Cyclic antibacterial peptide derivative and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to design, preparation and application of an antibacterial peptide derivative. According to the invention, P-07 antibacterial peptide is taken as a structural template, and a series of cyclopeptide derivatives with symmetrical tail ends and containing beta-corner structures are designed and prepared. The cyclic peptide derivative disclosed by the invention has relatively high antibacterial activity and safety, and has a good application prospect in the antibacterial aspect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a cyclic antimicrobial peptide derivative and its application in antibacterial applications. Background Technology

[0002] Currently, the spread of multidrug-resistant bacteria caused by antibiotic overuse has become a global public health crisis, urgently requiring the development of novel antimicrobial drugs. Unlike traditional antibiotics with specific molecular targets, antimicrobial peptides primarily exert their bactericidal effect by physically disrupting bacterial membranes. This mechanism of action gives them broad-spectrum anti-drug-resistant properties and makes them less likely to induce bacterial resistance, demonstrating enormous application potential. However, antimicrobial peptides are easily degraded by proteases in the physiological environment, leading to rapid inactivation during circulation, which limits their clinical application.

[0003] P-07 (KIKIKPWWWPKIKIK-NH2) is a line with the central β -A core of the turn sequence (PWWWP) flanked by symmetrically arranged alternating cationic / hydrophobic residue extension fragments (KIKIK). β - Corner-turn antimicrobial peptide. This antimicrobial peptide exerts broad-spectrum antimicrobial activity through a membrane disruption mechanism: β - The continuously distributed tryptophan residues (WWW) in the corner region can strongly anchor to the bacterial membrane surface through hydrophobic and hydrogen bonding interactions with bacterial membrane phospholipids via their indole ring side chains. Meanwhile, the terminal lysine residues, due to their positive charge, electrostatically attract negatively charged membrane components, while isoleucine synergistically promotes peptide adsorption and insertion onto the membrane through hydrophobic interactions. P-07 not only exhibits strong anti-drug-resistant bacterial activity but also shows low hemolytic and cytotoxic properties. Furthermore, the combination of P-07 and levofloxacin effectively inhibits bacteremia infections caused by drug-resistant Escherichia coli, demonstrating high clinical application potential. Currently, the anti-enzymatic stability of P-07 needs further improvement. Summary of the Invention

[0004] To improve the resistance to enzymatic hydrolysis of P-07, this invention employs a cyclization and D-amino acid substitution strategy. β The antimicrobial peptide P-07 with a turn-off underwent structural optimization. Specifically, the cyclization strategy and the D-amino acid substitution strategy can interfere with protease recognition and degradation by shielding protease cleavage sites and altering the chiral environment of key amino acids, thereby enhancing the antimicrobial peptide's resistance to enzymatic degradation.

[0005] This invention provides a class of P-07-derived antimicrobial peptides having an amino acid sequence as shown in any one of general formulas (1), (2), or (3):

[0006] According to an embodiment of the present invention, ZZ is selected from...β - Corner sequence WWW or PWWWP; According to embodiments of the present invention, each X1 may be the same or different, and is independently selected from basic amino acids or basic amino acid derivatives, for example, it may be selected from lysine (Lys, K). According to embodiments of the present invention, each X2 may be the same or different, and is independently selected from hydrophobic amino acids or hydrophobic amino acid derivatives, for example, it may be selected from isoleucine (Ile, I). According to embodiments of the present invention, each Y may be the same or different and is independently selected from amino acids or amino acid derivatives that can be used to form covalent crosslinks between side chains, such as cysteine ​​(Cys, C), glutamic acid (Glu, E), and lysine (Lys, K). The covalent crosslinks between side chains are selected from disulfide bonds or amide bonds, wherein the side chains of two cysteines form disulfide bonds, and the side chain of one glutamic acid forms an amide bond with the side chain of one lysine.

[0007] According to an embodiment of the present invention, the amino acid or amino acid derivative is L-type or D-type.

[0008] According to an embodiment of the present invention, the N-terminal group of the antimicrobial peptide is a free amino group or is acetylated; for example, the N-terminal group is an acetamino group.

[0009] According to an embodiment of the present invention, the C-terminal group of the antimicrobial peptide is a carboxyl group or is amidated; for example, the C-terminal group is amidated to become an amide group.

[0010] According to an embodiment of the present invention, the antimicrobial peptide is derived from P-07 and obtained through D-amino acid substitution optimization and cyclization treatment.

[0011] According to a preferred embodiment of the present invention, the antimicrobial peptide has the amino acid sequence shown in Table 1: Table 1. Antimicrobial peptide sequences and molecular weights according to preferred embodiments of the present invention.

[0012] Note: a D-type amino acids are represented by lowercase letters; b Circulation sites are indicated by an asterisk (*).

[0013] According to embodiments of the present invention, the antimicrobial peptide further comprises a pharmaceutically acceptable salt selected from one or more of the following: acetate, acrylate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, bisulfite, tartrate, bromide, butyrate, butyn-1,4-dicarboxylate, camphorate, camphorsulfonate, hexanoate, octanoate, chlorobenzoate, chloride, citrate, cyclopentanepropionate, decanoate, diglucuronate, dihydrogen phosphate, dinitrobenzoate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucohepate, glycerophosphate, glycolate, hemisulfate, heptasulfate, 1,6-dicarboxylate, hydroxybenzoate, γ-hydroxy Butyrate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isobutyrate, lactate, maleate, malonate, methanesulfonate, mandelate, metaphosphate, methoxybenzoate, methyl benzoate, monohydrogen phosphate, 1-naphthalenesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmitate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, pyrosulfonate, pyrophosphate, propynate, phthalate, phenylacetate, phenylbutyrate, propanesulfonate, salicylate, succinate, sulfate, sulfite, octanoate, sebate, sulfonate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and xylenesulfonate.

[0014] According to an embodiment of the present invention, the antimicrobial peptide is synthesized by solid-phase synthesis.

[0015] According to an embodiment of the present invention, the purity of the antimicrobial peptide is >95%.

[0016] According to an embodiment of the present invention, the antimicrobial peptide has antimicrobial activity against Gram-positive and / or Gram-negative bacteria. The Gram-positive and / or Gram-negative bacteria are selected from at least one of Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, or multidrug-resistant strains thereof.

[0017] According to an embodiment of the present invention, the antimicrobial peptide has low hemolytic activity and high antimicrobial selectivity.

[0018] According to an embodiment of the present invention, the antimicrobial peptide has low cytotoxicity.

[0019] According to an embodiment of the present invention, the antimicrobial peptide has high stability, such as high salt tolerance, high enzyme stability, and high plasma stability.

[0020] According to an embodiment of the present invention, the antimicrobial peptide has high bactericidal ability.

[0021] According to an embodiment of the present invention, the antimicrobial peptide has low resistance induction potential.

[0022] According to an embodiment of the present invention, the antimicrobial peptide interacts with LPS on the bacterial membrane surface, disrupting the stability of the outer membrane and the cytoplasmic membrane, thereby achieving its bactericidal effect.

[0023] According to an embodiment of the present invention, the antimicrobial peptide has in vivo safety and high in vivo antimicrobial activity.

[0024] The present invention also provides an antimicrobial drug comprising a therapeutically effective amount of the said antimicrobial peptide.

[0025] According to an embodiment of the present invention, the antibacterial drug is a topical preparation, an oral preparation, an injectable preparation, or an inhaled preparation.

[0026] According to embodiments of the present invention, the oral preparation is selected from tablets, capsules, granules, powders, pills, oral liquids, syrups, or drops; the injectable preparation is selected from injection solutions, powders for injection, or concentrated solutions; the external preparation is selected from ointments, creams, gels, patches, sprays, or lotions; and the inhaled preparation is selected from inhaled powders, aerosols, or sprays.

[0027] According to an embodiment of the present invention, the antibacterial drug further includes at least one pharmaceutically acceptable excipient.

[0028] According to embodiments of the present invention, the excipients include buffers, preservatives, stabilizers, binders, lubricants, chelating agents, dispersants, disintegrants, flavoring agents, diluents, surfactants, sweeteners, and / or colorants.

[0029] The present invention also provides the use of the antimicrobial peptide in the preparation of antimicrobial drugs.

[0030] According to an embodiment of the present invention, the antimicrobial agent is used to prevent or treat infections caused by pathogens (e.g., skin wound infections). According to an embodiment of the present invention, the pathogens are selected from Gram-positive or Gram-negative bacteria, such as at least one selected from Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, or multidrug-resistant strains thereof.

[0031] The present invention also provides a composition in which the active ingredients comprise a therapeutically effective amount of the said antimicrobial peptide and an antibacterial antibiotic.

[0032] According to embodiments of the present invention, the composition is used to prevent or treat infections caused by pathogens (e.g., skin infections, urinary tract infections, or bloodstream infections).

[0033] According to an embodiment of the present invention, the antimicrobial peptide is preferably antimicrobial peptide PT-17.

[0034] According to embodiments of the present invention, the antibiotic is selected from at least one of polypeptide antibiotics, penicillin antibiotics, quinolone antibiotics, cephalosporin antibiotics, and rifamycin antibiotics. For example, the antibiotic is selected from at least one of polymyxin B, amoxicillin, levofloxacin, cefoperazone, and rifampin.

[0035] According to an embodiment of the present invention, the pathogen is selected from Gram-positive or Gram-negative bacteria, such as at least one of Staphylococcus aureus, Escherichia coli, or their multidrug-resistant strains.

[0036] Beneficial effects The P-07 cyclized antimicrobial peptide of this invention is constructed through disulfide / lactam cyclization and D-amino acid substitution optimization, exhibiting stability. β The angular conformation and enhanced stability exhibit excellent broad-spectrum activity against drug-resistant bacteria and high safety. For example, it shows strong antibacterial activity against multidrug-resistant strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, while also possessing low hemolytic properties and good in vivo safety. As an antibacterial agent with high bactericidal efficiency, the antimicrobial peptide PT-17 exerts its bactericidal effect by binding to bacterial lipopolysaccharides, disrupting membrane integrity, and inducing membrane depolarization. In a systemic infection model of drug-resistant Escherichia coli, it showed significantly better therapeutic effects than the parent peptide P-07 and polymyxin B, and no obvious toxicity was observed. Attached Figure Description

[0037] Figure 1 shows the high performance liquid chromatograms of antimicrobial peptides PT-01~PT-17; Figure 2 shows the mass spectra of antimicrobial peptides PT-01~PT-17; Figure 3 (A) Hemolytic activity of antimicrobial peptides PT-01~PT-17 and polymyxin B on human erythrocytes; (B) Cytotoxicity of antimicrobial peptides PT-01~PT-17 on HEK-293T and LO2 cells; Figure 4(A) The minimum inhibitory concentrations (MICs) of antimicrobial peptides P-07, PT-08, and PT-17 against multidrug-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Escherichia coli (MEC) in the presence of salt ions. The concentrations of NaCl, KCl, MgCl2, and FeCl3 used were 150 mM, 4.5 mM, 1 mM, and 4 μM, respectively. (B) The MICs of antimicrobial peptides P-07, PT-08, and PT-17 against MRS and MEC after incubation with 50% plasma for 1, 6, 12, and 24 h, respectively. (C) The residual rates of antimicrobial peptides P-07, PT-08, and PT-17 after incubation with 0.2 mg / mL chymotrypsin and 0.2 mg / mL trypsin at 37°C for 0.5, 1, 2, 3, and 6 h, respectively. Figure 5 The study aimed to investigate the bactericidal kinetics of P-07 and PT-17 at twice the minimum inhibitory concentration (MIC) against multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli. It also aimed to investigate the induction of drug resistance in Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 in the presence of sub-inhibitory concentrations of peptides and antibiotics. Figure 6 The diagrams show the synergistic antibacterial activity of (A) PT-17 in combination with polymyxin B and amoxicillin against Staphylococcus aureus ATCC 25923; (B) PT-17 in combination with polymyxin B and amoxicillin against Escherichia coli ATCC 25922; (C) PT-17 in combination with polymyxin B and antibiotics against multidrug-resistant Staphylococcus aureus; and (D) PT-17 in combination with polymyxin B and antibiotics against multidrug-resistant Escherichia coli. Figure 7 (A) Fluorescence confocal microscopy image of multidrug-resistant Staphylococcus aureus treated with 2× minimum inhibitory concentrations of P-07 and PT-17 for 2 h (scale bar: 40 μm); (B) Fluorescence confocal microscopy image of multidrug-resistant Escherichia coli treated with 2× minimum inhibitory concentrations of P-07 and PT-17 for 2 h (scale bar: 40 μm); (C) Transmission electron microscopy image of multidrug-resistant Staphylococcus aureus treated with 2× minimum inhibitory concentrations of P-07 and PT-17 for 2 h (scale bar: 500 nm); (D) Transmission electron microscopy image of multidrug-resistant Escherichia coli treated with 2× minimum inhibitory concentrations of P-07 and PT-17 for 2 h (scale bar: 500 nm). Figure 8The objectives are: (A) the effect of LPS on the activity of 1× minimum inhibitory concentration (MIC) of P-07, PT-17 and polymyxin B against multidrug-resistant Escherichia coli; (B) the outer membrane permeation effect of 4×, 2× and 1× MIC of P-07, PT-17 and polymyxin B on multidrug-resistant Escherichia coli; (C) the cell membrane depolarization effect of P-07, PT-17 and polymyxin B on multidrug-resistant Escherichia coli; and (D) the cell membrane depolarization effect of P-07, PT-17 and polymyxin B on multidrug-resistant Staphylococcus aureus. Figure 9 (A) Survival curves of mice after a single intravenous injection of different doses of P-07 (A), PT-17 (B), and polymyxin B (C); (D) Median lethal doses of P-07, PT-17, and polymyxin B. Figure 10 (A) Body weight change curves of mice in different groups during the 5-day intravenous injection period; (B) Major organ indices of mice after 5 days of intravenous injection. Organ index = organ weight (g) / body weight (g) × 100%; (C) Hematological parameters of mouse blood samples collected after 5 days of intravenous injection; (IJ) Biochemical parameters of mouse blood samples collected after 5 days of intravenous injection; Figure 11 (A) Schematic diagram of the treatment process; (B) Curve of mouse body weight change during 3 days of intravenous injection; (C) Bacterial load in major organs (liver, spleen, lung, kidney) of mice collected on day 4 post-infection; (D) Pathological sections of major organs (heart, liver, spleen, lung, kidney) of mice collected on day 4 post-infection after H&E staining (scale bar: 200 μm). Detailed Implementation

[0038] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0039] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0040] Example 1: Synthesis, identification, and purification of antimicrobial peptides: 1. Solid-phase synthesis of linear peptides: All linear peptides were synthesized using a Liberty Blue automated microwave peptide synthesizer (CEM, USA). Rink-Amide AM resin was used for solid-phase synthesis of C-terminal amidated peptides. Piperidine / DMF (20%, v / v) solution was used as the deprotection agent. Fmoc-protected amino acids were coupled one by one to the resin using DIC / Oxyma as the coupling agent.

[0041] 2. Disulfide bond cyclization: The peptide resin (1 equivalent) was transferred to a peptide synthesis vessel equipped with a filter. A solution of I2 (8 equivalents) in DMF (10 mL) was added to the vessel, and the mixture was gently stirred at 25°C for 6 hours. After the reaction, the resin was washed with DMF (3 × 4 mL) and DCM (3 × 4 mL). Finally, the N-terminal Fmoc groups were removed using a piperidine / DMF (20%, v / v) solution, followed by washing with DMF (3 × 4 mL) and DCM (3 × 4 mL).

[0042] 3. Lactam bond cyclization: The allyl oxycarbonyl (Alloc) and O-allyl (OAll) protecting groups on lysine and glutamic acid residues were removed, respectively. The peptide resin (1 equivalent) was suspended in a solution of triphenylsilanol (20 equivalents) in DCM (5 mL), stirred under N2 for 15 min, and then washed with DMF (3 × 4 mL) and DCM (3 × 4 mL). The peptide resin (1 equivalent) was then suspended in a solution of Pd(PPh3)4 (0.15 equivalents) in dry DMF (5 mL), stirred under N2 in the dark for 15 min, and then washed with DMF (3 × 4 mL) and DCM (3 × 4 mL). The Alloc deprotection procedure was repeated three times. Free amines and carboxylic acids with side chains were coupled using PyAOP (3 equivalents), HOAt (3 equivalents), and DIEA (6 equivalents) dissolved in DMF / DCM (1:1, v / v). The reaction system was gently stirred under N2 for 12 h. After the reaction, the resin was washed with DMF (3×4 mL) and DCM (3×4 mL). Finally, the N-terminal Fmoc group was removed with piperidine / DMF (20%, v / v) solution, followed by washing with DMF (3×4 mL) and DCM (3×4 mL).

[0043] 4. Pyrolysis and freeze-drying: The peptides were lysed using a TFA / TIS / H2O lysis buffer (95:2.5:2.5, v / v / v). The lysis reaction was carried out at 0°C for 0.5 h, followed by a further 2.5 h at 25°C. The crude peptides were purified by precipitation with pre-cooled diethyl ether (0°C), followed by filtration, redissolving in acetonitrile / water solution (1:1, v / v), and lyophilized. The HPLC methods for peptide purification are shown in Table 2-1. Peptide purity was determined by analytical HPLC (LC-20AP, Shimadzu, Japan) using a C18 column (Kromasil, Sweden) with a gradient elution program at a flow rate of 1.0 mL / min. Mobile phase A consisted of water containing 0.1% (v / v) TFA, and mobile phase B consisted of acetonitrile / water (70:30, v / v) containing 0.1% (v / v) TFA. The purity of each peptide was confirmed to be >95% based on peak area normalization. The high-performance liquid chromatograms of antimicrobial peptides PT-01~PT-17 are shown below. Figures 1-1 to 1-17 As shown, the mass spectra of antimicrobial peptides PT-01~PT-17 are as follows: Figures 2-1 to 2-17 As shown in Table 2-2, the high-performance liquid chromatography (HPLC) analysis results of antimicrobial peptides PT-01 to PT-17 are shown in Table 2-2.

[0044] Table 2-1 High-performance liquid chromatography (HPLC) procedures for the purification of peptide compounds

[0045] Table 2-2 High-performance liquid chromatography analysis results of antimicrobial peptides

[0046] Freeze-drying: Collect the crude peptide solution and transfer it to a centrifuge tube. Freeze-dry the solution rapidly under liquid nitrogen conditions to obtain crude peptide solid powder.

[0047] Example 2: Evaluation of the in vitro antimicrobial activity of antimicrobial peptides: The minimum inhibitory concentration (MIC) of P-07 and its derivative peptides in multidrug-resistant strains of Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii was determined using the dilution method.

[0048] The bacterial strain was incubated overnight at 37°C with shaking at 150 rpm. The bacterial suspension was then transferred to fresh medium and allowed to grow until logarithmic growth was achieved. The MIC of the peptide was determined using the standard broth dilution method: the bacterial suspension was diluted to 1 × 10⁻⁶. 6CFU / mL was prepared for use. The peptide solution was serially diluted 2-fold with culture medium to 1-256 μM. 50 μL of bacterial suspension was mixed with an equal volume of peptide solution and added to a 96-well plate. The plate was incubated at 37°C with shaking for 180 r / min for 18 h. After incubation, bacterial growth in the 96-well plate was observed. The minimum peptide concentration corresponding to the well with clear liquid was the peptide's MIC. The experimental results are shown in Table 3. The cyclic antimicrobial peptide PT-17, optimized with D-amino acid substitution, maintained strong antimicrobial activity against both Gram-positive and Gram-negative bacteria.

[0049] Table 3 MIC values ​​of P-07 and its derivatives

[0050] Example 3 Hemolytic Experiment: The hemolytic activity of antimicrobial peptides was assessed using human erythrocytes. Fresh erythrocytes were washed three times with PBS and resuspended in a cell suspension (8%). v / v The uniformly dispersed cell suspension was rapidly added to sterile 96-well clear plates, with five replicates per concentration, 50 μL per well. Then, 50 μL of peptide solution was added to each well. Untreated erythrocytes served as a negative control, and Triton-X100-treated erythrocytes served as a positive control. After incubation, the plates were centrifuged at 1800 r / min for 5 min, and 50 μL of supernatant was added to each well to a new 96-well clear plate. The absorbance at 490 nm was measured using a microplate reader. The hemolysis rate was calculated using the following formula: Hemolytic rate = [OD] 490 nm (treatment) - OD 490 nm (negative control) ] / [OD 490 nm (positive control) - OD 490 nm (negative control) ] × 100%. OD 490 nm (treatment) : Indicates the absorbance value corresponding to the cell pore for drug delivery. OD 490 nm (positive control) : Indicates the absorbance value corresponding to the positive control well. OD 490 nm (negative control) : Indicates the absorbance value corresponding to the negative control well. The experimental results are shown in Figure 3 (A) Even at the highest test concentration of 256 μM, all tested cyclic peptides showed low hemolytic activity (<10%), with PT-08 and PT-17 exhibiting relatively high antimicrobial selectivity.

[0051] Example 4: In vitro cell proliferation toxicity experiment: The in vitro cytotoxicity of antimicrobial peptides was assessed using human renal epithelial cells (HEK-293T) and human hepatocytes (LO2). Cells were cultured to the logarithmic growth phase, and a uniformly dispersed cell suspension was rapidly added to sterile 96-well plates, with five replicates per concentration, 100 μL per well (approximately 10,000 cells per well), and incubated at 37°C for 24 h. After 24 h, the original culture medium was aspirated from the plates, and the prepared peptide solutions of each concentration were added. The negative control group received blank culture medium (without cells), and the positive control group received blank culture medium with the same cell count as the drug-treated groups. Incubation was continued at 37°C for 24 h. After incubation, the 96-well plates were removed, the original culture medium was discarded, and CCK-8 working solution was added. Incubation was continued for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated based on the values. The cell viability calculation formula is as follows: Cell survival rate = [OD] 450 nm (treatment) - OD 450 nm (negative control) ] / [OD 450 nm (positive control) - OD 450 nm (negative control) ] × 100%. OD 450 nm (treatment) : Indicates the absorbance value corresponding to the cell pore for drug delivery. OD 450 nm (positive control) : Indicates the absorbance value corresponding to the positive control well. OD 450 nm (negative control) : Indicates the absorbance value corresponding to the negative control well. The experimental results are shown in Figure 3 (B) The antimicrobial peptide PT-17 showed low cytotoxicity to HEK-293T and LO2 cells.

[0052] Example 5 Stability Test 1. Salt sensitivity test: The salt susceptibility of PT-08 and PT-17 to multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli was analyzed by systematically evaluating their MICs under different physiological saline conditions. The concentrations of NaCl, KCl, MgCl2, and FeCl3 were 150 mM, 4.5 mM, 1 mM, and 4 μM, respectively. Figure 4 As shown in (A), PT-08 and PT-17 exhibited effective salt tolerance. In all selected salt solutions, the MIC of PT-17 remained unchanged against multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli, while the MIC of PT-08 showed almost no change, and the MIC against multidrug-resistant Staphylococcus aureus increased only by a factor of two.

[0053] 2. Stability testing: In plasma stability assays, the MICs of antimicrobial peptides P-07, PT-08, and PT-17 against multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli were tested before and after treatment with 50% plasma. Results are as follows: Figure 4 As shown in (B), the MICs of P-07, PT-08, and PT-17 against both pathogens remained unchanged after 1 h of plasma treatment. However, P-07 showed a significant loss of activity under prolonged incubation. In contrast, PT-08 and PT-17 maintained their initial MICs for 6 h and 12 h, respectively. PT-17 exhibited excellent plasma stability, with its MIC only doubling after 24 h of plasma treatment. The experimental results indicate that cyclization and D-amino acid substitution significantly enhanced the activity of P-07, PT-08, and PT-17 against both pathogens in plasma. β - Metabolic resistance of corner antimicrobial peptides.

[0054] In the enzyme stability assay, P-07, PT-08, and PT-17 were incubated with chymotrypsin (0.2 mg / mL) and trypsin (0.2 mg / mL) at 37°C for different times (0.5, 1, 2, 3, 6 h). The half-lives of the peptides in chymotrypsin and trypsin were then calculated by HPLC analysis. The results are as follows: Figure 4 As shown in (C), the stability of the above antimicrobial peptidases is the same as that in plasma (PT-17>PT-08>P-07), confirming that both lactam cyclization and D-amino acid substitution can improve their enzyme stability. Among them, the half-life of PT-17 in chymotrypsin is 54.3 times that of P-07 and 25.5 times that of trypsin.

[0055] Example 6: Bactericidal Kinetics Experiment Using P-07 and PBS as control groups, the killing kinetics of multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli were investigated at 2×MIC. Figure 5 As shown in (A), both P-07 and PT-17 exhibited rapid and time-dependent killing kinetics, completely eradicating both pathogens within 6 hours. This confirms the effective bactericidal ability of PT-17.

[0056] Example 7: Induction of Drug Resistance Experiment Using polymyxin B, amoxicillin, and amoxicillin-containing PT-17 as positive controls, the potential of PT-17 to induce bacterial resistance was investigated through continuous and repeated drug treatments on Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. This study employed a dynamic concentration gradient method to assess the activity of antibiotics and peptides. The upper and lower limits of the concentration gradient were not fixed but were adjusted based on the real-time measurement results of the MIC during the test, and the range was typically set from 1 / 4 × MIC. original To n×MIC original.like Figure 5 As shown in (B), after 20 consecutive passages, the MICs of PT-17 against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 29522 remained stable (≤ 2-fold change), indicating a low tendency to induce resistance in these strains. Polymyxin B only caused moderate (4-fold) or less MIC increases during consecutive passages; in contrast, amoxicillin induced significant resistance, increasing the MICs of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 29522 by 8-fold and 32-fold, respectively. Notably, the combination of PT-17 and amoxicillin effectively inhibited the emergence of resistance. This synergistic effect may be due to the membrane-disrupting effect of PT-17, which enhances amoxicillin penetration by impairing membrane integrity, thereby promoting its acquisition of penicillin-binding protein (PBP). These findings highlight the potential of antimicrobial peptide-antibiotic combinations to counteract the evolution of resistance.

[0057] Example 8: Combined Drug Use Experiment The combined effects of PT-17 with other antibacterial agents were evaluated using a checkerboard method. The synergistic efficacy of the drug combination was quantitatively assessed using the fractional inhibition concentration index (FICI). Figure 6 As shown, the combined effects of PT-17 with various antimicrobial agents exhibit different patterns against different bacterial strains. For Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, PT-17 showed an additive effect when combined with polymyxin B or amoxicillin. For multidrug-resistant Staphylococcus aureus, PT-17 showed a synergistic effect with amoxicillin, while maintaining an additive effect with polymyxin B, levofloxacin, or cefoperazone. For multidrug-resistant Escherichia coli, PT-17 showed a synergistic effect with rifampin or cefoperazone, but an additive effect with polymyxin B or levofloxacin. In general, PT-17, when used in combination with other antimicrobial agents, showed synergistic or additive antimicrobial effects against both standard and multidrug-resistant bacterial strains. This combination strategy can reduce the dosage of clinical antimicrobial agents, delay the onset of resistance, and improve treatment efficacy.

[0058] Example 9: Study on the antibacterial mechanism To systematically elucidate the membrane-disrupting effect of PT-17, bacterial morphology was analyzed using confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Lipopolysaccharide (LPS) competitive inhibition experiments were used to further evaluate LPS binding capacity. Meanwhile, bacterial membrane integrity was quantitatively assessed by measuring outer membrane permeability and membrane depolarization.

[0059] 1. CLSM Analysis: CLSM was used to study the bacterial membrane disruption effect of PT-17. P-07 and PBS served as positive and negative controls, respectively. Multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli cells were treated with a 2×MIC peptide for 2 h, followed by 4',6-diamidinyl-2-phenylindole (DAPI) and propidium iodide (PI) staining. DAPI permeated all bacterial cells, staining DNA blue regardless of membrane integrity, while PI selectively bound to DNA and emitted red fluorescence only when membrane damage occurred. Figure 7 As shown in (A) and (B), the multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli bacterial cells in the control group showed strong blue fluorescence but almost no red fluorescence, indicating that the bacterial membranes were intact. In contrast, PT-17 and P-07 treatments produced strong red fluorescence in most bacteria, indicating that they have a strong ability to disrupt bacterial membranes.

[0060] 2. TEM analysis: To further evaluate the bacterial membrane disruption effect of PT-17, P-07 and PBS were used as positive and negative controls, respectively. The morphology of multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli cells before and after PT-17 treatment was directly observed using TEM. Both pathogens were treated with a 2×MIC peptide for 2 h. Figure 7 As shown in (C) and (D), PBS-treated bacterial cells maintained their intact cell morphology, characterized by smooth cell membranes without structural damage. In contrast, bacteria treated with PT-17 and P-07 exhibited severe membrane damage, manifested as significant membrane rupture, pore formation, cell shrinkage, and leakage of cytoplasm.

[0061] 3. LPS competitive inhibition assay: LPS is a key anionic component of the outer membrane of Gram-negative bacteria and a major target of many cationic AMPs (such as polymyxin B). The affinity of antimicrobial peptides for LPS significantly controls their interactions with Gram-negative bacterial membranes. Therefore, we used a competitive inhibition assay to quantify the LPS-binding abilities of PT-17, P-07, and polymyxin B. Figure 8 As shown in (A), the bactericidal activity of all three antimicrobial agents decreased sharply at their respective LPS neutralization thresholds (PT-17: 16 μg / mL, P-07: 16 μg / mL, and polymyxin B: 32 μg / mL). This is because excess LPS competitively occupies AMP binding sites, thereby neutralizing their antimicrobial function. The LPS binding capacity order is as follows: PT-17 ≈ P-07 > polymyxin B.

[0062] 4. Outer membrane permeability measurement: The outer membrane permeability of multidrug-resistant *E. coli* treated with PT-17 was assessed using N-phenyl-1-naphthylamine (NPN). P-07 and polymyxin B were used as positive controls, and PBS as a negative control. The hydrophobic NPN dye partitioned into the hydrophobic outer membrane upon rupture, resulting in a significant increase in fluorescence intensity. Figure 8 As shown in (B), PT-17, P-07, and polymyxin B significantly increased the outer membrane permeability of multidrug-resistant Escherichia coli cells in a dose-dependent manner within 1 min. Notably, PT-17 elicited a greater NPN fluorescence intensity than its parent peptide P-07 and polymyxin B at 1 × MIC to 4 × MIC, demonstrating its superior ability to disrupt the outer membrane of multidrug-resistant Escherichia coli.

[0063] 5. Measurement of cell membrane depolarization: The membrane depolarization activity of PT-17 against multidrug-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli was quantified using a cytoplasmic membrane depolarization assay. P-07 and polymyxin B were used as positive controls, and PBS as a negative control. Figure 8 As shown in (C), all three peptides induced rapid cell membrane depolarization in multidrug-resistant Escherichia coli within 1 min, with the depolarization effect in the order of PT-17 > P-07 > polymyxin B. For multidrug-resistant Staphylococcus aureus, such as Figure 8 As shown in (D), both PT-17 and P-07 induced rapid cytoplasmic membrane depolarization within 1 min, with PT-17 exhibiting stronger depolarizing activity than P-07 at 2 × MIC and 4 × MIC. Conversely, polymyxin B failed to induce significant depolarization at the tested concentrations, consistent with its limited antibacterial efficacy against multidrug-resistant Staphylococcus aureus. In conclusion, PT-17 demonstrated a strong ability to disrupt the normal bacterial membrane potential, confirming its potent membrane-disrupting activity.

[0064] In summary, PT-17 initially interacts with LPS on the bacterial membrane surface, subsequently disrupting the stability of the outer membrane and cytoplasmic membrane to achieve its bactericidal effect.

[0065] Example 10 In vivo safety experiment Assess the acute toxicity of PT-17 in mouse models, such as Figure 9 As shown, male KM mice (n=5) were administered a single intravenous injection of P-07 (1–5 mg / kg), PT-17 (10–50 mg / kg), and polymyxin B (5–15 mg / kg) via the tail vein, and mortality rates were recorded for each group. The LD50 was calculated for P-07 (2.9 mg / kg), PT-17 (38.0 mg / kg), and polymyxin B (12.0 mg / kg). Compared with P-07 and polymyxin B, PT-17 showed excellent in vivo safety.

[0066] To further explore the safety of PT-17, repeated-dose toxicity assays were performed over 5 days, with the antibiotic administered intravenously once daily. Twenty male KM mice were randomly assigned to four groups (n=5): control group (saline), PT-17 (5 mg / kg), PT-17 (10 mg / kg), and polymyxin B group (5 mg / kg). Figure 10 As shown in (A), the weight change curves of the three treatment groups were comparable to those of the control group. Five days later, the major organs and whole blood of the mice were harvested. Figure 10 As shown in (B), there were no significant differences in organ indices of major organs between the treatment and control groups, indicating no evidence of treatment-induced organ hypertrophy or atrophy. Complete blood count (CBC) analysis is as follows: Figure 10 (CH) showed that key hematological parameters (including red blood cell count (RBC), mean corpuscular volume (MCV), hematocrit (HCT), hemoglobin (HGB), and mean corpuscular hemoglobin (MCH)) and platelet count (PCT) were not significantly different between the treatment and control groups. That is, at the test dose, neither PT-17 nor polymyxin B produced detectable toxicity to the hematopoietic system. Serum biochemical analysis is as follows: Figure 10 As shown in (IJ), there were no significant differences in alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels between the treatment and control groups, indicating that neither PT-17 nor polymyxin B induced detectable hepatotoxicity or nephrotoxicity at the test doses. Overall, PT-17 (5, 10 mg / kg) and polymyxin B (5 mg / kg) did not induce significant toxicological responses, confirming their favorable in vivo safety profile.

[0067] Example 11 Evaluation of in vivo antibacterial activity The in vivo antibacterial efficacy of PT-17 was studied using a mouse model of multidrug-resistant Escherichia coli infection. Twenty-five male KM mice were randomly divided into five groups (n=5): an uninfected group, a model group (untreated), a PT-17 (5 mg / kg) group, a PT-17 (10 mg / kg) group, and a polymyxin B (5 mg / kg) group. Figure 11 As shown in (A), mice were given a fixed dose once daily. Figure 11 As shown in (B), uninfected mice exhibited continuous weight gain over 3 days, while all infected mice experienced initial weight loss followed by gradual weight recovery starting from day 2. Notably, all treatment groups demonstrated accelerated weight recovery kinetics compared to the model group, indicating improved survival. On day 4, mice were sacrificed for organ collection to assess bacterial load and for H&E staining. Bacterial load analysis is as follows: Figure 11As shown in (C), compared with the model group, the colonization of major organs (liver, spleen, lung, kidney) was significantly reduced in the PT-17 (5, 10 mg / kg) and polymyxin B (5 mg / kg) groups. Notably, the bacterial clearance effect of the PT-17 (10 mg / kg) group was superior to that of the PT-17 (5 mg / kg) and polymyxin B (5 mg / kg) groups. The PT-17 (5 mg / kg) and polymyxin B (5 mg / kg) groups showed comparable organ bacterial loads.

[0068] like Figure 11 (D) shows that H&E staining was performed to examine the histopathological changes of the major organs (liver, spleen, lung, and kidney) in mice. Histopathological analysis further validated the therapeutic effects of PT-17 (5, 10 mg / kg) and polymyxin B (5 mg / kg). Compared with the uninfected group, the model group showed significant histopathological changes in different organs and tissues: hepatocyte degeneration and inflammatory infiltration in the liver, lymphoid follicle hyperplasia and phagocyte aggregation in the spleen, interstitial congestion, alveolar collapse with extensive lung inflammation, and glomerular hypertrophy in the kidney. In contrast, both PT-17 (5, 10 mg / kg) and polymyxin B (5 mg / kg) significantly improved these pathological changes, as evidenced by the restoration of radial hepatocyte arrangement, reduction of splenic lymphoid hyperplasia, normalization of alveolar structure, reduction of pulmonary edema and inflammation, and normalization of glomerular size. In summary, PT-17 demonstrated effective treatment for systemic infections caused by multidrug-resistant Escherichia coli at doses of 5 and 10 mg / kg, with the high-dose regimen (10 mg / kg) showing superior bacterial clearance efficiency compared to the positive control polymyxin B (5 mg / kg).

Claims

1. A class of P-07-derived antimicrobial peptides having an amino acid sequence as shown in general formula (1), (2) or (3): ZZ is selected from β - Corner sequence WWW or PWWWP; Each X1 may be the same or different, and is independently selected from basic amino acids or basic amino acid derivatives, for example, it may be selected from lysine (Lys, K). Each X2 may be the same or different, and is independently selected from hydrophobic amino acids or hydrophobic amino acid derivatives, for example, it may be selected from isoleucine (Ile, I). Each Y may be the same or different, and is independently selected from amino acids or amino acid derivatives that can be used to form covalent crosslinks between side chains, such as cysteine ​​(Cys, C), glutamic acid (Glu, E), and lysine (Lys, K).

2. The antimicrobial peptide according to claim 1, characterized in that, The amino acid or amino acid derivative is of the L-type or D-type.

3. The antimicrobial peptide according to claim 1 or 2, characterized in that, The N-terminal group of the antimicrobial peptide is a free amino group or is acetylated; for example, the N-terminal group is an acetamino group. Preferably, the C-terminal group of the antimicrobial peptide is a carboxyl group or is amidated; for example, the C-terminal group is amidated to form an amide group.

4. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide has the amino acid sequence shown in the table below: Note: a D-type amino acids are represented by lowercase letters; b Circulation sites are indicated by an asterisk (*); Preferably, the antimicrobial peptide further includes a pharmaceutically acceptable salt thereof.

5. The method for preparing the antimicrobial peptide according to any one of claims 1-4, wherein the peptide is prepared by solid-phase synthesis.

6. An antimicrobial agent comprising a therapeutically effective amount of the antimicrobial peptide according to any one of claims 1-4; Preferably, the antibacterial drug is a topical preparation, an oral preparation, an injectable preparation, or an inhaled preparation; Preferably, the oral preparation is selected from tablets, capsules, granules, powders, pills, oral liquids, syrups, or drops; the injectable preparation is selected from injection solutions, powders for injection, or concentrated solutions; the external preparation is selected from ointments, creams, gels, patches, sprays, or lotions; and the inhaled preparation is selected from inhaled powders, aerosols, or sprays. Preferably, the antibacterial drug further includes at least one pharmaceutically acceptable excipient; Preferably, the excipients include buffers, preservatives, stabilizers, binders, lubricants, chelating agents, dispersants, disintegrants, flavoring agents, diluents, surfactants, sweeteners, and / or colorants.

7. The use of the antimicrobial peptide according to any one of claims 1-4 in the preparation of antimicrobial drugs.

8. The application according to claim 7, characterized in that, The antibacterial drug is used to prevent or treat infections caused by pathogens, such as skin wound infections; The pathogens are selected from Gram-positive or Gram-negative bacteria, such as at least one of Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, or their multidrug-resistant strains.

9. A composition wherein the active ingredient comprises a therapeutically effective amount of the antimicrobial peptide and antibacterial antibiotic as described in any one of claims 1-4; Preferably, the combined antimicrobial drug is used to treat infections caused by pathogens, such as skin wound infections; Preferably, the antibiotic is selected from polypeptide antibiotics, penicillin antibiotics, etc. β - At least one of the following: lactam antibiotics, quinolone antibiotics, cephalosporin antibiotics, and semi-synthetic carbapenem antibiotics; Preferably, the pathogen is selected from Gram-positive or Gram-negative bacteria, such as at least one of Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella pneumoniae, Streptococcus, Bacillus, or multidrug-resistant strains thereof.

10. The composition according to claim 9, characterized in that, The antimicrobial peptide is antimicrobial peptide PT-17.

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