β-sheet self-assembly antimicrobial peptide regulated by hydrogen bonds and its preparation method and application

Abstract

The present invention discloses a β-folded self-assembly antimicrobial peptide regulated by hydrogen bonds, a preparation method and an application thereof, and its amino acid sequence is shown in SEQ ID NO.1. The antimicrobial peptide KVL-1 is designed using a "++ABBBCCCD++" template, wherein "+" is lysine, "A" and "D" are serine and tryptophan with side chains capable of forming hydrogen bonds, and "B" and "C" are valine and leucine that are easy to form a β-folded structure. The overall structure is a Bola structure with hydrophilic ends and a hydrophobic core. The antimicrobial peptide KVL-1 exhibits β-folded characteristics in PBS / SDS solution, and has a critical aggregation concentration of 12.66 μM in PBS. At high concentrations, it can form a nanochain assembly composed of spherical units arranged in a nanostructured structure, has a strong inhibitory effect on a variety of common pathogens, has no obvious hemolytic activity and cytotoxicity, and has a simple preparation process and low cost, and has the potential to be used as an antibiotic substitute.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a beta-sheet self-assembly antimicrobial peptide KVL-1 regulated by hydrogen bonds, a preparation method and an application thereof. Background Art

[0002] Antimicrobial peptides fulfill the dual role of killing pathogens and regulating immune responses within the innate immune system, while also exhibiting a low risk of drug resistance. Increasingly, research has shown that, in addition to utilizing natural antimicrobial peptides, the de novo design of short peptides has been employed to confer a high degree of programmability upon antimicrobial peptides. By appropriately manipulating the amino acid configuration, it is possible to create artificial peptides or proteins with desired functions and mitigate or overcome inherent drawbacks that hinder clinical trial progress, such as potential toxicity and physiological instability. Notably, the recent development of supramolecular self-assembling nanopeptides has demonstrated significant advantages in enhancing the antimicrobial activity, in vivo stability, and bioavailability of traditional antimicrobial peptides. Driven and stimulated by non-covalent interactions (such as hydrogen bonds, hydrophobic interactions, and electrostatic forces), peptide molecules can spontaneously assemble into microscopically regular nanostructured aggregates, such as nanofibers, particles, and micelles. Therefore, it is crucial to understand the relationship between intermolecular forces and self-assembly behavior in nanopeptides to construct highly effective and functionally stable peptide-based antimicrobial agents. Summary of the Invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide a β-sheet self-assembling antimicrobial peptide KVL-1 regulated by hydrogen bonds, which can inhibit the growth of a variety of common pathogenic bacteria at low concentrations, is basically non-toxic, has abundant intermolecular hydrogen bonds, and can self-assemble into regular nanochain structures in phosphate buffered saline (PBS).

[0004] The technical solution adopted by the present invention is as follows: a β-sheet self-assembling antimicrobial peptide KVL-1 regulated by hydrogen bonds, whose amino acid sequence is shown in SEQ ID NO.1, wherein its C-terminus is amidated with -NH2.

[0005] Furthermore, the aforementioned antimicrobial peptide KVL-1, which is self-assembled by hydrogen bonding, has a molecular formula as shown in formula (I):

[0006]

[0007] Furthermore, the present invention also provides a self-assembly method for the antimicrobial peptide KVL-1 as described above, wherein the self-assembly conditions are as follows: dissolving the polypeptide in PBS at a concentration of 12.66 μM-256 μM, ultrasonically treating for 50 min at a frequency of 40 kHz and a power of 250 W, and incubating at 37° C. for 12 h.

[0008] Another object of the present invention is to provide a method for preparing the antimicrobial peptide KVL-1, which self-assembles through hydrogen bonding. The overall peptide chain layout is designed to have a central Bola structure with hydrophilic ends and a hydrophobic core. This structure generally favors peptide self-assembly in water. Furthermore, hydrophobic amino acids, which are prone to occurring in the β-sheet region, are used to promote the formation of regular nanostructures. Amino acids with polar groups in their side chains can act as hydrogen bond donors or acceptors, stabilizing the protein structure. Therefore, a self-assembling antimicrobial peptide template "++ABBBCCCD++" was designed, in which: (1) "+" selected lysine with positive charge in a neutral environment to meet the electrostatic attraction required for contact with bacterial membranes; (2) "A" and "D" selected serine and tryptophan with side chains capable of forming hydrogen bonds to stabilize the secondary structure, while tryptophan provides additional hydrophobicity to the peptide chain; (3) "B" and "C" selected valine and leucine that are prone to appear in the hydrophobic core region of the β-sheet to provide sufficient hydrophobicity for the antimicrobial peptide, thereby enhancing its ability to destroy bacterial membranes. The final amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and the C-terminus is amidated with -NH2 to improve metabolic stability. The polypeptide was prepared by solid-phase synthesis, separated and purified by high-performance liquid chromatography, and finally freeze-dried to obtain a high-purity target polypeptide. After minimum inhibitory activity test, hemolytic activity test, cytotoxicity test, circular dichroism test, and nanocharacterization analysis test, it was finally named antimicrobial peptide KVL-1.

[0009] Another object of the present invention is to provide a use of the above-mentioned hydrogen-bonded β-sheet self-assembly antimicrobial peptide KVL-1 in the preparation of a drug for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

[0010] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli, Salmonella or Pseudomonas aeruginosa.

[0011] Furthermore, the Gram-positive bacteria mentioned above are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

[0012] The present invention also provides a drug suitable for treating infections caused by Gram-negative bacteria and / or Gram-positive bacteria, wherein the drug contains the antimicrobial peptide KVL-1 as described above.

[0013] The present invention has the following advantages and beneficial effects: The antimicrobial peptide KVL-1 exhibits strong inhibitory effects against common Gram-negative and Gram-positive bacteria at low concentrations, including Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis. No hemolysis or cytotoxicity was observed within the effective range, and the critical aggregation concentration was 12.66 μM. Circular dichroism analysis revealed that KVL-1 exhibited a typical β-pleated secondary structure. Above this concentration, it can form regularly ordered nanochain assemblies through intermolecular forces, demonstrating its potential as an antibiotic alternative. The antimicrobial peptide KVL-1 of the present invention has a short peptide chain, resulting in a simple preparation process, low synthesis cost, and a short cycle time. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the reverse phase high performance liquid chromatogram of the antimicrobial peptide KVL-1;

[0015] Figure 2 is the mass spectrum of the antimicrobial peptide KVL-1;

[0016] Figure 3 is the hemolytic activity graph of the antimicrobial peptide KVL-1;

[0017] Figure 4 is the cytotoxicity graph of the antimicrobial peptide KVL-1;

[0018] Figure 5 is the circular dichroism spectrum of the antimicrobial peptide KVL-1;

[0019] Figure 6 is the fluorescence spectrum of the antimicrobial peptide KVL-1;

[0020] Figure 7 is the critical aggregation concentration diagram of the antimicrobial peptide KVL-1;

[0021] Figure 8 This is a transmission electron microscopy image of the self-assembled antimicrobial peptide KVL-1 nanostructure. DETAILED DESCRIPTION

[0022] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0023] Example 1

[0024] The self-assembling antimicrobial peptide template "++ABBBCCCD++" designed for the antimicrobial peptide KVL-1 adopts a Bola structure with hydrophilic ends and a hydrophobic core. This design facilitates the self-assembly of the peptide in water. The specific design is as follows:

[0025] (1) “+” selects lysine, which is positively charged in a neutral environment and binds to the bacterial membrane through electrostatic attraction;

[0026] (2) “A” and “D” are serine and tryptophan, respectively, whose side chains have the ability to form hydrogen bonds. The secondary structure is stabilized by hydrogen bonds, and tryptophan can provide additional hydrophobicity;

[0027] (3) “B” and “C” use valine and leucine, which are easy to form β-folds, to enhance hydrophobicity, thereby improving the ability of antimicrobial peptides to destroy bacterial membranes.

[0028] The overall design stabilizes the structure through a hydrophobic core and hydrogen bonds, promoting self-assembly and enhancing antibacterial efficacy. The sequence of the antimicrobial peptide KVL-1 is shown in Table 1.

[0029] The amino acid sequence of the antimicrobial peptide KVL-1 is as follows:

[0030]

[0031] Table 1 Amino acid sequence of antimicrobial peptide KVL-1

[0032]

[0033] The molecular formula is shown in formula (I):

[0034]

[0035] Example 2

[0036] Synthesize linear peptides, and the peptides are synthesized from C-terminus to N-terminus.

[0037] 1. Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 mL reactor and soak it with 50 mL of dichloromethane (DCM).

[0038] 2. After 2 hours, wash the resin with nitrogen-dimethylformamide (DMF) 3 times the volume of the resin, and then dry it. Repeat this process four times. Drain the resin and set it aside for later use.

[0039] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash four times with 3 times the volume of DMF and then drain.

[0040] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0041] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxybenzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0042] 6. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for 0.5 hours, then wash it four times with DMF 3 times the volume of the resin and drain it for use.

[0043] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash with DMF four times and then drain.

[0044] 8. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0045] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50 mL centrifuge tube, add 25 mL of DMF to dissolve them, then add 2.5 mL of DIC and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0046] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops each of test A and test B, react at 100°C for 1 minute). If the resin is colorless, it means the reaction is complete; if the resin has color, it means the condensation is incomplete, and the reaction should be continued.

[0047] 11. After the reaction is complete, wash the resin four times with DMF and drain. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF and drain it to check whether the protection is removed.

[0048] 12. Follow steps 9-11 to connect the following amino acids.

[0049] 13. After the last amino acid is attached, remove the protection, wash four times with DMF, and then drain the resin with methanol. Then use 95% cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) to cut the peptide from the resin (add 10 mL of cutting solution per gram of resin) and centrifuge four times with ice ether (cutting solution: ether = 1:9). Finally, separate and purify by HPLC and freeze-dry to obtain a peptide with a purity of >95%. Figure 1-2 shown.

[0050] 14. Purification conditions:

[0051] (1) Stationary phase: C18;

[0052] (2) Configuration of mobile phase: Pump A: V(TFA) / V(water) = 1 / 1000; Pump B: V(TFA) / V(acetonitrile) = 1 / 1000; flow rate: 10 mL / min; retention time: between 20-30 min, prepared twice.

[0053] Example 3

[0054] Determination of antibacterial activity of antimicrobial peptide KVL-1

[0055] The susceptibility of antimicrobial drugs was tested by referring to the broth microdilution method with slight modifications. Under sterile conditions, the strains stored in a -20°C freezer were revived and passaged. The bacteria were cultured in MHB broth medium until the logarithmic growth phase and the turbidity of the bacterial solution was diluted to OD 600 =0.38-0.4. KVL-1 was diluted to a gradient concentration (0.25-128 μM) with 0.2% (w / v) bovine serum albumin solution in a 96-well plate. Each test well consisted of 50 μL of polypeptide solution and 50 μL of bacterial solution diluted 1000 times again. 100 μL of normally grown bacterial solution was used as a positive control, and sterile MHB medium was used as a negative control, both of which verified the accuracy and reliability of the test. After incubation at 37°C for 18-24 hours, the absorbance value of each well was measured at 492 nm using an Infinite M200 pro microplate reader (Tecan). When the absorbance value was close to that of the negative control and there was no visible bacterial growth, the concentration was determined as the minimum inhibitory concentration (MIC) of KVL-1. The MIC values ​​represent the results of six independent experiments. The results are shown in Table 2.

[0056] Table 2 Minimum inhibitory concentration of antimicrobial peptide KVL-1 (μM)

[0057]

[0058] As can be seen from Table 2, the antimicrobial peptide KVL-1 exhibited significant antibacterial activity against both common Gram-negative and Gram-positive bacteria, demonstrating its potential as a broad-spectrum antibacterial agent.

[0059] Example 4

[0060] Determination of hemolytic activity of antimicrobial peptide KVL-1

[0061] The destructive effect of the antimicrobial peptide KVL-1 on the membrane of human red blood cells (hRBCs) was evaluated by the level of hemoglobin release. The lowest concentration that caused 10% red blood cell death was defined as the minimum hemolytic concentration (MHC). 10 ). Fresh blood was centrifuged (1000g, 10min) and red blood cells were collected. Washed three times with PBS to remove residual plasma proteins and other small molecule compounds, and the red blood cells were diluted to 1% (v / v) in PBS. 50μL of gradient concentration (2-64μM) of antimicrobial peptide KVL-1 was added to a 96-well plate, and 50μL of red blood cells were added to each well. 0.1% TritonX-100-treated red blood cells and healthy red blood cells were used as positive and negative controls, respectively. After incubation at 37°C for 1h, the 96-well plate was centrifuged as a whole (1000g, 10min) and an equal amount of supernatant was drawn into a new plate, and the absorbance value of each well was measured at 570nm using a Varioskan LUX microplate reader (Thermo Fisher). The experiment was repeated three times independently. The measurement results are shown in Figure 3 The hemolysis rate was calculated by the following formula:

[0062]

[0063] The results showed that no significant hemolysis was observed within the effective antibacterial concentration range of the antimicrobial peptide KVL-1, using 10% hemolysis as the evaluation standard. This indicates that the antimicrobial peptide KVL-1 has a very low destructive effect on the red blood cell membrane and exhibits good safety. The therapeutic index is calculated using the MHC 10 In this study, no hemolytic toxicity was observed when the test concentration was 64 μM, so 128 μM was used as the MHC. 10 The final therapeutic index was 23.35, as shown in Table 3.

[0064] Table 3 Hemolytic activity and therapeutic index of antimicrobial peptide KVL-1

[0065]

[0066] Example 5

[0067] Determination of cytotoxicity of antimicrobial peptide KVL-1

[0068] The cytotoxicity of the antimicrobial peptide KVL-1 to mouse mononuclear macrophages (RAW 264.7) was determined using the MTT assay. Briefly, cells cryopreserved in liquid nitrogen were revived and subcultured until the density reached 3–5 × 10 5 cells / ml to ensure that the cells are in good growth condition. Inoculate 50μL of cell suspension per well in a 96-well plate and culture overnight at 37°C and 5% CO2. Add 50μL of gradient concentration (2-64μM) of antimicrobial peptide KVL-1 and continue to culture for 4h. Then add 5mg / mL of MTT solution to each well and incubate for 3h. Untreated normal cells and cell-free culture medium were used as positive and negative controls, respectively. After centrifuging the 96-well plate (1000g, 10min), carefully remove the supernatant and dissolve the purple precipitate with 100μL of dimethyl sulfoxide (DMSO). After 15min, the precipitate was completely dissolved and the absorbance value was measured at 570nm. The experiment was repeated three times independently. The calculation formula for cell survival rate is the same as the hemolysis rate. The measurement results are shown in Figure 4 .

[0069] like Figure 4 As shown in the data, when the concentration of antimicrobial peptide KVL-1 was in the range of 2-64 μM, the survival rate of RAW 264.7 cells was close to 100%, indicating that within this concentration range, the antimicrobial peptide KVL-1 had no significant effect on cell activity and had good biocompatibility.

[0070] Example 6

[0071] Secondary structure analysis of antimicrobial peptide KVL-1

[0072] Circular dichroism (CD) spectroscopy was used to analyze the secondary structure changes of the antimicrobial peptide KVL-1 in different environments. The antimicrobial peptide KVL-1 was diluted to 75 μM in PBS and sodium dodecyl sulfate (SDS, 30 mM) solution. A Chirascan circular dichroism spectrometer (Applied Photophysics) was used to scan in the wavelength range of 195-260 nm, and the difference in absorption coefficient (Δε) between left-handed and right-handed circularly polarized light was recorded. The experiment used a quartz cuvette with an optical path of 0.5 mm. Each sample was scanned repeatedly 3 times, and the final spectrum was averaged. The average residue ellipticity (θ M ) is calculated by the following formula: M =(θ obs ×1000) / (c×l×n), where θ obs is the observed ellipticity (mdeg) after buffer background correction, c is the peptide concentration (mM), l is the optical path length (mm), and n is the number of amino acid residues. Figure 5 .

[0073] like Figure 5 As shown, the circular dichroism spectra of the antimicrobial peptide KVL-1 in PBS and SDS exhibit similar characteristics. Under both conditions, the spectra exhibit a positive peak at approximately 200 nm (PBS: 201 nm; SDS: 199 nm) and a negative peak at 219 nm, consistent with a typical β-sheet structure. This result validates the β-sheet conformational assumption adopted in the design of the antimicrobial peptide KVL-1 and demonstrates that it possesses the secondary structural foundation for the formation of a stable nanostructure.

[0074] Example 7

[0075] Determination of critical aggregation concentration of antimicrobial peptide KVL-1

[0076] The fluorescent probe 8-aniline-1-naphthalenesulfonic acid (1,8-ANS) was dissolved in dimethylformamide (DMF) to a final concentration of 2 mM. 100 μL of the antimicrobial peptide KVL-1 at different concentrations (2-256 μM) was added to a 96-well plate and aged overnight at 37°C. Subsequently, 1 μL of 1,8-ANS solution was added to each well and incubated in the dark for 20 minutes. The negative control was a water environment without peptide. The fluorescence spectrum was collected using a SpectraMaxiD3 multifunctional microplate reader (Molecular Devices) with a step size of 10 nm. The excitation wavelength was set to 369 nm and the emission wavelength range was 420-550 nm. In the Origin software, the logarithm of the antimicrobial peptide KVL-1 concentration and the corresponding fluorescence intensity peak at 480-490 nm were used to draw a scatter plot and calculate the critical aggregation concentration (CAC), which represents the critical point at which the KVL-1 monomer molecules begin to aggregate. The measurement results are shown in Figure 6-7 .

[0077] As the concentration of the antimicrobial peptide KVL-1 increased, the fluorescence intensity showed a significant increase, with a distinct peak at 480-490 nm. Curve fitting determined the CAC value of the antimicrobial peptide KVL-1 to be 12.66 μM. This result suggests that when the concentration of the antimicrobial peptide KVL-1 exceeds 12.66 μM, its monomeric molecules can spontaneously aggregate to form macromolecules in aqueous solution.

[0078] Example 8

[0079] Nanostructure Observation of Antimicrobial Peptide KVL-1

[0080] Transmission electron microscopy observation: The antimicrobial peptide KVL-1 was diluted in PBS to a final concentration of 256 μM and 128 μM, sonicated for 50 min at a frequency of 40 kHz and a power of 250 W, and aged at 37°C for 12 h. The sample was deposited on a carbon-supported copper grid and observed using a Hitachi H-7800 transmission electron microscope (Hitachi, Japan) at 100 kV using 1% phosphotungstic acid negative staining for 2 seconds. The measurement results are shown in Figure 8 .

[0081] like Figure 8 As shown, transmission electron microscopy observations show that the antimicrobial peptide KVL-1 can form regular nanochain assemblies in PBS solution. These assemblies are composed of orderly arranged spherical structural units with smooth edges, indicating the formation of a uniformly distributed hydrogen bond network between the polypeptide monomers.

Claims

1. A β-sheet self-assembling antimicrobial peptide KVL-1 regulated by hydrogen bonds, characterized in that: Its amino acid sequence is shown in SEQ ID NO. 1, wherein its C-terminus is amidated with -NH2, and its molecular formula is shown in formula (I).

2. The self-assembly method of the antimicrobial peptide KVL-1 β-sheet self-assembly regulated by hydrogen bonds according to claim 1, characterized in that: The self-assembly conditions were as follows: the peptide was dissolved in phosphate buffer at a concentration of 12.66 μM–256 μM, ultrasonically treated, and incubated at 37°C for 12 h.

3. The method for preparing a β-sheet self-assembling antimicrobial peptide KVL-1 regulated by hydrogen bonds according to claim 1, characterized in that: The method is as follows: a peptide chain uses a template "++ABBBCCCD++", where "+" selects a positively charged lysine in a neutral environment to satisfy the electrostatic attraction necessary for contact with bacterial membranes; "A" and "D" select serine and tryptophan, whose side chains have hydrogen bonding ability to stabilize the secondary structure, while tryptophan provides additional hydrophobicity to the peptide chain; "B" and "C" select valine and leucine, which are prone to appear in the hydrophobic core region of the β-sheet, to provide sufficient hydrophobicity to the antimicrobial peptide, thereby enhancing its ability to destroy bacterial membranes; the amino acid sequence of the final peptide is shown in SEQ ID NO. 1, and the C-terminus is amidated with -NH2. The peptide is prepared by solid-phase synthesis, separated and purified by high-performance liquid chromatography, and then subjected to minimum inhibitory activity, hemolytic activity, cytotoxicity, circular dichroism, and nanocharacterization analysis tests, and finally named antimicrobial peptide KVL-1.

4. Use of the hydrogen-bond-regulated β-pleated self-assembling antimicrobial peptide KVL-1 according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria; the Gram-negative bacteria are Escherichia coli, Salmonella, or Pseudomonas aeruginosa; and the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

5. A drug for treating infections caused by Gram-negative bacteria and / or Gram-positive bacteria, comprising the antimicrobial peptide KVL-1 according to claim 1.

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