Hybrid peptide targeting bacteria FtsZ and cell membrane as well as preparation method and application of hybrid peptide

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hybrid peptide targeting bacteria FtsZ and cell membranes as well as a preparation method and application of the hybrid peptide. The amino acid sequence of the hybrid peptide is as shown in SEQ ID NO. 1-21. According to the hybrid peptide, SulA (88-106), A3 and B1 designed and synthesized by a research group are used as mother peptides, and 21 hybrid peptides are designed and synthesized based on four modification strategies of fatty acid connection, cyclization, dimerization and cell-penetrating peptide connection. The hybrid peptide provided by the invention shows more excellent broad-spectrum antibacterial activity, the FtsZ protein and cell membrane double-target effect is strongest, the toxicity is low, and the in-vivo pharmacodynamic effect is good. The hybrid peptide provided by the invention provides a new thought for solving the drug resistance problem, and provides a candidate drug for the development of novel double-target antibacterial peptides.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a hybrid peptide that targets bacterial FtsZ and cell membranes, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The advent of antibiotics was a milestone in human development, greatly improving survival rates from bacterial infections and enhancing quality of life. However, with the widespread use and overuse of antibiotics across various fields, bacterial resistance has emerged, and infections caused by drug-resistant strains pose an increasingly serious threat to humanity. In response to the severe development of drug resistance, there is an urgent need to find new antibacterial strategies or develop novel antibacterial drugs.

[0004] Antimicrobial peptides (AMPs) are small peptides that play a crucial role in the host's innate immunity against a variety of microorganisms, including bacteria, fungi, parasites, and viruses. AMPs exhibit advantages by acting on multiple targets on the plasma membrane and intracellular targets of pathogenic bacteria, and possess potent activity against drug-resistant bacteria. Therefore, AMPs offer a novel antibiotic alternative. Filament temperature-sensitive protein Z (FtsZ) is a GTPase closely related to bacterial cell division, playing a key role in regulating the formation of the Z-loop structure essential for bacterial cell division. This Z-loop serves as the initiation point for cell division, guiding the synthesis of new cell membrane components. Since bacteria are unlikely to alter their cell membrane composition through a single mutation, drugs targeting the cell membrane are less likely to induce resistance, making them potential targets for antibiotic development.

[0005] SulA protein is transcriptionally regulated by the SOS response system and is part of the bacterial SOS response. It is induced to express during DNA damage, pausing cell division by inhibiting FtsZ assembly to buy time for DNA repair. SulA(88-106), as a target peptide for FtsZ, exhibits poor antibacterial activity but good targeting specificity to FtsZ. It possesses some membrane permeability and an α-helical structure, but is not an α-helical peptide and therefore cannot effectively enter the cell to exert its antibacterial effect. Existing technologies include peptides A-3 and B-1, which show good antibacterial activity. While FtsZ protein has strong targeting specificity, its effect on the cell membrane needs further improvement. Summary of the Invention

[0006] In view of this, the present invention provides a hybrid peptide targeting bacterial FtsZ and cell membrane, its preparation method, and its application. The hybrid peptide provided by the present invention exhibits superior broad-spectrum antibacterial activity, with strong dual-target effects on both FtsZ protein and cell membrane, low toxicity, and good in vivo efficacy; it provides a new approach to solving drug resistance problems and offers candidate drugs for the development of novel dual-target antimicrobial peptides.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a hybrid peptide that targets bacterial FtsZ and the cell membrane, the hybrid peptide having an amino acid sequence as shown in any of SEQ ID NO. 1-21.

[0009] This invention uses SulA(88-106), A-3, and B-1 as template peptides. It selects four modification methods—fatty acid modification, cyclization, dimerization, and linking to transmembrane peptides—to enhance the dual-target effect on FtsZ and the cell membrane. The resulting hybrid peptides are named and have the following amino acid sequences:

[0010] C 10 -SulA(88-106):C 10 -THEWLRRAGLNRERILLLQ(SEQ ID NO.1);

[0011] C 14 -SulA(88-106):C 14 -THEWLRRAGLNRERILLLQ(SEQ ID NO.2);

[0012] C 16 -SulA(88-106):C 16 -THEWLRRAGLNRERILLLQ(SEQ ID NO.3);

[0013] Cyclo-SulA(88-106):

[0014] SulA(88-106)-Ahx-SulA(88-106):THEWLRRAGLNRERILLLQ-C6-THEWLRRAGLNRERILLLQ (SEQ ID NO. 5);

[0015] TAT-SulA(88-106):YGRKKRRQRRRTHEWLRRAGLNRERILLLQ(SEQ ID NO.6);

[0016] R9-SulA(88-106):RRRRRRRRRGGGTHEWLRRAGLNRERILLLQ(SEQ ID NO.7);

[0017] C 10 -A3:C 10 -THRWLRRLLLNRRRILLLQ(SEQ ID NO.8);

[0018] C 14 -A3:C 14 -THRWLRRLLLNRRRILLLQ(SEQ ID NO.9);

[0019] C 16 -A3:C 16 -THRWLRRLLLNRRRILLLQ(SEQ ID NO.10);

[0020] Cyclo-A3:

[0021] A3-Ahx-A3:THRWLRRLLLNRRRILLLQ-C6-THRWLRRLLLNRRRILLLQ(SEQ ID NO.12);

[0022] TAT-A3:YGRKKRRQRRRTHRWLRRLLLNRRRILLLQ(SEQ ID NO.13);

[0023] R9-A3:RRRRRRRRRGGGTHRWLRRLLLNRRRILLLQ(SEQ ID NO.14);

[0024] C 10 -B1:C 10 -THRWLRRLLRNRRRILRLQ(SEQ ID NO.15);

[0025] C 14 -B1:C 14 -THRWLRRLLRNRRRILRLQ(SEQ ID NO.16);

[0026] C 16 -B1:C 16 -THRWLRRLLRNRRRILRLQ(SEQ ID NO.17);

[0027] Cyclo-B1:

[0028] B1-Ahx-B1:THRWLRRLLRNRRRILRLQ-C6-THRWLRRLLRNRRRILRLQ (SEQ ID NO. 19);

[0029] TAT-B1: YGRKKRRQRRRTHRWLRRLLRNRRRILRLQ (SEQ ID NO. 20);

[0030] R9-B1:RRRRRRRRRGGGTHRWLRRLLRNRRRILRLQ (SEQ ID NO. 21).

[0031] Preferably, the heteropeptide has the amino acid sequence shown in SEQ ID NO.14 or SEQ ID NO.21.

[0032] The amino acid sequence of the heteropeptide has at least 80% homology with SEQ ID NO.1-21; more preferably, at least 90% homology; most preferably, at least 95% homology; such as at least 96%, 97%, 98%, or 99% homology.

[0033] In a second aspect, the present invention provides a conjugate comprising the heteropeptide described in the first aspect and a modified portion.

[0034] Preferably, the modified portion is optionally connected to the N-terminus or C-terminus of the heteropeptide via a linker;

[0035] Preferably, the modified portion includes a targeting portion, a fluorescent dye, and a protein tag;

[0036] Preferably, the targeting portion is a ligand, receptor, or antibody;

[0037] Preferably, the fluorescent dye is FITC;

[0038] Preferably, the protein tag is His, Flag, GST, MBP, HA, Myc, GFP, or biotin.

[0039] Thirdly, the present invention provides a nucleic acid encoding the heterozygous peptide described in the first aspect or the conjugate described in the second aspect.

[0040] Fourthly, a pharmaceutical composition comprising the heteropeptide described in the first aspect or the conjugate described in the second aspect.

[0041] In one or more specific embodiments of the present invention, the composition may further include other antibacterial agents, wherein the other antibacterial agents include penicillin antibiotics, cephalosporin antibiotics, erythromycin antibiotics, tetracycline antibiotics, aminoglycoside antibiotics, macrolide antibiotics and / or polypeptide antibiotics.

[0042] Alternatively, the broad-spectrum antibacterial drug or composition may include pharmaceutically acceptable excipients;

[0043] Preferably, the pharmaceutically acceptable excipients include pharmaceutically acceptable carriers, excipients, and diluents.

[0044] In one or more specific embodiments of the present invention, the drug is an oral drug or a topical drug; the oral drug is an oral preparation or an injection; the topical drug includes patches and ointments.

[0045] Fifthly, the present invention provides a bactericide comprising the heteropeptide described in the first aspect or the conjugate described in the second aspect.

[0046] In a sixth aspect, the present invention provides a method for preparing the heterozygous peptide described in the first aspect, using SulA(88-106), A-3 and B-1 as template peptides, and selecting four modification methods, namely fatty acid modification, cyclization, dimerization and linking to membrane-penetrating peptides, to obtain the heterozygous peptide.

[0047] The Fmoc-XS1 and XS2 were coupled to the intermediate XS-Tag and processed to obtain the heteropeptide C. 10 -SulA(88-106).

[0048] The Fmoc-XS1 and XS2-1 were coupled to the intermediate XS-Tag and then processed to obtain the heteropeptide C. 14 -SulA(88-106).

[0049] The Fmoc-XS1 and XS2-2 were coupled to the intermediate XS-Tag and then processed to obtain the heteropeptide C. 16 -SulA(88-106).

[0050] The Fmoc-XS1 and Fmoc-XS3 were coupled to the intermediate XS-Tag and then processed to obtain the heterozygous peptide Cycol-SulA (88-106).

[0051] The Fmoc-XS1, Fmoc-XS4, Fmoc-XS5, Fmoc-XS1 and Fmoc-XS3 were coupled to the intermediate XS-Tag and processed to obtain the heterozygous peptide SulA(88-106)-Ahx-SulA(88-106).

[0052] The Fmoc-XS1, Fmoc-XS3, Fmoc-XS6 and Fmoc-XS7 were coupled to the intermediate XS-Tag and then processed to obtain the heterozygous peptide TAT-SulA (88-106).

[0053] The Fmoc-XS1, Fmoc-XS3, Fmoc-XS8 and Fmoc-XS9 were coupled to the intermediate XS-Tag and then processed to obtain the heterozygous peptide R9-SulA(88-106).

[0054] The Fmoc-XA1 and Fmoc-XA2 were coupled to the intermediate XA-Tag and then processed to obtain the heterozygous peptide Cycol-A3.

[0055] The Fmoc-XA1, Fmoc-XA2, Fmoc-XS6 and Fmoc-XS7 were coupled to the intermediate XA-Tag and then processed to obtain the heterozygous peptide TAT-A3.

[0056] The Fmoc-XA1, Fmoc-XA2, Fmoc-XS8 and Fmoc-XS9 were coupled to the intermediate XA-Tag and then processed to obtain the heterozygous peptide R9-A3.

[0057] The Fmoc-XB1 and Fmoc-XA2 were coupled to the intermediate XB-Tag and then processed to obtain the heterozygous peptide Cycol-B1.

[0058] The Fmoc-XB1, Fmoc-XA2, Fmoc-XS6 and Fmoc-XS7 were coupled to the intermediate XA-Tag and then processed to obtain the heterozygous peptide TAT-B1.

[0059] The Fmoc-XB1, Fmoc-XA2, Fmoc-XS8 and Fmoc-XS9 were coupled to the intermediate XA-Tag and then processed to obtain the heterozygous peptide R9-B1.

[0060] In one or more specific embodiments of the present invention, peptides are synthesized in fragments using a combination of liquid-phase synthesis and solid-phase synthesis, and then purified using reversed-phase high-performance liquid chromatography to obtain the hybrid peptides. The basic fragment structures and sequences used are as follows:

[0061] Fragment XS-Tag structure: Glu(tBu)-Arg(Pbf)-Ile-Leu-Leu-Leu-Gln(Trt)-O-Tag;

[0062] Fragment Fmoc-XS1 structure: Fmoc-Arg(Pbf)-Ala-Gly-Leu-Asn(Trt)-Arg(Pbf)-OH;

[0063] Fragment XS2 structure: C 10 -Thr(tBu)-His(Trt)-Glu(tBu)-Trp(Boc)-Leu-Arg(Pbf)-OH;

[0064] Fragment XS2-1 structure: C 14 -Thr(tBu)-His(Trt)-Glu(tBu)-Trp(Boc)-Leu-Arg(Pbf)-OH;

[0065] Fragment XS2-2 structure: C 16 -Thr(tBu)-His(Trt)-Glu(tBu)-Trp(Boc)-Leu-Arg(Pbf)-OH;

[0066] Fragment Fmoc-XS3 structure: Fmoc-Thr(tBu)-His(Trt)-Glu(tBu)-Trp(Boc)-Leu-Arg(Pbf)-OH;

[0067] Fragment Fmoc-XS4 structure: Fmoc-Ahx-Thr(tBu)-His(Trt)-Glu(tBu)-Trp(Boc)-Leu-Arg(Pbf)-OH;

[0068] Fragment Fmoc-XS5 structure: Fmoc-Glu(tBu)-Arg(Pbf)-Ile-Leu-Leu-Leu-Gln(Trt)-OH;

[0069] Fragment Fmoc-XS6 structure: Fmoc-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-OH;

[0070] Fragment Fmoc-XS7 structure: Fmoc-Tyr(tBu)-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-OH;

[0071] Fragment Fmoc-XS8 structure: Fmoc-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Gly-Gly-Gly-OH;

[0072] Fragment Fmoc-XS9 structure: Fmoc-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-OH;

[0073] Fragment XA-Tag structure: Arg(Pbf)-Arg(Pbf)-Ile-Leu-Leu-Leu-Gln(Trt)-O-Tag;

[0074] Fragment Fmoc-XA1 structure: Fmoc-Arg(Pbf)-Leu-Leu-Leu-Asn(Trt)-Arg(Pbf)-OH;

[0075] Fragment Fmoc-XA2 structure: Fmoc-Thr(tBu)-His(Trt)-Arg(Pbf)-Trp(Boc)-Leu-Arg(Pbf)-OH;

[0076] Fragment XB-Tag structure: Arg(Pbf)-Arg(Pbf)-Ile-Leu-Arg(Pbf)-Leu-Gln(Trt)-O-Tag;

[0077] Fragment Fmoc-XB1 structure: Fmoc-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Asn(Trt)-Arg(Pbf)-OH.

[0078] A general synthetic method for coupling solid-phase resins with amino acids: Weigh CTC Resin, Fmoc-AA-OH, and DIPEA into a solid-phase synthesis tube, add DCM to dissolve them, and react with a vortex mixer for 3 hours (Note: For amino acids with large side chains and protecting groups such as Fmoc-Arg(Pbf)-OH and Fmoc-His(Trt)-OH, coupling can be performed twice). Add MeOH and DIPEA and react for 20 minutes to cover the remaining active sites of the resin. Remove the solid-phase synthesis tube, filter, and wash alternately with DMF and DCM.

[0079] A general synthetic method for removing the Fmoc-protecting group from the amino terminus through solid-phase synthesis: Place the intermediate with the Fmoc-protecting group in a solid-phase synthesis tube, add PIP and DMF, shake and react for 20 min, detect with ninhydrin solution, filter, and wash with DMF and DCM alternately.

[0080] Solid-phase synthesis of coupled amino acid residues: Fmoc-AA-OH, DIPEA, HBTU, and HoBt were added sequentially to a synthesis tube, followed by DMF. The reaction was carried out at room temperature for 1 hour, with monitoring using ninhydrin solution. Note: Amino acids such as Fmoc-Arg(Pbf)-OH, with their large side chains and protecting groups, can be coupled multiple times.

[0081] General synthesis method for solid-phase cleavage resin: Treat with 10 ml DCM:TFA = 100:1 for 5 min (3 times) at room temperature, combine the concentrated filtrates, precipitate with cold isopropyl ether, and filter; or evaporate to dryness multiple times, then dissolve in acetonitrile:water = 1:1 and freeze dry.

[0082] A general synthetic method for coupling intermediate fragments in liquid phase synthesis: Dissolve the amino-terminal naked Tag-peptide chain in a mixed solvent of THF and DMF, and add the Fmoc-protected intermediate fragment to be coupled, HoAt, HATU and DIPEA in sequence with stirring. React at room temperature to obtain the target product coupled with a new fragment.

[0083] A general synthetic method for removing the Fmoc-protecting group from Tag-long polypeptide fragments by liquid phase synthesis: Dissolve the Fmoc-protected Tag-long polypeptide chain in THF, add PIP and DBU sequentially with stirring, and react at room temperature to obtain the Tag-long polypeptide fragment without the Fmoc-protecting group.

[0084] A general synthetic method for removing the carrier and amino acid side chain protecting groups from Tag-fully protected polypeptide chains in liquid phase: The Tag-fully protected polypeptide chains obtained by coupling all intermediates are dissolved in a mixed solution of TFA:TIS:H2O = 38:1:1 and reacted under stirring at room temperature to obtain crude peptides.

[0085] In a seventh aspect, the present invention provides the use of the heteropeptide described in the first aspect in any one or more of the following:

[0086] (1) As a broad-spectrum antibacterial drug or composition;

[0087] (2) As a rapid bactericide;

[0088] (3) As a pharmaceutically acceptable carrier or additive.

[0089] Preferably, the drug is an oral or topical drug; or, the oral drug is an oral preparation or an injection; or, the topical drug includes patches and ointments.

[0090] Preferably, the bacteria include Gram-positive bacteria and Gram-negative bacteria;

[0091] Preferably, the Gram-positive bacteria include one or more of Bacillus pumilus, Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, and Enterococcus faecium; and / or, the Gram-negative bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, or Klebsiella pneumoniae.

[0092] Preferably, when the heterozygous peptide is Cyclo-SulA(88-106) (SEQ ID NO.4), the bacterium is Staphylococcus aureus.

[0093] Preferably, when the heterozygous peptide is SulA(88-106)-Ahx-SulA(88-106) (SEQ ID NO.5), the bacterium is Staphylococcus epidermidis.

[0094] Preferably, when the heterozygous peptide is TAT-SulA(88-106) (SEQ ID NO.6), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, Acinetobacter baumannii, or Pseudomonas aeruginosa.

[0095] Preferably, when the heterozygous peptide is R9-SulA(88-106) (SEQ ID NO.7), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, Acinetobacter baumannii, or Pseudomonas aeruginosa.

[0096] Preferably, the hybrid peptide is C 10 When -A3 (SEQ ID NO. 8) is used, the bacteria are one or more of Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Klebsiella pneumoniae, Acinetobacter baumannii, or Escherichia coli.

[0097] Preferably, the hybrid peptide is C 14 When -A3 (SEQ ID NO. 9) is used, the bacteria is one or more of Staphylococcus aureus, Enterococcus faecalis, or Acinetobacter baumannii.

[0098] Preferably, the hybrid peptide is C 16 When -A3 (SEQ ID NO.10) is used, the bacteria is Acinetobacter baumannii.

[0099] Preferably, when the heterozygous peptide is Cyclo-A3 (SEQ ID NO.11), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0100] Preferably, when the heterozygous peptide is A3-Ahx-A3 (SEQ ID NO.12), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0101] Preferably, when the heterozygous peptide is TAT-A3 (SEQ ID NO.13), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0102] Preferably, when the heterozygous peptide is R9-A3 (SEQ ID NO.14), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0103] Preferably, the hybrid peptide is C 10 When -B1 (SEQ ID NO.15), the bacteria is one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0104] Preferably, the hybrid peptide is C 14 When -B1 (SEQ ID NO.16), the bacteria is one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0105] Preferably, the hybrid peptide is C 16 When -B1 (SEQ ID NO.17) is selected, the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0106] Preferably, when the heterozygous peptide is Cyclo-B1 (SEQ ID NO.18), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0107] Preferably, when the heterozygous peptide is B1-Ahx-B1 (SEQ ID NO.19), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0108] Preferably, when the heterozygous peptide is TAT-B1 (SEQ ID NO.20), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0109] Preferably, when the heterozygous peptide is R9-B1 (SEQ ID NO.21), the bacteria are one or more of Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0110] The hybrid peptides obtained in this invention enhance the dual-target effects on the cell membrane and FtsZ, further improving antibacterial activity.

[0111] In an eighth aspect, the present invention provides a targeting peptide that targets FtsZ protein and cell membrane SulA (88-106), containing any of the amino acid sequences shown in SEQ ID NO. 1-21.

[0112] Preferably, the FtsZ protein is derived from Bacillus subtilis.

[0113] It should be noted that the heterozygous peptides SEQ ID NO.1-21 disclosed in this invention have the functions of bacterial FtsZ and cell membrane SulA (88-106). Therefore, liposomes, vesicles, exosomes or cells that achieve targeting functions by containing the above sequences are all within the protection scope of this invention; at the same time, FtsZ antibodies containing the above sequences are also within the protection scope of this invention.

[0114] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0115] (1) The heteropeptides of the present invention are based on SulA (88-106), A3 and B1 obtained in this study as parent peptides. They are further modified by fatty acid modification, cyclization, dimerization and connection of membrane-penetrating peptides to obtain 21 heteropeptides. Among them, R9-B1 has excellent broad-spectrum antibacterial activity, good safety, strong cell membrane and FtsZ dual target activity. It is a novel dual-target antimicrobial peptide with potential for further research and development. It provides a new idea for solving the drug resistance problem and provides a candidate drug for the development of novel dual-target antimicrobial peptides.

[0116] (2) The heterozygous peptide of the present invention has broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria after antibacterial activity and targeting assays. It has strong targeting to bacterial cell membranes and FtsZ, low toxicity, and excellent preliminary in vivo efficacy.

[0117] (3) The heteropeptide R9-B1 is composed of 31 amino acids. It is synthesized by a solid-liquid combination method, which saves raw materials and time. The purification method is mature and the synthesis cost is low. Attached Figure Description

[0118] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0119] Figure 1 The morphology of B. pumilus CMCC63202 was observed under an optical microscope for the heterozygous peptides TAT-SulA(88-106), R9-SulA(88-106), TAT-A3, R9-A3, TAT-B1, and R9-B1. (A) Bacterial morphology under normal conditions; (B) Bacterial morphology in the presence of TAT-SulA(88-106); (C) Bacterial morphology in the presence of R9-SulA(88-106); (D) Bacterial morphology in the presence of A3; (E) Bacterial morphology in the presence of R9-A3; (F) Bacterial morphology in the presence of B1; (G) Bacterial morphology in the presence of R9-B1.

[0120] Figure 2 The figure shows the polymerization kinetics results of the heterozygous peptides R9-A3, TAT-B1, and R9-B1 FtsZ.

[0121] Figure 3 Figure 1 shows the FtsZ polymerization results observed under transmission electron microscopy for the heteropeptides R9-A3, TAT-B1, and R9-B1; (A) Polymerization state under normal conditions; (B) Bacterial morphology in the presence of A3; (C) Bacterial morphology in the presence of R9-A3; (D) Polymerization state in the presence of B1; (E) Polymerization state in the presence of TAT-B1; (F) Polymerization state in the presence of R9-B1.

[0122] Figure 4 Figure 1 shows the cell membrane depolarization results of the heteropeptides TAT-A3 and R9-A3. (A): ATCC25923, (B): ATCC19606.

[0123] Figure 5 Figure 1 shows the cell membrane depolarization results of the heteropeptides TAT-B1 and R9-B1. (A): ATCC25923, (B): ATCC19606.

[0124] Figure 6 The image shows the results of scanning electron microscopy observation of bacterial morphology of the heterozygous peptides R9-A3 and R9-B1.

[0125] Figure 7Figure 1 shows the results of the drug resistance induction experiments of the heterozygous peptides R9-A3 and R9-B1. (A): S. aureus ATCC25923, (B): A. baumnnii ATCC19606.

[0126] Figure 8 Figure 1 shows the results of the acute toxicity test on the large wax moth; (A) growth status after 5 days of R9-B1 treatment; (B) growth status after 5 days of B1 treatment; (C) survival rate after 5 days of R9-B1 treatment; (D) survival rate after 5 days of B1 treatment.

[0127] Figure 9 Preliminary in vivo pharmacodynamic experiments of the heterozygous peptide R9-B1; (A) Wound healing within 8 days; (B) Blood colony count after treatment under different conditions; (C) Skin colony count after treatment under different conditions; (D) Heat map of colony count in the heart, liver, spleen, lungs and kidneys. Detailed Implementation

[0128] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Information on the strains used in this invention:

[0129] B. subtilis ATCC9372: Bacillus subtilis ATCC 9372, a penicillin-sensitive strain;

[0130] B. pumilus CMCC63202: Bacillus pumilus CMCC63202, a penicillin-sensitive strain;

[0131] S. aureus ATCC25923: Staphylococcus aureus ATCC25923, erythromycin-sensitive strain;

[0132] E. faecalis ATCC29212: Enterococcus faecalis ATCC29212, a vancomycin-sensitive strain;

[0133] S. pyogenes EMA-S: Streptococcus pyogenes EMA-S, a clinically isolated erythromycin-sensitive strain;

[0134] E. faecium ATCC19434: Enterococcus faecium ATCC19434, a vancomycin-sensitive strain;

[0135] S. epidermidis: Staphylococcus epidermidis, a penicillin-resistant strain isolated clinically;

[0136] S. aureus CI: Staphylococcus aureus CI, a penicillin-resistant strain isolated clinically;

[0137] S. aureus ATCC31007: Staphylococcus aureus ATCC31007, a penicillin-resistant isolate;

[0138] S. pyogenes EMA-R: Streptococcus pyogenes EMA-R, a clinically isolated erythromycin-resistant strain;

[0139] S. aureus ATCC43300: Staphylococcus aureus ATCC43300, a methicillin-resistant strain;

[0140] E. faecalis ATCC51299: Enterococcus faecalis ATCC51299, a vancomycin-resistant strain;

[0141] E. faecium ATCC51559: Enterococcus faecium ATCC51559, a vancomycin-resistant strain;

[0142] E. coli ATCC25922: Escherichia coli ATCC25922;

[0143] P. aeruginosa ATCC27853: Pseudomonas aeruginosa ATCC 27853;

[0144] K. pneumoniae ATCC BAA 1902: Klebsiella pneumoniae ATCC BAA1902;

[0145] A. baumnnii ATCC19606: Acinetobacter baumannii ATCC19606;

[0146] E. coli BW25113: Escherichia coli BW25113.

[0147] Example 1: Preparation of 2,4-bis(eicosethoxy)benzyl alcohol (Tag support)

[0148]

[0149] Anhydrous potassium carbonate (6.00 g, 43.40 mmol) was weighed and dissolved in 30 mL of DMF. 2,4-Dihydroxybenzaldehyde (0.60 g, 4.34 mmol) and 1-bromodocosahexadecane (3.49 g, 8.88 mmol) were added sequentially with stirring. The reaction was carried out under nitrogen protection at 70 °C for 12 h. After the reaction was complete, the reaction solution was poured into a reaction flask containing 300 mL of purified water and stirred at 25 °C for 1 h, producing a pink precipitate. This precipitate was filtered to obtain a pink solid. The pink solid was slurried in 200 mL of anhydrous methanol and filtered to obtain a brownish-red solid, which was then dried under vacuum for 3 h. This brownish-red solid was then dissolved in a 9:1 THF:MeOH mixture (60 mL), and sodium borohydride (0.40 g, 10.57 mmol) was added with stirring. The reaction was carried out at 40 °C for 3 h. Then, 2 mL of purified water was added to the reaction solution to react with the unreacted sodium borohydride. The mixture was then filtered, and the filtrate was concentrated to about 30 mL. Methanol (300 mL) was then added, and a white precipitate was formed. The precipitate was filtered and dried under vacuum to obtain a white product, namely Tag-OH (2.29 g), with a yield of 69.8%.

[0150] Example 2: Synthesis of fragment XS-Tag

[0151] (1) Synthesis of Fmoc-Gln(Trt)-O-Tag

[0152]

[0153] Weigh 2.27 g (3 mmol) of Tag-OH and dissolve it in 60 mL of DCM. Then, add Fmoc-Gln(Trt)-OH (2.20 g, 3.6 mmol), DIC (567.90 mg, 4.5 mmol), and DMAP (73.30 mg, 0.6 mmol) sequentially. React at 40 °C for 30 min, and monitor the reaction progress by TLC. After complete reaction of Tag-OH, concentrate the reaction solution, add acetonitrile to precipitate the product, filter, wash the filter cake 2-3 times with acetonitrile, and vacuum dry the filter cake to obtain the white product Fmoc-Gln(Trt)-O-Tag.

[0154] (2) Synthesis of NH2-Gln(Trt)-O-Tag

[0155]

[0156] Fmoc-Gln(Trt)-O-Tag (3.0 mmol, 1.0 eq) was dissolved in 60 mL of THF, and PIP (4.5 mmol, 1.5 eq) and DBU (0.6 mL, 1%) were added sequentially. The reaction was carried out at room temperature for 10 min. The reaction was monitored by TLC until it was complete. The solution was concentrated, and acetonitrile was added to precipitate the product. The pH was adjusted to about 7 by adding 6 mol / L hydrochloric acid. The product was obtained by filtration. The filter cake was slurryed and washed 2-3 times, filtered, and the product was dried under vacuum to obtain the white solid NH2-Gln(Trt)-O-Tag product.

[0157] (3) Synthesis of Fmoc-Leu-Gln(Trt)-O-Tag

[0158]

[0159] The NH2-Gln(Trt)-O-Tag (3.0 mmol, 1.0 eq) obtained in the previous step was dissolved in a THF:DMF = 9:1 mixed solvent (60 mL). Fmoc-Leu-OH (3.6 mmol, 1.2 eq), HBTU (3.6 mmol, 1.2 eq), HOBt (3.6 mmol, 1.2 eq), and DIPEA (15.0 mmol, 5.0 eq) were added sequentially. The reaction was carried out at room temperature for 30 min. The reaction was monitored by TLC until it ended. The mixture was concentrated, acetonitrile was precipitated, and the mixture was filtered. The filter cake was washed 2-3 times, and finally filtered to obtain the white solid product Fmoc-Leu-Gln(Trt)-O-Tag.

[0160] (4) Synthesis of fragment XS-Tag

[0161]

[0162] The product from the previous step, Fmoc-Leu-Gln(Trt)-O-Tag, was linked sequentially through multiple coupling and deprotection methods using (2) Fmoc protecting group removal and (3) amino acid coupling to obtain a white solid, XS-Tag (4.26 g), with a yield of 65.3%.

[0163] Example 3: Synthesis of Fmoc-XS1 fragment

[0164] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase fragment synthesis, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, and Fmoc-Arg(Pbf)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid product, Fmoc-XS1 (346.8 mg), with a yield of 41.9%.

[0165]

[0166] Example 4: Synthesis of fragment XS2

[0167] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase synthesis of fragments, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Thr(tBu)-OH, and C were sequentially coupled. n H 2n O2 was then used for resin cutting to obtain a white solid product XS2-1 (400.0 mg), with a yield of 45.5%. A white solid product XS2-2 (462.0 mg) was obtained, with a yield of 51.8%.

[0168]

[0169]

[0170] Example 5: Synthesis of Fmoc-XS3 fragment

[0171] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase synthesis of fragments, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, and Fmoc-Thr(tBu)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XS3 (368.5 mg), with a yield of 41.7%.

[0172]

[0173] Example 6: Synthesis of Fmoc-XS4 fragment

[0174] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase fragment synthesis, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Ahx-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XS4 (549.3 mg), with a yield of 58.4%.

[0175]

[0176] Example 7: Synthesis of Fmoc-XS5 fragment

[0177] Starting with Fmoc-Gln(Trt)-OH, and following general methods for solid-phase fragment synthesis, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Glu(tBu)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XS5 (482.3 mg), with a yield of 58.3%.

[0178]

[0179]

[0180] Example 8: Synthesis of Fmoc-XS6 fragment

[0181] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase synthesis of fragments, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, and Fmoc-Arg(Pbf)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XS6 (550.0 mg), with a yield of 49.0%.

[0182]

[0183] Example 8: Synthesis of Fmoc-XS7 fragment

[0184] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase synthesis of fragments, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, and Fmoc-Tyr(tBu)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XS7 (526.0 mg), with a yield of 58.8%.

[0185]

[0186] Example 9: Synthesis of Fmoc-XS8 fragment

[0187] Starting with Fmoc-Gly-OH, and following general methods for solid-phase synthesis of fragments, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Arg(Pbf)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XS8 (267.0 mg), with a yield of 32.7%.

[0188]

[0189]

[0190] Example 10: Synthesis of Fmoc-XS9 fragment

[0191] Starting with Fmoc-Arg(Pbf)-OH, and following a general method for solid-phase synthesis of fragments, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Arg(Pbf)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XS9 (426.0 mg), with a yield of 31.7%.

[0192]

[0193] Example 11: Synthesis of fragment XA-Tag

[0194] Following the synthesis method of XS-Tag, Fmoc-Gln(Trt)-OH was used as the starting material and successively linked with Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Arg(Pbf)-OH to obtain a white solid XA-Tag (4.43 g), with a yield of 61.64%.

[0195]

[0196] Example 12: Synthesis of Fmoc-XA1 fragment

[0197] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase fragment synthesis, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH and Fmoc-Arg(Pbf)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XA1 (408.8 mg), with a yield of 46.7%.

[0198]

[0199]

[0200] Example 13: Synthesis of Fmoc-XA2 fragment

[0201] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase synthesis of fragments, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, and Fmoc-Thr(tBu)-OH were sequentially coupled, followed by resin cleavage to obtain a white solid Fmoc-XA2 (413.1 mg), with a yield of 46.6%.

[0202]

[0203] Example 14: Synthesis of fragment XB-Tag

[0204] Following the synthesis method of XS-Tag, starting with Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Arg(Pbf)-OH were coupled sequentially to obtain a white solid XB-Tag (5.12 g), with a yield of 63.4%.

[0205]

[0206]

[0207] Example 15: Synthesis of Fmoc-XB1 fragment

[0208] Starting with Fmoc-Arg(Pbf)-OH, and following general methods for solid-phase fragment synthesis, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, and Fmoc-Arg(Pbf)-OH were sequentially coupled, followed by resin cleavage to obtain the fragment Fmoc-XB1 (511.6 mg), with a yield of 50.0%.

[0209]

[0210]

[0211] Example 16: C n Synthesis of -SulA(88-106)(n=10,14,16)

[0212]

[0213] (1) Fragment coupling

[0214] Fragments Fmoc-XS1 (0.23 mmol, 1.5 eq), HATU (0.23 mmol, 1.5 eq), HOAt (0.23 mmol, 1.5 eq), and DIPEA (0.75 mmol, 5.0 eq) were weighed and dissolved in a 9:1 THF:DMF mixed solution (10 mL). Then fragment XS-Tag (0.15 mmol, 1.0 eq) was added. The mixture was stirred at room temperature for 30 min. The reaction was monitored by TLC until it ended. The reaction solution was concentrated, and an acetonitrile:pure water mixed solution precipitated out. The solution was filtered and dried to obtain the intermediate product.

[0215] (2) Removal of Fmoc protecting groups

[0216] The fragment to be deprotected from the Fmoc group obtained in the previous step was dissolved in 10 mL of THF. PIP (3.0 mmol, 20.0 eq) and DBU (1.3 mmol, 8.7 eq) were added under stirring at room temperature. The reaction was carried out for 10 min. The reaction was monitored by TLC until it ended. The reaction solution was concentrated and acetonitrile:pure water = 10:1 mixed solution was precipitated. 6 mol / L hydrochloric acid was added dropwise to adjust the pH to about 7. The solution was filtered and dried under vacuum to obtain the intermediate product.

[0217] (3) Residual fragment coupling and deprotection

[0218] XS2 is connected to the fragment obtained in the previous step according to (1) the fragment coupling method and (2) the Fmoc protection base removal method.

[0219] (4) Removal of carrier and side chain protecting groups

[0220] Finally, the fragment to be processed was dissolved in a mixed solution of TFA:H2O:TIS = 38:1:1 (10 mL), and reacted at room temperature for about 3 hours. The reaction solution was filtered through diatomaceous earth, and cold isopropyl ether was added to the filtrate to precipitate the product. After filtration and vacuum drying, a pale yellow crude peptide product C was obtained. 10 -SulA(88-106) (259.7 mg), crude product yield 68.5%. Pale yellow crude peptide C was obtained. 14 -SulA(88-106)(209.7mg), crude product yield 54.1%. Pale yellow crude peptide C was obtained. 16 -SulA(88-106)(202.9mg), crude product yield 51.8%.

[0221] Example 17: Synthesis of Cyclo-SulA(88-106)

[0222]

[0223] Using XS-Tag as the starting material, refer to C n The synthesis method of -SulA(88-106) (n=10,14,16) involves sequentially coupling with fragments Fmoc-XS1 and Fmoc-XS3 to obtain the fragment NH2-Thr(tBu)-His(Trt)-Glu(tBu)-Trp(Boc)-Leu-Arg(Pbf)-Arg(Pbf)-Ala-Gly-Leu-Asn(Trt)-Arg(Pbf)-Glu(tBu)-Arg(Pbf)-Ile-Leu-Leu-Leu-Gln(Trt)-O-Tag. Dissolve it in 20 mL of DCM, then add 2 mL of TFE and 0.2 mL of TFA sequentially. React at room temperature for 30 min. Monitor the reaction progress by TLC. A large amount of purple powdery solid was observed. Filter with diatomaceous earth, concentrate the filtrate, add cold isopropyl ether to precipitate, filter, and dry to obtain the fragment NH2-Thr(tBu)-His(Trt)-Glu(tBu)-Trp(Boc)-Leu-Arg(Pbf)-Arg(Pbf)-Ala-Gly-Leu-Asn(Trt)-Arg(Pbf)-Glu(tBu)-Arg(Pbf)-Ile-Leu-Leu-Leu-Gln(Trt)-OH.

[0224] Next, the product from the previous step (0.05 mmol, 1.0 eq) was dissolved in a THF:DMF = 9:1 mixed solution (76 mL). DMT-MM (0.23 mmol, 5.0 eq) and DIPEA (0.91 mmol, 20.0 eq) were added under stirring at 40 °C. After the reaction was completed as monitored by TLC, the reaction solution was concentrated, and an appropriate amount of an acetonitrile:pure water = 10:1 mixed solution was added to precipitate crystals. The crystals were filtered, washed, and dried under vacuum to obtain a cyclic peptide product containing a side-chain protecting group. Subsequent removal of the side-chain protecting group yielded a white crude peptide Cycol-SulA (88-106) (210.2 mg), with a crude product yield of 59.5%.

[0225] Example 18: Synthesis of SulA(88-106)-Ahx-SulA(88-106)

[0226]

[0227] Using fragment XS-Tag as the starting material, refer to C n The synthesis method of -SulA(88-106) (n=10,14,16) involved sequentially coupling with fragments Fmoc-XS1, Fmoc-XS4, Fmoc-XS5, Fmoc-XS1 and Fmoc-XS3 to obtain a white crude peptide SulA(88-106)-Ahx-SulA(88-106) (204.1 mg), with a crude product yield of 28.1%.

[0228] Example 19: Synthesis of TAT-SulA(88-106)

[0229]

[0230] Using fragment XS-Tag as the starting material, refer to C n The synthesis method of -SulA(88-106) (n=10,14,16) involved sequentially coupling with fragments Fmoc-XS1, Fmoc-XS3, Fmoc-XS6 and Fmoc-XS7 to obtain the white crude peptide TAT-SulA(88-106) (320.6 mg), with a crude product yield of 54.6%.

[0231] Example 20: Synthesis of R9-SulA(88-106)

[0232]

[0233] Using fragment XS-Tag as the starting material, refer to C nThe method for synthesizing -SulA(88-106) (n=10,14,16) involved sequentially coupling it with fragments Fmoc-XS1, Fmoc-XS3, Fmoc-XS8 and Fmoc-XS9 to obtain a white crude peptide R9-SulA(88-106) (319.9 mg), with a crude product yield of 54.0%.

[0234] Example 21: Synthesis of Cyclo-A3

[0235]

[0236] Starting with fragment XA-Tag, and following the synthesis method of Cyclo-SulA (88-106), fragments Fmoc-XA1 and Fmoc-XA2 were sequentially coupled, followed by decoupling, cyclization, and removal of side-chain protecting groups to obtain a white crude peptide Cyclo-A3 (147.4 mg), with a crude product yield of 39.2%.

[0237] Example 22: Synthesis of TAT-A3

[0238]

[0239] Using fragment XA-Tag as the starting material, refer to C n The method for synthesizing -SulA(88-106) (n=10,14,16) involved sequentially coupling it with fragments Fmoc-XA1, Fmoc-XA2, Fmoc-XS6 and Fmoc-XS7 to obtain a white crude peptide TAT-A3 (336.1 mg), with a crude product yield of 55.1%.

[0240] Example 23: Synthesis of R9-A3

[0241]

[0242] Using fragment XA-Tag as the starting material, refer to C n The method for synthesizing -SulA(88-106) (n=10,14,16) involved sequentially coupling it with fragments Fmoc-XA1, Fmoc-XA2, Fmoc-XS8 and Fmoc-XS9 to obtain a white crude peptide R9-A3 (324.8 mg), with a crude product yield of 52.8%.

[0243] Example 24: Synthesis of Cyclo-B1

[0244]

[0245] Starting with fragment XB-Tag, and following the synthesis method of Cyclo-SulA (88-106), fragments Fmoc-XB1 and Fmoc-XA2 were sequentially coupled, followed by decoupling, cyclization, and removal of side-chain protecting groups to obtain a white crude peptide Cyclo-B1 (119.4 mg), with a crude product yield of 30.7%.

[0246] Example 25: Synthesis of TAT-B1

[0247]

[0248] Using fragment XB-Tag as the starting material, refer to C n The synthesis method of -SulA(88-106) (n=10,14,16) was used to sequentially couple it with fragments Fmoc-XB1, Fmoc-XA2, Fmoc-XS6 and Fmoc-XS7 to obtain white crude peptide TAT-B1 (285.3 mg), with a crude product yield of 45.8%.

[0249] Example 26: Synthesis of R9-B1

[0250]

[0251] Using fragment XB-Tag as the starting material, refer to C n The synthesis method of -SulA(88-106) (n=10,14,16) involved sequentially coupling with fragments Fmoc-XB1, Fmoc-XA2, Fmoc-XS8 and Fmoc-XS9 to obtain a white crude peptide R9-B1 (267.6 mg), with a crude product yield of 42.6%.

[0252] Mass spectrometry analysis was performed on the heteropeptides prepared in the above examples, and the results are shown below:

[0253] C 10 -SulA(88-106):MALDI-TOF(m / z):calculated for C 115 H 194 N 36 O 28 [M+H] + =2529.498,found=2529.015.

[0254] C 14 -SulA(88-106):MALDI-TOF(m / z):calculated for C 119 H 202 N 36 O 28 [M+H] +=2585.560,found=2585.400。

[0255] C 16 -SulA(88-106):MALDI-TOF(m / z):calculated for C 121 H 206 N 36 O 28 [M+H] + =2614.211,found=2614.053。

[0256] Cycol-SulA(88-106):ESI-MS(m / z):calculated for C 105 H 174 N 36 O 26 [M+2H] 2+ =1178.68,found=1178.90。

[0257] SulA(88-106)-Ahx-SulA(88-106):MALDI-TOF(m / z):calculated forC 216 H 361 N 73 O 54 [M+H] + =4844.789,found=4844.004。

[0258] TAT-SulA(88-106):MALDI-TOF(m / z):calculated for C 169 H 292 N 68 O 40 [M+H] + =3916.302,found=3916.577。

[0259] R9-SulA(88-106):MALDI-TOF(m / z):calculated for C 165 H 293 N 75 O 39 [M+H] + =3951.336,found=3951.501。

[0260] C 10 -A3:ESI-MS(m / z):calculated for C 124 H218 N 42 O 24 [M+4H] 4+ =670.94,found=671.30。

[0261] C 14 -A3:ESI-MS(m / z):calculated for C 128 H 226 N 42 O 24 [M+4H] 4+ =684.95,found=685.35。

[0262] C 16 -A3:SI-MS(m / z):calculated for C 130 H 230 N 42 O 24 [M+4H] 4+ =691.96,found=692.40。

[0263] Cycol-A3:ESI-MS(m / z):calculated for C 114 H 198 N 42 O 22 [M+2H] 2+ =1255.29,found=1255.39。

[0264] A3-Ahx-A3:ESI-MS(m / z):calculated for C 234 H 409 N 85 O 46 [M+2H] 2+ =736.18,found=736.55。

[0265] TAT-A3:MALDI-TOF(m / z):calculated for C 178 H 316 N 74 O 36 [M+H] + =4067.525,found=4067.668。

[0266] R9-A3:MALDI-TOF(m / z):calculated for C 174 H 317 N 81 O35 [M+H] + =4102.559,found=4102.522。

[0267] C 10 -B1:ESI-MS(m / z):calculated for C 124 H 220 N 48 O 24 [M+3H] 3+ =922.92,found=923.35。

[0268] C 14 -B1:ESI-MS(m / z):calculated for C 128 H 228 N 48 O 24 [M+5H] 5+ =565.37,found=565.70。

[0269] C 16 -B1:ESI-MS(m / z):calculated for C 130 H 232 N 48 O 24 [M+4H] 4+ =950.95,found=951.40。

[0270] Cycol-B1:ESI-MS(m / z):calculated for C 114 H 200 N 48 O 22 [M+4H] 4+ =649.41,found=649.84。

[0271] B1-Ahx-B1:ESI-MS(m / z):calculated for C 234 H 413 N 97 O 46 [M+8H] 8+ =665.79,found=666.60。

[0272] TAT-B1:MALDI-TOF(m / z):calculated for C 178 H 318 N 80 O 36 [M+H]+ =4154.562,found=4154.405.

[0273] R9-B1:MALDI-TOF(m / z):calculated for C 174 H 319 N 87 O 35 [M+H] + =4189.597,found=4190.216.

[0274] Example 27: Determination of Minimum Inhibitory Concentration (MIC)

[0275] Initial activity screening was performed using MIC (micro-detection index) assay. The assay method was as follows:

[0276] (1) Add 190 μL of broth to the first column of the 96-well plate and 100 μL of broth to the second to 12th columns.

[0277] (2) Add 10 μL of the prepared heteropeptide solution with a concentration of 5120 μg / mL to the first column of the 96-well plate.

[0278] (3) Using the two-fold dilution method, after thoroughly mixing the broth-based heterozygous peptide mixture in column 1 of the 96-well plate, 100 μL was added to column 2, mixed thoroughly, and then added to column 3. This process was repeated until column 10. After mixing column 10, 100 μL was discarded. That is, the heterozygous peptide concentrations from column 1 to column 10 were 256, 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 μg / mL, respectively.

[0279] (4) Add 5 μL of the prepared bacterial solution to each of the 1st to 11th wells of the 96-well plate. Column 11 is the positive control, containing bacteria but no heterozygous peptides. Column 12 is the negative control, containing no bacterial solution and no heterozygous peptides.

[0280] (5) Place the 96-well plate in a 37℃ incubator and incubate for 18-24 hours.

[0281] (6) The concentration of the sample in the well before turbidity appears in the 96-well plate is the MIC value of the heteropeptide. If the MIC value cannot be read at the measured concentration, the concentration can be adjusted. For example, if the 10th well is still not turbid, it can be diluted twofold to determine the concentration where turbidity exists.

[0282] The MIC results for Gram-positive susceptible strains are shown in Table 1:

[0283] Table 1. MICs of heterozygous peptides against Gram-positive susceptible strains

[0284]

[0285] The MIC results for Gram-positive drug-resistant strains are shown in Table 2:

[0286] Table 2. MICs of heterozygous peptides against Gram-positive drug-resistant strains

[0287]

[0288] The MIC results for Gram-negative strains are shown in Table 3:

[0289] Table 3. MICs of heterozygous peptides against Gram-negative drug-resistant strains

[0290]

[0291] MIC assay results showed that TAT-SulA(88-106) and R9-SulA(88-106) had improved antibacterial activity compared to the parent peptide SulA(88-106), but their activity against Gram-negative bacteria was poor. TAT-A3 and R9-A3 had significantly improved activity against Gram-negative bacteria compared to the parent peptide A3. TAT-B1 and R9-B1 had significantly improved activity against both the tested Gram-positive drug-resistant bacteria and Gram-negative bacteria, and their antibacterial performance was the best in this series.

[0292] Example 28

[0293] Bacterial morphology was observed using an optical microscope. TAT-SulA(88-106), R9-SulA(88-106), R9-A3, and R9-B1, which exhibited good antibacterial activity, were selected for FtsZ targeting studies. Measurement methods:

[0294] (1) The MIC value of the heterozygous peptide against B. pumilus CMCC63202 was determined according to the MIC determination method;

[0295] (2) Read the MIC value, select the first turbid well, i.e. 1 / 2 × MIC concentration, and mix it well;

[0296] (3) Take 10 μL and add it to pure water to dilute it about 10 times. Take 10 μL and place it on a glass slide. Observe the morphology of the bacteria under an optical microscope.

[0297] (4) Take another 10 μL of the negative blank group well, dilute it, take 10 μL and place it on a glass slide, and observe the bacterial morphology under an optical microscope;

[0298] (5) Comparison of bacterial morphology between the experimental group and the blank control group.

[0299] Figure 1The morphology of *B. pumilus* CMCC63202 was observed under an optical microscope for the heterozygous peptides TAT-SulA(88-106), R9-SulA(88-106), TAT-A3, R9-A3, TAT-B1, and R9-B1. (A) Bacterial morphology under normal conditions; (B) Bacterial morphology in the presence of TAT-SulA(88-106); (C) Bacterial morphology in the presence of R9-SulA(88-106); (D) Bacterial morphology in the presence of A3; (E) Bacterial morphology in the presence of R9-A3; (F) Bacterial morphology in the presence of B1; (G) Bacterial morphology in the presence of R9-B1. Figure 1 As shown, compared with the control group, the bacterial morphology was significantly longer, affecting the normal division of bacteria, which preliminarily verified its targeting of FtsZ protein.

[0300] Example 29

[0301] FtsZ polymerization kinetics experiment, determination method:

[0302] (1) Dilute the 5120 μg / mL hybrid peptide stock solution to 640 and 320 μg / mL and set aside for later use;

[0303] (2) Turn on the fluorescence spectrophotometer and preheat it;

[0304] (3) Take 146.6 μL of polymerization buffer, 3.2 μL of sample, and 8.6 μL of FtsZ protein and add them to a microcuvette. Mix well and incubate at 37°C for 5-10 min. Then add 1.6 μL of GTP solution and measure quickly.

[0305] (4) Save and process the data.

[0306] Figure 2 The graph shows the polymerization kinetics results of the heterozygous peptides R9-A3, TAT-B1, and R9-B1 FtsZ. Figure 2 As shown, R9-A3, TAT-B1, and R9-B1 all reduced fluorescence intensity, indicating their inhibition of FtsZ polymerization. R9-A3 significantly reduced light scattering intensity, with its inhibitory strength being similar to that of the A3 group and stronger than the parent peptide SulA (88-106), suggesting that R9-A3 maintains the excellent FtsZ targeting of A3 and has a strong inhibitory effect on FtsZ protein polymerization. TAT-B1 caused only a small increase in light scattering intensity, similar to the B1 group. R9-B1 showed the lowest fluorescence intensity at a concentration of 12.8 μg / mL, indicating the strongest ability to inhibit FtsZ protein polymerization. Furthermore, at a concentration of 6.4 μg / mL, its FtsZ protein targeting was still stronger than that of SulA (88-106) (12.8 μg / mL).

[0307] Transmission electron microscopy observation of FtsZ protein morphology

[0308] Determination method: (1) Take 146.6 μL of polymerization buffer, 3.2 μL of sample, and 8.6 μL of FtsZ protein into a centrifuge tube, mix well, incubate at 37℃ for 20 min, then add 1.6 μL of GTP solution, mix well, and continue incubating at 37℃ for 30 min; (2) Add an appropriate amount of the mixed solution obtained in (1) to the copper mesh to completely wet the copper mesh, remove the copper mesh and let it air dry naturally; (3) Add an appropriate amount of 1% phosphotungstic acid to stain for 1 min; (4) Add purified water to wash 5-6 times and air dry naturally.

[0309] Figure 3 Figures show the results of FtsZ polymerization experiments observed under transmission electron microscopy for the heterozygous peptides R9-A3, TAT-B1, and R9-B1; (A) Polymerization state under normal conditions; (B) Bacterial morphology in the presence of A3; (C) Bacterial morphology in the presence of R9-A3; (D) Polymerization state in the presence of B1; (E) Polymerization state in the presence of TAT-B1; (F) Polymerization state in the presence of R9-B1. Figure 3 As shown, although the protein in the R9-A3 group could not effectively polymerize into protofilaments like the A3 group, the protofilaments in the R9-A3 group were significantly thinner than those in the control group, indicating that R9-A3 has a strong targeting ability for FtsZ and effectively inhibits FtsZ polymerization. Although the protein in the TAT-B1 treatment formed protofilaments, they were significantly thinner than those in the control group, and basically consistent with the B1 group. After R9-B1 treatment, the protofilaments were noticeably thinner and shorter than those in the previous three groups, further verifying that R9-B1 enhances the targeting ability of FtsZ protein.

[0310] Cell membrane depolarization experiment

[0311] Determination method: (1) Resuscitate Staphylococcus aureus ATCC25923 and Acinetobacter baumannii ATCC19606, passage them, and set them aside for use;

[0312] (2) Take logarithmic growth cycle bacteria and place them in a mixed solution of 5mM glucose: 5mM HEPES (pH=7.2) = 1:1. Centrifuge at 3500 r / min for 5 min, discard the supernatant, wash twice with a mixed solution of 5mM glucose: 5mM HEPES (pH=7.2) = 1:1, and then resuspend in a mixed solution of 5mM glucose: 5mM HEPES (pH=7.2): 100mM KCl = 1:1:1 for later use;

[0313] (3) Staphylococcus aureus ATCC25923: Take a black 96-well plate, add 150 μL of the bacterial solution obtained in (2) and 50 μL of 8 μM diSC35 to the well, mix well, and incubate at 37℃ for 30 min. Acinetobacter baumannii ATCC19606: Take a black 96-well plate, add 150 μL of the bacterial solution obtained in (2), 50 μL of 8 μM diSC35 and 50 μL of 200 μM EDTA to the well in sequence, mix well, and incubate at 37℃ for 60 min;

[0314] (4) Place the plate in the microplate reader and set the measurement conditions: excitation wavelength 622nm, slit width 10nm; emission wavelength 670nm, slit width 5nm. Measure every 2 minutes for a total of 8 minutes.

[0315] (5) After measurement, add 10 μL of the heterozygous peptide to be tested or the control drug, and add 10 μL of DMSO for the blank control. Set up three parallel experiments. Mix well and measure under the same conditions for 12 min using an ELISA reader.

[0316] (6) Save the data and plot it.

[0317] Figure 4 Figure 1 shows the results of cell membrane depolarization experiments of the hybrid peptides TAT-A3 and R9-A3. (A): ATCC25923, (B): ATCC19606. Figure 5 Figure 1 shows the cell membrane depolarization results of the hybrid peptides TAT-B1 and R9-B1. (A): ATCC25923, (B): ATCC19606. Figure 4 and 5 As shown, all tested heterozygous peptides enhanced fluorescence intensity in a concentration-dependent manner. For Staphylococcus aureus ATCC25923, at a concentration of 2×MIC, R9-A3 exhibited stronger cell membrane depolarization than TAT-A3, but weaker than A3. At a concentration of 1×MIC, R9-A3 showed stronger cell membrane depolarization than both TAT-A3 and A3. At a concentration of 2×MIC, R9-B1 increased fluorescence intensity the most, followed by TAT-B1, both higher than B1. At a concentration of 1×MIC, R9-B1 increased fluorescence intensity higher than TAT-B1, and also higher than the fluorescence intensity of group B1 at a concentration of 2×MIC. For Acinetobacter baumannii ATCC19606, at both measured concentrations, the intensity of cell membrane depolarization was R9-A3 > TAT-A3 > A3, but all were lower than 1% Triton X-100. Both TAT-B1 and R9-B1 increased fluorescence intensity; at 2×MIC concentration, R9-B1 increased fluorescence intensity the most, followed by TAT-B1. At 1×MIC concentration, the intensity remained R9-B1 > TAT-B1 > B1.

[0318] Bacterial morphology observed by scanning electron microscopy

[0319] Determination method: (1) Staphylococcus aureus ATCC25923 and Acinetobacter baumannii ATCC19606 were revived, passaged into liquid culture medium, and cultured for 20 h;

[0320] (2) Dilute the liquid culture medium with broth to OD. 600 =0.05, incubated at 37℃ until the logarithmic growth phase, then further diluted with broth to OD. 600 =0.05, to be used;

[0321] (3) Take 1 mL of bacterial culture and put it into a 1.5 mL centrifuge tube. Add the corresponding heterozygous peptide stock solution to make the final concentration the MIC concentration. Add the corresponding amount of DMSO to the blank control group and incubate at 37℃ for 24 h.

[0322] (4) Centrifuge at 5000 r / min for 5 min, discard the supernatant, and wash twice with PBS buffer solution for 15 min each time;

[0323] (5) Add 1 mL of 2.5% glutaraldehyde solution, mix well, and fix at 4℃ for 4 h;

[0324] (6) Centrifuge at 5000 r / min for 5 min, discard the supernatant, add PBS buffer solution and wash twice, 15 min each time;

[0325] (7) Elute with a gradient of ethanol (30%, 50%, 70%, 90%, 100%) for 10 min per gradient, then centrifuge at 5000 r / min for 3 min;

[0326] (8) Add an appropriate amount of acetone to convert ethanol 2-3 times, each time for 20-30 minutes, centrifuge at 5000 r / min for 3 minutes, dry it, and wait for sample loading.

[0327] Figure 6 The image shows the results of scanning electron microscopy (SEM) observations of bacterial morphology of the heterozygous peptides R9-A3 and R9-B1. Figure 6 As shown, the effects on Staphylococcus aureus ATCC25923 were as follows: the control group had a smooth surface and remained intact spherical. After R9-A3 treatment, a large number of bacteria ruptured, causing leakage of contents and severely damaging cell morphology, which was more severe than after A3 treatment. After R9-B1 treatment, a large number of bacteria ruptured, causing leakage of contents and severely damaging cell morphology. The effects on Acinetobacter baumannii ATCC19606 were as follows: the control group remained intact coccobacillus-like. After R9-A3 treatment, some cells ruptured, causing leakage of contents, and some began to shrink, resulting in morphological damage, which was more pronounced than after A3 treatment. After R9-B1 treatment, some bacterial cells ruptured, and some began to shrink, resulting in morphological damage.

[0328] Induction of drug resistance experiment

[0329] Determination method: (1) Resuscitate Staphylococcus aureus ATCC25923 and Acinetobacter baumannii ATCC19606, passage them, and set them aside for use;

[0330] (2) The MIC values ​​of R9-A3 and the control drug were determined according to the MIC determination method;

[0331] (3) Mix the 1 / 2×MIC well, take 10μL onto an agar plate, spread it evenly with a spreader, and incubate at 37℃ for 18-24h.

[0332] (4) Take out the colonies with good growth status, and continue to measure the MIC value according to the MIC determination method. The subsequent operation is the same as (3), and repeat for fifteen generations.

[0333] (5) Plot the MIC values ​​of the fifteenth generation.

[0334] Figure 7 Figure 1 shows the results of drug resistance induction experiments for the heterozygous peptides R9-A3 and R9-B1. (A): *S. aureus* ATCC25923; (B): *A. baumnnii* ATCC19606. Figure 7 As shown, R9-A3 is unlikely to induce resistance in *S. aureus* ATCC25923 and *A. baumnnii* ATCC19606, but its MIC is unstable. For *S. aureus* ATCC25923, R9-B1 exhibits excellent resistance, with its MIC value fluctuating within a 2-fold range over fifteen generations. In contrast, the control drug vancomycin shows a resistance development trend—vancomycin maintains a stable MIC value in the first eleven generations, but increases to four times the initial value in the twelfth generation, and then maintains this level, indicating the beginning of resistance formation. Ciprofloxacin, on the other hand, shows a 4-fold increase in MIC value in the third generation, indicating resistance development; the MIC value increases to eight times the original value in the seventh generation; and with prolonged time, its MIC value increases rapidly, reaching 64 times the original value in the thirteenth generation, exhibiting a typical exponential resistance development trend. The above data indicate that R9-B1 is significantly superior to the control drugs ciprofloxacin and vancomycin in delaying the development of bacterial resistance, successfully maintaining the good resistance advantage of peptide B1. For Acinetobacter baumannii ATCC19606, the MIC value of R9-B1 changed within a 2-fold range, indicating good resistance. The MIC value of the control drug ciprofloxacin began to increase in the third generation, indicating resistance development, and the 13th generation showed a 16-fold increase. For polymyxin B, the MIC values ​​changed within a 2-fold range for the first five generations, but increased rapidly in the sixth generation, becoming the 16-fold increase. In other words, R9-B1 is superior to the control drugs ciprofloxacin and polymyxin B, maintaining the good resistance of B1.

[0335] Acute toxicity test of the large wax moth

[0336] Determination method: (1) Dissolve R9-B1 in purified water and prepare concentrations of 50, 100, 200, 500, 1000 and 2000 mg / kg for later use;

[0337] (2) 10 μL of the heterozygous peptide aqueous solution was injected into the left posterior abdominal leg of the larvae of the giant wax moth using an insulin needle. Ten larvae were used for each concentration. The control group was injected with an equal dose of PBS buffer solution. The trauma group was injected only through the larval epidermis to simulate injection trauma.

[0338] (3) Observe for 5 consecutive days and record the larval mortality rate and abnormal behavior daily;

[0339] (4) Calculate LD 50 , draw a diagram.

[0340] Figure 8 The diagram shows the results of an acute toxicity test on the large wax moth; (A) growth status after 5 days of R9-B1 treatment; (B) growth status after 5 days of B1 treatment; (C) survival rate after 5 days of R9-B1 treatment; (D) survival rate after 5 days of B1 treatment. Figure 8 As shown, after 5 consecutive days of observation following injection, the R9-B1 group remained normal and exhibited no abnormal behavior at all tested concentrations within 5 days. Generally, an LD50 > 2000 mg / kg indicates low toxicity, meaning R9-B1 has high safety. This experiment further demonstrates the in vivo safety of R9-B1; therefore, it was selected for preliminary in vivo efficacy determination in this study.

[0341] Preliminary in vivo efficacy test, determination method:

[0342] (1) Select 2-week-old mice and feed them for 3-5 days before the experiment to allow them to adapt to their living environment;

[0343] (2) Dissolve R9-B1 and the control drug in physiological saline and DMSO (DMSO ratio less than 10%) to prepare the required concentration and set aside for use;

[0344] (3) Resuscitate Staphylococcus aureus ATCC25923, passage it, and prepare a solution with physiological saline at a concentration of 6×10⁻⁶. 8 CFU / mL bacterial suspension, ready for use;

[0345] (4) Mice were stunned with ether, and the hair on the back of the mice was removed with a cotton swab dipped in depilatory cream. A wound of about 10 mm was made with surgical scissors, 50 μL of bacterial solution was added, and the wound was bandaged. The infection was allowed to continue for 2 hours. After successful infection, 50 μL of heterozygous peptide and control drug solution were added to the wound, while the control group was given an equal dose of physiological saline, and the wound was bandaged.

[0346] (5) Observe the size of the mouse wound and the mouse's condition for 8 consecutive days, and take photos and record them at regular intervals every day;

[0347] (6) After taking photos on the 8th day, blood was taken from the mouse eyeballs, and the procedure was the same as the hemolysis test. The mice were then euthanized and dissected.

[0348] (7) Dilute the blood with physiological saline, take 20 μL and spread it evenly in a culture dish; select representative wound skin and fix it in paraformaldehyde, then stain and section it; grind the remaining wound skin and dilute it, take 20 μL and spread it evenly in a culture dish; grind the internal organs and dilute it, take 20 μL and spread it evenly in a culture dish; incubate all plates at 37℃ for 18-24 h.

[0349] (8) Count the colonies on the plate and plot them.

[0350] Figure 9 Preliminary in vivo pharmacodynamic experiments of the heterozygous peptide R9-B1; (A) wound healing within 8 days; (B) blood colony counts after treatment under different conditions; (C) skin colony counts after treatment under different conditions; (D) heatmap of colony counts in the heart, liver, spleen, lungs, and kidneys. Figure 9 As shown, in the PBS group, white pus appeared on the wound starting from day 1, and after 5 days, scabs slowly formed on day 6. Although the wound area decreased, it still could not heal completely. In the linezolid group, there was less white pus, and the wound area was significantly smaller than in the PBS group from day 3, with scabs forming on day 4. The wound healing and wound area in the 2 μM / kg R9-B1 group were basically the same as in the 5 μM / kg linezolid group, but significantly improved compared to the PBS group. In the 5 μM / kg R9-B1 group, scabs began to form on day 1, and the wound area was reduced by half. Its effect was comparable to that of the linezolid combined with the low-concentration R9-B1 group on day 3, demonstrating significant therapeutic efficacy. Figures B, C, and D show that the bacterial count in blood, skin, and internal organs was 5 μM / kg LIN > 2 μM / kg R9-B1 > 5 μM / kg R9-B1, exhibiting a concentration-dependent effect. This indicates that R9-B1 has good therapeutic effects on blood, skin, and internal organs, and is superior to the control drug linezolid.

[0351] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hybrid peptide targeting bacterial FtsZ and cell membrane, characterized in that, The heteropeptide has an amino acid sequence as shown in any of SEQ ID NO. 1-21.

2. A conjugate, characterized in that, The conjugate comprises the heteropeptide of claim 1 and the modified portion.

3. The conjugate as described in claim 2, characterized in that, Optionally, the modified portion is connected to the N-terminus or C-terminus of the heteropeptide via a linker; preferably, the modified portion includes a targeting portion, a fluorescent dye, and a protein tag; preferably, the targeting portion is a ligand, a receptor, or an antibody; preferably, the fluorescent dye is FITC; preferably, the protein tag is His, Flag, GST, MBP, HA, Myc, GFP, or biotin.

4. A nucleic acid encoding the heteropeptide of claim 1 or the conjugate of claim 2.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the heteropeptide of claim 1 or the conjugate of claim 2.

6. The method for preparing the heteropeptide as described in claim 1, characterized in that, Using SulA(88-106), A-3, and B-1 as template peptides, four modification methods were selected: fatty acid modification, cyclization, dimerization, and linking to membrane-penetrating peptides, to obtain hybrid peptides.

7. The use of the heteropeptide as described in claim 1 in any one or more of the following: (1) As a broad-spectrum antibacterial drug or composition; (2) As a rapid bactericide; (3) As a pharmaceutically acceptable carrier or additive.

8. The application as described in claim 7, characterized in that, The bacteria include Gram-positive and Gram-negative bacteria; preferably, the Gram-positive bacteria include one or more of Bacillus pumilus, Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, and Enterococcus faecium; and / or, the Gram-negative bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, or Klebsiella pneumoniae; or, the drug is an oral or topical drug; preferably, the oral drug is an oral preparation or an injection, and the topical drug includes patches and ointments.

9. A targeting peptide that targets FtsZ protein and cell membrane SulA (88-106), characterized in that, It contains any of the amino acid sequences shown in SEQ ID NO. 1-21.

10. A bactericide, characterized in that, Includes the heteropeptide of claim 1 or the conjugate of claim 2.

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