Micromolecular antibacterial polypeptide, preparation method therefor and use thereof

Abstract

Provided is an antibacterial peptide having an amino acid sequence X1KRFKKFFX2KLKKWV-NH2, wherein X1 is selected from any one or of amino acids A, C, D, E, F, G, H, K, L, N, M, P, Q, R, S, I, V, W, Y, and T or is absent; and X2 is an amino acid having an aromatic side chain or an amino acid having an alkaline side chain. In view of defects such as poor gastrointestinal fluid stability of antibacterial peptides in the prior art, a brand-new sequence design scheme and preparation method for an antibacterial peptide are provided. By means of a complete or partial D-amino acid substitution or structural derivatization of a peptide sequence, a series of antimicrobial peptides having stronger antibacterial activity and gastrointestinal stability as compared with the prior art are obtained, such that the antibacterial peptides have a wider application range and higher development potential value.

Description

Small molecule antimicrobial peptides, preparation methods and applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2024105212819, filed with the Chinese Patent Office on April 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of antimicrobial drug technology, specifically to a small molecule antimicrobial polypeptide, its preparation method, and its application. Background Technology

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] Existing technologies (Chinese patent applications: CN202310880462.6 and CN202310880156.2) aim to improve the activity of *Rhizoctonia solani* antimicrobial peptides and reduce their synthesis costs. These technologies involve modifying and optimizing the amino acid sequence and structure of the *Rhizoctonia solani* antimicrobial peptides to obtain mutant antimicrobial peptides with broad-spectrum and high activity. The technologies also provide methods for preparing and applying these mutant antimicrobial peptides. However, the antimicrobial peptides disclosed in these existing technologies suffer from poor stability in gastrointestinal fluids.

[0009] Summary of the Invention

[0010] To address the aforementioned technical issues, this disclosure aims to improve the activity of *Rhizoctonia solani* antimicrobial peptides, reduce their synthesis costs, and enhance their stability in gastrointestinal fluids. It involves replacing or structurally derivatizing the nucleotide sequences and structures of existing antimicrobial peptides with all or part of the D-amino acids to obtain mutant antimicrobial peptides that simultaneously possess broad-spectrum high activity and gastrointestinal fluid stability. The disclosure also provides a method for preparing and applying this mutant antimicrobial peptide.

[0011] On the one hand, this disclosure provides an antimicrobial peptide having an amino acid sequence X1KRFKKFFX2KLKKWV-NH2, wherein X1 is selected from any one of amino acids A, C, D, E, F, G, H, K, L, N, M, P, Q, R, S, I, V, W, Y, and T or is omitted; and X2 is an amino acid with an aromatic side chain or an amino acid with a basic side chain.

[0012] In some embodiments, the amino acids with aromatic side chains in the aforementioned antimicrobial peptides are amino acids Y, W, and F, and the amino acids with basic side chains are R or K.

[0013] In some embodiments, the aforementioned antimicrobial peptide has an amino acid sequence selected from any one of SEQ ID NO. 1-21.

[0014] In some preferred embodiments, the aforementioned antimicrobial peptide comprises an amino acid sequence as shown in SEQ ID NO:16.

[0015] In some embodiments, the N-terminus of the aforementioned antimicrobial peptide contains a modifying functional group selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, C8-C16 acyl, 2-morpholinoacetyl, pyrazinyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

[0016] In some preferred embodiments, the aforementioned modified functional group is an acetyl group.

[0017] In some embodiments, the aforementioned antimicrobial peptide contains at least one D-type or β-type amino acid.

[0018] In some preferred embodiments, the aforementioned antimicrobial peptide has an acetyl group at its N-terminus, and all of its amino acids are D-type amino acids.

[0019] In some preferred embodiments, the aforementioned antimicrobial peptide has an acetyl group at its N-terminus, and all of its amino acids, except for isoleucine (I), which is L-type, are D-type amino acids.

[0020] On the other hand, this disclosure provides a method for preparing an antimicrobial peptide, characterized in that a solid-phase synthesis or liquid-phase synthesis method is used to sequentially couple amino acids with side-chain protecting groups according to the amino acid sequence, and then sequentially remove the side-chain protecting groups, extract and purify to obtain the aforementioned antimicrobial peptide.

[0021] In some embodiments, the aforementioned method for preparing antimicrobial peptides further includes a step of modifying the N-terminus of the solid-phase synthesized antimicrobial peptides, wherein the functional group used for the aforementioned N-terminal modification is selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, C8-C16 acyl, 2-morpholinoacetyl, pyrazinyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

[0022] In some embodiments, at least one of the amino acids used in the solid-phase synthesis of the aforementioned antimicrobial peptide preparation method is a D-type or β-type amino acid.

[0023] On the other hand, this disclosure provides the use of the aforementioned antimicrobial peptide or the antimicrobial peptide prepared by the aforementioned method in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, radiation dermatitis prevention and treatment drugs, bacterial / fungal or mixed vaginal disease prevention and treatment drugs, preservatives, animal feed, hygiene and disinfection products or cosmetic precursors.

[0024] On the other hand, this disclosure provides an antimicrobial pharmaceutical preparation containing the aforementioned antimicrobial peptide or an antimicrobial peptide prepared using the aforementioned method for preparing the antimicrobial peptide.

[0025] In some embodiments, the effective dose of the antimicrobial peptide contained in the aforementioned antimicrobial drug preparation is 0.01 to 512 μg / ml, preferably 0.125 to 256 μg / ml.

[0026] In some preferred embodiments, the types of pathogenic microorganisms for the aforementioned antibacterial effect include bacteria and / or fungi.

[0027] In some preferred embodiments, the aforementioned bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and / or,

[0028] Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Helicobacter pylori, Klebsiella pneumoniae, or Acinetobacter baumannii; and / or,

[0029] Anaerobic bacteria, including Propionibacterium acnes, Bacteroides fragilis, and Gardnerella vaginalis.

[0030] In some preferred embodiments, the aforementioned fungi include Candida albicans, Candida glabrata, Candida parapsilosis, and Aspergillus fumigatus.

[0031] In some embodiments, the aforementioned pathogenic microorganisms are drug-resistant and resistant to at least one of the following antibiotics: β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampin.

[0032] In some embodiments, the dosage form of the aforementioned antibacterial drug preparation includes oral preparations, topical preparations, or injections.

[0033] In some embodiments, the aforementioned oral formulations include granules, tablets, pastes, or oral solutions.

[0034] In some embodiments, the aforementioned topical preparations include ointments, gels, suppositories, medicated bath solutions, hoof bath solutions, or sprays.

[0035] The beneficial effects achieved by this disclosure include at least the following:

[0036] The antimicrobial peptides disclosed herein exhibit stronger antimicrobial activity compared to most other derived peptides and antimicrobial peptides disclosed in existing reports.

[0037] The antimicrobial peptide provided in this disclosure exhibits superior gastrointestinal fluid stability compared to most other derived peptides and previously reported antimicrobial peptides. Gastric and intestinal fluid stability tests were conducted using HPLC analysis. The results showed that the antimicrobial peptide provided in this disclosure showed no significant degradation within 24 hours under simulated gastric and intestinal fluid conditions at 37°C, demonstrating excellent gastrointestinal fluid stability.

[0038] The antibacterial activity was evaluated using a variety of known drug-resistant strains. The results showed that the antimicrobial peptides provided in this disclosure have broad-spectrum antibacterial activity against drug-resistant strains, with a more comprehensive antibacterial effect and a wider range of applicable environments.

[0039] Furthermore, the safety of the antimicrobial peptides provided in this disclosure was evaluated using hemolytic activity. The results showed that the preferred antimicrobial peptides of this disclosure did not exhibit hemolytic toxicity at the highest concentration of 256 μg / ml, indicating high safety. Attached Figure Description

[0040] Figure 1 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-A synthesized in Example 1.

[0041] Figure 2 shows the mass spectrum of the antimicrobial peptide WZ-16-A synthesized in Example 1.

[0042] Figure 3 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-C synthesized in Example 2.

[0043] Figure 4 shows the mass spectrum of the antimicrobial peptide WZ-16-C synthesized in Example 2.

[0044] Figure 5 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-D synthesized in Example 3.

[0045] Figure 6 shows the mass spectrum of the antimicrobial peptide WZ-16-D synthesized in Example 3.

[0046] Figure 7 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-E synthesized in Example 4.

[0047] Figure 8 shows the mass spectrum of the antimicrobial peptide WZ-16-E synthesized in Example 4.

[0048] Figure 9 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-F synthesized in Example 5.

[0049] Figure 10 shows the mass spectrum of the antimicrobial peptide WZ-16-F synthesized in Example 5.

[0050] Figure 11 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-G synthesized in Example 6.

[0051] Figure 12 shows the mass spectrum of the antimicrobial peptide WZ-16-G synthesized in Example 6.

[0052] Figure 13 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-H synthesized in Example 7.

[0053] Figure 14 shows the mass spectrum of the antimicrobial peptide WZ-16-H synthesized in Example 7.

[0054] Figure 15 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-K synthesized in Example 8.

[0055] Figure 16 shows the mass spectrum of the antimicrobial peptide WZ-16-K synthesized in Example 8.

[0056] Figure 17 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-L synthesized in Example 9.

[0057] Figure 18 shows the mass spectrum of the antimicrobial peptide WZ-16-L synthesized in Example 9.

[0058] Figure 19 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-N synthesized in Example 10.

[0059] Figure 20 shows the mass spectrum of the antimicrobial peptide WZ-16-N synthesized in Example 10.

[0060] Figure 21 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-M synthesized in Example 11.

[0061] Figure 22 shows the mass spectrum of the antimicrobial peptide WZ-16-M synthesized in Example 11.

[0062] Figure 23 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-P synthesized in Example 12.

[0063] Figure 24 shows the mass spectrum of the antimicrobial peptide WZ-16-P synthesized in Example 12.

[0064] Figure 25 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-Q synthesized in Example 13.

[0065] Figure 26 shows the mass spectrum of the antimicrobial peptide WZ-16-Q synthesized in Example 13.

[0066] Figure 27 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-R synthesized in Example 14.

[0067] Figure 28 shows the mass spectrum of the antimicrobial peptide WZ-16-R synthesized in Example 14.

[0068] Figure 29 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-S synthesized in Example 15.

[0069] Figure 30 shows the mass spectrum of the antimicrobial peptide WZ-16-S synthesized in Example 15.

[0070] Figure 31 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-I synthesized in Example 16.

[0071] Figure 32 shows the mass spectrum of the antimicrobial peptide WZ-16-I synthesized in Example 16.

[0072] Figure 33 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-V synthesized in Example 17.

[0073] Figure 34 shows the mass spectrum of the antimicrobial peptide WZ-16-V synthesized in Example 17.

[0074] Figure 35 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-W synthesized in Example 18.

[0075] Figure 36 shows the mass spectrum of the antimicrobial peptide WZ-16-W synthesized in Example 18.

[0076] Figure 37 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-Y synthesized in Example 19.

[0077] Figure 38 shows the mass spectrum of the antimicrobial peptide WZ-16-Y synthesized in Example 19.

[0078] Figure 39 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-Del synthesized in Example 20.

[0079] Figure 40 shows the mass spectrum of the antimicrobial peptide WZ-16-Del synthesized in Example 20.

[0080] Figure 41 is a high-performance liquid chromatogram of the antimicrobial peptide WZ-16-T synthesized in Example 21.

[0081] Figure 42 shows the mass spectrum of the antimicrobial peptide WZ-16-T synthesized in Example 21.

[0082] Figure 43 is a high-performance liquid chromatogram of the antimicrobial peptide Ac-WZ16D1 synthesized in Example 22.

[0083] Figure 44 shows the mass spectrum of the antimicrobial peptide Ac-WZ16D1 synthesized in Example 22.

[0084] Figure 45 is a high-performance liquid chromatogram of the antimicrobial peptide Ac-WZ16D2 synthesized in Example 23.

[0085] Figure 46 shows the mass spectrum of the antimicrobial peptide Ac-WZ16D2 synthesized in Example 23.

[0086] Figure 47 shows the bactericidal curve test results of antimicrobial peptides Ac-WZ16D2 and PL-18.

[0087] Figure 48 shows the complete bactericidal curve evaluation results of the antimicrobial peptide Ac-WZ16D2.

[0088] Figure 49 shows the comparison results of the stability of antimicrobial peptide Ac-WZ16 with artificial gastric juice compared with Ac-WZ16D1 and Ac-WZ16D2.

[0089] Figure 50 shows the comparison results of the stability of antimicrobial peptide Ac-WZ16 with artificial intestinal fluid, Ac-WZ16D1, and Ac-WZ16D2.

[0090] Figure 51 shows the comparison of mouse plasma stability of antimicrobial peptides Ac-WZ16, Ac-WZ16D1, and Ac-WZ16D2.

[0091] Figure 52 shows the experimental results of hemolytic activity of antimicrobial peptides Ac-WZ16D1 and Ac-WZ16D2. Detailed Implementation

[0092] 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.

[0093] 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.

[0094] 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.

[0095] definition

[0096] 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.

[0097] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0098] 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".

[0099] 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.

[0100] As used in this article, the term "drug 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 the 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] Detailed implementation plan

[0105] On the one hand, this disclosure provides an antimicrobial peptide having an amino acid sequence X1KRFKKFFX2KLKKWV-NH2, wherein X1 is selected from any one of amino acids A, C, D, E, F, G, H, K, L, N, M, P, Q, R, S, I, V, W, Y, and T or is omitted; and X2 is an amino acid with an aromatic side chain or an amino acid with a basic side chain.

[0106] In some embodiments, the amino acids with aromatic side chains in the aforementioned antimicrobial peptides are amino acids Y, W, and F, and the amino acids with basic side chains are R or K.

[0107] In some embodiments, the aforementioned antimicrobial peptide has an amino acid sequence selected from any one of SEQ ID NO. 1-21.

[0108] In some preferred embodiments, the aforementioned antimicrobial peptide comprises an amino acid sequence as shown in SEQ ID NO:16.

[0109] In some embodiments, the N-terminus of the aforementioned antimicrobial peptide contains a modifying functional group selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, C8-C16 acyl, 2-morpholinoacetyl, pyrazinyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

[0110] In some preferred embodiments, the aforementioned modified functional group is an acetyl group.

[0111] In some embodiments, the aforementioned antimicrobial peptide contains at least one D-type or β-type amino acid.

[0112] In some preferred embodiments, the aforementioned antimicrobial peptide has an acetyl group at its N-terminus, and all of its amino acids are D-type amino acids.

[0113] In some preferred embodiments, the aforementioned antimicrobial peptide has an acetyl group at its N-terminus, and all of its amino acids, except for isoleucine (I), which is L-type, are D-type amino acids.

[0114] On the other hand, this disclosure provides a method for preparing an antimicrobial peptide, characterized in that a solid-phase synthesis or liquid-phase synthesis method is used to sequentially couple amino acids with side-chain protecting groups according to the amino acid sequence, and then sequentially remove the side-chain protecting groups, extract and purify to obtain the aforementioned antimicrobial peptide.

[0115] In some embodiments, the aforementioned method for preparing antimicrobial peptides further includes a step of modifying the N-terminus of the solid-phase synthesized antimicrobial peptides, wherein the functional group used for the aforementioned N-terminal modification is selected from 5-isoxazole-5-formyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, C8-C16 acyl, 2-morpholinoacetyl, pyrazinyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

[0116] In some embodiments, at least one of the amino acids used in the solid-phase synthesis of the aforementioned antimicrobial peptide preparation method is a D-type or β-type amino acid.

[0117] On the other hand, this disclosure provides the use of the aforementioned antimicrobial peptide or the antimicrobial peptide prepared by the aforementioned method in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, radiation dermatitis prevention and treatment drugs, bacterial / fungal or mixed vaginal disease prevention and treatment drugs, preservatives, animal feed, hygiene and disinfection products or cosmetic precursors.

[0118] On the other hand, this disclosure provides an antimicrobial pharmaceutical preparation containing the aforementioned antimicrobial peptide or an antimicrobial peptide prepared using the aforementioned method for preparing the antimicrobial peptide.

[0119] In some embodiments, the effective dose of the antimicrobial peptide contained in the aforementioned antimicrobial drug preparation is 0.01 to 512 μg / ml, preferably 0.125 to 256 μg / ml.

[0120] In some preferred embodiments, the types of pathogenic microorganisms for the aforementioned antibacterial effect include bacteria and / or fungi.

[0121] In some preferred embodiments, the aforementioned bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and / or,

[0122] Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Helicobacter pylori, Klebsiella pneumoniae, or Acinetobacter baumannii; and / or,

[0123] Anaerobic bacteria, including Propionibacterium acnes, Bacteroides fragilis, and Gardnerella vaginalis.

[0124] In some preferred embodiments, the aforementioned fungi include Candida albicans, Candida glabrata, Candida parapsilosis, and Aspergillus fumigatus.

[0125] In some embodiments, the aforementioned pathogenic microorganisms are drug-resistant and resistant to at least one of the following antibiotics: β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampin.

[0126] In some embodiments, the dosage form of the aforementioned antibacterial drug preparation includes oral preparations, topical preparations, or injections.

[0127] In some embodiments, the aforementioned oral formulations include granules, tablets, pastes, or oral solutions.

[0128] In some embodiments, the aforementioned topical preparations include ointments, gels, suppositories, medicated bath solutions, hoof bath solutions, or sprays.

[0129] The embodiments of this disclosure 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 this disclosure. The scope of this disclosure may include some embodiments having a combination of all or some of the described features. Where specific techniques or conditions are not specified in the examples, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0130] In this disclosure, 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, stability evaluation of artificial gastric juice, intestinal juice, and plasma, and evaluation of hemolytic activity.

[0131] For example:

[0132] The MIC determination method is as follows:

[0133] (1) Preparation of CAMHB medium: Weigh 22.5g of CAMHB medium powder (Shenzhen Haibo Biotechnology Co., Ltd., catalog 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 the 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 disclosure is used for the determination of antimicrobial peptide MIC. As its alternative medium, MHB medium or LB medium can also be selected.

[0134] (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.

[0135] (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.

[0136] (4) Preparation of positive control drugs: BF-30 (Jier Biochemical (Shanghai) Co., Ltd., purity >99%) and PL-18 (Jier Biochemical (Shanghai) Co., Ltd., purity >99%) were prepared into stock solutions with sterile 0.9% (w / v) NaCl, respectively, and prepared into a maximum concentration of 512 μg / ml using 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.

[0137] (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).

[0138] (6) Specific operating steps

[0139] Step 1: Preparation and Reagent Preparation

[0140] Wipe the work surface with alcohol and prepare pre-sterilized pipette tips, pipettes, EP tubes, petri dishes, racks, capped 96-well plates, 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. Add 25.6μl of the stock solution to 474.4μl of CAMHB medium to obtain the maximum treatment concentration of 512μg / ml.)

[0141] Step 2: Double dilution method for sample addition

[0142] 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 in this disclosure, 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, discard 100 μl. 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 hours.

[0143] Step 3: Observation and Judgment

[0144] 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 clear 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.

[0145] The method for MBC determination is as follows:

[0146] (1) Preparation of TSB agar medium: Weigh 30g of TSB medium powder and 15g of agar, mix them, add 1000mL of distilled water, sterilize and set aside for use.

[0147] (2) Preparation of TSB agar plates: Cool the sterilized TSB medium to about 45°C (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 minutes). Then, turn the plate upside down so that the lid is down and the bottom is up.

[0148] (3) Mix the MIC and subsequent 2x, 4x MIC up to the maximum concentration of the microbial culture by pipetting and aspirating 100 μl 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, observe the lowest drug concentration in the corresponding culture tube for each inoculum with a colony count <0.1%. This is the minimum bactericidal concentration (MBC) of the drug.

[0149] The stability testing methods for artificial gastric and intestinal fluids are as follows:

[0150] Add 40 μl of the sample to be tested (62.5 mg / ml) to 1.96 ml of artificial gastric fluid (Yuan Ye, R30387) or artificial intestinal fluid (Yuan Ye, R30384), vortex for 10 seconds to mix; incubate at 37 °C for 0 h, 0.5 h, 1 h, 2 h, 4 h, 24 h, and 48 h; at each time point, take 200 μl of gastric fluid, add 800 μl of acetonitrile-methanol (v:v = 1:1), mix well; centrifuge at 13000 rpm for 10 min at 4 °C; take the supernatant for HPLC analysis (the concentration of the sample on the HPLC is 0.25 mg / ml).

[0151] The HPLC method is as follows:

[0152] The plasma stability test method is as follows:

[0153] Add 2 μl of sample (1 mg / mL) to 198 μl of mouse plasma, vortex rapidly for 10 seconds to mix, and aliquot 20 μl into 7 tubes. Incubate at 37 °C for 0 h, 0.5 h, 1 h, 2 h, 4 h, 24 h, and 48 h, respectively. At each time point, take a sample and add 80 μl of acetonitrile-water (v:v = 1:2) containing the internal standard Exatecan (ChemExpress, HY-13631A, 104 ng / mL). Centrifuge at 13000 rpm for 10 min at 4 °C. The supernatant solution is directly analyzed by LC-MS / MS (final sample concentration is 2 μg / mL).

[0154] The LC-MS / MS method is as follows:

[0155] The methods for evaluating hemolytic activity are as follows:

[0156] 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 no longer appears 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 hour. After incubation, it was centrifuged at 1000×g for 3 minutes, 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.

[0157] The strains used in this disclosure are as follows:

[0158] The standard strains of *Escherichia coli* (ATCC25922) and *Enterococcus faecalis* (ATCC29212) were obtained from the ATCC official website. *Staphylococcus aureus* (SA113), *Pseudomonas aeruginosa* (PA2237), *Klebsiella pneumoniae* (K2044), *Acinetobacter baumannii* (Ab2201), *Staphylococcus aureus* (YUSA132), and *Staphylococcus aureus* (YUSA139) are all deposited in the Department of Infectious Diseases, Nanshan People's Hospital, Shenzhen, at No. 89 Taoyuan Road, Nanshan District, Shenzhen. The accession numbers correspond to the specific strain numbers.

[0159] Candida albicans ATCC90028, Aspergillus flavus ATCC204304, and Bacteroides fragilis ATCC25285 were provided by Beina Chuanglian Biotechnology Co., Ltd. Gardnerella vaginalis ATCC14018 and JDN-2023-D2-001 were provided by Nanjing Canchen Microbial Technology Co., Ltd. Propionibacterium acnes ATCC11827 and CC-2023-D2-001, Candida albicans BN-2023-D2-002 and BN-2023-D2-003, Candida glabrata GH-2023-D2-001 and GH-2023-D2-002, Candida parapsilosis JPH-2023-D2-001 and JPH-2023-D2-002, and Aspergillus fumigatus YQ-2023-D2-001 and YQ-2023-D2-002 were all provided by the Institute of Dermatology, Chinese Academy of Medical Sciences.

[0160] Helicobacter pylori ATCC 700392 (NCTC 26695) was donated by Professor Bi Hongkai's research group at Nanjing Medical University, China; Helicobacter pylori SS1 and CS01 were donated by Professor Liu Jing's research group at ShanghaiTech University; and QYZ-003 and QYZ-004 were provided by Qingyuan Traditional Chinese Medicine Hospital in Guangzhou. All strains were identified by the donors through morphological observation, Gram staining, and biochemical reactions, and were stored at -80℃ in 65% BHI / 25% glycerol / 10% FBS (v / v / v).

[0161] Example 1: Synthesis of antimicrobial peptide AKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-A SEQ ID NO.1)

[0162] (1) Peptide synthesis

[0163] On Rink Amide MBHA resin (Jier Biochemical (Shanghai) Co., Ltd., 49006), following the core peptide sequence of the antimicrobial peptide WZ-16-A, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-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-Ala-OH were sequentially coupled. After synthesis, the peptide resin was obtained by sequentially washing with DMF, DCM, and methanol and then drying.

[0164] (2) Cleavage and peptide purification

[0165] 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-ethylenedioxy)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-16-A peptide. The crude peptide was purified by HPLC (purification conditions as follows) to obtain refined WZ-16-A peptide. The HPLC results are shown in Figure 1, with a purity of 98.58%. The MS detection conditions are as follows, and the detection results are shown in Figure 2. + calc.for C 100 H 161 N 29 O 15 [M+2H] 2+ :1005.found:1005, successfully synthesized antimicrobial peptide WZ-16-A.

[0166] HPLC conditions:

[0167] MS conditions:

[0168] Example 2: Synthesis of antimicrobial peptide CKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-C SEQ ID NO.2)

[0169] The antimicrobial peptide CKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-C. The HPLC and MS detection results of WZ-16-C are shown in Figures 3 and 4, respectively. The purity of the prepared WZ-16-C was 98.13%, and the ES... + calc.for C 100 H 161 N 29 O 15 S1[M+2H] 2+ :1021.found:1021.

[0170] Example 3: Synthesis of antimicrobial peptide DKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-D SEQ ID NO.3)

[0171] The antimicrobial peptide DKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-D. The HPLC and MS detection results of WZ-16-D are shown in Figures 5 and 6, respectively. The purity of the prepared WZ-16-D was 96.07%, and the ES... + calc.for C 101 H 161 N 29 O 17 [M+2H] 2+ :1027.found:1027.

[0172] Example 4: Synthesis of antimicrobial peptide EKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-E SEQ ID NO.4)

[0173] The antimicrobial peptide EKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-E. The HPLC and MS detection results of WZ-16-E are shown in Figures 7 and 8, respectively. The purity of the prepared WZ-16-E was 98.79%, and the ES... + calc.for C 102 H 163 N 29 O 17 [M+2H] 2+ :1035.found:1035.

[0174] Example 5: Synthesis of antimicrobial peptide FKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-F SEQ ID NO.5)

[0175] The antimicrobial peptide FKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-F. The HPLC and MS detection results of WZ-16-F are shown in Figures 9 and 10, respectively. The purity of the prepared WZ-16-E was 100%, and the ES... + calc.for C 106 H 165 N 29 O 15 [M+2H] 2+ :1043.found:1043.

[0176] Example 6: Synthesis of antimicrobial peptide GKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-G SEQ ID NO.6)

[0177] The antimicrobial peptide GKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-G. The HPLC and MS detection results of WZ-16-G are shown in Figures 11 and 12, respectively. The purity of the prepared WZ-16-G was 100%, and the ES... + calc.for C 99 H 159 N 29 O 15 [M+2H] 2+ :998.found:998.

[0178] Example 7: Synthesis of antimicrobial peptide HKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-H SEQ ID NO.7)

[0179] The antimicrobial peptide HKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-H. The HPLC and MS detection results of WZ-16-H are shown in Figures 13 and 14, respectively. The purity of the prepared WZ-16-H was 98.53%, and the ES... + calc.C 103 H 163 N 31 O 15 [M+2H] 2+ :1039.found:1039.

[0180] Example 8: Synthesis of antimicrobial peptide KKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-K SEQ ID NO.8)

[0181] The antimicrobial peptide KKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-K. The HPLC and MS detection results of WZ-16-K are shown in Figures 15 and 16, respectively. The purity of the prepared WZ-16-K was 99.58%, and the ES... + calc.C 103 H 168 N 30 O 15 [M+2H] 2+ :1034.found:1034.

[0182] Example 9: Synthesis of antimicrobial peptide LKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-L SEQ ID NO.9)

[0183] The antimicrobial peptide LKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-L. The HPLC and MS detection results of WZ-16-L are shown in Figures 17 and 18, respectively. The purity of the prepared WZ-16-L was 99.71%, and the ES... + calc.C 103 H 167 N 29 O 15 [M+2H] 2+ :1027.found:1027.

[0184] Example 10: Synthesis of antimicrobial peptide NKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-N SEQ ID NO.10)

[0185] The antimicrobial peptide NKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-N. The HPLC and MS detection results of WZ-16-N are shown in Figures 19 and 20, respectively. The purity of the prepared WZ-16-N was 99.93%, and the ES... + calc.C 101 H 162 N 30 O 16 [M+2H] 2+ :1027.found:1027.

[0186] Example 11: Synthesis of antimicrobial peptide MKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-M SEQ ID NO.11)

[0187] The antimicrobial peptide MKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-M. The HPLC and MS detection results of WZ-16-M are shown in Figures 21 and 22, respectively. The purity of the prepared WZ-16-M was 99.75%, and the ES... + calc.C 102 H 165 N 29 O 15 S1[M+2H] 2+ :1036.found:1036.

[0188] Example 12: Synthesis of antimicrobial peptide PKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-P SEQ ID NO.12)

[0189] The antimicrobial peptide PKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-P. The HPLC and MS detection results of WZ-16-P are shown in Figures 23 and 24, respectively. The purity of the prepared WZ-16-P was 98.48%, and the ES... + calc.C 102 H 163 N 29 O 15 [M+2H] 2+ :1018.found:1018.

[0190] Example 13: Synthesis of antimicrobial peptide QKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-Q SEQ ID NO.13)

[0191] The antimicrobial peptide QKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-Q. The HPLC and MS detection results of WZ-16-Q are shown in Figures 25 and 26, respectively. The purity of the prepared WZ-16-Q was 96.76%, and the ES... + calc.C 102 H 164 N 30 O 16 [M+2H] 2+ :1034.found:1034.

[0192] Example 14: Synthesis of antimicrobial peptide RKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-R SEQ ID NO.14)

[0193] The antimicrobial peptide RKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-R. The HPLC and MS detection results of WZ-16-R are shown in Figures 27 and 28, respectively. The purity of the prepared WZ-16-E was 99.39%, and the ES... + calc.C 103 H 168 N 32 O 15 [M+2H] 2+ :1048.found:1048.

[0194] Example 15: Synthesis of antimicrobial peptide SKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-S SEQ ID NO.15)

[0195] The antimicrobial peptide SKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-S. The HPLC and MS detection results of WZ-16-S are shown in Figures 29 and 30, respectively. The purity of the prepared WZ-16-S was 99.69%, and the ES... + calc.C 100 H 161 N 29 O 16 [M+2H] 2+ :1014.found:1014.

[0196] Example 16: Synthesis of antimicrobial peptide IKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-I SEQ ID NO.16)

[0197] The antimicrobial peptide IKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-I. The HPLC and MS detection results of WZ-16-I are shown in Figures 31 and 32, respectively. The purity of the prepared WZ-16-I was 98.30%, and the ES... + calc.C 103 H 167 N 29 O 15 [M+2H] 2+ :1026.found:1026.

[0198] Example 17: Synthesis of antimicrobial peptide VKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-V SEQ ID NO.17)

[0199] The antimicrobial peptide VKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-V. The HPLC and MS detection results of WZ-16-V are shown in Figures 33 and 34, respectively. The purity of the prepared WZ-16-V was 99.90%, and the ES... + calc.C 102 H 165 N 29 O 15 [M+2H] 2+ :1020.found:1020.

[0200] Example 18: Synthesis of antimicrobial peptide WKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-W SEQ ID NO.18)

[0201] The antimicrobial peptide WKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-W. The HPLC and MS detection results of WZ-16-W are shown in Figures 35 and 36, respectively. The purity of the prepared WZ-16-W was 100%, and the ES... + calc.C 108 H 166 N 30 O 15 [M+2H] 2+ :1063.found:1063.

[0202] Example 19: Synthesis of antimicrobial peptide YKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-Y SEQ ID NO.19)

[0203] The antimicrobial peptide YKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-Y. The HPLC and MS detection results of WZ-16-Y are shown in Figures 37 and 38, respectively. The purity of the prepared WZ-16-Y was 99.83%, and the ES... + calc.C 106 H 165 N 29 O 16 [M+2H] 2+ :1052.found:1052.

[0204] Example 20: Synthesis of antimicrobial peptide KRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-Del SEQ ID NO.20)

[0205] The antimicrobial peptide KRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-Del. The HPLC and MS detection results of WZ-16-Del are shown in Figures 39 and 40, respectively. The purity of the prepared WZ-16-Del was 98.18%, and the ES... + calc.C 97 H 156 N 28 O 14 [M+2H] 2+ :970.found:970.

[0206] Example 21: Synthesis of antimicrobial peptide TKRFKKFFRKLKKWV-NH2 (hereinafter referred to as WZ-16-T SEQ ID NO.21)

[0207] The antimicrobial peptide TKRFKKFFRKLKKWV-NH2 was synthesized according to Example 1 and named WZ-16-T. The HPLC and MS detection results of WZ-16-T are shown in Figures 41 and 42, respectively. The purity of the prepared WZ-16-T was 97.63%, and the ES... + calc.C 101 H 163 N 29 O 16 [M+2H] 2+ :1021.found:1021.

[0208] Example 22: Comparison of antimicrobial peptides' antibacterial activity against different pathogen strains

[0209] This embodiment tested the antimicrobial activity of 21 antimicrobial peptides synthesized in Examples 1-21 against 6 pathogens. As shown in Table 1, the tested peptides showed good inhibitory activity against various strains, with most having MIC values ​​of 8-32 ug / mL. It can be seen that the type of the first amino acid at the N-terminus (hereinafter referred to as X1) has little effect on the antimicrobial activity, but the absence (serial number 20) will reduce the antimicrobial activity against some strains.

[0210] Table 1. Comparison of antimicrobial peptides' antibacterial activity against different pathogen strains

[0211] *Strains: Enterococcus faecalis ATCC29212 (standard strain), Staphylococcus aureus SA113 (standard strain), Klebsiella pneumoniae K2044 (standard strain), Pseudomonas aeruginosa PA2237 (clinical strain), Escherichia coli ATCC25922 (standard strain), Acinetobacter baumannii Ab2201 (clinical strain)

[0212] Example 23: Synthesis of an N-terminal acetyl-modified antimicrobial peptide containing some D-amino acids

[0213] This embodiment refers to the amino acid sequence of the antimicrobial peptide WZ-16-I synthesized in Example 16, and combines the synthesis method of Example 1 to selectively introduce acetyl groups at the N-terminus of the antimicrobial peptide, and perform all or part of the D-type amino acid substitution or structural derivation on its peptide sequence.

[0214] (1) Peptide synthesis

[0215] On Rink Amide MBHA resin (Jier Biochemical (Shanghai) Co., Ltd., 49006), with SEQ ID NO.22 as the core peptide sequence, Fmoc-D-Val-OH / Fmoc-Val-OH, Fmoc-D-Trp(Boc)-OH / Fmoc-Trp(Boc)-OH, Fmoc-D-Lys(Boc)-OH / Fmoc-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH / Fmoc-Lys(Boc)-OH, Fmoc-D-Leu-OH / Fmoc-Leu-OH were sequentially coupled. Fmoc-D-Lys(Boc)-OH / Fmoc-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH / Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe-OH / Fmoc-Phe-OH, Fmoc-D-Phe-OH / Fmoc-Phe-OH, Fmoc-D-Lys(Boc)-OH / Fmoc-Lys(Boc)-OH, Fmoc-D-Lys(Bo c)-OH / Fmoc-Lys(Boc)-OH, Fmoc-D-Phe-OH / Fmoc-Phe-OH, Fmoc-D-Arg(Pbf)-OH / FmocArg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH / Fmoc-Lys(Boc)-OH, Fmoc-D-Ile-OH / Fmoc-Ile-OH, the nomenclature, modifications and D / L configurations of the amino acids used are as follows:

[0216] WZ-16: All L-type amino acids;

[0217] WZ-16D: All D-type amino acids;

[0218] Ac-WZ16: N-terminal acetyl modification, all L-type amino acid;

[0219] Ac-WZ16D1: N-terminal acetyl modification, all-D-type amino acid;

[0220] Ac-WZ16D2: N-terminal acetyl modification, except for L-isoleucine (I), all other amino acids are D-type amino acids;

[0221] After synthesis, acetic anhydride and pyridine were added to complete the acetylation modification. The mixture was then washed with DMF, DCM and methanol in sequence, and dried to obtain the peptide resin.

[0222] (2) Cleavage and peptide purification

[0223] 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-ethylenedioxy)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-16-D1 peptide. The crude peptide was purified by HPLC (purification conditions as follows) to obtain Ac-WZ16D1 refined peptide. The HPLC results are shown in Figure 43, with a purity of 99.58%. The MS detection conditions were the same as in Case 1, and the detection results are shown in Figure 44. + calc.C 105 H 169 O 16 N 29 [M+2H] 2+ :1047.found:1047, successfully synthesized the antimicrobial peptide Ac-WZ16D1.

[0224] HPLC conditions:

[0225] The HPLC and MS detection results of WZ-16-D2 are shown in Figures 45 and 46, respectively. The purity of the prepared Ac-WZ16D2 was 100%, and the ES... + calc.C 105 H 169 O 16 N 29 [M+2H] 2+ :1048.found:1048.

[0226] Example 24: Antimicrobial activity test of D-type and L-type antimicrobial peptides

[0227] The antibacterial activity of the polypeptide synthesized in Example 23 against eight pathogenic bacteria was tested and compared, and the results are shown in Table 2.

[0228] Table 2. Comparison of antibacterial activity between type D and type L of WZ-16 or Ac-WZ16

[0229] Data on the antimicrobial activity of D-type and L-type antimicrobial peptides showed that replacing all amino acids in the WZ-16 sequence from L-type to D-type did not significantly alter or slightly improve the antimicrobial activity. Acetylation modification of the N-terminus of WZ-16 or WZ-16D improved the overall antimicrobial activity to some extent (Ac-WZ16 vs WZ-16; Ac-WZ16D1 vs WZ-16D). Furthermore, the MIC values ​​of the antimicrobial peptide Ac-WZ16D2 (after replacing D-type isoleucine with L-type amino acids) against the eight tested strains remained largely consistent, indicating that the chiral type of the first amino acid at the N-terminus had no significant impact on antimicrobial activity.

[0230] As shown in Table 3, the bactericidal activities of the above D-type and L-type antimicrobial peptides against four strains of Staphylococcus aureus SA113, Pseudomonas aeruginosa PA2237, Escherichia coli ATCC25922 and Enterococcus faecalis ATCC29212 were compared. All three peptides showed significant bactericidal activity against these four test strains, and the bactericidal activities of the D-type and L-type were comparable.

[0231] Table 3. Comparison of bactericidal activities between L-type and D-type Ac-WZ16

[0232] To investigate the time- and dose-dependent effects of the antibacterial activity of Ac-WZ16D2 and compare its activity with that of Yangshen antimicrobial peptide PL-18 (patent CN102219831B, for the treatment of bacterial / fungal vaginitis, suppository), the bactericidal curves of the two peptides against four bacterial strains were determined at a 4X MIC concentration. As shown in Figure 47, at a 4X MIC concentration, Ac-WZ16D2 rapidly killed Staphylococcus aureus SA113, Pseudomonas aeruginosa PA2237, Escherichia coli ATCC25922, and Enterococcus faecalis ATCC29212. After 2–4 hours, colony formation was virtually undetectable, demonstrating a certain time-dependent effect. Its bactericidal efficacy was comparable to or better than that of the Phase I candidate drug PL-18.

[0233] Subsequently, a complete bactericidal curve evaluation of Ac-WZ16D2 against Staphylococcus aureus SA113 and Escherichia coli ATCC25922 was conducted. As shown in Figure 48, Ac-WZ16D2 at concentrations ≥2*MIC can kill Staphylococcus aureus and Escherichia coli relatively quickly (≤8h), and the bactericidal effect is significantly concentration-dependent; the higher the concentration, the stronger the killing power against pathogens and the faster the killing speed, exhibiting a concentration-dependent effect. Concentrations below 0.5*MIC show no significant bacteriostatic effect.

[0234] Example 25: Stability test of antimicrobial peptides in gastric and intestinal fluids

[0235] The gastric and intestinal stability of the antimicrobial peptide Ac-WZ16 was analyzed by HPLC. The results showed that Ac-WZ16 degraded by 98.7% and 99.5% respectively in 5 minutes under simulated gastric and intestinal fluid conditions at 37℃. This indicates that Ac-WZ16 based on natural amino acids (L-type) has particularly poor gastric and intestinal fluid stability and cannot be used for gastrointestinal administration. However, Ac-WZ16 based on D-amino acids, whether it is the all-D-type Ac-WZ16D1 or Ac-WZ16D2 containing a single L-type isoleucine, showed excellent stability in simulated gastric and intestinal fluids, with no significant degradation within 24 hours under simulated gastric and intestinal fluid conditions at 37℃ (Figures 49 and 50).

[0236] Furthermore, mouse plasma stability experiments showed similar results: the half-life (t1 / 2) of Ac-WZ16 in mouse plasma was approximately 1 hour, while Ac-WZ16D1 and Ac-WZ16D2 plasma showed high stability, with t1 / 2 exceeding 48 hours (Figure 51). Therefore, the D-type antimicrobial peptides exhibited superior stability.

[0237] Based on these results, the inhibitory activity of Ac-WZ162 against Helicobacter pylori, a common bacterium in gastritis, was tested. As shown in Table 4, Ac-WZ162 showed significant antibacterial effects against two drug-sensitive Helicobacter pylori strains (ATCC 700392, SS1) and three drug-resistant strains (CS01, QYZ-003, and QYZ-004), with MICs of 32–64 ug / ml. This was superior to the activity of two clinical candidate drugs, BF-30 (patent WO2016201972A1, for the treatment of bacterial vaginosis, effervescent tablets) and PL-18, suggesting that Ac-WZ16D2 may be used for the treatment of Helicobacter pylori infection.

[0238] Considering the potential applications of antimicrobial peptides in acne and Gardnerella vaginalis vaginitis, the antimicrobial activity of the three peptides against three anaerobic bacteria—Propionibacterium acnes, Gardnerella vaginalis, and Bacteroides fragilis—was further tested. As shown in Table 4, Ac-WZ16D2 exhibited excellent antimicrobial activity against both standard and clinical Propionibacterium acnes, with a MIC of 4 μg / ml, superior to BF30 and PL-18 (MICs of 8–16 μg / ml), suggesting that Ac-WZ16D2 may be suitable for acne treatment. Ac-WZ16D2 also showed strong antimicrobial activity against Gardnerella vaginalis, effective against both standard and clinical strains, comparable to or better than BF30 and PL-18, indicating that this antimicrobial peptide can also be used to treat Gardnerella vaginalis vaginitis. Furthermore, all three peptides showed good activity against the opportunistic pathogen Bacteroides fragilis ATCC25285, with a MIC of 8 μg / ml, comparable to the antimicrobial peptides BF30 and PL-18.

[0239] Table 4. Antimicrobial activity (MIC) of Ac-WZ16 D2 and its clinical antimicrobial peptides against various pathogens.

[0240] Given that traditional antimicrobial peptides generally possess antifungal activity, and considering that PL-18 is undergoing Phase I clinical trials for both bacterial and fungal vaginitis, the inhibitory effect of Ac-WZ16D2 on fungi was also tested. As shown in Table 4, Ac-WZ16D2 exhibited significant antifungal effects against yeasts and fungi (Candida albicans, Candida glabrata, and Candida parapsilosis), with MICs ranging from 16 to 64 ug / ml, demonstrating antifungal activity comparable to or superior to PL-18; BF30 was ineffective against fungi. Furthermore, Ac-WZ16D2 also showed some antifungal activity against Aspergillus fumigatus, while BF30 and PL-18 were ineffective. Therefore, Ac-WZD2 possesses the potential to be used as an antifungal drug (e.g., for the treatment of fungal vaginitis).

[0241] Example 26: Detection of the hemolytic activity of antimicrobial peptides against human erythrocytes

[0242] The hemolytic activity of the antimicrobial peptides Ac-WZ16D1, Ac-WZ16D2, and Yangshen PL-18 obtained in the above examples was tested according to the aforementioned hemolytic activity evaluation method, with Triton-X100 as a positive control. The concentrations of the antimicrobial peptides ranged from 0.25 to 256 μg / ml.

[0243] Similar to the previous results for the L-type antimicrobial peptide Ac-WZ16 (Chinese Patent Application No.: CN202310880156.2, Example 9), Ac-WZ16D2 showed no hemolytic activity and good safety. As shown in Figure 52, Ac-WZ16D1 and Ac-WZ16D2 showed no hemolytic toxicity at various test concentrations, including a maximum concentration of 256 ug / ml and a 2-fold dilution, proving that the antimicrobial peptides provided in the above examples are safe and effective.

[0244] Although specific embodiments of this disclosure 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 such changes are all within the scope of protection of this disclosure.

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide has an amino acid sequence X1KRFKKFFX2KLKKWV-NH2, wherein X1 is selected from any one of amino acids A, C, D, E, F, G, H, K, L, N, M, P, Q, R, S, I, V, W, Y, and T or is omitted; X2 is an amino acid with an aromatic side chain or an amino acid with a basic side chain.

2. The antimicrobial peptide according to claim 1, characterized in that, The amino acids with aromatic side chains are amino acids Y, W, and F, and the amino acids with basic side chains are R or K.

3. The antimicrobial peptide according to claim 1 or 2, characterized in that, The antimicrobial peptide has an amino acid sequence selected from any one of SEQ ID NO.1-21; Preferably, the antimicrobial peptide comprises an amino acid sequence as shown in SEQ ID NO:

16.

4. The antimicrobial peptide according to any one of claims 1-3, 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, C8-C16 acyl, 2-morpholinoacetyl, pyrazinyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl. Preferably, the modifying functional group is an acetyl group.

5. The antimicrobial peptide according to any one of claims 1-4, characterized in that, The antimicrobial peptide contains at least one D-type or β-type amino acid; Preferably, the antimicrobial peptide has an acetyl group at its N-terminus, and all of its amino acids are D-type amino acids; More preferably, the antimicrobial peptide has an acetyl group at its N-terminus, and all of its amino acids, except for isoleucine (I), which is L-type, are D-type amino acids.

6. The method for preparing the antimicrobial peptide according to any one of claims 1-3, 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.

7. The preparation method according to claim 6, 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, C8-C16 acyl, 2-morpholinoacetyl, pyrazinyl, bis(toluenesulfonyl) or 2-methylthiazol-5-formyl.

8. The preparation method according to claim 6 or 7, characterized in that, At least one of the amino acids used in solid-phase synthesis is a D-type or β-type amino acid.

9. The use of the antimicrobial peptide according to any one of claims 1-5 or the antimicrobial peptide obtained by the preparation method according to any one of claims 6-8 in the preparation of antimicrobial drugs, anti-infective drugs, wound repair products, acne treatment drugs, radiation dermatitis prevention and treatment drugs, bacterial / fungal or mixed vaginal disease prevention and treatment drugs, preservatives, animal feed, hygiene and disinfection products or cosmetic precursors.

10. An antibacterial drug preparation, characterized in that, The antimicrobial drug preparation contains the antimicrobial peptide according to any one of claims 1-5 or the antimicrobial peptide prepared by the preparation method according to any one of claims 6-8.

11. The antibacterial drug formulation according to claim 10, characterized in that, The effective dose of the antimicrobial peptide is 0.01–512 μg / ml, preferably 0.125–256 μg / ml.

12. The antibacterial drug formulation according to claim 10 or 11, 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, including Staphylococcus aureus or Enterococcus faecalis; and / or, Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Helicobacter pylori, Klebsiella pneumoniae, or Acinetobacter baumannii; and / or, Anaerobic bacteria, including Propionibacterium acnes, Bacteroides fragilis, and Gardnerella vaginalis; Preferably, the fungi include Candida albicans, Candida glabrata, Candida parapsilosis, and Aspergillus fumigatus.

13. The antibacterial drug formulation according to claim 12, 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.

14. The antibacterial drug formulation according to claim 13, 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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