β-Hairpin Self-Assembly Capture of Antimicrobial Peptides and Its Application

Abstract

The present invention discloses a β-hairpin structure self-assembly capture antimicrobial peptide and its application, and relates to the field of biomedicine technology. The amino acid sequence of the β-hairpin structure self-assembly capture antimicrobial peptide is shown in any one of SEQ ID NO.3-6. The present invention selects the broad-spectrum antimicrobial short peptide Jelleine-1 (J-1) derived from honeybee royal jelly as a template peptide, designs and modifies it according to the amino acid sequence characteristics of the short peptide J-1, obtains a structural unit with alternating hydrophilic and hydrophobic amino acids, and uses it as a self-assembly peptide fiber skeleton. D The β-hairpin peptides designed with two turn sequences, PG and RRRF, possess dual antimicrobial and bacterial capture properties. The β-hairpin structure provided by this invention, which self-assembles and captures antimicrobial peptides, can form a fibrous network structure with excellent antibacterial efficacy, high stability, and biocompatibility, providing effective technical support for the development of new antimicrobial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a beta-hairpin structure self-assembly capturing antimicrobial peptide and application thereof. Background Art

[0002] Antimicrobial peptides, as important components of the innate immune system, exert their antimicrobial effects through physical mechanisms such as altering membrane permeability and disrupting cell membrane integrity, and are considered promising alternatives to traditional antibiotics. However, natural AMPs suffer from a range of issues, including weak bioactivity, high toxicity, and immature stability, which severely hinder their application. In recent years, advances in molecular design and optimization strategies, as well as the rapid rise of nanotechnology, have demonstrated the potential to improve the biological and chemical properties of AMPs, making their application possible.

[0003] Hydrogen bonding, ionic bonding, hydrophobic bonding, and π-π stacking interactions between amino acids are the primary driving forces of self-assembly. Supramolecular self-assembly is considered a method for enhancing the antimicrobial activity and stability of antimicrobial peptides. This invention aims to develop nanostructured antimicrobial peptides with a propensity for self-assembly, hoping to provide technical support for preventing antibiotic resistance and developing effective clinical anti-infective alternatives. Summary of the Invention

[0004] The present invention aims to provide a β-hairpin structure for self-assembly and capture of antimicrobial peptides and its application to address the aforementioned problems of the prior art. The β-hairpin structure provided by the present invention can form a fibrous network structure with excellent antibacterial efficacy, high stability, and biocompatibility, providing effective technical support for the development of new antimicrobial drugs.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a β-hairpin structure self-assembly capture antimicrobial peptide J-1-pG, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a β-hairpin structure self-assembly capture antimicrobial peptide J-1-RF, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The present invention also provides a β-hairpin structure self-assembly capture antimicrobial peptide JR-pG, the amino acid sequence of which is shown in SEQ ID NO.5.

[0009] The present invention also provides a β-hairpin structure self-assembly capture antimicrobial peptide JR-RF, the amino acid sequence of which is shown in SEQ ID NO.6.

[0010] The present invention also provides the use of the above-mentioned β-hairpin structure self-assembled capture antimicrobial peptide J-1-pG, β-hairpin structure self-assembled capture antimicrobial peptide J-1-RF, β-hairpin structure self-assembled capture antimicrobial peptide JR-pG or β-hairpin structure self-assembled capture antimicrobial peptide JR-RF in the preparation of self-assembled capture peptide fibers with antimicrobial function.

[0011] The present invention also provides a self-assembling capture peptide fiber with antibacterial function, wherein the self-assembling capture peptide fiber is obtained by self-assembling the above-mentioned β-hairpin structure self-assembling capture antimicrobial peptide J-1-pG, β-hairpin structure self-assembling capture antimicrobial peptide J-1-RF, β-hairpin structure self-assembling capture antimicrobial peptide JR-pG or β-hairpin structure self-assembling capture antimicrobial peptide JR-RF.

[0012] The present invention also provides an antimicrobial peptide JR, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] The present invention also provides the use of the above-mentioned β-hairpin structure self-assembled capture antimicrobial peptide J-1-pG, β-hairpin structure self-assembled capture antimicrobial peptide J-1-RF, β-hairpin structure self-assembled capture antimicrobial peptide JR-pG, β-hairpin structure self-assembled capture antimicrobial peptide JR-RF, self-assembled capture peptide fiber or antimicrobial peptide JR in the preparation of antimicrobial agents.

[0014] Furthermore, the pathogens targeted by the antibacterial agent include Escherichia coli, Enterobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessus or Bacillus cereus.

[0015] The present invention also provides an antimicrobial agent, the active ingredients of which include the above-mentioned β-hairpin structure self-assembled capture antimicrobial peptide J-1-pG, β-hairpin structure self-assembled capture antimicrobial peptide J-1-RF, β-hairpin structure self-assembled capture antimicrobial peptide JR-pG, β-hairpin structure self-assembled capture antimicrobial peptide JR-RF, self-assembled capture peptide fiber or antimicrobial peptide JR.

[0016] The present invention discloses the following technical effects:

[0017] The present invention selects Jelleine-1 (J-1), a broad-spectrum antibacterial peptide derived from bee royal jelly, as a template peptide, designs and modifies it according to the amino acid sequence characteristics of the short peptide J-1, obtains a structural unit with alternating hydrophilic and hydrophobic amino acids, and uses it as a self-assembling peptide fiber skeleton. DThe β-hairpin peptides, designed with two turn sequences, PG and RRRF, possess dual antimicrobial and bacterial capture properties. The self-assembling, captured antimicrobial peptides in this invention can form a fibrous network structure, exhibiting excellent antimicrobial efficacy, high stability, and biocompatibility, providing effective technical support for the development of novel antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 HPLC chromatograms of template peptide J-1 and designed peptides J-1-pG and J-1-RF;

[0020] Figure 2 HPLC chromatograms of designed peptides JR, JR-pG, and JR-RF;

[0021] Figure 3 Mass spectra of template peptide J-1 and designed peptides J-1-pG and J-1-RF;

[0022] Figure 4 Mass spectra of designed peptides JR, JR-pG, and JR-RF;

[0023] Figure 5 Circular dichroism spectra of template peptide J-1 and designed peptides J-1-pG, J-1-RF, JR, JR-pG, and JR-RF in three different environments;

[0024] Figure 6 Statistical graph of the toxicity test results of template peptide J-1 and designed peptides J-1-pG, J-1-RF, JR, JR-pG and JR-RF on RAW264.7 macrophages;

[0025] Figure 7 The results of the critical aggregation concentration determination of β-hairpin are shown in Figure 2. A is the fluorescence intensity of hairpin peptide J-1-RF at different concentrations; B is the linear fitting graph of hairpin peptide J-1-RF concentration and fluorescence intensity; C is the fluorescence intensity of hairpin peptide JR-pG at different concentrations; D is the linear fitting graph of hairpin peptide JR-pG concentration and fluorescence intensity;

[0026] Figure 8 Transmission electron micrographs of β-hairpin J-1-RF at different concentrations;

[0027] Figure 9 Atomic force microscopy for β-hairpin J-1-RF;

[0028] Figure 10 The results of the bacterial capture ability test of β-hairpin are shown in Figure 1. A is the cuvette observation diagram; and B is the statistical diagram of the CFU value of Escherichia coli. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1 Polypeptide sequence design and physicochemical parameter analysis

[0035] The invention selects the broad-spectrum antimicrobial peptide Jelleine-1 (i.e. J-1) from honeybee royal jelly as a template, replaces the first amino acid at its N-terminus with arginine, and constructs a β-sheet structure sequence with alternating hydrophilic and hydrophobic amino acids as a β-hairpin structure self-assembling peptide fiber skeleton;D The PG and RRRF turn sequences were used for sequence design, and a total of four β-hairpin peptides (J-1-pG, J-1-RF, JR-pG, and JR-RF) were obtained. The physicochemical parameters and molecular structures of all tested peptides are shown in Tables 1 and Figure 1 shown.

[0036] Table 1 Amino acid sequence and physicochemical parameters of target peptides

[0037]

[0038]

[0039] Note: a Actual molecular mass was determined by mass spectrometry; b Isoelectric point was calculated using the online tool PepCalc.com-Peptidecalculator; p stands for D P, namely D-proline.

[0040] Hydrophilic cationic residues and hydrophobic residues play an important role in electrostatic interactions with negatively charged bacterial membranes and in promoting the penetration of peptide sequences into the hydrophobic domains of bacterial membranes, respectively. The number of positive charges on the designed peptides in the study was increased compared to the template peptides, providing appropriate electrostatic interactions for antibacterial effects.

[0041] Example 2 Synthesis and Identification of Polypeptides

[0042] 1. Synthesis of antimicrobial peptides

[0043] The peptides shown in Table 1 were synthesized using solid phase synthesis:

[0044] Weigh the resin, add dichloromethane and soak for 5 minutes, wash with N,N-dimethylformamide and then remove the dichloromethane, then use the prepared deprotecting agent to remove the Fmoc protecting group on the resin (decapping), the decapping time is 20 minutes; weigh the second amino acid at the C-terminus, add the condensing agent, N,N-dimethylformamide, and place it in the reaction column for reaction (airing); use the ninhydrin method for detection, the test result is that the solution is bright yellow, the resin is transparent, and there is no variegated color; use the deprotecting solution to remove the Fmoc protecting group on the second amino acid, wash with N,N-dimethylformamide 6 times, repeat the operation for the remaining amino acids until the last amino acid; use the deprotecting solution to remove the last Fmoc protecting group, and then wash with N,N-dimethylformamide; after the reaction is completed, wash and shrink with dichloromethane and methanol respectively; cut the polypeptide with a cleavage solution, and precipitate the liquid with ether; analyze with a mass spectrometer (MS) and a high performance liquid chromatography (HPLC) for purification.

[0045] 2. Purification of antimicrobial peptides

[0046] (1) Pre-analysis: Take a sample and place it in a 0.5 mL tube, dissolve it in ultrapure water, filter it with a 0.45 μm membrane, and then analyze it by rapid gradient HPLC (10-100%).

[0047] (2) Sample treatment: Add 200 mg of sample to a 20 mL beaker, followed by 15 mL of H2O and 5 mL of methanol. Ultrasonicate the sample until it is completely dissolved, then filter the solution through a 0.45 μm membrane.

[0048] (3) High Performance Liquid Chromatography (HPLC): Samples were collected from 0 to 25 minutes. Pump A was 100% acetonitrile plus 0.1% trifluoroacetic acid, and pump B was 100% water plus 0.1% trifluoroacetic acid. The collected fractions were analyzed by HPLC to check purity.

[0049] The chromatograms of the template peptide and the designed peptide after purification are shown in Figure 2. Figure 1-Figure 2 The results showed that each designed peptide had a distinct absorption peak. The purity of the purified peptide reached over 95%.

[0050] 3. Identification of antimicrobial peptides

[0051] The purified peptides were collected and the target peaks were identified by LC 6000 reverse phase preparative chromatography and Waters 2000 mass spectrometer, and the actual peptide molecular weight and purity were finally analyzed. Figure 3-Figure 4 As shown. The results showed that the actual relative molecular mass was basically consistent with the theoretical value, indicating that all peptides were successfully synthesized. The results showed that the actual relative molecular mass was basically consistent with the theoretical value, indicating that all peptides were successfully synthesized.

[0052] Example 3 Determination of the secondary structure of antimicrobial peptides

[0053] 1. Determination of the secondary structure of antimicrobial peptides

[0054] Circular dichroism (CD) was used to determine the secondary structures of the antimicrobial peptides (shown in Table 1).

[0055] The structure of the peptide was simulated in three different solution environments (water, prokaryotic cell membrane, and biomembrane hydrophobic environment). The peptide was dissolved in 10mM PBS buffer solution (pH = 7.4), 30mM sodium dodecyl sulfate (SDS), and 50% trifluoroethanol (TFE) to prepare a peptide solution with a final concentration of 150μM. The results were scanned at room temperature using a circular dichroism spectrometer within the wavelength range of 190nm to 250nm at a scanning rate of 1nm / second. Figure 5 shown.

[0056] In PBS buffer, J-1 exhibits a negative absorption peak at 198 nm, indicating a random coil conformation. However, in SDS buffer, JR, J-1-pG, JR-pG, and JR-RF exhibit positive absorption peaks at 203 nm and negative absorption peaks around 216 nm, indicating a typical β-sheet structure. This suggests that the alternating arrangement of hydrophilic and hydrophobic amino acids facilitates the formation of the peptide's β-sheet conformation in a simulated hydrophobic environment.

[0057] Example 4 Determination of antibacterial activity

[0058] Minimum inhibitory concentration (MIC) determination: Antimicrobial activity was determined using the doubling microdilution method. Test strains were cultured to the logarithmic growth phase. Different concentrations of peptides (shown in Table 1) and bacterial suspension were added to rows 1-11 of a 96-well plate and incubated at 37°C for 16 hours. Bacterial suspension and culture medium without peptide served as positive and negative controls, respectively. The MIC values of the peptides were expressed as the critical concentration at which no turbidity or precipitation was observed. The results are shown in Tables 2 and 3.

[0059] Table 2 MIC values of short peptides against Gram-negative bacteria (μM)

[0060]

[0061]

[0062] Table 3 MIC (μM) of short peptides against Gram-positive bacteria

[0063]

[0064] The five derived peptides (JR, J-1-pG, J-1-RF, JR-pG and JR-RF) all exhibited broad-spectrum antibacterial activity, and their antibacterial activity was significantly enhanced compared with that of the template peptide. Among them, the designed peptides JR and J-1-RF showed enhanced antibacterial activity, which may be due to the appropriate increase in the number of positive charges.

[0065] Example 5 Cytotoxicity Assay

[0066] The cytotoxicity of peptides J-1, JR, J-1-pG, J-1-RF, JR-pG and JR-RF was tested:

[0067] The cytotoxicity of peptides in mouse macrophages (RAW 264.7) was assessed using a CCK-8 assay. Cells were seeded into individual wells of a 96-well plate and exposed to varying concentrations of peptide solutions for 12 hours at 37°C in a 5% CO2 atmosphere. After the incubation period, 10 μL of CCK-8 solution was added to each well. The cells were allowed to stand for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Positive controls included cells without peptide treatment, and negative controls included complete culture medium.

[0068] like Figure 6 As shown in the results, the cell survival rate gradually decreased with increasing experimental concentration. At a concentration of 4 μM, the cell survival rate of melittin was less than 20%. In contrast, the cell survival rate of the peptide designed by the present invention was still greater than 80% at a test concentration of 64 μM. These results demonstrate that the series of derivative peptides designed by the present invention have good biocompatibility.

[0069] Example 6 Critical Aggregation Concentration (CAC) Determination

[0070] Determination of the CAC of peptides J-1, JR, J-1-pG, J-1-RF, JR-pG, and JR-RF using the ANS fluorescence assay:

[0071] ANS was dissolved in dimethylformamide at a concentration of 1 mM, and 1 μL of this solution was then added to 100 μL of peptides at different concentrations. The mixed solution was transferred to a quartz cuvette and measured using an F-4500 fluorescence spectrophotometer (Hitachi, Japan), and fluorescence spectra were collected from 420 to 550 nm under 360 nm excitation. The CAC was determined by a matching curve obtained by plotting the ANS fluorescence intensity at 475 nm against different peptide concentrations. The results are shown in Figure 2. Figure 7 shown.

[0072] The results showed that the critical aggregation concentrations of the two hairpin peptides, J-1-RF and JR-pG, were 12.45 μM and 13.03 μM, respectively. Therefore, above this critical concentration, hairpin peptides can self-assemble into nanostructures, thereby achieving excellent bioactivity and stability.

[0073] Example 7 Characterization of the Self-Assembled Capture Peptide Fiber Structure

[0074] To observe the self-assembly structure of the β-hairpin peptide, negative staining transmission electron microscopy and atomic force microscopy were used to observe the microstructure of the peptide and evaluate its ability to form a fiber structure. The steps are as follows:

[0075] 10 μL of different concentrations of hairpin peptide J-1-RF solution were respectively taken and evenly coated on a copper grid (400 square mesh), adsorbed for 5 minutes, stained with 0.1% phosphotungstic acid for 30 seconds, dried at room temperature for 15 minutes, and observed using a transmission electron microscope. The results are as follows Figure 8 shown.

[0076] The results showed that when the concentration of J-1-RF was 12 μM, the nanofibers observed by transmission electron microscopy were only short and thin, indicating that they were in the early stages of aggregation. When the concentration increased to 32 μM and 128 μM, the hairpin peptide J-1-RF further self-assembled into supramolecular nanostructures. The antiparallel β-sheet dominated and drove the formation of twisted and dense fiber structures, indicating concentration-driven self-assembly. Atomic force microscopy was used to investigate the formation of fiber morphology of J-1-RF hairpin peptide (32 μM), and the same results were obtained ( Figure 9 ).

[0077] Example 8 Bacterial Agglutination Test

[0078] To evaluate the ability of antimicrobial peptides to capture pathogens, peptides J-1, JR, J-1-RF, and JR-pG were mixed with Escherichia coli culture solutions in sterile cuvettes, and bacterial agglutination was observed. The supernatant of the culture solution was removed and the viable bacteria were counted after gradient dilution.

[0079] The specific test steps are as follows:

[0080] (1) Bacterial preparation: Escherichia coli ATCC 25922 was used as the model strain, and a bacterial suspension grown to the logarithmic phase was obtained. The suspension was centrifuged at 3000 rpm for 5 min and washed three times with HEPES buffer (pH = 7.0).

[0081] (2) Co-culture of peptide and bacteria: OD 600 = 0.25 in 3 mL of bacterial solution in an ethanol-sterilized cuvette, add 32 μM nanopeptide solution, and let it stand at room temperature for 8 h. Observe the bacterial agglutination state and take pictures. The group without peptide treatment was used as the control group.

[0082] (3) Result evaluation: 50 μL of bacterial supernatant was aspirated at 0, 30, 60, and 90 min, and serially diluted with 450 μL of autoclaved PBS. 100 μL of the dilution was evenly inoculated onto MHA plates. The cells were incubated overnight at 37°C. The number of colonies in each sample was counted, and the CFU value was calculated for each sample. The experiment was repeated three times.

[0083] The results are as follows Figure 10As shown in the results, it was found that with the increase of time, β-hairpin induced the rapid agglutination and sedimentation of Escherichia coli, and the total number of colonies in the supernatant was significantly reduced. Among them, J-1-RF could basically induce the complete sedimentation of bacteria after 90 minutes, and had a strong bacteria capture effect.

[0084] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A β-hairpin structure self-assembly captures antimicrobial peptide J-1-pG, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. A use of the β-hairpin structure self-assembly capturing antimicrobial peptide J-1-pG as claimed in claim 1 in the preparation of an antimicrobial agent, characterized in that: The pathogens targeted by the antibacterial agent are Escherichia coli, Enterobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessus or Bacillus cereus.

3. An antibacterial agent, characterized in that The active ingredient comprises the beta-hairpin structure self-assembled capture antimicrobial peptide J-1-pG according to claim 1.

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