083] As shown in Table 2, the antimicrobial peptide H-08 exhibited good antimicrobial activity against the above eight strains.

[0084] Example 4 Hemolytic Experiment

[0085] Fresh human blood was collected in 5 mL blood collection tubes containing heparin anticoagulant, then transferred to 15 mL sterile centrifuge tubes and centrifuged at 1500 rpm and 4°C for 5 min. After centrifugation, the lower layer of red blood cells was collected, and a suitable amount of PBS was added for gentle pipetting and washing (the operation must be very gentle to prevent red blood cell breakage and hemolysis). The cells were then centrifuged again at 1800 rpm and 4°C for 5 min. This washing process was repeated 2-3 times to obtain red blood cells. The red blood cells were then diluted to a 5% (v / v) red blood cell / PBS cell suspension for later use. PBS was poured into a sterile sample loading dish, and 50 μL of PBS was pipetted into rows 2-8 of a 96-cell culture plate. Each pure peptide powder was dissolved in PBS to prepare an 800 μM solution. 50 μL of the peptide solution was pipetted into the first well of a 96-well plate. Using a two-fold dilution method, 50 μL of the peptide solution was added from the first well to the second well, and the mixture was thoroughly mixed. This process was repeated until the eighth well was reached. Finally, 50 μL of excess solution was discarded. 50 μL of erythrocyte suspension was pipetted into each well of the 96-well plate. The plate was capped and gently shaken to mix. The plate was incubated at 37°C for 1 hour. Triple-well plates of the same peptide solution at the same concentration were prepared. PBS was used as a negative control, and Triton X-100 was used as a positive control. After incubation, the 96-well plate was centrifuged at 1500 rpm for 5 minutes. 50 μL of the supernatant was pipetted into a new, sterile, flat-bottomed 96-well plate. After the operation, the absorbance (OD) value at a wavelength of 405 nm was measured using a SpectraMax iD5 microplate reader in detectable absorbance mode. The percentage of red blood cell hemolysis was calculated using the following formula:

[0086] Percentage of hemolyzed red blood cells = [OD(pep) - OD(neg) / OD(pos) - OD(neg)] × 100%

[0087] OD(pep): Represents the absorbance value corresponding to the cell pore used for drug delivery.

[0088] OD(neg): Indicates the absorbance value of the negative control well.

[0089] OD(pos): Represents the absorbance value of the positive control well.

[0090] Experimental results are as follows Figure 3 As shown, H-08 exhibits lower hemolytic toxicity compared to P-α-02-B. Even at a concentration of 200 μM, the hemolysis rate of H-08 is only 7%.

[0091] Example 5 Stability Test

[0092] The chymotrypsin was dissolved in ultrapure water to a final concentration of 0.02 mg / mL. A 1 mM antimicrobial peptide was mixed with the chymotrypsin solution at a 1:1 volume ratio and incubated at 37°C for 6 hours. The peptide-protease mixture was collected at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h. The mixture was then heated at 100°C for 15 minutes to inactivate the protease. The degradation of the antimicrobial peptide under chymotrypsin treatment was determined by HPLC, with the experiment repeated three times in triplicate. Simultaneously, the degradation of the peptide by trypsin and proteinase K was also tested. A 0.25 mg / mL trypsin solution and a 4 mg / mL proteinase K solution were co-incubated with a 1 mM peptide solution at a 1:1 volume ratio at 37°C for 2 hours. The peptide-protease mixture was collected at 0 h and 2 h. The mixture was then heated at 100°C for 15 minutes to inactivate the protease. The degradation of the antimicrobial peptide under two protease treatment conditions was determined by HPLC, with each experiment repeated three times in triplicate. In addition, to evaluate the stability of the antimicrobial peptide under physiological conditions, the minimum inhibitory concentration (MIC) of the peptide against Staphylococcus aureus and Escherichia coli was determined in the presence of different salts (150 mM NaCl, 4 μM FeCl3, 1 mM MgCl2, and 4.5 mM KCl). This experiment was repeated three times in triplicate.

[0093] Experimental results are as follows Figure 4 As shown, the antimicrobial peptide H-08 exhibits the best stability in the presence of chymotrypsin, with a half-life of 11.78 h. H-08 also shows good stability in the presence of trypsin and proteinase K, with the remaining proportion of H-08 after co-incubation with these two enzymes for 2 h exceeding 70%. In the salt stability experiment, the MIC of H-08 changed relatively little under the presence of four different salts.

[0094] Example 6: Determination of sterilization curve

[0095] Staphylococcus aureus and Escherichia coli were used as model bacteria, and the strains were revived and grown to the logarithmic growth phase. A sample solution with a concentration of 2×MIC was mixed with an equal volume of bacterial suspension and incubated (37℃, 180 rpm). At time points of 0, 30, 60, 120, 240, 360, and 1440 min, 20 μL of the mixture was taken and diluted with culture medium. The diluted solution (100 μL) was evenly spread onto sterilized MH plates, with three sub-plates for each concentration. PBS and polymyxin B were used as negative and positive controls, respectively. The spread MH plates were incubated at 37℃ for 24 h, and the number of colonies on the plates was counted to calculate the sterilization efficiency of the samples.

[0096] Experimental results are as follows Figure 5As shown, H-08 exhibited good bactericidal effects, killing Staphylococcus aureus and Escherichia coli within 24 hours and 6 hours, respectively.

[0097] Example 7 In vivo safety experiment

[0098] Male Kunming mice (18-22g) were used for acute toxicity experiments. Mice were randomly divided into three groups: a PBS group, five H-08 dosage groups (10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg, 50mg / kg), and five polymyxin B dosage groups (5mg / kg, 7.5mg / kg, 10mg / kg, 12.5mg / kg, 15mg / kg), with eight mice in each group. All dosage solutions were administered to the mice via tail vein injection. Mice survival rates and weight changes were recorded for 10 consecutive days, and the median lethal dose (LD50) was calculated. To further verify the toxicity of H-08, 18 male Kunming mice (18-22g) were randomly divided into three groups: a normal control group, a control group, and an H-08 group. Mice in the normal group received no treatment. Mice in the control group received a tail vein injection of 100 μL of physiological saline, while mice in the H-08 group received an intravenous injection of 100 μL of H-08 (10 mg / kg). Throughout the experiment, the weight, behavior, and survival status of each mouse were recorded daily. On day 12 post-injection, blood was collected from the orbital cavity, and the mice were then sacrificed. The major organs (heart, liver, spleen, lung, and kidney) were collected for hematoxylin and eosin (H&E) staining.

[0099] Experimental results are as follows Figure 6 As shown, all mice survived when H-08 was administered at doses of 10 mg / kg and 20 mg / kg, but began to die at a dose of 30 mg / kg; mice also died at a dose of 5 mg / kg for polymyxin B. Calculations showed that the median lethal dose (LD50) of H-08 was significantly higher than that of polymyxin B. In subsequent experiments verifying the toxicity of H-08, there were no significant differences in organ indices and blood biochemical parameters among the normal group, control group, and H-08 group mice. Organ tissue sections (heart, liver, spleen, lung, and kidney) from the H-08 group mice did not show obvious physiological abnormalities, indicating that this dose (10 mg / kg) was relatively safe in mice.

[0100] Example 8: In vivo antibacterial activity experiment

[0101] Establishing a bacterial infection model: Male Kunming mice weighing 18-22g were selected and injected intraperitoneally with cyclophosphamide solution (150mg / kg and 100mg / kg) on ​​the first and fourth days, respectively, to induce immunosuppression in the mice. Staphylococcus aureus was then inoculated with physiological saline solution (100μL, 4.3×10⁻⁶). 9A mouse model of Staphylococcus aureus infection was established by intraperitoneal injection of CFU / mL into each mouse. The mice were then randomly divided into four groups (H-08 group, P-α-02-B group, control group, and normal group), receiving different antibiotics within safe dosage ranges. One hour after inoculation, mice in the treatment groups were administered 100 μL of 10 mg / kg H-08 and 100 μL of 10 mg / kg P-α-02-B via tail vein injection, while the control group received 100 μL of physiological saline. Mice in the normal group were not inoculated. A second dose was administered at the same dosage 12 hours later. Each group received treatment for three consecutive days, twice daily, with a 12-hour interval between doses. After three days of treatment, the mice were sacrificed, and major organs were harvested for bacterial load counting (liver, spleen, lung, kidney) and H&E staining (heart, liver, spleen, lung, kidney). Throughout the experiment, the weight and survival status of each mouse were recorded daily.

[0102] Experimental results are as follows Figure 7 As shown, in the control group, the survival rate of mice after infection was only 46.7%. The survival rates of mice in the H-08 group and the P-α-02-B group were higher than those in the control group, at 73.3% and 60%, respectively, indicating that H-08 has a better therapeutic effect. Bacterial smear experiments on mouse organs showed that the Staphylococcus aureus load in the major organs of the H-08 group mice was significantly lower than that in the control group mice. Histopathological changes in mouse organs (heart, liver, spleen, lung, and kidney) were examined by hematoxylin and eosin (H&E) staining. The control group mice showed severe inflammatory cell infiltration, diffuse congestion, and edema in the pulmonary interstitium. In contrast, the physiological state of the H-08 group mice was significantly improved, similar to the normal group. In conclusion, the antimicrobial peptide H-08 exhibits good therapeutic effects in vivo.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antibacterial peptide derivative, characterized in that: The antibacterial peptide derivative is as follows (1) (13) any one of polypeptides: (1) Ac-GLPZOWLZOFWNOIOZ-NH2 (2) Ac-GIZOWLZOFWNOIOZ-NH2 (3) Ac-GLZOWIZOFWNOIOZ-NH2 (4) Ac-GIZOWIZOFWNOIOZ-NH2 (5) Ac-GIPZOWLZOFWNOIOZ-NH2 (6) Ac-GLPZOWIZOFWNOIOZ-NH2 (7) Ac-GIPZOWIZOFWNOIOZ-NH2 (8) Ac-GiZOWLZOFWNOIOZ-NH2 (9) Ac-GLZOWiZOFWNOIOZ-NH2 (10) Ac-GiZOWiZOFWNOIOZ-NH2 (11) Ac-GiPZOWLZOFWNOIOZ-NH2 (12) Ac-GLPZOWiZOFWNOIOZ-NH2 (13) Ac-GiPZOWiZOFWNOIOZ-NH2 I represents L-form of isoleucine, L represents L-form of leucine, I represents D-form of leucine, i represents D-form of isoleucine, O represents ornithine, and Z represents homoarginine.

2. The antimicrobial peptide derivative of claim 1, wherein: X is -CH2-; Y is -CH2-; Z is -CH2-; and R is -CH3. The antibacterial peptide derivative can be used alone or in combination or in the form of a multimer.

3. The antimicrobial peptide derivative of claim 1, wherein: X is -CH2-; Y is -CH2-; Z is -CH2-; and R is -CH3. The antibacterial peptide derivative also includes a modified antibacterial peptide derivative.

4. The antimicrobial peptide derivative of claim 1, wherein: X is -CH2-; Y is -CH2-; Z is -CH2-; and R is -CH3. The antibacterial peptide derivative can be fused with other proteins, or the antibacterial peptide derivative can be linked with a polymer or a carrier.

5. The antimicrobial peptide derivative of claim 1, wherein: X is -CH2-; Y is -CH2-; Z is -CH2-; and R is -CH3. The antibacterial peptide derivative is obtained by chemical synthesis or by recombinant technology.

6. The antimicrobial peptide derivative of claim 1, wherein: X is -CH2-; Y is -CH2-; Z is -CH2-; and R is -CH3. The antibacterial peptide derivative has at least one of the following properties: (1) antibacterial activity; (2) low hemolytic toxicity; (3) high stability and anti-degradation performance; (4) in vivo safety.

7. A pharmaceutical composition, characterized by: The antibacterial peptide derivative of any one of claims 1-6, and a pharmaceutically acceptable carrier or excipient.

8. A nucleic acid encoding the antibacterial peptide derivative of any one of claims 1-6.

9. A vector containing the nucleic acid of claim 8.

10. A host cell containing the nucleic acid of claim 8, the vector of claim 9, or expressing the antibacterial peptide derivative of any one of claims 1-6.

11. Use of the antibacterial peptide derivative of any one of claims 1-6, the pharmaceutical composition of claim 7, the nucleic acid of claim 8, the vector of claim 9, or the host cell of claim 10 in the preparation of an antibacterial drug. The antibacterial drug can be used for the treatment of drug-resistant bacterial infectious diseases. The drug-resistant bacteria are drug-resistant bacteria of Staphylococcus aureus, Enterococcus faecium, Bacillus mycoides, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Citrobacter freundii, and Klebsiella pneumoniae.

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