Broad-spectrum low-drug-resistant antibacterial peptide with antibacterial motif modification and application thereof

Abstract

This invention discloses a class of broad-spectrum, low-drug-resistance antimicrobial peptides modified with antimicrobial motifs and their applications. The antimicrobial peptide is obtained by adding an antimicrobial "FLPII" motif to the N-terminus of the original antimicrobial peptide XWKWKWKWKK (X = W or K) and then amidating the C-terminus. Its structural formula is: FLPIIWWKWKWKWKK-NH2, denoted as FWK; or FLPIIKWKWKWKWKK-NH2, denoted as FKK. Antimicrobial tests and in vivo antimicrobial activity experiments show that the antimicrobial peptides have the advantages of strong antimicrobial activity and a broad antimicrobial spectrum. Induction of drug resistance experiments show that the antimicrobial peptides of this invention all have low drug resistance incidence, and have good application prospects in the preparation of clinical antimicrobial drugs, especially for the treatment of lung infections caused by Klebsiella pneumoniae. They can be used to prepare drugs for treating lung infections caused by Klebsiella pneumoniae.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and relates to a class of broad-spectrum, low-drug-resistance antimicrobial peptides modified with antimicrobial motifs. This invention also relates to the application of these antimicrobial peptides in the preparation of clinical antimicrobial drugs. Background Technology

[0002] In recent years, the overuse of antibiotics has led to increased microbial resistance, creating an unprecedented and urgent problem in the field of public health (Nature Medicine, 2018, 24, 1097-1103). According to the latest survey results from the US Centers for Disease Control and Prevention, bacteria are the main cause of serious infections (such as lung and wound infections) that threaten human health and safety. These persistent and difficult-to-treat infections not only place a heavy burden on society but also pose a huge challenge to the healthcare system (Jama, 2013, 310(16), 1661-1663).

[0003] Antimicrobial peptides (AMPs) are a class of biomolecules found in the innate immune system of organisms in nature. Their ability to bind to negatively charged lipids can lead to bacterial membrane permeation. Furthermore, numerous studies have shown that AMPs also possess the ability to target internal mechanisms (Antibiotics, 2024, 13(3), 202). In this context, their induction of cell death may involve interactions with DNA / RNA, adversely affecting protein synthesis and enzyme activity within the cell, or preventing cell wall / membrane formation.

[0004] However, the application of natural antimicrobial peptides has some limitations, such as metabolic instability, high production costs, and the potential to cause hemolytic side effects. Furthermore, there is a growing consensus that the clinical use of AMPs with sequences too close to human AMPs may impair natural defenses and pose a threat to public health. Therefore, researchers have begun developing non-natural (or synthetic) peptides to expand the AMP arsenal (Science, 2020, 1; 368(6490):eaau5480.). Based on this, the applicant designed and synthesized a class of highly active antimicrobial peptides that exhibit advantages in broadly inhibiting bacterial activity and are effective in treating lung infections caused by Klebsiella pneumoniae. This research is expected to provide candidate drugs for novel antibiotics and shows promising potential in the preparation of clinical antimicrobial drugs. Summary of the Invention

[0005] One of the objectives of this invention is to provide a class of broad-spectrum, low-drug-resistance antimicrobial peptides modified with antimicrobial motifs and their applications.

[0006] A second objective of this invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of clinical antimicrobial drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] I. Structural Design of Broad-Spectrum, Low-Drug-Resistance Antimicrobial Peptides Modified with Antimicrobial Motifs

[0009] The present invention provides a class of broad-spectrum, low-drug-resistance antimicrobial peptides modified with antimicrobial motifs. These peptides are obtained by adding an antimicrobial "FLPII" motif to the N-terminus of the original antimicrobial peptide XWKWKWKWKK (X = W or K), thereby increasing antimicrobial activity. The C-terminus is then amidated to maintain a high net positive charge. The structural formula is: FLPIIWWKWKWKWKK-NH2, denoted as FWK; or FLPIIKWKWKWKWKK-NH2, denoted as FKK. FKK is preferred.

[0010] The aforementioned broad-spectrum, low-drug-resistance antimicrobial peptides with antimicrobial motif modifications were all prepared using classical solid-phase synthesis methods.

[0011] II. Application of Broad-Spectrum, Low-Drug-Resistance Antimicrobial Peptides Modified with Antimicrobial Motifs in the Preparation of Clinical Antimicrobial Drugs

[0012] 1. In vitro antibacterial test

[0013] The minimum inhibitory concentration (MIC) of antimicrobial peptides against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii) was determined using the classic micro-dilution serial two-fold method. The antibiotics methicillin, polymyxin B, cephalothin, gentamicin, stretchtomycin, and rifampicin were used as positive controls. The experiment was repeated in triplicate, and the results are shown in Table 1.

[0014] Table 1. Minimum inhibitory concentrations of the antimicrobial peptides of this invention against common standard strains.

[0015]

[0016] Table 1 shows that the antimicrobial peptides of this invention exhibit strong inhibitory effects against both Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii), demonstrating broad-spectrum antimicrobial activity. Among them, FKK showed superior antimicrobial activity. Therefore, using FKK as a representative, salt sensitivity tests, LPS binding tests, induction of drug resistance tests, scanning electron microscopy tests, and experiments on the treatment of Klebsiella pneumoniae-induced bacterial pneumonia in mice were conducted.

[0017] 2. Salt sensitivity test

[0018] The instability of peptides hinders their clinical application. Therefore, this study investigates the effect of salt ions in the human physiological environment on the antibacterial activity of peptides to assess their salt ion stability (a change in antibacterial activity within 4-fold is considered good salt ion stability). Vancomycin and polymyxin B were used as quality assurance controls.

[0019] Table 2. Minimum inhibitory concentrations of the antimicrobial peptides of this invention against common standard strains under salt ion conditions.

[0020]

[0021]

[0022] like Figure 1 As shown, in a NaCl salt environment, the activity of FKK against Klebsiella pneumoniae and Staphylococcus aureus decreased by 2-fold. The activity of vancomycin against Staphylococcus aureus also decreased by 2-fold in a NaCl salt environment. Conversely, the activity of polymyxin B against Klebsiella pneumoniae increased by 2-fold in ZnCl2 solution. These results indicate that the activity changes of the designed antimicrobial peptides under different salt ion conditions were all within 4-fold, demonstrating that the antimicrobial peptides of this invention have good salt ion stability.

[0023] 2. LPS combined experiment

[0024] Endotoxins are a major component of the outer membrane of Gram-negative bacteria and a target of polymyxin B. Therefore, the applicant designed a competitive inhibition experiment to verify whether the antimicrobial peptides of this invention can act on endotoxins by incubating with different concentrations of endotoxins. The results are shown in [Figure number missing]. Figure 1 .

[0025] Figure 1 The results showed that endotoxin had a dose-dependent inhibitory effect on the antibacterial activity of antimicrobial peptides FKK and polymyxin B. The binding efficiency of FKK to endotoxin (128 μg / mL) was lower than that of polymyxin B to endotoxin (256 μg / mL), indicating that FKK can interact with endotoxin.

[0026] 3. Induction of drug resistance experiment

[0027] The most significant advantage of antimicrobial peptides compared to traditional antibiotics is their low tendency to induce drug resistance. Due to their membrane-dissolving mechanism, bacteria find it difficult to alter their cell membrane composition and thus develop resistance. Therefore, to investigate whether the antimicrobial peptides of this invention possess this characteristic of low resistance development, the applicant determined the antimicrobial activity of the antimicrobial peptide FKK and the antibiotic (gentamicin / polymyxin B) against Staphylococcus aureus ATCC 25923 and Klebsiella pneumoniae ATCC 700603 after continuous treatment for 15 days.

[0028] Generally, fluctuations in the MIC (microinjection ratio) within the range of 1-4 times are considered normal. If the MIC is greater than 4 times, it indicates that antibiotic resistance has developed. Figure 2 As shown, the traditional antibiotic gentamicin developed resistance to Staphylococcus aureus (S. aureus) ATCC 25923 after 8 days of continuous treatment, and the MIC against S. aureus increased by 512-fold after 15 days of continuous treatment. The polypeptide antibiotic polymyxin B showed no significant change in antibacterial activity against Klebsiella pneumoniae (K. pneumoniae) ATCC 700603 after 15 days of continuous treatment. The polypeptide antibiotic vancomycin also showed no significant change in antibacterial activity against S. aureus ATCC 25923 after 15 days of continuous treatment. However, the antimicrobial peptide FKK of this invention did not develop resistance to either S. aureus ATCC 25923 or Klebsiella pneumoniae (K. pneumoniae) ATCC 700603 after 15 days of continuous treatment, indicating that the antimicrobial peptide of this invention has a significant advantage in terms of resistance compared to traditional antibiotics.

[0029] 5. Scanning electron microscopy experiment

[0030] In this experiment, the applicant used scanning electron microscopy (SEM) to visually observe the changes in bacterial morphology caused by the action of antimicrobial peptides. The specific experimental method is as follows: Staphylococcus aureus and Klebsiella pneumoniae grown overnight were diluted in MH medium to a concentration of 10 × 10⁻⁶. 8 CFU / mL, centrifuged at 10000 rpm for 5 min, washed twice with PBS, and resuspended half-volume in PBS. 500 μL of bacterial culture was mixed with 500 μL of peptide solution (final peptide concentration 2×MIC), and incubated at 37℃ for 30 min and 2 h, respectively. After incubation, the sample was centrifuged at 10000 rpm for 10 min, fixed overnight at 4℃ with 500 μL of 2.5% glutaraldehyde, washed twice with PBS, and eluted with a gradient of ethanol (ethanol concentrations of 50%, 70%, 80%, 90%, and 100%). After adding ethanol and standing for 10 min, the sample was centrifuged again. After ethanol gradient elution, 150 μL of tert-butanol was added and incubated at room temperature for 2 h. After centrifugation, the bacterial pellet was placed on a round glass slide. The sample was dried, sputter-coated with gold, and analyzed using an Apreo S scanning electron microscope (Thermo Fisher Scientific, MA, USA). The results are shown in the figure. Figure 3 .

[0031] Depend on Figure 3It was observed that normal Klebsiella pneumoniae had a smooth and intact surface. However, after 30 minutes of treatment with the antimicrobial peptides FKK and polymyxin B, Klebsiella pneumoniae began to show surface shrinkage, leakage of cell contents, and loss of intact cell morphology, indicating that the antimicrobial peptides of the present invention were more effective than polymyxin B. Staphylococcus aureus, after 30 minutes of treatment with the antimicrobial peptides FKK and vancomycin, mainly exhibited bacterial aggregation, and with prolonged treatment time, caused even larger-scale changes in bacterial cell morphology. Scanning electron microscopy results clearly showed that the antimicrobial peptides of the present invention had a much stronger membrane-disrupting ability against Klebsiella pneumoniae and Staphylococcus aureus than polymyxin B and vancomycin.

[0032] 7. Experiment on antimicrobial peptide treatment of Klebsiella pneumoniae-induced bacterial pneumonia in mice

[0033] Male BALB / c mice, weighing 18-22g, were fed according to the experimental animal ethics management regulations of Lanzhou University. They were divided into 6 groups of 8 mice each: normal control group, model group, positive control group (Polymyxin B, PB-L: 5mg / kg, PB-H: 10mg / kg), and FKK treatment group (FKK-L: 5mg / kg, FKK-H: 10mg / kg). After anesthetizing with pentobarbital, each mouse was administered a loading dose of Klebsiella pneumoniae (in physiological saline, 50μL, 3×10⁻⁶) via nebulizer to the bronchi of the lungs. 9 A bacterial pneumonia model was established using CFU / mL. Six hours later, the group receiving the drug received the first dose intraperitoneally. Twelve hours later, the group received the second dose intraperitoneally. After 24 hours, the group was sacrificed, and the left lung was homogenized. The homogenate (concentration 1 mg tissue / mL) and its dilution were plated for counting. Figure 4 The number of bacteria on the agar plates for each group of mice was recorded and statistically analyzed. Figure 5 ("***": P < 0.001 "ns": No statistically significant difference).

[0034] Figure 5 Based on the counting results, FKK showed a significant therapeutic effect on bacterial pneumonia caused by Klebsiella pneumoniae in mice, and there was no statistically significant difference in the therapeutic effect compared with Polymyxin B. Both were effective in treating bacterial pneumonia caused by Klebsiella pneumoniae.

[0035] The beneficial effects of this invention are as follows:

[0036] This invention adds an antibacterial "FLPII" motif to the N-terminus of the original antimicrobial peptide XWKWKWKWKK (X = W or K) to increase its antimicrobial activity, and amidates the C-terminus to maintain a high net positive charge, resulting in a broad-spectrum, low-resistance antimicrobial peptide with an antimicrobial motif modification. Antimicrobial tests and in vivo antimicrobial activity experiments demonstrate that the antimicrobial peptide possesses strong antimicrobial activity and a broad antimicrobial spectrum. Induction of resistance experiments show that the antimicrobial peptides of this invention all exhibit low resistance rates, showing great promise for the preparation of clinical antimicrobial drugs, especially for treating lung infections caused by Klebsiella pneumoniae, and are expected to become candidate drugs for novel antibiotics. Attached Figure Description

[0037] Figure 1 This invention provides an experiment demonstrating the binding of the antimicrobial peptide to LPS.

[0038] Figure 2 The antimicrobial peptides of this invention exhibit antimicrobial activity against Staphylococcus aureus ATCC 25923 and Klebsiella pneumoniae ATCC 700603 after continuous action for 15 days.

[0039] Figure 3 Electron micrographs of the antimicrobial peptide FKK and antibiotic of this invention;

[0040] Figure 4 This is a plate count image of diluted fluid from the left lung of a mouse used to treat bacterial pneumonia in mice, based on the antimicrobial peptide FKK and antibiotics of this invention.

[0041] Figure 5 This is a bacterial statistical chart showing the effects of the antimicrobial peptide FKK and antibiotics of this invention on the treatment of bacterial pneumonia in mice.

[0042] Figure 6 This is the mass spectrum of the antimicrobial peptide FKK of this invention;

[0043] Figure 7 This is the mass spectrum of the antimicrobial peptide FWK of this invention. Detailed Implementation

[0044] The synthesis of the broad-spectrum antibacterial peptide with low toxicity of the present invention will be further illustrated below through specific embodiments.

[0045] Example 1: Synthesis of antimicrobial peptide FKK

[0046] (1) Resin activation and pretreatment

[0047] Weigh 0.465 g of MBHA resin (0.43 mmol / g) and add it to a polypeptide solid-phase synthesizer. After swelling in DCM for 30 min and washing with DMF, identify the resin using the ninhydrin colorimetric method. If it is colorless, the resin is normal.

[0048] (2) Synthesis of Fmoc-FKK-MBHA

[0049] The swollen resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc protecting group. The indole-based resin was blue-purple when it turned out. Three times the excess of Lys, three times the excess of HOBt and HBTU, and six times the excess of DIEA were dissolved in redistilled DMF and added to the synthesizer. The mixture was stirred for 1 hour. After the reaction time was reached, the indole-based resin turned colorless and transparent, indicating successful condensation, yielding Fmoc-Lys-MBHA.

[0050] Following the above method, Lys, Trp, Lys, Trp, Lys, Trp, Lys, Trp, Lys, Ile, Ile, Pro, Leu, Phe are condensed sequentially to obtain Fmoc-Phe-Leu-Pro-Ile-Ile-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Lys-MBHA.

[0051] Similarly, following the above method, condense Lys, Trp, Lys, Trp, Lys, Trp, Lys, Trp, Trp, Ile, Ile, Pro, Leu, Phe sequentially to obtain Fmoc-Phe-Leu-Pro-Ile-Ile-Trp-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Lys-MBHA.

[0052] (3) Peptide cleavage

[0053] The obtained Fmoc-Phe-Leu-Pro-Ile-Ile-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Lys-MBHA or Fmoc-Phe-Leu-Pro-Ile-Ile-Trp-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-Lys-MBHA were washed with DMF solution containing 20% ​​piperidine to remove the Fmoc protecting group, and then washed twice with DCM and methanol respectively. The resin was thoroughly dried under vacuum. 10 mL of cleavage reagent (TFA:Tris:water = 9.5:0.25:0.25 (v:v:v)) was added and reacted for 3 h. After extraction with diethyl ether, the mixture was freeze-dried.

[0054] (4) Peptide purification

[0055] The RP-HPLC purification conditions were: mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile, using linear gradient elution. The target peak eluent was collected, lyophilized, and the antimicrobial peptides FKK and FWK were obtained. The mass spectrum of FKK is shown below. Figure 6As shown, the FWK mass spectrum is as follows: Figure 7 As shown.

Claims

1. A class of broad-spectrum, low-drug-resistance antimicrobial peptides modified with antimicrobial motifs, characterized by: The antimicrobial peptide is obtained by adding an antimicrobial "FLPII" motif to the N-terminus of the original antimicrobial peptide XWKWKWKWKK and then amidating the C-terminus. Its structural formula is: FLPIIWWKWKWKWKK-NH2, and the amino acid sequence is shown in SEQ ID No. 1, denoted as: FWK; or FLPIIKWKWKWKWKK-NH2, as shown in SEQ ID No. 2, denoted as: FKK.

2. The broad-spectrum, low-drug-resistance antimicrobial peptide with antimicrobial motif modification as described in claim 1, characterized in that, The antimicrobial peptide has the structural formula FLPIIKWKWKWKWKK-NH2, denoted as FKK.

3. The application of the broad-spectrum, low-drug-resistance antimicrobial peptide modified with antimicrobial motifs as described in claim 1 or 2 in the preparation of antimicrobial drugs, characterized in that, The bacteria inhibited by the antibacterial drug are Gram-positive or Gram-negative bacteria; the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis and / or Bacillus subtilis, and the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and / or Acinetobacter baumannii.

4. The use of the broad-spectrum, low-drug-resistance antimicrobial peptide modified with the antimicrobial motif as described in claim 1 or 2 in the preparation of a drug for treating lung infections caused by Klebsiella pneumoniae.

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