Nano antibacterial peptide based on leucine zipper as well as preparation method and application of nano antibacterial peptide
By designing the leucine zipper structure of the antimicrobial peptide KLL4, the problems of high toxicity and poor biocompatibility in the self-assembly process of existing antimicrobial peptides were solved. Stable nanostructures were formed at low concentrations, showing strong antibacterial activity against a variety of bacteria and good biocompatibility.
Patent Information
- Application Number
- CN202511526585.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-24
- Publication Date
- 2026-02-06
AI Technical Summary
Existing antimicrobial peptides suffer from high toxicity and poor biocompatibility during self-assembly, making it difficult to form stable nanostructures at low concentrations, thus limiting their antimicrobial activity.
A nano-antimicrobial peptide KLL4 based on leucine zipper was designed. A Leu zipper structure was formed by a specific amino acid sequence (such as SEQ ID No. 1), and Lys and Glu were combined to form a salt bridge. Trp was added to increase hydrophobicity. The nanostructure was prepared by solid-phase chemical synthesis and self-assembled.
It forms a stable nanostructure at low concentrations, exhibiting significant antibacterial activity against both Gram-positive and Gram-negative bacteria, good biocompatibility and stability, and maintaining strong antibacterial activity under different conditions.
Smart Images
Figure CN121471323A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a leucine zipper-based nano-antimicrobial peptide, its preparation method, and its application. Background Technology
[0002] Antimicrobial peptides, as key components of the body's innate immune system, are considered the most promising antibiotic alternatives due to their unique membrane-breaking and bactericidal mechanisms, which make them less likely to induce bacterial resistance. The self-assembly of antimicrobial peptides into nanostructures can enhance their activity and stability, reduce bacterial resistance, and advance the antibiotic-to-antibiotic transition. Although chemical hydrophobic groups are frequently used in the design of self-assembled antimicrobial peptides, they often have significant toxicity. Therefore, developing assembled antimicrobial peptides with good biocompatibility and stability is particularly essential. Summary of the Invention
[0003] In view of the above shortcomings, the purpose of this invention is to provide a nano-antimicrobial peptide based on leucine zipper, which can be assembled into a nanostructure at low concentration and has strong antibacterial activity, good biocompatibility and stability.
[0004] The technical solution adopted in this invention is as follows: a nano-antimicrobial peptide KLL4 based on leucine zipper, the amino acid sequence of which is shown in SEQ ID No.1.
[0005] Furthermore, the molecular formula of the leucine zipper-based nano-antimicrobial peptide KLL4, as described above, is shown in formula (I): Formula (I).
[0006] The present invention also provides a method for preparing the leucine zipper-based nano-antimicrobial peptide KLL4 as described above, the steps of which are as follows:
[0007] S1. Using the α-helical heptapeptide repeat sequence (abcdefg)4 as a base, Leu was selected to be placed at positions a and d to form a Leu zipper structure, which facilitates the formation of the α-helical structure; Lys was selected to be placed at positions b and c to provide positive charge; Lys and Glu were selected to be placed at positions e and g, respectively, to form salt bridges and promote polypeptide oligomerization; Trp was selected to be placed at position f to disrupt perfect amphiphilicity while increasing hydrophobicity and improving the therapeutic index. Based on this, the sequence was repeated 4 times to achieve a sufficient number of positive charges. The resulting polypeptide sequence is shown in SEQ ID No. 1.
[0008] S2. The polypeptide is synthesized using a solid-phase chemical synthesis method, and then purified by reversed-phase high-performance liquid chromatography and identified by mass spectrometry to complete the preparation of the polypeptide. The polypeptide is then subjected to nano-morphological characterization, antibacterial ability detection, and biocompatibility detection, and finally named antimicrobial peptide KLL4.
[0009] The present invention also provides a self-assembly method for the leucine zipper-based nano-antimicrobial peptide KLL4 as described above, wherein the self-assembly conditions are: a concentration of 20.84-256 μM and incubation at 37°C for 24 hours to self-assemble into a nanostructure.
[0010] The present invention also provides the use of the leucine zipper-based nanopeptide KLL4 as described above in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.
[0011] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis.
[0012] Furthermore, the Gram-negative bacteria mentioned are Escherichia coli, Pseudomonas aeruginosa, or Salmonella typhimurium.
[0013] The present invention also provides a medicament suitable for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, said medicament containing a leucine zipper-based nanopeptide KLL4 as described above.
[0014] The beneficial effects and advantages of this invention are as follows: The antimicrobial peptide KLL4 of this invention is composed of natural amino acids and can self-assemble into a nanostructure. It exhibits significant inhibitory effects on Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium). High concentrations of antimicrobial peptide KLL4 show good biocompatibility with human erythrocytes and porcine jejunal epithelial cells IPEC-J2. Furthermore, antimicrobial peptide KLL4 maintains strong antibacterial activity under serum, acidic, and alkaline conditions. In summary, antimicrobial peptide KLL4 is a self-assembled antimicrobial peptide with high application value. Attached Figure Description
[0015] Figure 1 This is the mass spectrum of the antimicrobial peptide KLL4;
[0016] Figure 2 The chromatogram of the antimicrobial peptide KLL4 is shown.
[0017] Figure 3 The graph shows the critical aggregation concentration and linear fitting value of the critical aggregation concentration for the antimicrobial peptide KLL4, where a is the ANS fluorescence spectrum of the antimicrobial peptide KLL4 and b is the critical aggregation concentration.
[0018] Figure 4 This is a negative staining image of the antimicrobial peptide KLL4;
[0019] Figure 5 The graph shows the determination of the hemolytic activity of the antimicrobial peptide KLL4.
[0020] Figure 6This is a graph showing the assay of the cytotoxicity of the antimicrobial peptide KLL4;
[0021] Figure 7 This is a graph showing the binding capacity of the antimicrobial peptide KLL4 to LPS.
[0022] Figure 8 Figure showing the effect of antimicrobial peptide KLL4 on the outer membrane permeability of Escherichia coli; Detailed Implementation
[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0024] Example 1
[0025] Design of the antimicrobial peptide KLL4: Based on the α-helical heptapeptide repeat sequence abcdefg, Leu was placed at positions a and d to form a Leu zipper structure, facilitating the formation of the α-helical structure; Lys was placed at positions b and c to provide positive charge; Lys and Glu were placed at positions e and g, respectively, to form salt bridges and promote peptide oligomerization. Trp was placed at position f to disrupt perfect amphiphilicity while increasing hydrophobicity and improving the therapeutic index. The sequence was repeated four times to achieve a sufficient number of positive charges, resulting in the amino acid sequence of the antimicrobial peptide KLL4, as shown in Table 1.
[0026] Table 1. Gene sequence of antimicrobial peptide KLL4
[0027] peptides amino acid sequence molecular weight Net charge KLL4 LKKLKWE LKKLKWE LKKLKWE LKKLKWE 3722.54 +8
[0028] The molecular structure is shown in formula (I):
[0029]
[0030] Formula (I).
[0031] Example 2
[0032] Synthesis of antimicrobial peptide KLL4 using solid-phase chemical synthesis:
[0033] 1. Weigh an appropriate amount of 2-CTC resin and place it in dichloromethane (DCM) for swelling treatment, soaking for 1 hour. Subsequently, use dimethylformamide (DMF) to clean the resin three times to remove surface impurities.
[0034] 2. Accurately weigh 1 equivalent (eq) of the first protected amino acid and 1.5 equivalents of diisopropylethylamine (DIEA), and use DMF as a solvent to mix with the resin and react for 2 hours.
[0035] 3. After the reaction is complete, the resin is dried and then washed three times with DMF. Subsequently, a mixed solution of methanol and DIEA is added to carry out the end-capping reaction for 1 hour.
[0036] 4. After the end-capping reaction is complete, clean the resin and use a 20% piperidine DMF solution for deprotection treatment to remove the Fmoc protecting group. The reaction time is 10 minutes, and the operation is repeated twice.
[0037] 5. After washing the resin, add 3 equivalents of the second amino acid, 3 equivalents of 1-hydroxybenzotriazole (HOBT) and 3 equivalents of dicyclohexylcarbodiimide (DIC), and use DMF as solvent to carry out the coupling reaction for 1.5 hours.
[0038] 6. Repeat steps 4 and 5 to sequentially connect the following amino acid residues until the last amino acid residue at the N-terminus is connected, and remove the Fmoc protecting group at the N-terminus.
[0039] 7. After completing the connection of all amino acids, wash the resin and dry it.
[0040] 8. The resin was cleaved using a mixed solution of 95% trifluoroacetic acid (TFA), 2% triisopropylsilane (Tis), 2% ethylenedithiol (EDT) and 1% water, while the protecting groups of the peptide side chains were removed. The reaction time was 2 hours.
[0041] 9. After the reaction is complete, filter the resin, wash the filtrate with ice-cold ether, then centrifuge to collect the precipitate and obtain the crude polypeptide.
[0042] 10. Finally, the crude peptide was purified by liquid chromatography and the final antimicrobial peptide KLL4 was obtained by freeze drying.
[0043] The final mass spectrum of the antimicrobial peptide KLL4 is as follows: Figure 1 As shown; the chromatogram of the antimicrobial peptide KLL4 is as follows. Figure 2 As shown.
[0044] Example 3
[0045] Nanoscale characterization of antimicrobial peptide KLL4:
[0046] Critical aggregation concentration (CAC) determination: The critical aggregation concentration (CAC) was determined using a 1-aniline-8-naphthalenesulfonic acid (ANS) fluorescent probe. 1 µL of ANS (final concentration 1 mM, dissolved in 100% DMF) was added to peptides of different concentrations dissolved in phosphate buffer and incubated at 37°C for 24 h. The mixed samples were transferred to 96-well plates, and fluorescence spectroscopy was performed using a microplate reader with excitation wavelength of 369 nm and emission wavelengths of 440 nm–550 nm. The CAC value of the peptides was then calculated using Origin software. The results are shown below. Figure 3 .
[0047] from Figure 3 (A) It can be seen that as the peptide concentration increases, the fluorescence intensity of the antimicrobial peptide KLL4 increases sharply in the range of 440 nm to 550 nm, indicating the presence of nanomolecules in the solution and preliminarily determining the formation of nanostructures. Subsequent fitting analysis using Origin software, such as... Figure 3 As shown in (B), the CAC value of the antimicrobial peptide KLL4 is 20.84 μM.
[0048] Example 4
[0049] Negative staining: After incubation at 128 μM for 12 h, 20 μL of the antimicrobial peptide KLL4 solution was placed on a copper grid plate for 2 min, and stained with 0.1% phosphotungstic acid for 10 s. Finally, the morphology of the air-dried peptide was examined using a Hitachi H-7650 transmission electron microscope (Hitachi H-7650, Japan). The results are shown below. Figure 4 Dense and clear nanostructures were observed in the solution of the antimicrobial peptide KLL4.
[0050] Example 5
[0051] In vitro hemolytic activity, cytotoxicity, bactericidal activity, and stability assays of antimicrobial peptide KLL4:
[0052] 1. Hemolytic Activity Assay: 1 mL of fresh blood from healthy individuals was collected, centrifuged (4 ℃, 1000×g, 5 min), washed three times with PBS (10 mM) buffer, the supernatant was discarded, and the precipitated blood cells were collected and resuspended in PBS (10 mM) buffer to 10 mL. Then, an equal volume of the diluted red blood cell suspension was placed in a 96-well plate and mixed with different concentrations of the antimicrobial peptide KLL4 solution. After incubation at 37 ℃ for 1 h, the plate was centrifuged (1000×g, 10 min), and the supernatant was transferred to a new 96-well plate. Wells treated with 0.1% Triton X-100 served as positive controls, and wells not treated with antimicrobial peptide KLL4 served as negative controls. The absorbance was measured using a microplate reader at a wavelength of 570 nm. The concentration of antimicrobial peptide KLL4 that caused 50% hemolytic activity was defined as cytotoxic. The test results are attached. Figure 5 .
[0053] pass Figure 5 It can be seen that the antimicrobial peptide KLL4 did not cause hemolytic toxicity even at a high concentration (128 μM), which indicates that KLL4 has good biocompatibility.
[0054] 2. Cytotoxicity assay: IPEC-J2 porcine jejunal epithelial cells, revived from liquid nitrogen, were inoculated into a culture medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies, and passaged at 37°C and 5% CO2. The cultured cells were then digested with 0.25% trypsin and adjusted to a culture medium volume of 2–4 × 10⁻⁶ cells / mL. 5 cells / mL. 50 µL of cell suspension was mixed with 50 µL of antimicrobial peptide KLL4 at different concentrations in a 96-well plate and incubated at 37°C and 5% CO2 for 16–18 h. Then, 50 µL of MTT (5 mg / mL) was added to each well, and incubation continued for 4 h. After incubation, the supernatant was discarded, and the crystals at the bottom of the wells were dissolved in 100 µL of DMSO. The absorbance of each well was measured at 570 nm using a microplate reader. Culture medium wells served as blank controls. Results are shown below. Figure 6 It can be seen that even after treatment with a high concentration of antimicrobial peptide KLL4 (64 μM), the survival rate of IPEC-J2 cells is greater than 90%, indicating that antimicrobial peptide KLL4 has good biocompatibility and has the potential to become an antibiotic alternative.
[0055] 3. Bactericidal activity assay: The minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined using the microdilution method. Different concentrations of peptide were added to BSA solution in 96-well plates, followed by the addition of an equal volume of solution with a final concentration of 1×10⁻⁶. 5 CFUmL -1 The final concentration range of the antimicrobial peptide KLL4 in the 96-well plate was 0.5 to 128 μM. After incubation for 24 hours, the absorbance was measured using a microplate reader at a wavelength of 600 nm. The results are shown in Table 2. It can be seen that the antimicrobial peptide KLL4 has bactericidal ability and exhibits strong antimicrobial activity against both Gram-negative and Gram-negative bacteria.
[0056] Table 2. Minimum inhibitory concentrations of antimicrobial peptide KLL4 against common pathogens.
[0057] strain Minimum inhibitory concentration (μM) Gram-negative bacteria E. coli 25922 2 E.coli K88 2 E. coli K99 2 S. typhimurium 14028 8 S. typhimurium C7731 4 P. aeruginosa 27853 2 Gram-positive bacteria S. aureus 29213 4 S.aureus 25923 4 S. aureus 43300 4 E.faecalis 29212 4 S.epidermidis 12228 4
[0058] 4. Stability Assay: The effects of serum, acid, and alkali on the antimicrobial activity of antimicrobial peptide KLL4 against E. coli 25922 were evaluated. The results are shown in Table 3. As can be seen from Table 3, antimicrobial peptide KLL4 maintains strong antimicrobial activity against E. coli 25922 under serum and different pH conditions, exhibiting a certain degree of stability.
[0059] Table 3. Minimum inhibitory concentrations of antimicrobial peptide KLL4 against Escherichia coli under serum, acidic, and alkaline conditions.
[0060] Test conditions Minimum inhibitory concentration (μM) Control 2 25% serum 2 50% serum 2 100% serum 4 pH=2 4 pH=12 2
[0061] Example 6:
[0062] Determination of the LPS binding capacity of antimicrobial peptide KLL4 and its effect on bacterial outer membrane permeability:
[0063] 1. The LPS binding ability testing method is as follows:
[0064] The binding affinity of the two parents to LPS was detected using a BODIPY-TR-cadaverine probe. E. coli O111:B4 LPS (50 μg / mL) and BC (5 μg / mL) were incubated in the dark for 4 h. The peptides (50 μL) were serially diluted with Tris buffer (pH 7.4) in 96-well plates, and an LPS-BC mixture was added to each well. Fluorescence intensity was measured using a fluorescence spectrophotometer at excitation wavelength of 580 nm and emission wavelength of 620 nm. Figure 7 It is evident that the antimicrobial peptide KLL4 possesses excellent LPS binding ability.
[0065] 2. The method for detecting bacterial outer membrane permeability is as follows:
[0066] Determination of bacterial outer membrane permeability: The effect of antimicrobial peptides on the outer membrane of *E. coli* ATCC25922 was detected using the cell membrane-sensitive fluorescent dye NPN. *E. coli* ATCC 25922 was cultured to the logarithmic growth phase, centrifuged (5000g, 5 min) to collect the cells, washed three times with 5 mM HEPES buffer (containing 5 mM glucose, pH 7.2), and reselected until OD600nm = 0.2. A final concentration of 10 μM NPN was added, and the cells were incubated at 37°C in the dark for 30 min. The bacterial culture was then added to 100 μL per well of a 96-well plate, and fluorescence intensity was immediately measured after adding various concentrations of antimicrobial peptides until fluorescence release stabilized. The excitation wavelength was set at 350 nm and the emission wavelength at 420 nm. Figure 8 It is evident that the antimicrobial peptide KLL4 disrupted the outer membrane of E. coli ATCC 25922 in a dose-dependent manner.
Claims
1. A nano-antimicrobial peptide KLL4 based on a leucine zipper, characterized in that, The amino acid sequence is shown in SEQ ID No.
1.
2. The nano-antimicrobial peptide KLL4 based on leucine zipper according to claim 1, characterized in that, The molecular formula is shown in formula (I): , formula (I).
3. The method for preparing the leucine-based nano-antimicrobial peptide KLL4 according to claim 1, characterized in that, The steps are as follows: S1. Using the α-helical heptapeptide repeat sequence (abcdefg)4 as a base, Leu was selected to be placed at positions a and d to form a Leu zipper structure, which facilitates the formation of the α-helical structure; Lys was selected to be placed at positions b and c to provide positive charge; Lys and Glu were selected to be placed at positions e and g, respectively, to form salt bridges and promote polypeptide oligomerization; Trp was selected to be placed at position f to disrupt perfect amphiphilicity while increasing hydrophobicity and improving the therapeutic index. Based on this, the sequence was repeated 4 times to achieve a sufficient number of positive charges. The resulting polypeptide sequence is shown in SEQ ID No.
1. S2. The polypeptide is synthesized using a solid-phase chemical synthesis method, and then purified by reversed-phase high-performance liquid chromatography and identified by mass spectrometry to complete the preparation of the polypeptide. The polypeptide is then subjected to nano-morphological characterization, antibacterial ability detection, and biocompatibility detection, and finally named antimicrobial peptide KLL4.
4. The self-assembly method of the leucine zipper-based nano-antimicrobial peptide KLL4 according to claim 1, characterized in that, Its self-assembly conditions are: concentration of 20.84-256 μM, incubation at 37℃ for 24 hours can self-assemble into nanostructures.
5. The use of the leucine zipper-based nano-antimicrobial peptide KLL4 according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.
6. The application according to claim 5, characterized in that: The Gram-positive bacteria mentioned are Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis.
7. The application according to claim 5, characterized in that: The Gram-negative bacteria mentioned are Escherichia coli, Pseudomonas aeruginosa, or Salmonella typhimurium.
8. A drug suitable for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, characterized in that, The drug contains a leucine-based nanopeptide KLL4 as described in claim 1.