An antimicrobial peptide and its application
By designing antimicrobial peptides E09 and E10 with D-type amino acid sequences, the problem of drug resistance in Gram-negative bacteria was solved, achieving efficient killing and good stability against multidrug-resistant strains, making them suitable for various application scenarios.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING UNIV OF TECH
- Filing Date
- 2026-06-18
- Publication Date
- 2026-07-17
AI Technical Summary
The problem of resistance to Gram-negative bacteria is serious among existing antibiotics, and there is a lack of highly effective and safe treatments. Furthermore, natural antimicrobial peptides are prone to developing resistance and have insufficient safety profiles.
Antimicrobial peptides with D-type amino acid sequences E09 and E10 were designed and synthesized using the Fmoc solid-phase chemical method. They exhibit good targeting biofilm activity and high stability, and can be applied to the treatment of various Gram-negative bacteria.
It exhibits broad-spectrum killing activity against multidrug-resistant strains such as multidrug-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa, with good stability, high biosafety, and low hemolytic toxicity. It is suitable for preparing antibacterial drugs, toiletries, food preservatives, and antibacterial agents for medical consumables.
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Abstract
Description
Technical Field
[0001] This invention relates to an antimicrobial peptide and its applications, and relates to the field of polypeptides. Background Technology
[0002] Bacterial infections are a global health concern, causing millions of deaths annually. Among the many pathogens causing clinical infections, Gram-negative bacteria are particularly challenging. The spread of antibiotic resistance is rapidly increasing worldwide, and its severity continues to escalate, becoming one of the most serious medical threats in the public health field. In the World Health Organization's list of priority drug-resistant pathogens, the ESKAPE pathogen consortium is a widely accepted group of "superbugs" that easily cause major nosocomial infections. This consortium includes Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. These are among the earliest identified key multidrug-resistant bacteria requiring effective treatment and are currently a major target of antimicrobial drug research.
[0003] Antimicrobial peptides (AMPs) are a class of short peptides containing 10-50 amino acids. As key components of the biological innate immune defense system, they are found in animals, plants, and microorganisms, and can also be synthesized artificially. Due to their unique physicochemical properties, they exhibit significant potential surpassing traditional antibiotics. AMPs possess advantages such as broad-spectrum antibacterial activity, highly efficient bactericidal activity, and low likelihood of inducing drug resistance. They primarily exert their effects by directly disrupting the integrity of the bacterial outer membrane and are considered the best alternative to traditional antibiotics. Therefore, developing novel antimicrobial peptides with high efficacy, protease stability, and safety as therapeutic agents for Gram-negative bacteria has become a current focus of breakthrough research in anti-infective drugs. Summary of the Invention
[0004] To address the aforementioned technical problems, the present invention aims to provide an antimicrobial peptide. The present invention also provides applications of this antimicrobial peptide, which exhibits strong antimicrobial activity, good biosafety, low hemolytic toxicity, and good stability.
[0005] To achieve the above objectives, the technical solution of the present invention is: an antimicrobial peptide, characterized in that its amino acid sequence and structural formula are as follows:
[0006] E09: wrrwrrwkr;
[0007] The structural formula is:
[0008] ;
[0009] Or E10: wkrwrrwwkr;
[0010] The structural formula is:
[0011] All amino acids are D-type amino acids. E09 and E10 are corresponding to SEQ ID No. 1 and SEQ ID No. 2 in the sequence listing, respectively.
[0012] The application of the aforementioned antimicrobial peptide in the preparation of antimicrobial drugs, wherein the bacteria are Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa.
[0013] The application of the aforementioned antimicrobial peptide in the preparation of antimicrobial drugs, wherein the bacteria are multidrug-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa.
[0014] The application of the aforementioned antimicrobial peptide in the preparation of personal care products, food preservatives, antimicrobial agents for medical consumables, and cosmetics.
[0015] Compared to traditional L-type antimicrobial peptides, D-type antimicrobial peptides exhibit better resistance to enzymatic degradation and greater stability. D-type antimicrobial peptides demonstrate excellent targeting properties against bacterial biofilms, capable of disrupting existing biofilms and inhibiting the development of antibiotic resistance. These antimicrobial peptides can be synthesized using the Fmoc solid-phase chemical method, which is relatively easy to synthesize.
[0016] Beneficial effects:
[0017] This invention relates to a D-type antimicrobial peptide designed based on the configuration of a natural antimicrobial peptide. This peptide exhibits excellent antimicrobial activity against a variety of pathogenic Gram-negative bacteria. It demonstrates broad-spectrum killing activity against multidrug-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, and standard strains of Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa, exhibiting good stability, good biocompatibility, and low hemolytic toxicity. At medium to high concentrations (32 μg / mL to 256 μg / mL), cell viability is good, with low cytotoxicity. The invention is highly operable and cost-effective. It addresses the problems of easy drug resistance and insufficient safety associated with natural AMP, and shows great promise for clinical application. Attached Figure Description
[0018] Figure 1 This is a high-performance liquid chromatogram of the antimicrobial peptide E09 of the present invention.
[0019] Figure 2 This is the mass spectrum of the antimicrobial peptide E09 of this invention.
[0020] Figure 3 This is a high-performance liquid chromatogram of the antimicrobial peptide E10 of the present invention.
[0021] Figure 4 This is the mass spectrum of the antimicrobial peptide E10 of the present invention.
[0022] Figure 5 This is a graph showing the results of the stability test of the antimicrobial peptide.
[0023] Figure 6 This is a graph showing the results of an antimicrobial peptide safety experiment.
[0024] Figure 7 This is a graph showing the hemolytic toxicity results of the antimicrobial peptides. Detailed Implementation
[0025] The present invention will be further described below with reference to the embodiments and accompanying drawings:
[0026] Example 1
[0027] Synthesis of antimicrobial peptides
[0028] Target product E09, sequence is
[0029] E09: wrrwrrwkr(dTrp-dArg-dArg-dTrp-dArg-dArg-dTrp-dLys-dArg).
[0030] The structural formula is:
[0031] .
[0032] The target product E10 has the following sequence:
[0033] E10:
[0034] wkrwrrwwkr(dTrp-dLys-dArg-dTrp-dArg-dArg-dTrp-dTrp-dLys-dArg).
[0035] The structural formula is:
[0036] .
[0037] E09 and E10 correspond to SEQ ID No. 1 and SEQ ID No. 2 in the sequence list, respectively.
[0038] Taking antibacterial nonapeptide E09 as an example, the solid-phase synthesis method is as follows:
[0039] Target sequence (N end → C end): dTrp-dArg-dArg-dTrp-dArg-dArg-dTrp-dLys-dArg.
[0040] 1. Resin activation and pretreatment
[0041] Weigh 1 g of dichloromethane resin (0.3 mmol / g) and place it in the reaction column of a peptide solid-phase synthesizer. Add an appropriate amount of dichloromethane and shake to swell for 30 min, then filter and drain the liquid. Dissolve 300 mg of Fmoc-dArg(Pbf)-OH in an appropriate amount of dichloromethane, add 193 mg of diisopropylethylamine, mix, and then add the mixture to the synthesizer to react with the resin for 2 h.
[0042] 2. Synthesis of peptide chains
[0043] The resin was treated with an N,N-dimethylformamide solution containing 20% piperidine to remove the Fmoc protecting group for 20-30 minutes. The resin color was tested using the ninhydrin colorimetric method; a blue-purple color indicated the protecting group had been removed. The resin was then washed. The desired amino acid to be added was dissolved and mixed with 137 mg of 1-hydroxybenzotriazole, 271 mg of O-benzotriazole-tetramethylurea hexafluorophosphate, and 193 mg of diisopropylethylamine in 20 mL of N,N-dimethylformamide. This mixture was then added to a synthesizer and condensed for 1 hour. The ninhydrin colorimetric method was used to test the resin; a colorless and transparent color indicated successful condensation. The feeding sequence for D-type amino acids is: Fmoc-dArg(Pbf)-OH-Fmoc-dLys(Boc)-OH (300mg)-Fmoc-dTrp(Boc)-OH (300mg)-Fmoc-dArg(Pbf)-OH (300mg)-Fmoc-dArg(Pbf)-OH (300mg)-Fmoc-dTrp(Boc)-OH (300mg)-Fmoc-dArg(Pbf)-OH (300mg)-Fmoc-dArg(Pbf)-OH (300mg)-Fmoc-dTrp(Boc)-OH (300mg)
[0044] After all amino acid condensation is complete, N,N-dimethylformamide containing 20% piperidine is added and reacted for 30 min to remove the Fmoc protecting group on the last D-Trp at the N-terminus. The mixture is then washed and dried for later use.
[0045] 3. Peptide cleavage
[0046] The peptide resin with side-chain protecting groups obtained in the above steps was used as a cleavage reagent in a mixed solution containing 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% H2O. The reaction was carried out at room temperature for 2-3 hours to cleave and deprotect the side chains. The lysate was collected by filtration, and a large amount of ice-cold ether was added to the filtrate to precipitate the peptide. After centrifugation, the precipitate was washed several times with ice-cold ether, reconstituted with water / acetonitrile, and then freeze-dried to obtain crude peptide lyophilized powder.
[0047] 4. Purification
[0048] The obtained crude peptide lyophilized powder was dissolved and then purified by preparative HPLC using a high-purity silica gel column. During purification, mobile phases A and B were 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution, respectively.
[0049] 5. Structural identification
[0050] The purified lyophilized peptide powder was dissolved in 50% acetonitrile solution, and its structure was identified by LC-MS (Liquid Chromatography-Mass Spectrometry). The molecular weight of the E09 antibacterial nonapeptide was determined to be 1486.30. The structural identification diagram is shown below. Figure 1 , Figure 2 The synthesis method for E10 is the same as described above. The structural identification diagram of E10 is shown below. Figure 3 , Figure 4 The molecular weight of the E10 antimicrobial peptide was measured to be 1644.10.
[0051] Experimental Example 1
[0052] Antimicrobial activity test of minimum inhibitory concentration (MIC) of antimicrobial peptides
[0053] Drug-resistant strains: Multidrug-resistant Acinetobacter baumannii (MDRAB) and carbapenem-resistant Pseudomonas aeruginosa (CRPA).
[0054] Standard strains: Escherichia coli (ATCC25922), Acinetobacter baumannii (ATCC19606), Klebsiella pneumoniae (ATCC700603), Enterobacter cloacae (ATCC13047), and Pseudomonas aeruginosa (ATCC27853).
[0055] The antibacterial activity test method is briefly described below:
[0056] According to the guidelines of the Clinical and Laboratory Standards Institute (CLSI), the antimicrobial activity of antimicrobial peptides was determined using the microplate serial dilution method. First, the bacterial culture was removed from a -80°C freezer and revived. The activated strain was then inoculated onto LB agar using the streak plate method and incubated overnight at 37°C. Single colonies from the agar plates were then inoculated onto MHB agar and incubated with shaking at 37°C for 18 hours to allow the colonies to reach the logarithmic growth phase (10⁻¹⁰). 8(CFU / mL). The bacterial culture was then centrifuged, the supernatant was discarded, and the culture was resuspended in MHB medium and diluted to 10⁻⁶. 5 CFU / mL. In sterile 96-well plates, serially diluted antimicrobial peptide solutions and equal volumes of diluted bacterial suspension were added sequentially (256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL), and incubated at 37°C for 18 h. The absorbance at 600 nm was measured using a multi-mode microplate reader. The lowest antimicrobial peptide concentration with an OD value close to that of the blank control group (sterile MHB medium) was selected as the MIC. The experiment was repeated three times.
[0057] Table 1. Antimicrobial activity of antimicrobial peptides E09 and E10
[0058]
[0059] The results are shown in Table 1. Antimicrobial peptides E09 and E10 have strong antimicrobial activity against Gram-negative bacteria, especially against ATCC25922 and ATCC700603, with antimicrobial activity reaching 2 μg / mL and 4 μg / mL, respectively.
[0060] Experiment Example 2: Stability Test of Antimicrobial Peptides
[0061] 1. Protease stability
[0062] The MIC values of antimicrobial peptides were determined by incubating them with different types of proteases to assess their protease stability under complex enzymatic environments. Antimicrobial peptide solutions diluted twofold were mixed with trypsin, pepsin, and proteinase K, respectively, to a final enzyme concentration of 1 mg / mL. The mixtures were incubated at 37°C for 1 h to promote the enzyme reaction. Subsequently, the samples were treated in a boiling water bath for 30 min to inactivate the proteases and terminate the reaction. Finally, the MIC values of the treated antimicrobial peptides against *Escherichia coli* were determined according to the same method used for MIC determination, assessing the antimicrobial peptides' resistance to enzymatic degradation. Untreated antimicrobial peptides served as a blank control, and the above experiments were independently repeated three times.
[0063] 2. Temperature stability
[0064] To assess the stability of antimicrobial peptides under temperature conditions, antimicrobial peptide solutions diluted twofold were treated in a water bath at different temperatures (0°C ice-water mixture, 37°C, 60°C, and 100°C) for 30 min. After cooling to room temperature, the MICs of the antimicrobial peptides treated at different temperatures against *Escherichia coli* were determined according to the method for determining MICs, thus evaluating the effect of temperature on antimicrobial peptides. Untreated antimicrobial peptides served as a blank control. The above experiments were independently repeated three times.
[0065] 3. Salt ion concentration stability
[0066] To assess the stability of antimicrobial peptides in physiological saline environments, antimicrobial peptide solutions diluted twofold were mixed with different salt ion solutions (150 mM NaCl, 6 mM NH4Cl, and 2.5 mM CaCl2) to simulate different ion concentrations in tissue fluids. The mixtures were incubated in a 37°C water bath for 30 min, and then the MICs of the antimicrobial peptides treated with different salt ion solutions against *Escherichia coli* were determined using the same method as for MIC determination, to evaluate the effect of different salt ion solutions on the antimicrobial peptides. Untreated antimicrobial peptides served as a blank control, and the above experiments were independently repeated three times.
[0067] 4. Serum stability
[0068] To assess the stability of antimicrobial peptides under different serum environments, different concentrations of fetal bovine serum (FBS) were added to MHB medium. Escherichia coli bacterial suspensions were mixed with different concentrations of FBS to achieve final concentrations of 0%, 5%, and 10% (v / v). The mixtures were incubated at 37°C for 30 min to simulate different serum concentrations. Subsequently, the MIC of the antimicrobial peptides treated with different serum concentrations against Escherichia coli was determined using the method for determining MIC, to evaluate the effect of different serum concentrations on the antimicrobial peptides. Antimicrobial peptides not treated with serum solution served as a blank control. The above experiments were independently repeated three times.
[0069] The results are as follows Figure 5 As shown, antimicrobial peptides E09 and E10 react with different proteases ( Figure 5 a) Temperature Figure 5 b) Physiological salt ion concentration ( Figure 5 c), and serum concentration ( Figure 5After treatment with d), the antimicrobial peptides maintained stable antimicrobial activity and exhibited high tolerance, demonstrating good biological stability. After co-incubation with different proteases, the antimicrobial peptides maintained relatively ideal antimicrobial activity, with their MIC values showing a two-fold increase. In different salt ion solutions, the MIC values of antimicrobial peptides E09 and E10 against Escherichia coli showed a two-fold increase, indicating good tolerance to antimicrobial activity. After water bath treatment at 100℃ for 1 hour, the MIC value of E09 increased fourfold, and the MIC value of E10 increased twofold, maintaining good antimicrobial activity in a relatively stable state. In different serum concentrations, when the FBS concentration reached 10%, the MIC values of antimicrobial peptides E09 and E10 increased by approximately two-fold, indicating good serum stability and suitability as candidate drugs with good safety.
[0070] Experimental Example 3: Hemolytic Toxicity of Antimicrobial Peptides
[0071] Sterile defibrinated sheep blood was centrifuged at 3000 rpm for 10 min at 4°C, the supernatant was discarded, and the blood was washed three times with sterile PBS and resuspended as a 3% (v / v) red blood cell suspension. The red blood cell suspension was incubated with antimicrobial peptide solutions of different concentrations at 37°C for 30 min, centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was transferred to a 96-well plate. The absorbance (OD570) at 57 nm was measured using a microplate reader, and the hemolytic activity was calculated. An equal volume of sterile PBS was added as a negative control, and an equal volume of 0.1% Triton-X100 was added as a positive control. The experiment was repeated three times, and the hemolytic activity was calculated. The formula for hemolytic activity is as follows:
[0072] Hemolytic activity = (OD) n -OD0) / (OD1-OD0)*100%
[0073] (where OD) n These are the OD values for the experimental group, OD0 is the OD value for the negative control group, and OD1 is the OD value for the positive control group.
[0074] See results Figure 6 As shown, antimicrobial peptides E09 and E10 exhibit low hemolytic toxicity to sheep erythrocytes, and at effective antibacterial concentrations, they cause almost no damage to erythrocytes. When the concentration reaches 256 µg / mL, the hemolysis rate of antimicrobial peptide E09 is 9.65%, and the hemolysis rate of antimicrobial peptide E10 is 7.80%, both showing relatively weak hemolytic effects, indicating that they have good cell compatibility.
[0075] Experimental Example 4: Cytotoxicity of Antimicrobial Peptides
[0076] The cytotoxic effects of antimicrobial peptides on cells from different sources were evaluated using the CCK-8 assay. Cell lines used included rat embryonic cardiomyocytes (H9c2 cells), African green monkey kidney cells (Vero cells), normal human hepatocytes (THLE-3 cells), and normal human colonic mucosal epithelial cells (NCM460 cells). Cells in the logarithmic growth phase were seeded into cell culture dishes and incubated overnight at 37°C with 5% CO2 to ensure complete adhesion. After discarding the culture medium and washing with sterile PBS, the cells were digested with 1 mL of 0.2% trypsin solution, and then the trypsin was discarded. Fresh culture medium was then added to dilute the cells to a final concentration of 2 × 10⁻⁶ cells / mL. 4 Cells / well. The antimicrobial peptide solution, diluted twofold, was added to 96-well plates containing cell suspension and incubated at 37°C and 5% CO2 for 2 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 2 hours. The absorbance (OD450) of each well was measured using a microplate reader at 450 nm, and cell viability was calculated for each group. Untreated cells served as a negative control, and cells containing only culture medium served as a blank control. The experiment was repeated three times, and cell viability was calculated for each group. The cell viability formula is as follows:
[0077] Cell viability = (OD) n - OD0) / (OD c - OD0) * 100%
[0078] (where OD) n These are the OD values of the experimental group of cells treated with OD0, which is the OD value of the culture medium only. c (This refers to the OD value of the untreated cell group.)
[0079] The results are as follows Figure 7 As shown, at the bactericidal concentration of the antimicrobial peptides, there was almost no damage to normal tissue cells, and the survival rate of normal cells was not affected. Within the tested concentration range of the antimicrobial peptides, neither of the two antimicrobial peptides showed significant cytotoxicity. At a high concentration of 256 µg / mL, the survival rate of all four cell types remained above 90%, with low toxicity and almost no toxicity. This indicates that the antimicrobial peptides have good biocompatibility and extremely high drug-likeness, making their antimicrobial activity even more significant.
[0080] This invention is not limited to the above embodiments. Those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this invention. The scope of this invention is defined by the claims and their equivalents.
Claims
1. An antimicrobial peptide, characterized in that, The amino acid sequence and structural formula are as follows: E09: wrrwrrwkr; The structural formula is: ； Or E10: wkrwrrwwkr; The structural formula is: All amino acids are D-type amino acids.
2. The use of the antimicrobial peptide of claim 1 in the preparation of an antimicrobial drug, wherein the bacteria are Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa.
3. The use of the antimicrobial peptide of claim 1 in the preparation of an antimicrobial drug, wherein the bacteria are multidrug-resistant Acinetobacter baumannii or carbapenem-resistant Pseudomonas aeruginosa.
4. The use of the antimicrobial peptide of claim 1 in the preparation of personal care products, food preservatives, antimicrobial agents for medical consumables, and cosmetics.