Small molecule antimicrobial peptides, preparation methods and applications

Abstract

This invention relates to the field of antimicrobial drug technology, and particularly to small molecule antimicrobial peptides, their preparation methods, and their uses. The antimicrobial peptides provided by this invention exhibit stronger antimicrobial activity compared to most other derived peptides and previously reported antimicrobial peptides. Evaluation of their bactericidal activity using various known drug-resistant strains showed that the antimicrobial peptides provided by this invention possess broad-spectrum antimicrobial activity against drug-resistant strains, offering a more comprehensive antimicrobial effect and a wider range of indications.

Description

[0001] This application is a divisional application. The original application was filed on July 18, 2023, with application number 2023108801562, and the invention title was "Small molecule antibacterial polypeptide, preparation method and use thereof". Technical Field

[0002] This invention relates to the field of antimicrobial drug technology, and in particular to small molecule antimicrobial peptides, their preparation methods, and their uses. Background Technology

[0003] In recent decades, the frequent and unregulated use of antibiotics has led to antibiotic resistance becoming a public health crisis. According to the latest survey by the U.S. Centers for Disease Control and Prevention (CDC), antibiotic resistance causes millions of illnesses worldwide each year, and the number of deaths due to antibiotic resistance is projected to reach tens of millions by 2050. Therefore, developing new antibacterial drugs has become a top priority in anti-infective treatment.

[0004] Antimicrobial peptides (AMPs) have shown unprecedented advantages as promising antimicrobial agents against multidrug-resistant bacteria. Unlike antibiotics, which interfere with the metabolic processes of pathogenic microorganisms, AMPs typically exert their antimicrobial effects by physically disrupting microbial cell membrane lipids and inducing leakage of cell contents; therefore, they have little impact on the probability of bacterial resistance evolution. Currently, due to their strong antimicrobial potential and unique mechanism of action, AMPs are considered ideal candidates to replace antibiotics.

[0005] Cathelicidin, an antimicrobial peptide from the banded krait, is a multifunctional family of antimicrobial peptides with broad-spectrum antimicrobial activity. It exhibits strong bactericidal activity against Gram-positive bacteria, Gram-negative bacteria, certain fungi, and viruses, and is also effective against many clinically resistant bacteria. BF30, an active polypeptide isolated from banded krait venom by Lai Ren et al. at the Kunming Institute of Zoology, Chinese Academy of Sciences in 2008, is a single polypeptide drug containing 30 amino acids, encoded by the cathelicidin gene of the banded krait. It is a linear polypeptide with an N-terminus α-helix, containing 30 amino acid residues and a molecular weight of 3636.24 Da.

[0006] Given that the antimicrobial peptide of the golden ring snake has high activity, is not prone to drug resistance, and has a broad spectrum of action, it has become an excellent lead molecule for screening new anti-infective drugs. However, the sequence of the golden ring snake antimicrobial peptide BF30 is relatively complex, the synthesis cost is high, and the antimicrobial activity still needs to be improved. Summary of the Invention

[0007] This invention aims to improve the activity of antimicrobial peptides from *Rhizoctonia solani* and reduce the synthesis cost of these peptides. It modifies and optimizes the amino acid sequence and structure of *Rhizoctonia solani* antimicrobial peptides to obtain mutant antimicrobial peptides with broad-spectrum and high activity. The invention also provides a method for preparing and using these mutant antimicrobial peptides.

[0008] The first aspect of the present invention provides an antimicrobial peptide having an amino acid sequence X1KRFKKFX2X3KLKKWV, wherein X1 is selected from nonpolar side-chain amino acids, X2 is selected from amino acids with aromatic side chains, and X3 is selected from amino acids with aromatic side chains or amino acids with basic side chains.

[0009] In an optional embodiment, X1 is selected from valine or isoleucine, X2 is selected from phenylalanine or tryptophan, and X3 is selected from phenylalanine, tryptophan, or arginine.

[0010] In an optional embodiment, the antimicrobial peptide has an amino acid sequence shown in any one of SEQ ID NO. 1 to 4.

[0011] In an optional embodiment, the N-terminus of the antimicrobial peptide contains a modifying functional group selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decacarbonyl, 2-morpholinylacetyl, pyrazinyl, dodecanoyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

[0012] In an optional embodiment, the modified antimicrobial peptide contains at least one D-type or β-type amino acid.

[0013] Secondly, the present invention provides a method for preparing the antimicrobial peptide described in any of the foregoing embodiments, which employs a solid-phase synthesis or liquid-phase synthesis method, wherein amino acids with side-chain protecting groups are sequentially coupled according to the amino acid sequence, and then the side-chain protecting groups are removed, extracted and purified sequentially to obtain the antimicrobial peptide.

[0014] In an optional embodiment, the method for preparing the antimicrobial peptide further includes a step of modifying the N-terminus of the solid-phase synthesized antimicrobial peptide, wherein the functional group used for the N-terminal modification is selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decacarbonyl, 2-morpholinylacetyl, pyrazinyl, dodecanoyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

[0015] In an optional embodiment, at least one of the amino acids used in solid-phase synthesis is a D-type or β-type amino acid.

[0016] Thirdly, the present invention provides the use of the antimicrobial peptide described in any of the foregoing embodiments or the antimicrobial peptide obtained by the preparation method described in any of the foregoing embodiments in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, radiation dermatitis prevention and treatment drugs, preservatives, animal feed or cosmetic precursors.

[0017] Fourthly, the present invention provides an antimicrobial drug formulation containing the antimicrobial peptide described in any one of the foregoing embodiments or an antimicrobial peptide prepared by any one of the foregoing embodiments.

[0018] In an optional embodiment, the effective dose of the antimicrobial peptide is 0.01~512 μg / ml, preferably 0.125~256 μg / ml.

[0019] In an optional embodiment, the types of pathogenic microorganisms for the antibacterial effect include bacteria and / or fungi.

[0020] Preferably, the bacteria include Gram-positive bacteria, such as Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ) or Enterococcus faecalis ( Enterococcus faecalis ); and / or, Gram-negative bacteria, such as Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Escherichia coli ( Escherichia coli Klebsiella pneumoniae ( Klebsiella pneumoniae ) or Acinetobacter baumannii ( Acinetobacter baumannii ).

[0021] In an optional embodiment, the pathogenic microorganism is drug-resistant and resistant to at least one of the following antibiotics: β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampin.

[0022] In optional embodiments, the dosage form of the antibacterial drug preparation includes oral preparations, topical preparations, or injections; the oral preparations include granules, tablets, pastes, or oral solutions; the topical preparations include ointments, gels, suppositories, medicated bath solutions, hoof bath solutions, or sprays.

[0023] The beneficial effects achieved by this invention include: The antimicrobial peptides provided by this invention exhibit stronger antibacterial activity compared to most other derived peptides and previously reported antimicrobial peptides. Evaluation of their bactericidal activity using various known drug-resistant strains showed that the antimicrobial peptides provided by this invention possess broad-spectrum antimicrobial activity against drug-resistant strains, offering a more comprehensive antimicrobial effect and wider applicability.

[0024] Furthermore, the safety of the antimicrobial peptides provided by the present invention was evaluated using hemolytic activity. The results showed that the preferred antimicrobial peptides of the present invention did not exhibit hemolytic toxicity at the highest concentration of 256 μg / ml, indicating high safety.

[0025] Therefore, this invention provides an antimicrobial peptide with stronger antibacterial activity, a wider range of applications, and greater potential development value. Attached Figure Description

[0026] Figure 1 The high-performance liquid chromatogram of the antimicrobial peptide WZ-2 synthesized in Example 1 is shown below. Figure 2 This is the mass spectrum of the antimicrobial peptide WZ-2 synthesized in Example 1; Figure 3 The high-performance liquid chromatogram of the antimicrobial peptide Ac-WZ-2 synthesized in Example 2 is shown below. Figure 4 This is the mass spectrum of the antimicrobial peptide Ac-WZ-2 synthesized in Example 2; Figure 5 The high-performance liquid chromatogram of the antimicrobial peptide WZ-4 synthesized in Example 3 is shown below. Figure 6 This is the mass spectrum of the antimicrobial peptide WZ-4 synthesized in Example 3; Figure 7 The high-performance liquid chromatogram of the antimicrobial peptide Ac-WZ-4 synthesized in Example 3 is shown below. Figure 8 The mass spectrum of the antimicrobial peptide Ac-WZ-4 synthesized in Example 3 is shown below. Figure 9 The high-performance liquid chromatogram of the antimicrobial peptide WZ-16 synthesized in Example 4 is shown below. Figure 10 The mass spectrum of the antimicrobial peptide WZ-16 synthesized in Example 4; Figure 11 The high-performance liquid chromatogram of the antimicrobial peptide Ac-WZ-16 synthesized in Example 4 is shown below. Figure 12 The mass spectrum of the antimicrobial peptide Ac-WZ-16 synthesized in Example 4 is shown. Figure 13 The high-performance liquid chromatogram of the antimicrobial peptide WZ-21 synthesized in Example 5 is shown below. Figure 14 The mass spectrum of the antimicrobial peptide WZ-21 synthesized in Example 5 is shown below. Figure 15 The high-performance liquid chromatogram of the antimicrobial peptide Ac-WZ-21 synthesized in Example 5 is shown below. Figure 16 The mass spectrum of the antimicrobial peptide Ac-WZ-21 synthesized in Example 5 is shown. Figure 17The figure shows the experimental results of the hemolytic activity of different antimicrobial peptides provided by this invention; Figure 18 The figure shows the experimental results of hemolytic activity of different antimicrobial peptides modified with acetyl groups provided by the present invention. Detailed Implementation

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0028] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below.

[0029] It should also be understood that in some methods described herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are listed, unless the context otherwise indicates.

[0030] definition As used herein, the terms “a” and “an” as well as “the” and similar pronouns indicate singular and plural, unless otherwise specified herein or the context clearly contradicts them.

[0031] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0032] As used herein, the conjunction term "and / or" between multiple elements is understood to include both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first element without the second. The second option refers to the applicability of the second element without the first. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the scope of meaning and thus satisfies the requirement of the term "and / or" as used herein. The concurrent applicability of multiple options is also understood to fall within the scope of the term's meaning and thus satisfies the requirement of the term "and / or".

[0033] As used herein, the term "antimicrobial peptide," also known as "antimicrobial polypeptide," is synonymous with "antimicrobial protein" or "antimicrobial protein," and is used herein to refer to polymers of amino acid residues that have antimicrobial, bacteriostatic, or bactericidal functions. This term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses variants of its conserved modifications.

[0034] As used in this article, the term "drug resistance," also known as antibiotic resistance, refers to the tolerance of microorganisms, parasites, and tumor cells to the effects of chemotherapy drugs. Once drug resistance develops, the effectiveness of chemotherapy drugs decreases significantly. Drug resistance can be classified into acquired resistance and natural resistance based on its cause. Pathogens in nature, such as a particular strain of bacteria, can also exhibit natural resistance. When antibiotics are used for a long period, the majority of sensitive strains are continuously killed, and resistant strains multiply in large numbers, replacing sensitive strains and causing the bacterial resistance rate to that drug to continuously increase. Currently, the latter is considered the main cause of the emergence of drug-resistant bacteria. To maintain the effectiveness of antibiotics, their rational use should be emphasized.

[0035] As used in this article, the term "MRSA" refers to methicillin-resistant Staphylococcus aureus, a drug-resistant strain in the general sense. It exhibits broad-spectrum resistance, being resistant to both β-lactam and cephalosporin antibiotics, and showing varying degrees of resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, and rifampin (WHO Priority Pathogens List for Novel Antibiotic Development: Very Important).

[0036] As used in this article, the term "MSSA" refers to methicillin-sensitive Staphylococcus aureus.

[0037] As used in this article, the term "D-amino acid," in contrast to L-amino acid, refers to two isomers of the same amino acid with different optical rotations. According to the Fischer projection, L-amino acids have the amino group on the left and D-amino acids have the amino group on the right. Typically, naturally occurring amino acids are L-amino acids, while D-amino acids must be obtained through artificial synthesis.

[0038] As used in this article, the term "β-amino acid" refers to an amino acid with its amino group bonded to a carbon atom at the β-position. The only commonly found naturally occurring β-amino acid is β-alanine. Although β-alanine is often used as a component of bioactive macromolecules, β-peptides are generally not found in nature. For this reason, β-peptide antibiotics are being used to address the problem of antibiotic resistance.

[0039] As used herein, the term "conservative substitution" or "conservative sequence modification" refers to a nucleotide and amino acid sequence modification that does not eliminate the binding of an antibody to an antigen encoded by a nucleotide sequence or containing an amino acid sequence. These conserved sequence modifications include conserved nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings described herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved sequence modifications include conserved amino acid substitutions, wherein an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues in anti-MASP-2 antibodies are preferably replaced by another amino acid residue from the same side chain family. Methods for identifying nucleotides and conserved amino acid substitutions that do not eliminate antigen binding are well known in the art.

[0040] Examples of amino acids that can undergo conserved substitution are shown in Table 1 below: Table 1 Examples of conserved substitutions of amino acids As used herein, the term "effective dose" is synonymous with "effective amount," referring to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0041] Detailed implementation plan In a first aspect, the present invention provides an antimicrobial peptide having an amino acid sequence X1KRFKKFX2X3KLKKWV, wherein X1 is selected from nonpolar side-chain amino acids, X2 is selected from amino acids with aromatic side chains, and X3 is selected from amino acids with aromatic side chains or amino acids with basic side chains.

[0042] In an optional embodiment, X1 is selected from valine or isoleucine, X2 is selected from phenylalanine or tryptophan, and X3 is selected from phenylalanine, tryptophan, or arginine.

[0043] In an optional embodiment, the antimicrobial peptide has an amino acid sequence shown in any one of SEQ ID NO. 1 to 4.

[0044] SEQ ID NO.1: VKRFKKFFWKLKKWV; SEQ ID NO.2: VKRFKKFWFKLKKWV; SEQ ID NO.3: IKRFKKFFRKLKKWV; SEQ ID NO.4: IKRFKKFFWKLKKWV.

[0045] SEQ ID NO.2 was obtained by conservative substitution at positions 8 and 9 of SEQ ID NO.1, SEQ ID NO.4 was obtained by conservative substitution at position 1 of SEQ ID NO.1, and SEQ ID NO.3 was obtained by non-conservative substitution at position 9 of SEQ ID NO.4. The core amino acid sequence of the antimicrobial peptide provided by this invention contains 15 amino acid residues. Dividing the core fragment of the antimicrobial peptide provided by this invention into three segments (proximal, mid, and distal) from the N-terminus to the C-terminus, it can be seen that the proximal segment is a tryptophan-free polypeptide fragment.

[0046] In an optional embodiment, the N-terminus of the antimicrobial peptide contains a modifying functional group selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decacarbonyl, 2-morpholinylacetyl, pyrazinyl, dodecanoyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

[0047] The aforementioned modified functional groups are additionally added to the N-terminus of the antimicrobial peptide for the purpose of improving its antibacterial activity. It is understood that other conventional modifying groups that can be used to improve the activity of antimicrobial peptides should also be understood to be within the scope of protection of this invention.

[0048] In an optional embodiment, the modified antimicrobial peptide contains at least one D-amino acid or β-amino acid.

[0049] Secondly, the present invention provides a method for preparing the antimicrobial peptide described in any of the foregoing embodiments, which employs a solid-phase synthesis or liquid-phase synthesis method, wherein amino acids with side-chain protecting groups are sequentially coupled according to the amino acid sequence, and then the side-chain protecting groups are removed, extracted and purified sequentially to obtain the antimicrobial peptide.

[0050] It is understandable that peptide synthesis is a process of repeatedly adding amino acids. Solid-phase synthesis generally proceeds from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus), significantly reducing the difficulty of product purification at each step. To prevent side reactions, the side chains of the amino acids participating in the reaction are protected, the carboxyl terminus is free, and it must be activated before the reaction. There are two solid-phase synthesis methods: Fmoc and tBoc. Liquid-phase synthesis mainly employs two strategies: stepwise synthesis and fragment combination. Stepwise synthesis is simple and rapid and has been used to synthesize various bioactive peptide fragments. Fragment combination provides the most promising route for synthesizing peptides containing more than 100 amino acids and has successfully synthesized many bioactive peptides. Its greatest advantage is ease of purification.

[0051] In an optional embodiment, the method for preparing the antimicrobial peptide further includes a step of N-terminal modification of the solid-phase synthesized antimicrobial peptide. The functional group used for the N-terminal modification is selected from 5-isoxazole-5-formyl (Ez), acetyl (Ac), 2-(1-imidazolyl)acetyl (Mz), benzoyl (Bz), decadecyl (Dec), 2-morpholinoacetyl (Ml), pyrazinyl (Pz), dodecyl (Dodec), bis(toluenesulfonyl) (Ts), or 2-methylthiazol-5-formyl (Sz). The structural formulas of each functional group are shown below: In an optional embodiment, at least one of the amino acids used in solid-phase synthesis is a D-type amino acid or a β-type amino acid.

[0052] Thirdly, the present invention provides the use of the antimicrobial peptide described in any of the foregoing embodiments or the antimicrobial peptide obtained by the preparation method described in any of the foregoing embodiments in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, preservatives, animal feed or cosmetic precursors.

[0053] Fourthly, the present invention provides an antimicrobial drug formulation containing the antimicrobial peptide described in any one of the foregoing embodiments or an antimicrobial peptide prepared by any one of the foregoing embodiments.

[0054] In an optional embodiment, the effective dose of the antimicrobial peptide is 0.01~512 μg / ml, preferably 0.125~256 μg / ml.

[0055] In an optional embodiment, the types of pathogenic microorganisms for the antibacterial effect include bacteria and / or fungi.

[0056] In an optional embodiment, the bacteria include Gram-positive bacteria, such as Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ) or Enterococcus faecalis ( Enterococcus faecalis); and / or, Gram-negative bacteria, such as Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Escherichia coli ( Escherichia coli Klebsiella pneumoniae ( Klebsiella pneumoniae ) or Acinetobacter baumannii ( Acinetobacter baumannii ).

[0057] In an optional embodiment, the pathogenic microorganism is drug-resistant and resistant to at least one of the following antibiotics: β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampin.

[0058] In optional embodiments, the dosage form of the antibacterial drug preparation includes oral preparations, topical preparations, or injections; the oral preparations include granules, tablets, pastes, or oral solutions; the topical preparations include ointments, gels, suppositories, medicated bath solutions, hoof bath solutions, or sprays.

[0059] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0060] In this invention, the detection method can be a conventional method in the art for detecting antimicrobial peptides and antimicrobial proteins, including but not limited to MIC determination, MBC determination, yield detection, purity detection, and hemolytic activity evaluation.

[0061] For example: The MIC determination method is as follows: (1) Preparation of CAMHB medium: Weigh 22.5g of CAMHB medium powder (Shenzhen Haibo Biotechnology Co., Ltd., product number: HB6231-1), add 1000mL of distilled water, sterilize and set aside for use; CAMHB medium contains beef extract powder and acid hydrolyzed casein, which can provide nitrogen source, vitamins and other growth factors required for bacterial growth; the soluble starch it contains provides carbon source and energy for bacteria; the calcium chloride it contains can adjust pH and is also an activator of certain enzymes. It is often used for rapid enrichment culture of aerobic microorganisms in clinical samples or other samples. This invention is used for the determination of antimicrobial peptide MIC. As its alternative medium, MHB medium or LB medium can also be selected.

[0062] (2) Sterilization of tools and equipment: Place the prepared CAMHB medium, 0.9% (w / v) NaCl, pipette tips, and centrifuge tubes into an autoclave at 121°C for 30 min. Place the pipette tips, centrifuge tubes, and test tubes into a drying oven for 24 h. Sterilize the 96-well plate under ultraviolet light on a clean bench for 30 min.

[0063] (3) Preparation of antibacterial peptide stock solution: The peptide samples were prepared into 10 mg / ml stock solutions using sterile 0.9% (w / v) NaCl. The maximum concentration of 512 μg / ml was prepared using CAMHB medium, and then diluted to 256 μg / ml, 128 μg / ml, 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, and 0.25 μg / ml according to the system.

[0064] (4) Preparation of positive control drugs: Daptomycin (Shanghai Yuanye Biotechnology Co., Ltd., B27420), BF-30 (Jier Biochemical (Shanghai) Co., Ltd., purity >99%), and ZY13 (Jier Biochemical (Shanghai) Co., Ltd., purity >99%) were prepared into stock solutions with sterile 0.9% (w / v) NaCl, and then diluted to a maximum concentration of 512 μg / ml in CAMHB medium. The solutions were then diluted to the corresponding drug concentrations of 256 μg / ml, 128 μg / ml, 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, and 0.25 μg / ml.

[0065] (5) Preparation of bacterial suspension: Gram-negative bacteria were shaken in advance on LB (Thermo Fisher Scientific (Hangzhou) Co., Ltd., 1278005) medium (lysozyme broth) and Gram-positive bacteria were shaken on TSB (Thermo Fisher Scientific (Hangzhou) Co., Ltd., CM0129) medium (tryptone soybean broth) at 220 rpm and 37°C overnight. The suspension was diluted 1:1000 before the experiment (the final concentration of the system was 1:2000).

[0066] (6) Specific operating steps Step 1: Preparation and Reagent Preparation Wipe the work surface with alcohol and prepare pre-sterilized pipette tips, pipettes, EP tubes, petri dishes, racks, 96-well plates with lids, reagents, CAMHB medium, 0.9% (w / v) NaCl, bacterial suspension, etc. (For example, dissolve 8mg of reagent in 800μl of 0.9% (w / v) NaCl to prepare a 10mg / ml stock solution. Take 25.6 μl of the stock solution and add it to 474.4 μl of CAMHB medium to obtain the maximum treatment concentration of 512 μg / ml.) Step 2: Double dilution method for sample addition Taking A1-12 of a 96-well plate as an example, add 200 μl of the test sample (control antibiotic, different antimicrobial peptide samples provided by this invention, etc.) at 512 μg / ml to A12, and add 100 μl of CAMHB medium to each of A1-11. Take 100 μl of the A12 solution, add it to A11 and mix by pipetting. Pipette 100 μl of the solution into A10 and mix by pipetting. Continue this operation until A2. After mixing A2 by pipetting, pipette 100 μl and discard it. Ensure that A1 is a blank control and does not contain any drugs. Then add 100 μl of bacterial culture by pipetting. Incubate in a bacterial incubator at 37°C and 62% humidity for 24 h.

[0067] Step 3: Observation and Judgment After 24 hours, remove the 96-well plate. Under a light source, hold the plate steadily overhead and observe the bacterial growth in each well with the naked eye. The test is only meaningful when there is obvious bacterial growth in the negative control wells (i.e., without the test sample). Clear, non-corporeal colonies in a bright CAMHB state are the MIC for that group (Note: OD values ​​can also be used for determination). When a single skipped well occurs in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple skipped wells occur, the results should not be reported, and the test should be repeated.

[0068] The method for MBC determination is as follows: (1) Preparation of TSB agar medium: Weigh 30 g of TSB medium powder and 15 g of agar, mix them, add 1000 mL of distilled water, sterilize and set aside for use.

[0069] (2) Preparation of TSB agar plates: Cool the sterilized TSB medium to about 45°C, pour about 15-20 mL into the petri dish, cover the dish with the lid, shake gently, and wait for the plate to cool and solidify (about 5-10 min). Then, turn the plate upside down so that the lid is down and the bottom is up.

[0070] (3) Mix the MIC and subsequent 2x, 4x MIC up to the maximum concentration of the microbial culture by pipetting and aspirating 100 μl of each solution onto pre-labeled TSB agar plates. Cover the plates and gently shake them back and forth until the bacterial culture covers the entire plate. Incubate overnight in a bacterial incubator. The next day, remove the plates and observe the lowest drug concentration in the corresponding culture tube for each inoculum amount where the number of colonies growing on each plate is <0.1%. This is the minimum bactericidal concentration (MBC) of the drug.

[0071] The methods for evaluating hemolytic activity are as follows: Collect blood from healthy rabbits (cynomolgus monkeys or human blood can also be used), place it in an Erlenmeyer flask containing glass beads, and shake for 10 minutes, or stir the blood with a glass rod to remove fibrinogen and obtain defibrinated blood. Add approximately 10 times the volume of 0.9% (w / v) sodium chloride solution, shake well, and centrifuge at 1000–1500 rpm for 15 minutes. Remove the supernatant, and wash the precipitated red blood cells 2–3 times with 0.9% (w / v) sodium chloride solution as described above, until the supernatant is no longer red. Prepare a 2% (w / v) suspension of the obtained red blood cells with 0.9% (w / v) sodium chloride solution for testing. Add 100 μL to a 96-well round-bottom polystyrene microplate. Add 100 μL of the test sample with an initial concentration of 256 µg / mL to each well, serially diluting twice. Use 1% (w / v) triton as a positive control and DMSO as a negative control. The mixture was then incubated at 37°C with shaking at 60 rpm for 1 h. After incubation, it was centrifuged at 1000 × g for 3 min, and 100 µL of supernatant was transferred to each well of a new 96-well plate. If the solution in the tube is clear red and there are no cells or only a small number of red blood cells remaining at the bottom, it indicates that hemolysis has occurred; if all the red blood cells have settled and the supernatant is colorless and clear, or if the supernatant is colorless and clear, it indicates that no hemolysis has occurred. Alternatively, the absorbance can be measured at A450 nm to determine the hemolysis.

[0072] The strains used in this invention are as follows: Common strains: Escherichia coli ( E. coli ATCC25922, Enterococcus faecalis ( E.faecalis The ATCC29212 standard strain was purchased from the official ATCC website. Staphylococcus aureus ( S. aureus SA113, Staphylococcus aureus ( S. aureus CHS101, Staphylococcus aureus ( S. aureus YUSA139, Staphylococcus aureus ( S. aureus YUSA145, Enterococcus faecalis ( E.faecalis 16C166, Pseudomonas aeruginosa ( P. aeruginosa PA2237, Pseudomonas aeruginosa ( P. aeruginosaATCC27853, Escherichia coli ( E. coli Eco2242, Escherichia coli ( E. coli ATCC25922, Klebsiella pneumoniae ( K. pneumoniae K2044, Acinetobacter baumannii ( A.baumannii Ab2201, Acinetobacter baumannii ( A.baumannii Ab2202 is deposited in the Department of Infectious Diseases, Nanshan People's Hospital, Shenzhen, at No. 89 Taoyuan Road, Nanshan District, Shenzhen. The accession number is the specific strain number.

[0073] Drug-resistant strains: Staphylococcus aureus ( S. aureus YUSA132, Staphylococcus aureus ( S. aureus YUSA139 is deposited in the Department of Infectious Diseases, Nanshan People's Hospital, Shenzhen, at No. 89 Taoyuan Road, Nanshan District, Shenzhen. The accession number is the specific strain number.

[0074] Example 1: Synthesis of antimicrobial peptide VKRFKKFFWKLKKWV (hereinafter referred to as WZ-2 SEQ ID NO.1) (1) Polypeptide synthesis On Rink Amide MBHA resin (Jier Biochemical (Shanghai) Co., Ltd., 49006), following the core peptide sequence of the antimicrobial peptide WZ-2, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, FmocArg(Pbf)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Val-OH were sequentially coupled. After synthesis, the peptide resin was obtained by sequentially washing with DMF, DCM, and methanol and then drying.

[0075] (2) Lysis and peptide purification The peptide resin obtained in step (1) was lysed using a lysis buffer containing TFA (trifluoroacetic acid, CAS#: 76-05-1, supplier: Shanghai Maclean Biochemical Technology Co., Ltd.), TIS (triisopropylsilane, CAS#: 6485-79-6, supplier: Shanghai Maclean Biochemical Technology Co., Ltd.), EDT (2,2′-(1,2-ethylenedioxydioxo)diethylthiol, CAS#: 14970-87-7, supplier: Shanghai Maclean Biochemical Technology Co., Ltd.) and H2O in a volume ratio of 91:3:3:3. The resin was then filtered off, washed with a small amount of TFA, and the filtrates were combined and added to anhydrous diethyl ether to precipitate a white solid. The solid was centrifuged, washed with anhydrous diethyl ether, and dried under vacuum to obtain crude WZ-2 peptide. The crude peptide was purified by HPLC (purification conditions as follows) to obtain refined WZ-2 peptide. The MS detection conditions were as follows, and the detection results are as follows. Figure 2 As shown, the antimicrobial peptide WZ-2 was successfully synthesized.

[0076] HPLC conditions: MS conditions: Example 2: Synthesis of N-terminal acetyl-modified antimicrobial peptide VKRFKKFFWKLKKWV (hereinafter referred to as Ac-WZ-2) On Rink Amide MBHA resin (Jier Biochemical (Shanghai) Co., Ltd., 49006), following the core peptide sequence of the antimicrobial peptide WZ-2, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Lys(Boc)-OH were sequentially coupled. After the synthesis of Fmoc-Val-OH, acetic anhydride and pyridine were added for acetylation modification. The mixture was then washed sequentially with DMF, DCM, and methanol, and dried to obtain the peptide resin. The purification and detection steps were the same as in Example 1. The HPLC and MS detection results are shown below. Figure 3 and Figure 4 As shown, the acetyl-modified antimicrobial peptide Ac-WZ-2 was successfully synthesized with a purity of 98.3%.

[0077] Example 3: Synthesis of antimicrobial peptides WZ-4 and Ac-WZ-4 (SEQ ID NO.2) Antimicrobial peptide VKRFKKFWFKLKKWV was synthesized according to Examples 1 and 2, respectively, and named WZ-4. An N-terminal acetyl-modified antimicrobial peptide VKRFKKFWFKLKKWV was named Ac-WZ-4. The HPLC and MS detection results of WZ-4 are as follows: Figure 5 and Figure 6 As shown in the figure. The HPLC and MS detection results of Ac-WZ-4 are as follows. Figure 7 and Figure 8 As shown.

[0078] The purity of the prepared WZ-4 was 98.4%, and the purity of Ac-WZ-4 was 98.3%.

[0079] Example 4: Synthesis of antimicrobial peptides WZ-16 and Ac-WZ-16 (SEQ ID NO.3) Antimicrobial peptide IKRFKKFFRKLKKWV was synthesized according to Examples 1 and 2, respectively, and named WZ-16. An N-terminal acetyl-modified antimicrobial peptide of IKRFKKFFRKLKKWV was named Ac-WZ-16. The HPLC and MS detection results of WZ-16 are as follows: Figure 9 and Figure 10 As shown in the figure. The HPLC and MS detection results of Ac-WZ-16 are as follows. Figure 11 and Figure 12 As shown. The purity of the prepared WZ-16 was 98.7%, and the purity of Ac-WZ-16 was 98.4%.

[0080] Example 5: Synthesis of antimicrobial peptides WZ-21 and Ac-WZ-21 (SEQ ID NO.4) Antimicrobial peptide IKRFKKFFWKLKKWV was synthesized according to Examples 1 and 2, respectively, and named WZ-21. An N-terminal acetyl-modified antimicrobial peptide IKRFKKFFWKLKKWV was named Ac-WZ-21. The HPLC and MS detection results of WZ-21 are as follows: Figure 13 and Figure 14 As shown in the figure. The HPLC and MS detection results of Ac-WZ-21 are as follows. Figure 15 and Figure 16 As shown. The purity of the prepared WZ-21 was 98.7%, and the purity of Ac-WZ-21 was 98.8%.

[0081] Example 6: Comparison of antibacterial activities of different antimicrobial peptides This embodiment compares the antimicrobial properties of antimicrobial peptide WZ-2 and antimicrobial peptide ZY-13 disclosed in patent CN103275190A, demonstrating that the antimicrobial peptide provided in Example 1 has superior antimicrobial activity.

[0082] Table 2 Comparison of antibacterial properties of WZ-2 and ZY13 against different pathogens Table 3 Comparison of the antibacterial effects of WZ-2 and ZY-13 against methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus. Table 4. Comparison of the antibacterial effects of WZ-2 and ZY-13 against methicillin-resistant Acinetobacter baumannii and methicillin-sensitive Acinetobacter baumannii. Table 5. Comparison of the antibacterial effects of WZ-2 and ZY-13 against methicillin-resistant Klebsiella pneumoniae and methicillin-sensitive Klebsiella pneumoniae. Table 6. Comparison of the antibacterial effects of WZ-2 and ZY-13 against methicillin-resistant Escherichia coli and methicillin-sensitive Escherichia coli. Table 7 Comparison of the antibacterial effects of WZ-2 and ZY-13 against methicillin-resistant and methicillin-sensitive Pseudomonas aeruginosa. As shown in Tables 2-7 above, WZ2 exhibits antibacterial and bactericidal activities comparable to or superior to ZY13 against various pathogenic bacterial standard strains. Furthermore, based on the systemic data on the antibacterial activity against Staphylococcus aureus, WZ2 is superior to the positive control drug ZY13 against both methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) (Table 3). Regarding the activity against other clinically derived strains, the antimicrobial peptide WZ2 shows overall superior antibacterial activity against Acinetobacter baumannii (Table 4), Klebsiella pneumoniae (Table 5), Escherichia coli (Table 6), and Pseudomonas aeruginosa (Table 7) compared to ZY13.

[0083] Example 7 This embodiment refers to the synthesis method of Example 1 to synthesize a variety of antimicrobial peptides with different amino acid sequences. The physicochemical parameters and antimicrobial activities of these antimicrobial peptides are compared with those of the antimicrobial peptides obtained in Examples 1, 3-5. The antimicrobial properties are also compared with those of antimicrobial peptide ZY-13 disclosed in CN103275190A, antimicrobial peptide ZY-4 disclosed in patent WO2016201972A1, BF-30 (IND obtained in 2018 for the treatment of bacterial vaginosis, effervescent tablets), ampicillin, vancomycin, and gentamicin. The results demonstrate that the antimicrobial peptides provided in Examples 1, 3-5 have superior antimicrobial activity.

[0084] Table 8 Comparison of amino acid sequences and physicochemical parameters of different antimicrobial peptides Table 9 Comparison of antibacterial activities of different antimicrobial peptides As can be seen from Tables 8 and 9 above, the antimicrobial peptides provided in Examples 1, 3-5 all exhibit superior antimicrobial activity compared to other antimicrobial peptides with similar amino acid sequences, the three previously reported antimicrobial peptides, and the three antibiotics.

[0085] Example 8 This embodiment refers to the amino acid sequences of various antimicrobial peptides synthesized in Example 7, and combines the synthesis method of Example 2. Acetyl groups are introduced at the N-terminus of these antimicrobial peptides for modification. Then, their bactericidal activity is compared with that of two clinical candidates, PL-5 (CAS #: 850761-47-6, currently in clinical phase II-III, for diabetic foot ulcers, spray) and PL-18 (patent CN102219831B, for the treatment of bacterial / fungal vaginitis, suppository). This demonstrates that the N-terminal modified antimicrobial peptides provided by this invention have superior antimicrobial activity (as shown in Table 10).

[0086] Table 10 Comparison of antimicrobial activities of different acetyl-modified antimicrobial peptides In addition, in this embodiment, the N-terminal modified acetyl group is replaced with other modified functional groups, or all antimicrobial peptides are synthesized from D-type amino acids. The resulting antimicrobial peptides all have antibacterial activity comparable to or better than that of the acetyl group (as shown in Table 11).

[0087] Table 11 Comparison of antimicrobial activities of antimicrobial peptides modified with different functional groups Example 9: Evaluation of hemolytic activity The antimicrobial peptides WZ-2, WZ-4, WZ-16, WZ-21, Ac-WZ-2, AC-WZ-4, Ac-WZ-16, Ac-WZ-21, ZY-13, and BF-30 obtained in the above examples were subjected to hemolytic activity experiments according to the above-described hemolytic activity evaluation method. The results are as follows: Figure 17 , Figure 18 As shown, the antimicrobial peptides WZ-2, WZ-4, WZ-16, WZ-21, Ac-WZ-2, AC-WZ-4, Ac-WZ-16, and Ac-WZ-21 provided by the present invention showed no hemolytic toxicity at the highest concentration of 256 μg / ml, proving that the antimicrobial peptides provided in the above embodiments are all safe and effective.

[0088] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

4.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide has a modified functional group at its N-terminus, which is selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decacarbonyl, 2-morpholinyl acetyl, pyrazinyl, dodecanoyl, p-toluenesulfonyl, or 2-methylthiazol-5-formyl.

3. The antimicrobial peptide according to claim 1, characterized in that, The modified antimicrobial peptide contains at least one D-type or β-type amino acid.

4. The method for preparing the antimicrobial peptide according to any one of claims 1 to 3, characterized in that, The antimicrobial peptide is obtained by sequentially coupling amino acids with side-chain protecting groups according to their amino acid sequence using solid-phase or liquid-phase synthesis methods, followed by removal of side-chain protecting groups, extraction, and purification.

5. The preparation method according to claim 4, characterized in that, It also includes a step of modifying the N-terminus of the solid-phase synthesized antimicrobial peptide, wherein the functional group used for the N-terminal modification is selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decacarbonyl, 2-morpholinoacetyl, pyrazinyl, dodecanoyl, p-toluenesulfonyl or 2-methylthiazol-5-formyl.

6. The preparation method according to claim 4 or 5, characterized in that, At least one of the amino acids used in solid-phase synthesis is a D-type or β-type amino acid.

7. Use of the antimicrobial peptide according to any one of claims 1 to 3 or the antimicrobial peptide obtained by the preparation method according to any one of claims 4 to 6 in the preparation of antimicrobial drugs, anti-infective drugs, wound repair products, acne treatment drugs, radiation dermatitis prevention and treatment drugs, preservatives, animal feed or cosmetic precursors.

8. An antibacterial drug preparation, characterized in that, The antimicrobial drug preparation contains the antimicrobial peptide according to any one of claims 1 to 3 or the antimicrobial peptide prepared by the preparation method according to any one of claims 4 to 6.

9. The antibacterial drug formulation according to claim 8, characterized in that, The effective dose of the antimicrobial peptide is 0.01~512 μg / ml, preferably 0.125~256 μg / ml.

10. The antibacterial drug preparation according to claim 8 or 9, characterized in that, The types of pathogenic microorganisms for which antibacterial action is achieved include bacteria and / or fungi; Preferably, the bacteria include Gram-positive bacteria and / or Gram-negative bacteria; Preferably, the Gram-positive bacterium is Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ) or Enterococcus faecalis ( Enterococcus faecalis ); Preferably, the Gram-negative bacterium is Pseudomonas aeruginosa (…). Pseudomonas aeruginosa ), Escherichia coli ( Escherichia coli Klebsiella pneumoniae ( Klebsiella pneumoniae ) or Acinetobacter baumannii ( Acinetobacter baumannii ).

11. The antibacterial drug formulation according to claim 10, characterized in that, The pathogenic microorganism is drug-resistant and resistant to at least one of the following antibiotics: β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampin.

12. The antibacterial drug formulation according to claim 11, characterized in that, The dosage forms of the antibacterial drug preparations include oral preparations, topical preparations, or injections; The oral preparations include granules, tablets, pastes, or oral solutions; The topical preparations include ointments, gels, suppositories, medicated bath solutions, hoof bath solutions, or sprays.

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