Antibacterial peptide derivative and application thereof

Abstract

The invention provides an antibacterial peptide derivative and application thereof. The antibacterial peptide derivative is any one of the following polypeptides (a)-(d): (a) polypeptides with an amino acid sequence as shown in a general formula (1) or a general formula (2): Ac-GX1aZOWX1bZOFWNOIOZ-NH2 (1) and Ac-GX2aPZOWX2bZOFWNOIOZ-NH2 (2), and the amino acid sequence of the polypeptides is shown in the general formula (1) or the general formula (2). The antibacterial peptide has excellent in-vitro antibacterial activity, relatively low hemolytic activity and relatively high anti-enzymolysis stability.

Description

Technical Field

[0001] The present invention relates to the technical field of biological peptides, and in particular to an antimicrobial peptide derivative and application thereof. Background Art

[0002] In recent years, antimicrobial peptides have attracted widespread attention due to their broad-spectrum antimicrobial activity and their low resistance to drug resistance in bacteria. They are considered to be a new generation of antimicrobial drugs that are expected to replace traditional antibiotics. Peptide P-α-02-B, a derivative of the natural antimicrobial peptide sC18, has good anti-resistant bacteria activity and has shown good effects in the treatment of bacteremia model mice. However, like most antimicrobial peptides, P-α-02-B has problems such as poor anti-enzymatic stability, which limits its clinical application. Therefore, how to improve the anti-enzymatic stability of P-α-02-B has become an urgent problem to be solved. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides an antimicrobial peptide derivative and its application, which solves the problem of instability of the antimicrobial peptide in the prior art.

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

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

[0006] Ac-GX 1 a ZOWX 1 b ZOFWNOIOZ-NH 2 (1)

[0007] Ac-GX 2 a PZOWX 2 b ZOFWNOIOZ-NH 2 (2)

[0008] X 1 a , X 1 b Same or Different, X 1 a , X 1 b Each of the amino acids is independently selected from one of L-isoleucine (I), D-leucine (l), and D-isoleucine (i), O represents ornithine, and Z represents homoarginine;

[0009] X2 a , X 2 b Same or Different, X 2 a , X 2 b are independently selected from one of L-leucine (L), L-isoleucine (I), D-leucine (l), and D-isoleucine (i), O represents ornithine, and Z represents homoarginine;

[0010] (b) a polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues of 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) A polypeptide having 80% or more, 85% or more, 90% or more, 95% or more, or 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 includes an amino acid sequence as shown in any one of SEQ ID NO:1-SEQ ID NO:13 in the following table, or a polypeptide having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with the amino acid sequence shown in any one of 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 an embodiment of the present invention, the strains include but are not limited to drug-resistant strains of Staphylococcus aureus, Enterococcus faecium, 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 has good antibacterial activity against the above-mentioned strains. In one embodiment of the present invention, the minimum inhibitory concentration of the antimicrobial peptide derivative H-08 against drug-resistant strains of Staphylococcus aureus, Enterococcus faecium, 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, 3.13 μM, 3.13 μM, 3.13 μM, respectively.

[0020] According to an embodiment of the present invention, the antimicrobial peptide derivative H-08 exhibits significant bactericidal effects on 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, compared with P-α-02-B, the antimicrobial peptide derivative has lower hemolytic toxicity.

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

[0023] According to an embodiment of the present invention, the antimicrobial peptide derivative has good in vivo safety and therapeutic effect against bacterial infection in vivo. In one embodiment of the present invention, the dosage of the antimicrobial peptide derivative is 10 mg / kg, showing good in vivo safety and efficacy.

[0024] According to an embodiment of the present invention, the antimicrobial peptide derivative can be used alone or in combination or in the form of a polymer, such as a peptide dimer, a peptide trimer, etc. The polymer can be composed of peptide derivatives with the same sequence or composed of several peptide derivatives with different sequences of general formula 1 or general formula 2.

[0025] According to an embodiment of the present invention, the antimicrobial peptide derivatives also include modified antimicrobial peptide derivatives.

[0026] According to an embodiment 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 a carrier, such as human serum albumin, humanized antibodies, liposomes, micelles, synthetic polymers, nanoparticles, and bacteriophages.

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

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

[0029] According to an embodiment 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.

[0030] According to an embodiment of the present invention, the pharmaceutical composition may be in the form of capsules, tablets, pills, drops, suppositories, powders, sprays, vaccines, ointments, pastes, creams, inhalants, patches, aerosols, and the like.

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

[0032] The third aspect of 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] The fifth aspect of 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 an antimicrobial peptide derivative, a pharmaceutical composition, and use of the above nucleic acid, vector or host cell in the preparation of antimicrobial drugs.

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

[0037] According to the embodiments of the present invention, the antimicrobial peptide derivatives and pharmaceutical compositions can be used to treat infectious diseases caused by drug-resistant bacteria.

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

[0039] The present invention transforms the antimicrobial peptide P-α-02-B through strategies such as proline insertion, isoleucine replacement, and D-amino acid replacement, and discovers a new class of antimicrobial peptides with a new sequence. The antimicrobial peptide has excellent in vitro antimicrobial activity, low hemolysis, and high anti-enzymatic stability. In particular, H-08 shows an in vivo therapeutic effect superior to that of P-α-02-B in the treatment of bacteremia mice. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0041] FIG2 is a mass spectrum of antimicrobial peptides H-01 to H-13; Figure 2A It is the antimicrobial peptide H-01; Figure 2B It is the antimicrobial peptide H-02; Figure 2C It is the antimicrobial peptide H-03; Figure 2D It is the antimicrobial peptide H-04; Figure 2E It is the antimicrobial peptide H-05; Figure 2F It is the antimicrobial peptide H-06; Figure 2G It is the antimicrobial peptide H-07; Figure 2H It is the antimicrobial peptide H-08; Fig.2I It is the antimicrobial peptide H-09; Figure 2J It is the antimicrobial peptide H-10; Figure 2K It is the antimicrobial peptide H-11; Figure 2L It is the antimicrobial peptide H-12; Figure 2M It is the antimicrobial peptide H-13.

[0042] Figure 3 This is the hemolytic results of the antimicrobial peptide P-α-02-B and its derivative peptides.

[0043] Figure 4The stability test results of the 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 the 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 curve of antimicrobial peptide H-08 against Staphylococcus aureus (left) and Escherichia coli (right).

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

[0046] Figure 7 The results of the in vivo antibacterial experiment of the antimicrobial peptide H-08 are shown in Figure 1. (A) Flow chart of the in vivo antibacterial experiment; (B) Survival curve of mice; (C) Weight change of mice; (D) Staphylococcus aureus load in the liver, spleen, lung, and kidney of mice; (E) HE staining of the heart, liver, spleen, lung, and kidney of mice. DETAILED DESCRIPTION

[0047] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations 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 included in the scope that the present invention is intended to protect.

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

[0049] "Derivative" refers to an antimicrobial peptide variant that replaces, deletes or adds one or more amino acid residues at a specific position of the antimicrobial peptide P-α-02-B polypeptide sequence, thereby affecting the biological activity (eg, antibacterial activity) of the antimicrobial peptide.

[0050] The term "modification" refers to the molecular engineering process of introducing non-amino acid derived functional chemical groups (such as pegylation, glycosylation or lipidation, etc.) into polypeptide molecules by covalent linkage or other chemical bonding methods, and ensuring that such structural modifications will not significantly affect its biologically active conformation and functional potency (such as antibacterial activity, etc.). Such modifications may be natural or non-natural. In certain embodiments, non-natural modifications may include adding protective or blocking groups to reactive portions, adding detectable labels (such as antibodies and / or fluorescent labels), adding or modifying glycosylation, or adding filling groups (such as PEG (pegylation)) and other changes known to those skilled in the art. In certain embodiments, non-natural modifications may be end-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 fluorescent marker proteins, such as (but not limited to) green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry. Conjugation of polyethylene glycol (PEG) to proteins has been used as a method to extend the circulation half-life of many pharmaceutical proteins.The term "modified" includes polypeptides that have been chemically modified by the covalent attachment of one or more PEG molecules.

[0051] "Percentage of amino acid sequence homology" 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 aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence homology can be achieved in various ways within the art, for example, using publicly available software such as BLAST or commercially available software such as DNASTAR. Two or more polypeptide sequences can be any value of 0-100% identity, or any integer value therebetween. 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 conservative 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 an antimicrobial peptide derivative described herein, are substituted with similar or conservative amino acid substitutions, and wherein the resulting peptide has at least one activity (e.g., antimicrobial effect) of a reference polypeptide, such as an antimicrobial peptide derivative described herein, the sequence of the polypeptide of the present disclosure is substantially homologous.

[0052] The therapeutically effective dose refers to the amount of active ingredient that improves symptoms or symptoms. 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 the dose to produce the desired therapeutic effect for the specific circumstances of the present invention. The drug dose is appropriately converted from animal studies to human studies (human equivalent dose, HED) by using the body surface area (BSA) normalization method. The relationship between animal and human doses (based on milligrams per square meter of body surface) is described by Reagan-Shaw et al. (2007. FASEB J. 22: 659-661).

[0053] Pharmaceutically acceptable salts can be used herein, for example, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. A full list of pharmaceutically acceptable salts is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0054] Example 1 Design and modification of antimicrobial peptides

[0055] In order to improve the anti-enzymatic stability of P-α-02-B, strategies such as proline insertion, isoleucine replacement, and D-amino acid replacement were adopted to design a series of P-α-02-B derivative peptides H-01 to H-13. Through structural prediction and sequence analysis, proline was inserted into the C-terminal side of the leucine at the 2nd position of the N-terminus of P-α-02-B to obtain H-01. In addition, L-isoleucine was used to replace the leucine at the 2nd and / or 6th position 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 at the 2nd and / or 6th position of the N-terminus of H-01 to obtain H-05, H-06 and H-07; D-isoleucine was used to replace the L-isoleucine in H-02 to H-07 to obtain H-08 to H-13. The synthesized P-α-02-B derivative peptides are shown in Table 1.

[0056] Table 1 Sequence and molecular weight of antimicrobial peptide P-α-02-B and its derivative peptides

[0057]

[0058]

[0059] i represents D-isoleucine, O represents ornithine, and Z represents homoarginine.

[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 0.5 g of ninhydrin and transfer to a 10 mL volumetric flask. Add anhydrous methanol to the mark and mix to obtain a ninhydrin solution.

[0064] (3) Pyridine solution: Re-evaporate pyridine to remove water.

[0065] (4) Peptide lysis buffer: This reagent is prepared before use. Add 45 mL of trifluoroacetic acid, 2.5 mL of m-cresol, 1.5 mL of anisole, and 0.5 mL of water into a 50 mL centrifuge tube and mix well to obtain a 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, 0.38 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 0.13 g of 1-hydroxy-7-azobenzotriazole (HOAT) in 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 the resin: Weigh 0.47 g (0.25 mmol) of Rink amide resin (loading: 0.53 mmol / g) and put it into the reactor. Add 10 mL of DMF and stir to swell for 30 min. After the resin swells, start the vacuum pump and drain the reaction solution.

[0069] (2) Deprotection: Add 10 mL of deprotection reagent and stir to react. After 5 minutes, drain and add 10 mL of deprotection reagent again and stir to react for 25 minutes to remove the Fmoc protecting group on the resin and expose the amino group. Wash the resin alternately with DMF and dichloromethane (DCM) three times each.

[0070] (3) Amino group detection: Use a capillary to dip a small amount of resin into a centrifuge tube, add one drop of ninhydrin solution and one drop of pyridine solution, place in a 100°C metal bath heating device and heat for 5 minutes, then take out and observe. If the resin shows blue, it means that the deprotection is successful and proceed to the next step; if there is no color change, the deprotection fails and add the deprotection reagent again and repeat the above steps.

[0071] (4) Condensation: After the amino acid and condensation reagent solution are fully mixed, they are put into the reactor, stirred, and reacted at room temperature for 1 hour. After the reaction is completed, the reaction liquid is drained, and the resin is washed with DMF and DCM. The detection operation is repeated. If the resin has no color reaction, the amino acid condensation is successful. If the resin shows blue, the condensation fails or the reaction is not complete. Repeat the condensation operation again until the condensation is successful. Then repeat the deprotection operation and couple the next amino acid until the last amino acid is condensed.

[0072] (5) Cleavage: Add anhydrous ether to the resin after the reaction is completed, wash it three times, and drain it. Transfer the resin to a round-bottom flask, add 15 mL of cleavage solution, and start stirring. Place in an ice bath 30 minutes before the reaction, remove the ice bath, and stir the reaction at room temperature for 3 hours. After the cleavage is completed, add anhydrous ether according to the mass of the polypeptide and stir rapidly for 0.5-1 hour. Let it stand until the polypeptide is completely precipitated, pour it into a G4 funnel, and drain it. Use ether to repeatedly wash the residual cleavage solution in the resin and polypeptide solid. Replace a new clean suction flask, and use double distilled water to dissolve the peptide on the funnel in small amounts multiple times until all the peptides in the funnel are dissolved and the remaining resin appears as loose sand.

[0073] (6) Lyophilization: The crude peptide solution in the filtration bottle was transferred to a centrifuge tube, frozen with liquid nitrogen, and the crude peptide was lyophilized for 48 hours using a lyophilizer to obtain crude peptide powders of H-01 to H-13.

[0074] 3. Analysis and purification

[0075] (1) Molecular weight determination: 0.1 mg of freeze-dried crude peptide powder was dissolved in 100 μL 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 the molecular weight was confirmed to be correct, the peptide was purified. As shown in Figure 2.

[0076] (2) Peptide purification: According to the physicochemical properties of each target peptide, select appropriate analysis and purification conditions, perform gradient elution at room temperature using a preparative high performance liquid chromatograph, collect the target peptide, perform purity analysis using an analytical high performance liquid chromatograph, collect peptide solutions with a purity greater than 95%, freeze-dry, obtain pure peptide powder, collect, weigh, seal in a centrifuge tube, and store at -40°C, as shown in Figure 1.

[0077] Chromatographic column selection: analytical HPLC column (C18), preparative HPLC column (C18).

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

[0079] Example 3 In vitro antibacterial activity assay

[0080] The minimum inhibitory concentration (MIC) of P-α-02-B and its derivative peptides in drug-resistant strains of Staphylococcus aureus, Enterococcus faecium, Bacillus mycoides, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Citrobacter freundii and Klebsiella pneumoniae was determined by the double dilution method. 50 μL of liquid culture medium was pipetted and added to the 1st to 8th rows of a 96-well plate in sequence. 50 μL of each antimicrobial peptide solution with a concentration of 400 μM was pipetted and added to each well in the first row. Then, 50 μL of peptide solution was pipetted from the first row to the second row by the double dilution method. After mixing, the peptide solution (50 μL) in the second row was pipetted to the third row. This operation was repeated to the 8th row, and 50 μL was pipetted out from the last row and discarded. Then, take a sterile sample tank, add the diluted bacterial solution (the dilution ratio of bacterial solution to culture medium is 1:1000), and use a pipette to draw 50μL and add it to the 1st to 8th rows of the 96-well plate in sequence. The drug solution and the bacterial solution are fully mixed and cultured in a 37℃ constant temperature shaking incubator at 180r / min for 18h, and the MIC value is observed and determined. The MIC value is defined as the lowest concentration of the peptide when the well plate is placed under a fluorescent lamp and the bacterial growth in the well is observed with the naked eye. The liquid in the well is clear and there is no visible bacterial growth. The experimental results are shown in Table 2.

[0081] Table 2 MIC values ​​of antimicrobial peptide P-α-02-B and its derivative peptides

[0082]

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

[0084] Example 4 Hemolytic Experiment

[0085] Collect fresh human blood in a 5mL blood collection tube with heparin anticoagulant, then transfer to a 15mL sterile centrifuge tube and centrifuge at 1500r / min and 4°C for 5min. After centrifugation, remove the lower layer of red blood cells, add an appropriate amount of PBS and gently blow and wash (the operation must be gentle to prevent red blood cells from breaking and hemolysis), then put it into the centrifuge again, centrifuge at 1800r / min and 4°C for 5min, repeat the above washing 2-3 times to obtain red blood cells. Then dilute the red blood cells into a 5% (v / v) red blood cell / PBS cell suspension for standby use. Pour PBS into a sterile sample tank, use a pipette to draw 50μL PBS and add it to the 2nd to 8th rows of the 96-pointed bottom 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 and added to the first row of the 96-well plate. 50 μL of the peptide solution was pipetted from the first row and added to the second row using the two-fold dilution method. The mixture was fully mixed and the operation was repeated to dilute to the eighth row in sequence. Finally, 50 μL of excess solution was discarded. 50 μL of red blood cell suspension was added to the 96-well plate using a pipettor, covered and gently shaken to mix, and the culture plate was cultured in a 37°C constant temperature incubator for 1 hour. Three parallel wells were set with the same peptide solution and the same concentration. PBS solution was set as a negative control group and Triton X-100 as a positive control group. After the culture was completed, the 96-well plate was placed in a centrifuge and centrifuged at 1500 r / min for 5 minutes. 50 μL of the supernatant was pipetted and placed in a new flat-bottomed sterile 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 the absorbance detection mode, and the percentage of red blood cell hemolysis was calculated according to the following formula:

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

[0087] OD(pep): indicates the absorbance value corresponding to the drug-dosing cell well

[0088] OD(neg): indicates the absorbance value corresponding to the negative control well

[0089] OD(pos): indicates the absorbance value corresponding to the positive control well

[0090] The experimental results are as follows Figure 3 As shown in the figure, H-08 exhibited lower hemolytic toxicity than P-α-02-B. Even at a concentration of 200 μM, the hemolytic rate of H-08 was only 7%.

[0091] Example 5 Stability Determination

[0092] Chymotrypsin was dissolved in ultrapure water to a final concentration of 0.02 mg / mL. The antimicrobial peptide with a concentration of 1 mM was mixed with the above chymotrypsin solution at a volume ratio of 1:1 and incubated at 37°C for 6 hours, and the peptide-protease mixture was taken at 0h, 0.5h, 1h, 2h, 4h and 6h. The mixture was heated at 100°C for 15 minutes to inactivate the protease. The degradation of the antimicrobial peptide under chymotrypsin treatment conditions was determined by HPLC. The experiment was repeated 3 times with 3 parallels each time. At the same time, the degradation of the peptide by trypsin and proteinase K was tested. Trypsin with a concentration of 0.25 mg / mL and proteinase K solution with a final concentration of 1 mM were co-incubated at 37°C for 2 hours at a volume ratio of 1:1, and the peptide-protease mixture was taken at 0h and 2h. The mixture was heated at 100°C for 15 minutes to inactivate the protease. The degradation of the antimicrobial peptides under the two protease treatment conditions was determined by HPLC. The experiment was repeated three times with three parallels each time. In addition, in order to evaluate the stability of the antimicrobial peptides under physiological conditions, different types of salts (150 mM NaCl, 4 μM FeCl 3 , 1 mM MgCl 2 The minimum inhibitory concentration of the polypeptide against Staphylococcus aureus and Escherichia coli was determined in the presence of 4.5 mM KCl and 4.5 mM KCl. The experiment was repeated 3 times, with 3 parallels each time.

[0093] The experimental results are as follows Figure 4 As shown in the figure, in the presence of chymotrypsin, the antimicrobial peptide H-08 has the best stability, with a half-life of up to 11.78h; in the presence of trypsin and proteinase K, H-08 also shows good stability, with the remaining proportion of H-08 exceeding 70% after incubation with these two enzymes for 2h; in the salt stability experiment, the MIC of H-08 has a small change multiple in the presence of four salts.

[0094] Example 6 Sterilization Curve Determination

[0095] Staphylococcus aureus and Escherichia coli were used as model bacteria. The strains were revived and grown to the logarithmic growth phase. The sample solution with a concentration of 2×MIC was mixed with an equal volume of bacterial solution and incubated (37°C, 180r / min). 20μL of the mixture was diluted with the culture medium at 0, 30, 60, 120, 240, 360 and 1440min, and the dilution (100μL) was evenly spread on the sterilized MH plate. Three sub-plates were set for each concentration, and PBS and polymyxin B were used as negative and positive controls respectively. The coated MH plate was placed in a 37°C constant temperature incubator for 24h, the number of colonies on the plate was counted, and the bactericidal efficiency of the sample was calculated.

[0096] The experimental results are as follows Figure 5 As shown, H-08 exhibited a good bactericidal effect, 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, and the mice were randomly divided into PBS group, 5 dose groups of H-08 (10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg, 50mg / kg), 5 dose groups of polymyxin B (5mg / kg, 7.5mg / kg, 10mg / kg, 12.5mg / kg, 15mg / kg), 8 in each group. All solutions of all dosage specifications were injected into the mice by tail vein injection. The survival rate of the mice was observed and the weight changes of the mice were recorded. The mice were observed for 10 consecutive days and the median lethal dose of the mice was calculated. At the same time, in order to further verify the toxicity of H-08, 18 male Kunming mice (18-22g) were selected and randomly divided into 3 groups, including a normal group, a control group and an H-08 group. The mice in the normal group were not treated, the mice in the control group were injected with 100 μL of normal saline via the tail vein, and the mice in the H-08 group were injected with 100 μL of H-08 (10 mg / kg) via the intravenous route. The body weight, behavior, and survival status of each mouse were recorded every day throughout the experiment. Blood was collected from the eye sockets on the 12th day after injection, and then the mice were killed, and the main organs (heart, liver, spleen, lung, and kidney) were collected for hematoxylin and eosin (H&E) staining.

[0099] The experimental results are as follows Figure 6 As shown, when the dose of H-08 was 10 mg / kg and 20 mg / kg, all mice survived. When the dose reached 30 mg / kg, mice began to die; when the dose of polymyxin B was 5 mg / kg, mice died. It was calculated that the median lethal dose of H-08 was much greater than that of polymyxin B. Subsequently, in experiments to further verify the toxicity of H-08, there were no significant differences in the organ indexes and blood biochemical indicators of mice in the normal group, control group, and H-08 group. The sections of the organ tissues (heart, liver, spleen, lung, and kidney) of mice in the H-08 group did not show obvious physiological lesions, which shows that this dose (10 mg / kg) is relatively safe in mice.

[0100] Example 8 In vivo antibacterial activity experiment

[0101] Constructing the infection model: Male Kunming mice weighing 18-22 g were selected and intraperitoneally injected with cyclophosphamide solution (150 mg / kg and 100 mg / kg) on ​​the first and fourth days, respectively, to induce immunosuppression in the mice. Staphylococcus aureus physiological saline solution (100 μL, 4.3×10 9CFU / mL) was intraperitoneally injected into each mouse to establish a mouse Staphylococcus aureus infection model. Subsequently, the mice were randomly divided into four groups (H-08 group, P-α-02-B group, control group, and normal group), and the mice were treated with different antibacterial agents within their safe dosage range. One hour after inoculation of bacteria, 100μL 10mg / kg H-08 and 100μL 10mg / kg P-α-02-B were given to the mice in the administration group by tail vein injection, and 100μL normal saline was given to the mice in the control group. The normal group of mice was not inoculated with bacteria. After 12 hours of administration, the second administration was performed at the same dose. Each group was administered for 3 consecutive days, twice a day, with an interval of 12 hours each time. After 3 consecutive days of administration, the mice were killed, and the main organs were taken 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 every day.

[0102] The experimental results are as follows Figure 7 As shown in the figure, in the control group, the survival rate of mice after infection was only 46.7%, while the survival rates of mice in the H-08 group and P-α-02-B group were higher than those in the control group, at 73.3% and 60%, respectively. It was concluded that H-08 had a good therapeutic effect. By taking the mouse organs for bacterial coating experiments, it can be concluded that the Staphylococcus aureus load in the main organs of the H-08 group mice was significantly lower than that of the control group mice; the histopathological changes of the mouse organs (heart, liver, spleen, lung, and kidney) were examined by hematoxylin-eosin (H&E) staining. The control group mice showed severe inflammatory cell infiltration, diffuse congestion, and edema in the lung interstitium. In contrast, the physiological state of the H-08 group mice was significantly improved, similar to that of the normal group. In summary, the antimicrobial peptide H-08 showed a good therapeutic effect in vivo.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An antimicrobial peptide derivative or a pharmaceutically acceptable salt thereof, characterized in that: The antimicrobial peptide derivative is any one of the following polypeptides (a)-(d): (a) A polypeptide having 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) X1 a 、X1 b Same or different, X1 a 、X1 b Each of the amino acids is independently selected from one of L-isoleucine (I), D-leucine (l), and D-isoleucine (i), O represents ornithine, and Z represents homoarginine; X2 a , X2 b Same or different, X2 a , X2 b are independently selected from one of L-leucine (L), L-isoleucine (I), D-leucine (l), and D-isoleucine (i), O represents ornithine, and Z represents homoarginine; (b) a polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence defined in (a); (c) a polypeptide obtained by modifying the N-terminus and / or C-terminus of the polypeptide defined in (a); (d) A polypeptide having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more homology with the amino acid sequence defined in (a).

2. An antimicrobial peptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antimicrobial peptide derivative includes an amino acid sequence as shown in any one of SEQ ID NO:1-SEQ ID NO:13 in the following table, or a polypeptide having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with the amino acid sequence as shown in any one of SEQ ID NO:1-SEQ ID NO:

13.

3. An antimicrobial peptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antimicrobial peptide derivatives can be used alone or in combination or in the form of a polymer; Preferably, the antimicrobial peptide derivatives also include modified antimicrobial peptide derivatives; Preferably, the antimicrobial peptide derivative can be fused with other proteins, or the antimicrobial peptide derivative can be connected with a polymer or a carrier; Preferably, the antimicrobial peptide derivative is obtained by chemical synthesis or by recombinant technology.

4. The antimicrobial peptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antimicrobial peptide derivative has at least one of the following characteristics: (1) Antibacterial activity; (2) Low hemolytic toxicity; (3) High stability and anti-degradation properties; (4) In vivo safety.

5. A pharmaceutical composition, characterized in that: The invention comprises the antimicrobial peptide derivative according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier or excipient.

6. A nucleic acid encoding the antimicrobial peptide derivative according to any one of claims 1 to 4.

7. A vector comprising the nucleic acid according to claim 6.

8. A host cell containing the nucleic acid according to claim 6, the vector according to claim 7, or expressing the antimicrobial peptide derivative according to any one of claims 1 to 4.

9. Use of the antimicrobial peptide derivative according to any one of claims 1 to 4, the pharmaceutical composition according to claim 5, the nucleic acid according to claim 6, the vector according to claim 7, and the host cell according to claim 8 in the preparation of antimicrobial drugs.

10. The use according to claim 9, characterized in that: The strains include, but are not limited to, drug-resistant strains of Staphylococcus aureus, Enterococcus faecium, Bacillus mycoides, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Citrobacter freundii, and Klebsiella pneumoniae; The antibacterial drug can be used to treat infectious diseases caused by drug-resistant bacteria.

Images