Composite antibacterial combined peptide capable of treating septicopyemia and application of composite antibacterial combined peptide

Abstract

The invention relates to the technical field of biology, and provides a composite antibacterial combined peptide capable of treating septicopyemia and application of the composite antibacterial combined peptide, and the amino acid sequence of the composite antibacterial combined peptide is one of the following sequences: Meta207: X1 WLRX1 LRRX2 X1 RRI, Meta220: X2 WRX3 LRRX3 LRRLLV, Meta221: IRWX1 RX1 LRRRLX1 X1, x1, X2 and X3 are random sites, X1 is L or I, X2 is V or L, and X3 is I or V; the Meta207, the Meta220 and the Meta221 are mixtures selected from all the random sites respectively. The composite peptide can have a powerful antibacterial effect on various bacteria, can be used for preparing related drugs or coatings of medical apparatuses and instruments for preventing or treating sepsis, septicopyemia and the like caused by bacterial infection, and has important guiding significance on diseases caused by bacterial infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a kind of complex antibacterial combination peptide for treating sepsis and application thereof. BACKGROUND

[0002] With the extensive use of antibiotics, the problem of bacterial resistance to antibiotics is increasingly serious. There is an urgent need for new antibiotics or antibiotic substitutes. Antimicrobial peptides are a class of natural or artificially synthesized polypeptides, usually with a short amino acid sequence (10-50), and have broad-spectrum antibacterial activity and strong bactericidal effect, playing an important role in the antibacterial function of the body's own immune system. Antimicrobial peptides exert antibacterial effect by lysing bacterial cell membranes, and are not prone to bacterial resistance. However, the synthesis of antimicrobial peptides is costly, which is not conducive to the widespread use of antimicrobial peptides.

[0003] Infectious diseases are common and frequently-occurring diseases, and children are particularly susceptible. Bacteria are the most common pathogenic microorganisms of infectious diseases. CN102887948A discloses an apolipoprotein E mimetic peptide drug with antibacterial and immunomodulatory effects. The apolipoprotein E mimetic peptide ApoE23 has the structure of SEQ ID NO: 1. In vivo and in vitro experiments have found that the apolipoprotein E mimetic peptide can down-regulate the expression of TNF-α, IL-6 and IL-10 in LPS-induced THP-1 cells and human peripheral blood mononuclear cells; it can significantly reduce the mortality rate of sepsis mice, reduce the concentration of TNF-α, IL-6 and LPS in the plasma of sepsis mice, and improve the inflammatory damage to the lungs, liver, small intestine and spleen of sepsis mice; it can kill Escherichia coli, Pseudomonas aeruginosa, pan-drug resistant Acinetobacter baumannii and Staphylococcus aureus. The apolipoprotein E mimetic peptide can be further prepared into polypeptide drugs against gram-negative bacilli and pan-drug resistant gram-negative bacilli, immunomodulatory polypeptide drugs and anti-sepsis drugs.

[0004] CN118453832A discloses the application of an immunomodulatory peptide in the preparation of drugs for preventing and treating bacterial infections. The immunomodulatory peptide RDP3 derived from rice can inhibit bacterial infection in mice, and to some extent, can reduce the inflammatory damage to the lung tissue of infected mice and reduce the inflammatory factor protein level in the serum of infected mice, and can also improve the survival rate of sepsis model mice. RDP3 exerts antibacterial effect by recruiting phagocytes to the infection site. Given the unique antibacterial mechanism of RDP3, it can provide a basis for the development of RDP3 as a polypeptide anti-infection drug, and provide a candidate molecule for the development of peptide antibiotics.

[0005] It is evident that research on peptide drugs with excellent antibacterial effects as antibiotic alternatives is of great significance to the biomedical field. The synthesis process of antibacterial peptides is simple, especially antibacterial peptides with broad-spectrum antibacterial activity, which provide a sustainable, cost-effective solution for the upgrading of antibacterial drugs and have important social significance. Summary of the Invention

[0006] This invention addresses the problem of antibiotic resistance by providing a class of complex antimicrobial peptides that can treat sepsis. These peptides exhibit potent antimicrobial effects against a variety of bacteria. The complex antimicrobial peptides of this invention can be used in the preparation of coatings for drugs or medical devices that prevent or treat sepsis and septicemia caused by bacterial infections, providing important guidance for various diseases caused by bacterial infections.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A class of complex antimicrobial combination peptides for treating sepsis, wherein the amino acid sequence of the complex antimicrobial combination peptide is one of the following: Meta207: X1WLRX1LRRX2X1RRI Meta220: X2WRX3LRX3LRRLLV Meta221: IRWX1RX1LRRRLX1X1 Among them, X1, X2 and X3 is a random site, X1 is L or I, X2 is V or L, and X3 is I or V; Meta207, Meta220, and Meta221 are mixtures of all selected random sites.

[0008] The present invention also provides the use of the aforementioned complex antimicrobial peptide for treating sepsis in the preparation of products or medicines for treating and / or preventing bacterial infections.

[0009] Preferably, the product includes one or more of the following: coatings for medical devices, coatings for implantable medical devices, drugs, dressings, food preservatives, and pet disinfectants.

[0010] The bacteria are Gram-positive and / or Gram-negative.

[0011] Preferably, the bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis. The antimicrobial combination peptide of the present invention is applicable to the prevention and treatment of infections caused by a variety of bacteria, possesses broad-spectrum antibacterial activity, and exhibits potent bactericidal effects against all of the above-mentioned bacteria.

[0012] Preferably, the antibacterial concentration of the complex antimicrobial peptide in the product or drug is above 2 µg / mL, more preferably above 4 µg / mL, or above 8 µg / mL.

[0013] The present invention also provides a nucleotide sequence encoding the aforementioned complex antimicrobial combination peptide, the nucleotide sequence comprising DNA and RNA.

[0014] The present invention also provides a type of coating for a composition or implantable device containing the aforementioned complex antimicrobial peptide for treating sepsis.

[0015] Preferably, the coating of the composition or implantable device comprises the composite antimicrobial peptide and antibiotic as described in claim 1; the antibiotic comprises any one of imipenem, ertapenem, and meropenem.

[0016] Preferably, the antibiotic is imipenem.

[0017] The mass ratio of the compound antibacterial peptide to the antibiotic is 1:(0.8-1.2). Preferably, the mass ratio is 1:1.

[0018] The composition or implantable device coating contains one or more of a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, or medium.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The composite antibacterial peptides of this invention have excellent broad-spectrum antibacterial and drug resistance properties, low biotoxicity, and have been shown in experiments to be effective in improving the survival rate of septicemia mouse models when used in combination with imipenem antibiotics. They can also be used as antibacterial coating materials on the surface of implantable devices to achieve long-acting release / sustaining release, providing a new direction for clinical medical research. Attached Figure Description

[0020] Figure 1 The ability of Meta207, Meta220 and Meta221 at different concentrations to target the outer membrane permeability of Acinetobacter baumannii pathogen.

[0021] Figure 2 The ability of Meta207, Meta220 and Meta221 to depolarize the plasma membrane of Acinetobacter baumannii pathogen at different concentrations.

[0022] Figure 3 Morphology of Acinetobacter baumannii after treatment with Meta207, Meta220 and Meta221 under TEM (size: 200 nm).

[0023] Figure 4Survival rate of mice 72 hours after treatment with PBS, Meta207, Meta220 and Meta221.

[0024] Figure 5 Mouse survival rate at 72 hours after treatment with PBS, 10 mg / kg Meta220, 5 mg / kg imipenem, 10 mg / kg imipenem, or a mixture of 5 mg / kg Meta220 and 5 mg / kg imipenem. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0026] All raw materials used in the following specific implementation methods were purchased from the market.

[0027] Example 1 The synthesis of antimicrobial combination peptides specifically includes: 1. Swelling of dendrites: Weigh 2-chlorotriphenylmethyl chloride resin (2-CTC) and soak it in dichloromethane (DCM) to swell the resin for 1 hour. Then wash the resin three times with dimethyl thionamide (DMF) to remove residual solvent and impurities.

[0028] 2. Connecting the first amino acid: Take one or two protected amino acids (e.g., 1 eq of amino acid A and 1 eq of amino acid B in a 1:1 molar ratio) and react them with 1.5 eq of diisopropylethylamine (DIEA) in DMF for 2 hours to connect these amino acids to the resin.

[0029] 3. End-capping: After the resin is dried, it is washed three times with DMF, and then methanol and DIEA are added for end-capping treatment. The reaction is carried out for 1 hour to protect the amino groups on the resin.

[0030] 4. Removal of Fmoc protecting group: Use 20% piperidine in DMF to remove the Fmoc protecting group, react for 10 minutes, and repeat twice to ensure complete removal of the protecting group.

[0031] 5. Linking new amino acids: After washing the resin, add 3 eq of one or two amino acids (e.g., amino acid C and amino acid D in a 1:1 molar ratio) and 3 eq of HOBT and 3 eq of DIC, and react in DMF for 1.5 hours to link these amino acids to the peptide chain.

[0032] 6. Repeat steps 4 and 5 until all the required amino acids are added, and remove the N-terminal Fmoc protecting group. Wash the resin and dry it to prepare for the subsequent deprotection step.

[0033] 7. Deprotection and peptide chain cleavage: The reaction was carried out using 95% trifluoroacetic acid (TFA), 2% Tis (thiazolidinone), 2% EDT (ethylenediaminetetraacetic acid), and 1% water for 2 hours to cleave the protecting groups of the resin and peptide side chains.

[0034] 8. Filtration and precipitation: Filter the resin, wash the filtrate with ice-cold ether, centrifuge and retain the precipitate, which is the crude product. After freeze-drying, the final random peptide mixture is obtained.

[0035] Example 2: Antimicrobial activity analysis of three AMPCs (antimicrobial combination peptides) Step 1: Inoculate the seven bacterial cultures [Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Acinetobacter baumannii ATCC 19606, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633] into sterile MHB medium and incubate overnight at 37°C and 250 rpm for 18 hours.

[0036] Step 2: Prepare the three antimicrobial peptides to 128 µg / mL with PBS, and then serially dilute them with PBS at 2-fold concentrations of 128, 64, 32, 16, 8, 4, 2, and 1 µg / mL. Add 50 µL of each concentration to the AH wells of a 96-well plate. Repeat each antimicrobial peptide combination three times, adding to wells 1-9.

[0037] Step 3: Dilute the seven cultured bacteria to 5 × 10⁵ CFU / mL with PBS. (Due to the large number of bacterial species, only one bacterial culture is used per plate; Staphylococcus aureus will be used as an example later.) Add 50 µL of bacterial dilution to each of the AF wells containing the existing antimicrobial peptide dilution. The peptide concentrations (µg / mL) at this point are shown in Table 1 below.

[0038] Table 1. Final concentrations of the antimicrobial combination peptides Meta207, Meta220, and Meta221 in the MIC assay, in µg / mL. Step 4: Add 100µL of PBS solution to wells A10-H10 as a negative control, and add 100µL of bacterial suspension (5×10⁵ CFU / mL) to wells A11-H11 as a positive control. Seal the 96-well plate with sealing film, place it in a self-sealing bag, and incubate overnight at 37˚C for 18 hours. Measure the OD600 values ​​of wells A1-G9 using a microplate reader. The minimum concentration corresponding to the lowest OD600 value in each antimicrobial combination peptide group is the minimum inhibitory concentration (MIC) of the corresponding bacteria. The results are shown in Table 2.

[0039] Table 2. MICs (µg / mL) of the antimicrobial combination peptides Meta207, Meta220, and Meta221 against seven pathogens. The antimicrobial combination peptides Meta207, Meta220, and Meta221 are compositions of antimicrobial peptides with multiple sequences. These compositions are obtained directly through synthesis, rather than by synthesizing multiple antimicrobial peptides and then mixing them. In the table, the amino acid sequences of the peptides are represented by one random amino acid within parentheses, such as (LV) indicating L or V. For example, the specific sequence of the antimicrobial combination peptides might be as follows: Meta207 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 1~16, specifically: SEQ ID NO.1: IWLRILRRLIRRI SEQ ID NO.2: IWLRILRRLLRRI SEQ ID NO.3: IWLRILRRVIRRI SEQ ID NO.4: IWLRILRRVLRRI SEQ ID NO.5: IWLRLLRRLIRRI SEQ ID NO.6: IWLRLLRRLLRRI SEQ ID NO.7: IWLRLLRRVIRRI SEQ ID NO.8: IWLRLLRRVLRRI SEQ ID NO.9: LWLRILRRLIRRI SEQ ID NO.10: LWLRILRRLLRRI SEQ ID NO.11: LWLRILRRVIRRI SEQ ID NO.12: LWLRILRRVLRRI SEQ ID NO.13: LWLRLLRRLIRRI SEQ ID NO.14: LWLRLLRRLLRRI SEQ ID NO.15: LWLRLLRRVIRRI SEQ ID NO.16: LWLRLLRRVLRRI Meta220 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 17~24, specifically: SEQ ID NO.17: VWRILRILRRLLV SEQ ID NO.18: VWRILRVLRRLLV SEQ ID NO.19: VWRVLRILRRLLV SEQ ID NO.20: VWRVLRVLRRLLV SEQ ID NO.21: LWRILRILRRLLV SEQ ID NO.22: LWRILRVLRRLLV SEQ ID NO.23: LWRVLRILRRLLV SEQ ID NO.24: LWRVLRVLRRLLV Meta221 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 25~40, specifically: SEQ ID NO.25: IRWLRILRRRLLL SEQ ID NO.26: IRWLRILRRRLLI SEQ ID NO.27: IRWLRILRRRLIL SEQ ID NO.28: IRWLRILRRRLII SEQ ID NO.29: IRWLRLLRRRLLL SEQ ID NO.30: IRWLRLLRRRLLI SEQ ID NO.31: IRWLRLLRRRLIL SEQ ID NO.32: IRWLRLLRRRLII SEQ ID NO.33: IRWIRILRRRLLL SEQ ID NO.34: IRWIRILRRRLLI SEQ ID NO.35: IRWIRILRRRLIL SEQ ID NO.36: IRWIRILRRRLII SEQ ID NO.37: IRWIRLLRRRLLL SEQ ID NO.38: IRWIRLLRRRLLI SEQ ID NO.39: IRWIRLLRRRLIL SEQ ID NO.40: IRWIRLLRRRLII Table 2 shows that the three antimicrobial combination peptides of the present invention all exhibit good antibacterial activity against seven different bacteria, demonstrating strong efficacy and broad applicability. Among them, Meta207 has a more significant antibacterial effect against Bacillus subtilis ATCC6633, with an MIC of only 2µg / mL.

[0040] Example 3: Hemolytic activity analysis of AMPCs 1. Blood samples are collected from the recipients via vein.

[0041] 2. Pre-cool the centrifuge (2000 rpm), aliquot the collected blood into 1.5 mL ep tubes, centrifuge at 2000 rpm and 4℃ for 5 min, discard the supernatant, wash the collected blood cells three times with PBS (washing ratio, PBS: plasma = 0.35: 0.65), then dilute with PBS to 2% of the original concentration, and slowly mix on a decolorizing shaker.

[0042] 3. Place 50 µL of the combined peptides at different concentrations prepared in Example 1 (the final concentration of the combined peptides was 128-1 µg / mL, serially diluted 2-fold) and an equal volume of red blood cell suspension into each well of a 96-well plate. Add 50 µL of PBS + 50 µL of red blood cell suspension as a negative control; add 50 µL of red blood cell suspension + 50 µL of 0.2% Tritonx-100 as a positive control. Incubate the 96-well plate in a 37°C incubator for 1 hour.

[0043] 4. After culturing for 1 hour, remove the 96-well plate and centrifuge at 1000 rpm and 4℃ for 5 min.

[0044] 5. After centrifugation, aspirate the supernatant from the solution and transfer it to a clean 96-well plate. Then, use a microplate reader to analyze the solution at 570 nm (OD500). 570 The hemolysis rate is calculated by measuring the absorbance value at a certain point.

[0045] Hemolysis rate (%) = [(sample OD)] 570 - Negative control OD 570 ) / (Positive control OD570 - Negative control OD 570 )]×100%.

[0046] The minimum hemolytic concentration (MHC) is the concentration at which the antimicrobial combination peptide causes a 10% hemolysis rate. This experiment was independently repeated three times, and the average value of the three tests was used to obtain Table 3.

[0047] Table 3. HC50 of AMPCs for human erythrocytes (hRBCs), in mg / L As shown in Table 3, the HC50 values ​​of the three antimicrobial combination peptides synthesized in this invention are all more than 20 times the working concentration, indicating high safety when used in vivo. Among them, Meta220 has a higher HC50 value, and its sequence contains 4 arginine residues, which carries less positive charge compared to the other two sequences (which contain 5 arginine residues), thus exhibiting higher safety.

[0048] Example 4: Investigation of the antibacterial mechanism of AMPCs I. Effect of NPN (1-naphthylaminobenzene) uptake assay on the effect of antimicrobial combination peptides on the extracellular membrane permeability of Acinetobacter baumannii.

[0049] Logarithmically growing microbial cells were harvested (5,000 rpm, 5 min) and diluted to OD 600 = 0.2 in 5 mM HEPES buffer (pH = 7.4, containing 5 mM glucose). The bacterial suspension was further incubated with 10 μM NPN in the dark for 30 min. Subsequently, different concentrations of peptides prepared in Example 1 were added (final peptide concentrations were 128–1 µg / mL, serially diluted 2-fold), and NPN fluorescence was detected (excitation λ = 350 nm, emission λ = 420 nm). The results are as follows: Figure 1 As shown, the fluorescence signal detected after treatment with AMPCs increased significantly, indicating that the permeability of the bacterial outer membrane was significantly enhanced, and that AMPCs exerted their bactericidal effect through membrane interaction mechanisms.

[0050] II. Effect of antimicrobial combination peptides on the depolarization of the inner membrane of Acinetobacter baumannii by staining with the cell fluorescent probe DiSC3-5.

[0051] Harvest bacteria in mid-log phase and wash three times with 5 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer, then resuspend in buffer containing 0.1% glucose to OD. 600The concentration was 0.07. DiSC3-5 was added to a final concentration of 0.4 μM, and the mixture was incubated at 37°C in the dark for 90 minutes. Subsequently, 300 mM KCl was added and incubated for 20 minutes, followed by treatment with different concentrations of peptide. Fluorescence (excitation wavelength 622 nm, emission wavelength 670 nm) was monitored until the fluorescence signal stabilized, and membrane potential changes were assessed. The results were recorded, background fluorescence was subtracted, and the data were normalized. Results are as follows: Figure 2 As shown, the fluorescence signal detected after treatment with AMPCs increased significantly, indicating that the inner membrane was significantly depolarized and lost its potential. AMPCs exerted their bactericidal effect through membrane interaction mechanisms.

[0052] III. Bacterial Agglutination Test The morphology of the peptides was observed using TEM. The peptide solution was diluted to 256 μg / mL in H₂O or 50% TFE and then placed on a carbon-coated surface of a copper grid (300 square mesh). After 1 min, the grid was stained three times with 10 μL of 1% uranyl acetate aliquots, washed three times with Milli-Q water to absorb excess dye, and the samples were air-dried for at least 15 min before imaging. Results are shown below. Figure 3 It can be seen that the bacteria in the control group have good morphology, while the cell membranes of the bacteria in the combined peptide treatment group are destroyed and their contents are spilled out, showing a significant bactericidal effect.

[0053] Example 5: In vivo activity test of AMPCs in mice I. The survival and efficacy experiments were conducted in two batches, each corresponding to one of two different regimens. The first regimen included PBS, 10 mg / kg Meta207, 10 mg / kg Meta220, and 10 mg / kg Meta221. The second regimen included PBS, 10 mg / kg Meta220, 5 mg / kg imipenem, 10 mg / kg imipenem, or a mixture of 5 mg / kg Meta220 and 5 mg / kg imipenem.

[0054] II. Establishment of a mouse model of sepsis: Mice were anesthetized with sodium pentobarbital (50 mg / kg). After thorough disinfection of the abdomen with 75% ethanol, a midline incision of approximately 1.5 cm was made. The cecum was exposed and ligated below the ileocecal valve at the midpoint. Then, the intestinal wall on both sides of the ligated segment was punctured three times with an 18G needle to express a minimal amount of fecal material. The cecum was carefully repositioned back into the abdominal cavity, and the abdominal wall was closed. The sham surgery involved laparotomy without ligation or puncture. Mice received subcutaneous injection of saline for fluid resuscitation.

[0055] III. Following CLP surgery, mice were intraperitoneally injected with an antibacterial agent containing Meta207, Meta220, and Meta221, or with PBS. Mice survival was monitored within 72 hours post-CLP surgery, and the results are as follows: Figure 4 and Figure 5 As shown, the survival rate of mice 72 hours after treatment with Meta207, Meta220 and Meta221 was significantly improved, with Meta220 and Meta221 showing better results.

[0056] like Figure 5 As shown, the combined action of the antimicrobial combination peptide and imipenem significantly improved and increased the survival rate of mice, indicating that the antimicrobial combination peptide can be used to replace part of the imipenem, significantly reducing the amount of imipenem required.

Claims

1. A class of complex antimicrobial combination peptides for treating sepsis, characterized in that, The amino acid sequence of the complex antibacterial peptide is one of the following: Meta207: X1WLRX1LRRX2X1RRI Meta220: X2WRX3LRX3LRRLLV Meta221: IRWX1RX1LRRRLX1X1 Among them, X1, X2 and X3 are random sites, X1 is L or I, X2 is V or L, and X3 is I or V; Meta207, Meta220 and Meta221 are mixtures of all selected random sites.

2. The use of the compound antimicrobial combination peptide for treating sepsis according to claim 1 in the preparation of products or drugs for treating and / or preventing bacterial infections.

3. The application according to claim 2, characterized in that, The products include one or more of the following: coatings for medical devices, coatings for implantable medical devices, dressings, food preservatives, and pet disinfectants.

4. The application according to claim 2, characterized in that, The bacteria are Gram-positive and / or Gram-negative.

5. The application according to claim 2, characterized in that, The bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis.

6. The nucleotide sequence encoding the complex antimicrobial combination peptide of claim 1, characterized in that, The nucleotide sequence includes DNA and RNA.

7. A coating of a composition or implantable device comprising the complex antimicrobial peptide of claim 1 for treating sepsis.

8. The coating of the composition or implantable device according to claim 7, characterized in that, The coating of the composition or implantable device comprises the composite antimicrobial peptide and antibiotic as described in claim 1; The antibiotics include any one of imipenem, ertapenem, and meropenem.

9. The coating of the composition or implantable device according to claim 8, characterized in that, The mass ratio of the compound antibacterial peptide to the antibiotic is 1:(0.8-1.2).

10. The coating of the composition or implantable device according to claim 7, characterized in that, The coating of the composition or implantable device contains one or more of a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, or medium.

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