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WO2025015978A1 - Polypeptide antibactérien micromoléculaire, son procédé de préparation et son utilisation - Google Patents

Polypeptide antibactérien micromoléculaire, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2025015978A1
WO2025015978A1 PCT/CN2024/086536 CN2024086536W WO2025015978A1 WO 2025015978 A1 WO2025015978 A1 WO 2025015978A1 CN 2024086536 W CN2024086536 W CN 2024086536W WO 2025015978 A1 WO2025015978 A1 WO 2025015978A1
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antimicrobial peptide
antimicrobial
preparation
antibacterial
bacteria
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PCT/CN2024/086536
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English (en)
Chinese (zh)
Inventor
占文虎
周方行
杨克树
林全浩
金东福
唐子涵
王维
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深圳碳云智肽药物科技有限公司
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Publication of WO2025015978A1 publication Critical patent/WO2025015978A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to the technical field of antibacterial drugs, and in particular to small molecule antibacterial polypeptides, preparation methods and uses thereof.
  • AMPs Antimicrobial peptides
  • AMPs Antimicrobial peptides
  • AMPs are considered to be ideal candidates to replace antibiotics due to their strong antimicrobial potential and unique mechanism of action.
  • Bungarus cathelicidin is a multifunctional antimicrobial peptide family with broad-spectrum antimicrobial activity. It has very strong bactericidal activity not only against Gram-positive bacteria, Gram-negative bacteria, certain fungi and viruses, but also against many clinical drug-resistant bacteria.
  • Bungarus cathelicidin (BF30) is an active peptide isolated from Bungarus venom by Lai Ren and others from the Kunming Institute of Zoology, Chinese Academy of Sciences in 2008. It is a single polypeptide drug containing 30 amino acids obtained by artificial synthesis. It is a straight-chain polypeptide with an ⁇ -helix at the N-terminus encoded by the Bungarus cathelicidin gene, containing 30 amino acid residues and a molecular weight of 3636.24Da.
  • the antimicrobial peptide from Bungarus fasciatus has high activity, is not prone to drug resistance, and has a broad spectrum of effects, it has become a very good lead molecule for screening new anti-infective drugs.
  • the sequence of the antimicrobial peptide from Bungarus fasciatus BF30 is relatively complex, the synthesis cost is high, and its antimicrobial activity still needs to be improved.
  • the present invention aims to improve the activity of Bungarus fasciatus antimicrobial peptides and reduce the synthesis cost of Bungarus fasciatus antimicrobial peptides, transforms and optimizes the amino acid sequence and structure of Bungarus fasciatus antimicrobial peptides, obtains mutant antimicrobial peptides with broad spectrum and high activity, and provides a preparation method and use of the mutant antimicrobial peptides.
  • the first aspect of the present disclosure provides an antimicrobial peptide having an amino acid sequence of X1KRFKKFX2X3KLKKWV , wherein X1 is selected from non-polar 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.
  • the X 1 is selected from valine or isoleucine
  • the X 2 is selected from phenylalanine or tryptophan
  • the X 3 is selected from phenylalanine, tryptophan or arginine.
  • the antimicrobial peptide has an amino acid sequence shown in any one of SEQ ID NO.1 to 4.
  • the N-terminus of the antimicrobial peptide contains a modified functional group, and the modified functional group is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, toluenesulfonyl or 2-methylthiazole-5-carbonyl.
  • the modified antimicrobial peptide contains at least one D-type or ⁇ -type amino acid.
  • the present disclosure provides a method for preparing the antimicrobial peptide described in any of the aforementioned embodiments, wherein amino acids with side chain protecting groups are coupled in sequence according to the amino acid sequence by solid phase synthesis or liquid phase synthesis, and then the side chain protecting groups are removed, extracted and purified in sequence to obtain the antimicrobial peptide.
  • the method for preparing the antimicrobial peptide further comprises the step of modifying the N-terminus of the solid phase synthesized antimicrobial peptide, and the functional group used for the N-terminal modification is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, toluenesulfonyl or 2-methylthiazole-5-carbonyl.
  • At least one of the amino acids used in the solid phase synthesis is a D-type or ⁇ -type amino acid.
  • the present disclosure provides the use of the antimicrobial peptide described in any of the preceding embodiments or the antimicrobial peptide obtained by the preparation method described in any of the preceding 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.
  • the present disclosure provides an antimicrobial drug preparation, which contains the antimicrobial peptide described in any one of the aforementioned embodiments or the antimicrobial peptide prepared by the preparation method described in any one of the aforementioned embodiments.
  • the effective dose of the antimicrobial peptide is 0.01 to 512 ⁇ g/ml, preferably 0.125 to 256 ⁇ g/ml.
  • the types of pathogenic microorganisms with antibacterial effects include bacteria and/or fungi.
  • the bacteria include Gram-positive bacteria, such as Staphylococcus aureus or Enterococcus faecalis; and/or, Gram-negative bacteria, such as Pseudomonas aer ⁇ ginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • Gram-positive bacteria such as Staphylococcus aureus or Enterococcus faecalis
  • Gram-negative bacteria such as Pseudomonas aer ⁇ ginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of ⁇ -lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides or rifampicin.
  • the dosage form of the antibacterial drug preparation includes an oral preparation, an external preparation or an injection; the oral preparation includes granules, tablets, pastes or oral solutions; the external preparation includes ointments, gels, suppositories, medicated baths, hoof baths or sprays.
  • the present disclosure provides a method for treating a condition associated with infection by pathogenic microorganisms, the method comprising administering the aforementioned antimicrobial peptide or antimicrobial drug preparation to a subject;
  • the pathogenic microorganisms include bacteria and/or fungi.
  • the bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and/or, Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • the pathogenic microorganism is resistant to antibiotics including ⁇ -lactam
  • antibiotics including ⁇ -lactam
  • ⁇ -lactam At least one of the following: oxaliplatin, cephalosporin, aminoglycoside, macrolide, tetracycline, fluoroquinolones, sulfonamides or rifampicin.
  • the present disclosure provides an antimicrobial peptide or an antimicrobial drug preparation for treating a disease associated with infection by a pathogenic microorganism.
  • the antimicrobial peptide is as described in the aforementioned aspects or obtained by the aforementioned preparation method, and the antimicrobial drug preparation is as described in the aforementioned aspects.
  • the pathogenic microorganisms include bacteria and/or fungi.
  • the bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and/or, Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of ⁇ -lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides or rifampicin.
  • the antimicrobial peptides provided by the present disclosure have stronger antibacterial activity than most other derivative peptides and antimicrobial peptides disclosed in existing reports.
  • the bactericidal activity was evaluated using a variety of strains known to be resistant to drugs. The results showed that the antimicrobial peptides provided by the present disclosure have broad-spectrum antibacterial activity against drug-resistant strains, with a more comprehensive antibacterial effect and a wider range of applicable scenarios.
  • the safety of the antimicrobial peptides provided by the present invention was evaluated by using hemolytic activity. It was determined that the preferred antimicrobial peptides of the present invention had no hemolytic toxicity at the highest concentration of 256 ⁇ g/ml, and had high safety.
  • the present disclosure provides an antimicrobial peptide with stronger antimicrobial activity, wider application range, and greater development potential value.
  • FIG1 is a high performance liquid chromatogram of the antimicrobial peptide WZ-2 synthesized in Example 1;
  • FIG2 is a mass spectrum of the antimicrobial peptide WZ-2 synthesized in Example 1;
  • FIG3 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-2 synthesized in Example 2;
  • FIG4 is a mass spectrum of the antimicrobial peptide Ac-WZ-2 synthesized in Example 2;
  • FIG5 is a high performance liquid chromatogram of the antimicrobial peptide WZ-4 synthesized in Example 3.
  • FIG6 is a mass spectrum of the antimicrobial peptide WZ-4 synthesized in Example 3.
  • FIG7 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-4 synthesized in Example 3;
  • FIG8 is a mass spectrum of the antimicrobial peptide Ac-WZ-4 synthesized in Example 3.
  • FIG9 is a high performance liquid chromatogram of the antimicrobial peptide WZ-16 synthesized in Example 4.
  • Figure 10 is a mass spectrum of the antimicrobial peptide WZ-16 synthesized in Example 4.
  • Figure 11 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-16 synthesized in Example 4.
  • Figure 12 is a mass spectrum of the antimicrobial peptide Ac-WZ-16 synthesized in Example 4.
  • Figure 13 is a high performance liquid chromatogram of the antimicrobial peptide WZ-21 synthesized in Example 5;
  • Figure 14 is a mass spectrum of the antimicrobial peptide WZ-21 synthesized in Example 5;
  • Figure 15 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-21 synthesized in Example 5;
  • Figure 16 is a mass spectrum of the antimicrobial peptide Ac-WZ-21 synthesized in Example 5;
  • FIG17 is a graph showing the hemolytic activity experimental results of different antimicrobial peptides provided by the present disclosure.
  • FIG. 18 is a graph showing the hemolytic activity experimental results of different antimicrobial peptides provided by the present disclosure after being modified with acetyl groups.
  • 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.
  • the conjunction term "and/or" between a variety of described elements is understood to include both single options and combined options.
  • the first option refers to the applicability of the first element without the second element.
  • the second option refers to the applicability of the second element without the first element.
  • the third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the meaning, thus satisfying the requirements of the term "and/or” used herein.
  • the concurrent applicability of multiple options is also understood to be the meaning of the term, thus satisfying the requirements of the term "and/or”.
  • antimicrobial peptide which may also be referred to as “antimicrobial polypeptide” is synonymous with “antimicrobial protein” and “antimicrobial protein”, and is used herein to refer to a polymer of amino acid residues having antimicrobial, bacteriostatic or bactericidal functions.
  • the term is applicable to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise stated, a specific polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • drug resistance also known as drug resistance
  • drug resistance refers to the tolerance of microorganisms, parasites and tumor cells to the effects of chemotherapeutic drugs. Once drug resistance occurs, the chemotherapeutic effect of the drug will be significantly reduced. Drug resistance can be divided into acquired resistance and natural resistance according to the cause of its occurrence. Pathogens in nature, such as a certain strain of bacteria, can also have natural drug resistance. When antibiotics are used for a long time, the majority of sensitive strains are constantly killed, and resistant strains multiply in large numbers, replacing sensitive strains, and the resistance rate of bacteria to this drug continues to increase. It is currently believed that the latter method is the main reason for the generation of drug-resistant bacteria. In order to maintain the effectiveness of antibiotics, attention should be paid to their rational use.
  • MRSA methicillin-resistant Staphylococcus aureus, a drug-resistant strain in the general sense. It has broad-spectrum drug resistance, is resistant to both ⁇ -lactam and cephalosporin antibiotics, and has varying degrees of resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, and rifampicin (WHO's list of priority pathogens for the development of new antibiotics: very important).
  • MSSA methicillin-sensitive Staphylococcus aureus
  • D-amino acid is opposite to L-amino acid, and refers to two isomers of the same amino acid with different optical rotations. According to the Fischer projection formula, the amino group on the left is the L-amino acid, and the amino group on the right is the D-amino acid. Generally, natural amino acids are L-amino acids, and D-amino acids need to be obtained through artificial synthesis.
  • ⁇ -amino acid refers to an amino acid in which the amino group is bound to the ⁇ -carbon atom.
  • ⁇ -amino acid The only common naturally occurring ⁇ -amino acid is ⁇ -alanine. Although ⁇ -alanine is often used as a component of biologically active macromolecules, ⁇ -peptides generally do not appear in nature. For this reason, ⁇ -peptide antibiotics are being used to address the problem of antibiotic resistance.
  • conservative substitutions or “conservative sequence modifications” of a sequence refers to nucleotide and amino acid sequence modifications that do not eliminate the binding of an antibody encoded by a nucleotide sequence or containing an amino acid sequence to an antigen.
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions.
  • modifications can be introduced into the sequence listing described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis).
  • Conservative sequence modifications include conservative amino acid substitutions, in which 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 already 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 uncharged 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 beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • amino acids with basic side chains e.g., lysine,
  • a predicted nonessential amino acid residue in an anti-MASP-2 antibody is preferably replaced with another amino acid residue from the same side chain family.
  • the term "effective dose” is synonymous with "effective amount” and refers to an amount of a substance, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or disorder.
  • the present disclosure provides an antimicrobial peptide having an amino acid sequence of X1KRFKKFX2X3KLKKWV , wherein X1 is selected from non-polar 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.
  • the X 1 is selected from valine or isoleucine
  • the X 2 is selected from phenylalanine or tryptophan
  • the X 3 is selected from phenylalanine, tryptophan or arginine.
  • the antimicrobial peptide has an amino acid sequence shown in any one of SEQ ID NO.1 to 4.
  • SEQ ID NO.1 VKRFKKFFWKLKKWV;
  • SEQ ID NO.3 IKRFKKFFRKLKKWV;
  • SEQ ID NO.4 IKRFKKFFWKLKKWV.
  • SEQ ID NO.2 is obtained by conservative substitution of SEQ ID NO.1 at position 8 and position 9
  • SEQ ID NO.4 is obtained by conservative substitution of SEQ ID NO.1 at position 1
  • SEQ ID NO.3 is obtained by non-conservative substitution of SEQ ID NO.4 at position 9.
  • the core amino acid sequence of the antimicrobial peptide provided by the present disclosure contains 15 amino acid residues, and 5 amino acid residues are a fragment. After the core fragment of the antimicrobial peptide provided by the present disclosure is divided into three fragments, namely, front, middle and back, from the N-terminus to the C-terminus, it can be seen that the front fragment is a polypeptide fragment without tryptophan.
  • the N-terminus of the antimicrobial peptide contains a modified functional group, and the modified functional group is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, toluenesulfonyl or 2-methylthiazole-5-carbonyl.
  • modified functional group is a modified functional group additionally added to the N-terminus of the antimicrobial peptide for the purpose of improving the antibacterial activity of the antimicrobial peptide. It is understandable that other conventional modified groups that can be used to improve the activity of the antimicrobial peptide should also be understood to be within the scope of protection of the present disclosure.
  • the modified antimicrobial peptide contains at least one D-type amino acid or ⁇ -type amino acid.
  • the present disclosure provides a method for preparing the antimicrobial peptide described in any of the aforementioned embodiments, wherein amino acids with side chain protecting groups are coupled in sequence according to the amino acid sequence by solid phase synthesis or liquid phase synthesis, and then the side chain protecting groups are removed, extracted and purified in sequence to obtain the antimicrobial peptide.
  • peptide synthesis is a process of repeated addition of amino acids.
  • the order of solid-phase synthesis is generally from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus), which can greatly reduce the difficulty of product purification at each step.
  • the side chains of the amino acids participating in the reaction are protected, the carboxyl terminus is free, and must be activated before the reaction.
  • Solid-phase synthesis methods namely Fmoc and tBoc.
  • Liquid-phase synthesis mainly has two strategies: stepwise synthesis and fragment combination.
  • Stepwise synthesis is simple and rapid, and is used for the synthesis of various biologically active peptide fragments; the fragment combination method provides the most promising route for the synthesis of peptides containing more than 100 amino acids, and has successfully synthesized a variety of biologically active peptides. Its biggest feature is that it is easy to purify.
  • the method for preparing the antimicrobial peptide further comprises the 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-carbonyl (Ez), acetyl (Ac), 2-(1-imidazolyl)acetyl (Mz), benzoyl (Bz), decacarbonyl (Dec), 2-morpholinoacetyl (Ml), pyrazinecarbonyl (Pz), dodecanoyl (Dodec), toluenesulfonyl (Ts) or 2-methylthiazole-5-carbonyl (Sz), and the structural formula of each functional group is as follows:
  • At least one of the amino acids used in the solid phase synthesis is a D-amino acid or a ⁇ -amino acid.
  • the present disclosure provides the use of the antimicrobial peptide described in any of the preceding embodiments or the antimicrobial peptide obtained by the preparation method described in any of the preceding embodiments in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, preservatives, animal feed or cosmetic precursors.
  • the present disclosure provides an antimicrobial drug preparation, which contains the antimicrobial peptide described in any one of the aforementioned embodiments or the antimicrobial peptide prepared by the preparation method described in any one of the aforementioned embodiments.
  • the effective dose of the antimicrobial peptide is 0.01 to 512 ⁇ g/ml, preferably 0.125 to 256 ⁇ g/ml.
  • the types of pathogenic microorganisms with antibacterial effects include bacteria and/or fungi.
  • the bacteria include Gram-positive bacteria, such as Staphylococcus aureus or Enterococcus faecalis; and/or, Gram-negative bacteria, such as Pseudomonas aer ⁇ ginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • Gram-positive bacteria such as Staphylococcus aureus or Enterococcus faecalis
  • Gram-negative bacteria such as Pseudomonas aer ⁇ ginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • the pathogenic microorganism is resistant to antibiotics including ⁇ -lactam
  • antibiotics including ⁇ -lactam
  • ⁇ -lactam At least one of the following: oxaliplatin, cephalosporin, aminoglycoside, macrolide, tetracycline, fluoroquinolones, sulfonamide or rifampicin.
  • the dosage form of the antibacterial drug preparation includes an oral preparation, an external preparation or an injection; the oral preparation includes granules, tablets, pastes or oral solutions; the external preparation includes ointments, gels, suppositories, medicated baths, hoof baths or sprays.
  • the present disclosure provides a method for treating a condition associated with infection by pathogenic microorganisms, the method comprising administering the aforementioned antimicrobial peptide or antimicrobial drug preparation to a subject;
  • the pathogenic microorganisms include bacteria and/or fungi.
  • the bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and/or, Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of ⁇ -lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides or rifampicin.
  • the present disclosure provides an antimicrobial peptide or an antimicrobial drug preparation for treating a disease associated with infection by a pathogenic microorganism.
  • the antimicrobial peptide is as described in the aforementioned aspects or obtained by the aforementioned preparation method, and the antimicrobial drug preparation is as described in the aforementioned aspects.
  • the pathogenic microorganisms include bacteria and/or fungi.
  • the bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and/or, Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.
  • the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of ⁇ -lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides or rifampicin.
  • the detection method may be a conventional method for detecting antimicrobial polypeptides and antimicrobial proteins in the art, including but not limited to MIC determination, MBC determination, yield detection, purity detection and hemolytic activity evaluation.
  • the MIC determination method is as follows:
  • CAMHB medium Preparation of CAMHB medium: Weigh 22.5 g of CAMHB medium powder (Shenzhen Haibo Biotechnology Co., Ltd., product number: HB6231-1), add 1000 mL of distilled water, and sterilize for later 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 contained provides carbon source and energy for bacteria; the calcium chloride contained can adjust pH and is also an activator of certain enzymes. It is often used for rapid bacterial proliferation and cultivation of aerobic microorganisms in clinical samples or other samples. The present invention is used for antimicrobial peptide MIC determination as an alternative culture medium.
  • the medium may also be MHB medium or LB medium.
  • bacterial suspension Gram-negative bacteria were shaken in advance with LB (Thermo Fisher Scientific, Hangzhou, 1278005) medium (lysate broth medium), and Gram-positive bacteria were shaken with TSB (Thermo Fisher Scientific, Hangzhou, CM0129) medium (tryptone soy broth medium), at 220 rpm, 37°C, overnight culture, and diluted 1:1000 before the experiment (the final concentration of the system was 1:2000).
  • LB Thermo Fisher Scientific, Hangzhou, 1278005
  • TSB Thermo Fisher Scientific, Hangzhou, CM0129
  • Step 1 Preparation and reagent preparation
  • Wipe the operating table with alcohol prepare the pre-sterilized gun tips, guns, EP tubes, culture dishes, racks, 96-well plates with lids, drugs, CAMHB culture medium, 0.9% (w/v) NaCl, bacterial solution and other items.
  • drugs CAMHB culture medium, 0.9% (w/v) NaCl, bacterial solution and other items.
  • CAMHB culture medium 0.9% (w/v) NaCl, bacterial solution and other items.
  • Step 2 Two-fold dilution method
  • A1 to 12 of the 96-well plate Take A1 to 12 of the 96-well plate as an example, add 200 ⁇ l 512 ⁇ g/ml test sample (control antibiotic drugs, different antimicrobial peptide samples provided by the present disclosure, etc.) to A12, and add 100 ⁇ l CAMHB culture medium to A1 to 11 respectively. Take 100 ⁇ l A12 solution, add A11 to mix by pipetting, aspirate 100 ⁇ l and add A10 to mix by pipetting, and continue this operation until A2. After A2 is mixed by pipetting, aspirate 100 ⁇ l and discard. Ensure that A1 is a blank control without adding any drugs. Then add 100 ⁇ l bacterial solution by gun. Put it in a bacterial incubator at 37°C and 62% humidity for 24 hours.
  • test sample control antibiotic drugs, different antimicrobial peptide samples provided by the present disclosure, etc.
  • Step 3 Observation and judgment
  • the MBC determination method is as follows:
  • TSB agar medium Weigh 30 g of TSB medium powder and 15 g of agar, mix, add 1000 mL of distilled water, and sterilize for later use.
  • TSB agar plates Cool the sterilized TSB medium to about 45°C, pour (about 15-20 mL) into a culture dish, cover the dish, and gently shake it. After the plate cools and solidifies (about 5-10 minutes), turn the plate upside down. Place the dish so that the lid is at the bottom and the bottom is at the top.
  • the hemolytic activity evaluation method is as follows:
  • red blood cells Take blood from healthy rabbits (cynomolgus monkeys or human blood can also be used), put it into a conical flask containing glass beads and shake for 10 minutes, or stir the blood with a glass rod to remove fibrinogen and make it defibrillated blood. Add about 10 times the amount of 0.9% (w/v) sodium chloride solution, shake well, centrifuge at 1000-1500 revolutions per minute for 15 minutes, remove the supernatant, and wash the precipitated red blood cells with 0.9% (w/v) sodium chloride solution 2-3 times according to the above method until the supernatant is no longer red. The obtained red blood cells are made into a 2% (w/v) suspension with 0.9% (w/v) sodium chloride solution for testing.
  • the solution in the test tube is clear red, with no cells or a small amount of red blood cells remaining at the bottom of the tube, it indicates that hemolysis has occurred; if all the red blood cells sink and the supernatant is colorless and clear, or the supernatant is color and clear, it indicates that no hemolysis has occurred. In addition, it can also be judged by measuring the absorbance at A450nm.
  • strains used in this disclosure are as follows:
  • Staphylococcus aureus (S.aureus) YUSA132 and Staphylococcus aureus (S.aureus) YUSA139 are both preserved in the Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, the preservation address is: No. 89, Taoyuan Road, Nanshan District, Shenzhen, and the preservation number is the specific strain number.
  • Example 1 Synthesis of antimicrobial peptide VKRFKKFFWKLKKWV (hereinafter referred to as WZ-2SEQ ID NO.1)
  • Rink Amide MBHA resin (Gill Biochemical (Shanghai) Co., Ltd., 49006), according to 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, Fmoc-Lys(Boc)-OH, and Fmoc-Val-OH were coupled in sequence.
  • the peptide resin obtained in step (1) was cleaved with a cleavage solution, wherein the cleavage solution contained 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)bis(ethanethiol, CAS#: 14970-87-7, supplier: Shanghai MacLean Biochemical Technology Co., Ltd.) and H 2 O in a volume ratio of 91:3:3:3.
  • TFA trifluoroacetic acid
  • TIS triisopropylsilane
  • EDT 2,2′-(1,2-ethylenedioxy)bis(ethanethiol, CAS#: 14970-87-7
  • supplier: Shanghai MacLean Biochemical Technology Co., Ltd. and H 2 O
  • the resin was then filtered off, the resin was washed with a small amount of TFA, the filtrate was combined, added to anhydrous ether to precipitate a white solid, centrifuged, the solid was washed with anhydrous ether, and vacuum dried to obtain a crude peptide WZ-2.
  • the crude peptide was purified by HPLC (purification conditions are as follows) to obtain refined peptide WZ-2.
  • the MS detection conditions are as follows. The detection results are shown in FIG2 , and the antimicrobial peptide WZ-2 was successfully synthesized.
  • Rink Amide MBHA resin (Gill Biochemical (Shanghai) Co., Ltd., 49006), according to 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, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, after the synthesis,
  • the purification and detection steps are the same as those in Example 1, and the HPLC and MS detection results are shown in Figures 3 and 4, respectively.
  • the acetyl-modified antimicrobial peptide Ac-WZ-2 was successfully synthesized with a purity of 98.3%.
  • the antimicrobial peptide VKRFKKFWFKLKKWV was synthesized with reference to Example 1 and Example 2, named WZ-4 and the N-terminal acetyl-modified antimicrobial peptide VKRFKKFWFKLKKWV, named Ac-WZ-4, and the HPLC and MS detection results of WZ-4 are shown in Figures 5 and 6, respectively.
  • the HPLC and MS detection results of Ac-WZ-4 are shown in Figures 7 and 8, respectively.
  • the purity of the prepared WZ-4 is 98.4%, and the purity of Ac-WZ-4 is 98.3%.
  • the antimicrobial peptide IKRFKKFFRKLKKWV was synthesized with reference to Example 1 and Example 2, named WZ-16 and the N-terminal acetyl-modified antimicrobial peptide IKRFKKFFRKLKKWV, named Ac-WZ-16, and the HPLC and MS detection results of WZ-16 are shown in Figures 9 and 10, respectively.
  • the HPLC and MS detection results of Ac-WZ-16 are shown in Figures 11 and 12, respectively.
  • the purity of the prepared WZ-16 is 98.7%, and the purity of Ac-WZ-16 is 98.4%.
  • Example 5 Synthesis of antimicrobial peptides WZ-21 and Ac-WZ-21 (SEQ ID NO.4)
  • the antimicrobial peptide IKRFKKFFWKLKKWV was synthesized with reference to Example 1 and Example 2, named WZ-21 and the N-terminal acetyl-modified antimicrobial peptide IKRFKKFFWKLKKWV, named Ac-WZ-21, and the HPLC and MS detection results of WZ-21 are shown in Figures 13 and 14, respectively.
  • the HPLC and MS detection results of Ac-WZ-21 are shown in Figures 15 and 16, respectively.
  • the purity of the prepared WZ-21 is 98.7%, and the purity of Ac-WZ-21 is 98.8%.
  • Example 1 compares the antimicrobial properties of the antimicrobial peptide WZ-2 and the antimicrobial peptide ZY-13 disclosed in patent CN103275190A, and proves that the antimicrobial peptide provided in Example 1 has better antimicrobial activity.
  • WZ2 As can be seen from Tables 2 to 7 above, the antibacterial and bactericidal activities of WZ2 against a variety of pathogenic bacteria standard strains are comparable to or better than those of ZY13. In addition, from the antibacterial activity data of the system's Staphylococcus aureus, WZ2 is superior to the positive drug ZY13 (Table 3), whether it is methicillin-sensitive (MSSA) or resistant Staphylococcus aureus (MRSA).
  • MSSA methicillin-sensitive
  • MRSA resistant Staphylococcus aureus
  • the antibacterial activity of the antimicrobial peptide WZ2 against Acinetobacter baumannii (Table 4), Klebsiella pneumoniae (Table 5), Escherichia coli (Table 6), and Pseudomonas aeruginosa (Table 7) is generally better than that of ZY13.
  • Example 1 the synthesis method of Example 1 is referred to to synthesize antimicrobial peptides with various amino acid sequences, and the physicochemical parameters and antimicrobial activities of these antimicrobial peptides are compared with those of the antimicrobial peptides obtained in Examples 1, 3 to 5, and the antimicrobial properties of the antimicrobial peptides ZY-13 disclosed in CN103275190A, the antimicrobial peptide ZY-4 disclosed in Patent WO2016201972A1, BF-30 (IND obtained in 2018, used for the treatment of bacterial vaginitis, effervescent tablets), Ampicillin, Vancomycin and Gentamicin are compared, proving that the antimicrobial peptides provided in Examples 1, 3 to 5 have better antimicrobial activity.
  • the amino acid sequences of various antimicrobial peptides synthesized in reference to Example 7 were combined with the synthesis method of Example 2 to introduce acetyl modification at the N-terminus of these antimicrobial peptides, and then compared with the bactericidal activity of two clinical candidate drugs 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), proving that the N-terminally modified antimicrobial peptides provided by the present disclosure have better antibacterial activity (as shown in Table 10).
  • the acetyl group modified at the N-terminus is replaced with other modified functional groups, or the antimicrobial peptides are synthesized entirely with D-amino acids.
  • the resulting antimicrobial peptides have an antibacterial activity equivalent to or better than that of the acetyl group (as shown in Table 11).
  • 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.
  • the results are shown in Figures 17 and 18. It can be seen that 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 disclosure had no hemolytic toxicity at the highest concentration of 256 ⁇ g/ml, which proves that the above examples
  • the antimicrobial peptides provided in the examples are all safe and effective.

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Abstract

La présente divulgation relève du domaine technique des médicaments antibactériens, et concerne en particulier un polypeptide antibactérien micromoléculaire, son procédé de préparation et son utilisation. Le peptide antibactérien selon la présente divulgation a une activité antibactérienne plus forte par comparaison avec la plupart des autres polypeptides dérivés et les polypeptides antibactériens divulgués dans les rapports existants. L'évaluation de l'activité bactéricide est réalisée à l'aide d'une pluralité de souches connues pour avoir une résistance aux médicaments, et les résultats montrent que le peptide antibactérien selon la présente divulgation a une activité antibactérienne à large spectre contre des souches résistantes aux médicaments et des effets antibactériens plus complets, et permet des indications plus larges.
PCT/CN2024/086536 2023-07-18 2024-04-08 Polypeptide antibactérien micromoléculaire, son procédé de préparation et son utilisation WO2025015978A1 (fr)

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