An anti-enzymatic antibacterial peptide Pba-Dab with nanostructure, its preparation method and application

Abstract

The present invention discloses an anti-proteolytic antibacterial peptide Pba-Dab with a nanostructure, its preparation method and application, belonging to the field of bioengineering. Its amino acid sequence is Pba-WWDabDabDab-NH2, wherein Pba is 1-pyrenebutanoic acid, W is tryptophan, and Dab is 2,4-diaminobutyric acid. Since W is the most hydrophobic among natural amino acids, WW is selected as the hydrophobic region of the peptide chain. On this basis, Pba is added as a hydrophobic scaffold to provide hydrophobic interaction, and at the same time Dab is provided to meet the need for positive charges of cationic peptides. The overall structure adopts a surfactant-like mode to promote the self-assembly process of short peptides. This antibacterial peptide has good biocompatibility, low hemolytic activity, and also has good ability to resist protease hydrolysis, and can maintain high antibacterial activity before and after protease treatment. It is one of the most favorable alternatives to antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to an anti-enzyme-degradable antibacterial peptide Pba-Dab with a nanostructure, and a preparation method and application thereof. Background Art

[0002] Due to their good antibacterial activity, unique antibacterial mechanism, and low tendency to develop drug resistance, antibacterial peptides are currently considered one of the best alternatives to antibiotics. However, most natural antibacterial peptides have many deficiencies, such as low biological potency, high cytotoxicity in vivo, and easy inactivation in the presence of serum, salt ions, strong acids, and proteases. When used as feed additives in the livestock industry, the low protease stability and high production cost of antibacterial peptides are undoubtedly the biggest obstacles to their popularization and application. Therefore, designing new synthetic peptides through artificial means is the best way to overcome the application difficulties of antibacterial peptides. Summary of the Invention

[0003] Based on the above deficiencies, the purpose of the present invention is to provide an anti-enzyme-degradable antibacterial peptide Pba-Dab with a nanostructure, which has low cytotoxicity to mammalian cells and strong resistance to proteases, salt ions, and serum in the physiological environment.

[0004] The technical solution adopted by the present invention is as follows: An anti-enzyme-degradable antibacterial peptide Pba-Dab with a nanostructure, whose amino acid sequence is Pba-WWDabDabDab-NH 2 , where the C-terminus is amidated, Pba is 1-pyrenebutanoic acid, W is tryptophan, and Dab is 2,4-diaminobutyric acid.

[0005] The molecular formula of the anti-enzyme-degradable peptide Pba-Dab with a nanostructure as described above is shown in formula (Ⅰ).

[0006]

[0007] Furthermore, the self-assembly conditions of the anti-enzyme-degradable antibacterial peptide Pba-Dab with a nanostructure as described above are: placed in a phosphate buffer solution with a concentration of 10 mM / L, the concentration of the antibacterial peptide Pba-Dab is 4.7 μg / mL, and incubated at 37 °C for 24 h.

[0008] Another object of the present invention is to provide a method for preparing an anti-proteolytic antibacterial peptide Pba-Dab with a nanostructure as described above, and the method is as follows: Select two tryptophans to form a hydrophobic region, and at the same time select 1-pyrenebutanoic acid Pba as a hydrophobic scaffold to provide hydrophobic interaction to promote its formation of nano self-assembly; Select unnatural amino acids: 2,4-diaminobutyric acid Dab is used to provide positive charges and avoid protease cleavage sites, and at the same time select three 2,4-diaminobutyric acids Dab as the hydrophilic region in the peptide chain. The polypeptide structure adopts a surfactant-like mode to promote the self-assembly process of the short peptide, and the obtained amino acid sequence is Pba-WWDabDabDab-NH 2 , and its C-terminus is amidated with an amino group; The polypeptide is synthesized by solid-phase chemical synthesis; Then, through the determination of the minimum aggregation concentration, antibacterial activity, hemolytic activity and protease stability, it is finally named anti-proteolytic peptide Pba-Dab.

[0009] Another object of the present invention is to provide the application of an anti-proteolytic antibacterial peptide Pba-Dab with a nanostructure as described above in the preparation of drugs for treating infectious diseases caused by Gram-negative bacteria and Gram-positive bacteria.

[0010] Furthermore, the Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium.

[0011] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis.

[0012] The present invention has the following advantages and beneficial effects: The synthesis process of the antibacterial peptide Pba-Dab of the present invention is simple and inexpensive. The peptide chain has only five amino acids, and at the same time has high biological activity and the characteristic of anti-protease hydrolysis. Through the unnatural amino acid Dab and the nano self-assembly system, it can effectively avoid protease cleavage sites and at the same time achieve the purpose of improving the protease resistance of the polypeptide. The prepared nano anti-proteolytic peptide is measured for aggregation degree, antibacterial activity, hemolytic activity and protease stability through tests such as minimum aggregation concentration, minimum inhibitory concentration, stability and hemolysis. It is found that in a phosphate buffer solution with a concentration of 10 mM / L, the antibacterial peptide Pba-Dab begins to aggregate into nanostructures when its concentration reaches 4.7 μg / mL; The antibacterial peptide Pba-Dab has a strong inhibitory effect on Gram-negative bacteria and Gram-positive bacteria, hardly has hemolysis on red blood cells, and has strong resistance under protease condition tests. The protease concentration is 10 mg / mL, has high application value, and the therapeutic index is 50.99, and has the application potential to become an alternative to antibiotics. Description of the Drawings

[0013] Figure 1 Reverse-phase high performance liquid chromatography chart of antibacterial peptide Pba-Dab;

[0014] Figure 2 Mass spectrometry of antibacterial peptide Pba-Dab;

[0015] Figure 3 Fluorescence spectrum of antibacterial peptide Pba-Dab;

[0016] Figure 4 Determination chart of critical aggregation concentration of antibacterial peptide Pba-Dab; Detailed implementation mode

[0017] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation modes of the present invention are not limited thereto.

[0018] Example 1

[0019] Design of antibacterial peptide Pba-Dab

[0020] (1) Select two tryptophans (WW) to form a hydrophobic region. At the same time, select 1-pyrenebutyric acid (Pba) as a hydrophobic scaffold to provide hydrophobic force to promote its formation of nano self-assembly; select 2,4-diaminobutyric acid (Dab) to provide positive charges to meet the basic conditions of cationic antibacterial peptides, and at the same time, it can also avoid protease cleavage sites;

[0021] (2) WW forms the hydrophobic region in the peptide chain. Select 1-pyrenebutyric acid (Pba) as the hydrophobic scaffold at the N-terminus of the peptide chain. DabDabDab is the hydrophilic region in the peptide chain. The polypeptide structure adopts a surfactant-like mode to promote the self-assembly process of the short peptide, and its C-terminus is amide amidated.

[0022] Table 1 Amino acid sequence of antibacterial peptide Pba-Dab

[0023]

[0024] Example 2

[0025] Synthesis of antibacterial peptide Pba-Dab by solid-phase chemical synthesis method

[0026] 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).

[0027] 2. After 2 hours, wash the resin with 3 times the resin volume of nitrogen-dimethylformamide (DMF), and then drain it. Repeat this four times. After draining the resin, set it aside for use.

[0028] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF 3 times the volume of the resin and then drain it.

[0029] 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.

[0030] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (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.

[0031] After 6.2 hours, a certain amount of acetic anhydride was used to cap the resin (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, and then washed four times with DMF 3 times the volume of the resin and dried for later use.

[0032] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it.

[0033] 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.

[0034] 9. Weigh an appropriate amount of the second amino acid and an appropriate amount of HOBT into a 50mL centrifuge tube, add 25mL of DMF to dissolve them, then add 2.5mL of DIC and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.

[0035] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, the condensation is incomplete and the reaction should continue.

[0036] 11. After the reaction is complete, wash the resin four times with DMF, then drain, add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain to check whether the protection is removed.

[0037] 12. Connect the subsequent amino acids in sequence according to Steps 9-11.

[0038] 13. After connecting the last amino acid, remove the protection, wash four times with DMF, and then dry the resin with methanol. Then cut the polypeptide from the resin using a 95% cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) (add 10 mL of the cutting solution per gram of resin), and centrifuge and sediment four times with ice-cold ether (cutting solution: ether = 1:9). Finally, separate and purify by HPLC, and then lyophilize to obtain a polypeptide with a certain purity.

[0039] 14. Purification conditions: stationary phase: C18;

[0040] Identification: Analyze the antibacterial peptide Pba-Dab obtained above by electrospray mass spectrometry (as Figure 1 shown), and the purity of the antibacterial peptide Pba-Dab is greater than 95% (as Figure 2 shown).

[0041] Example 3

[0042] Nanoscale Characterization of Antibacterial Peptide Pba-Dab - Determination of Critical Aggregation Concentration

[0043] The 1-anilino-8-naphthalene sulfonic acid (18-ANS) fluorescent probe can be used to determine the critical aggregation concentration (CAC) of the antibacterial peptide, thereby detecting the ability of the antibacterial peptide to form nanostructures. First, add 1 μL of 18-ANS (final concentration of 1 mM, dissolved in 100% DMF) to the peptide (dissolved in 10 mM / L PB buffer solution) at a concentration of 0.25 - 512 μg / mL. Subsequently, transfer the mixture of the peptide and 18-ANS to a 96-well plate, and after transfer, incubate in an incubator at 37 °C for 18 - 24 h. Then use an F-4500 fluorescence spectrophotometer (Hitachi, Japan), with an excitation wavelength of 369 nm and a fluorescence spectrum from an emission wavelength of 440 nm to 550 nm, to detect the fluorescence intensity at different peptide concentrations. Finally, calculate the CAC value of the polypeptide using Origin software based on the fluorescence intensity.

[0044] The results are as Figure 3 shown. As the peptide concentration increases, the fluorescence intensity of the antibacterial peptide Pba-Dab gradually increases in the range of emission wavelengths from 440 - 550 nm, indicating that there are relatively large aggregates in the solution, and it can be simply determined that nanostructures are formed. Subsequently, use Origin software to fit and analyze, and the CAC value of the antibacterial peptide Pba-Dab is 4.7 μg / mL (as Figure 4 ).

[0045] Example 4

[0046] Biological Activity Assay of Antibacterial Peptide Pba-Dab

[0047] 1. Determination of antibacterial activity: The minimum inhibitory concentration (MIC) of the peptide was determined by the standard microbroth dilution method. Bacteria in the logarithmic growth phase were selected and diluted with MHB medium to OD 600 = 0.08 - 0.1 (≈3×10 8 CFU / mL). Subsequently, the bacteria were diluted 1000-fold again with MHB medium, and then the bacteria (about 3×10 8 CFU / mL) were mixed with 0.2% BSA (bovine serum albumin) containing antibacterial peptide Pba-Dab (1×10 -6 -64×10 -6 mg / mL) at a ratio of 1:1 (50 μL + 50 μL) in a 96-well plate made of polypropylene material. The negative control was 100 μL of MHB without treated bacteria and antibacterial peptide Pba-Dab, and the positive control was bacteria not treated with antibacterial peptide Pba-Dab and MHB at a ratio of 1:1 (50 μL + 50 μL). Subsequently, the 96-well plate was incubated in an incubator at 37 °C for 18 - 24 hours. After incubation, detection was performed on an enzyme-linked immunosorbent assay (ELISA) reader at OD = 492 nm, and the MIC value was the minimum peptide concentration at which no bacterial growth was detected.

[0048] As can be seen from Table 2, antibacterial peptide Pba-Dab exhibits high antibacterial activity against common pathogenic bacteria.

[0049] Antibacterial Activity of Antibacterial Peptide Pba-Dab in Table 2

[0050]

[0051] 2. Determination of hemolytic activity: First, a fresh human red blood cell suspension was taken and centrifuged at 2700 r / min for ten minutes in a centrifuge. After centrifugation, it was resuspended 2 - 3 times with filtered PBS buffer solution (pH = 7.4), and then diluted 10-fold with PBS. The antibacterial peptide Pba-Dab with a final concentration of 1 - 64 μg / mL and the diluted red blood cell suspension were added to a 96-well plate in equal volumes (50 μL + 50 μL). The positive control was 0.1% Triton X-100 and the diluted red blood cell suspension (50 μL + 50 μL), and the negative control was the pure diluted red blood cell suspension (100 μL).

[0052] The 96-well plate was incubated in a constant temperature incubator at 37 °C for 1 hour, then centrifuged at 4 °C and 1000 g for ten minutes in a centrifuge. Subsequently, 50 μL of the supernatant was aspirated from each well of the centrifuged 96-well plate and transferred to a new 96-well plate at the same time. Finally, the absorbance value was detected at an OD value equal to 570 nm, and the hemolysis rate was calculated.

[0053] Hemolysis rate (%) = [(OD of sample 570 — OD of negative control 570 ) / (OD of positive control 570 — OD of negative control 570 )] × 100%.

[0054] The minimum hemolytic concentration is the concentration at which the antimicrobial peptide Pba-Dab causes a 15% hemolysis rate. The results are shown in Table 3.

[0055] Table 3 Geometric mean of the minimum inhibitory concentration, minimum hemolytic concentration, and therapeutic index of the antimicrobial peptide Pba-Dab

[0056]

[0057] Therapeutic index = minimum hemolytic concentration (μg / mL) / geometric mean of the minimum inhibitory concentration (μg / mL)

[0058] As can be seen from Table 3, Pba-Dab did not show hemolytic activity within this range and had a relatively high therapeutic index, with a therapeutic index of 50.99.

[0059] Example 5

[0060] Determination of the protease stability of the antimicrobial peptide Pba-Dab

[0061] Pepsin, chymotrypsin, and trypsin with a final concentration of 10 mg / mL were mixed with the peptide at 2 - 64 μg / mL in equal volumes, and then incubated in an incubator at 37°C for one hour. The untreated antimicrobial peptide Pba-Dab was used as a control. Finally, by comparing its minimum inhibitory concentration with that of the control group (the determination method was as described in the steps of Example 3.1 above), the anti-proteolytic ability of the antimicrobial peptide Pba-Dab was judged.

[0062] Table 4 Minimum inhibitory concentration of the antimicrobial peptide Pba-Dab against E. coli 25922 after protease treatment

[0063]

[0064] As can be seen from Table 4, under the conditions of pepsin and chymotrypsin, the antibacterial activity of the antimicrobial peptide Pba-Dab against E. coli 25922 did not change. Under the condition of trypsin, the MIC value of the antimicrobial peptide Pba-Dab became 16, and its MIC value changed by 2 times, but it still had relatively high antibacterial activity, indicating that the antimicrobial peptide Pba-Dab has relatively high protease stability.

Claims

1. A nanostructured antimicrobial peptide Pba-Dab, characterized in that: Its molecular formula is shown in formula (I), and its amino acid sequence is Pba-WWDabDabDab-NH2, wherein its C-terminus is amidated with amino, Pba is 1-pyrenebutyric acid, W is tryptophan, and Dab is 2,4-diaminobutyric acid.

2. The method for self-assembly of the nanostructured antimicrobial peptide Pba-Dab according to claim 1, characterized in that: The self-assembly conditions are as follows: the antimicrobial peptide Pba-Dab is placed in a phosphate buffer solution with a concentration of 10 mM / L, the concentration of the antimicrobial peptide Pba-Dab is 4.7 μg / mL, and incubated at 37° C. for 24 hours.

3. The method for preparing the nanostructured antimicrobial peptide Pba-Dab according to claim 1, characterized in that: The method is as follows: two tryptophans are selected to form a hydrophobic region, and 1-pyrenebutyric acid Pba is selected as a hydrophobic scaffold to provide hydrophobic force to promote the formation of nano self-assembly; a non-natural amino acid: 2,4-diaminobutyric acid Dab is selected to provide positive charge and avoid protease cleavage sites, and three 2,4-diaminobutyric acid Dabs are selected as hydrophilic regions in the peptide chain. The overall structure adopts a surfactant-like mode to promote the self-assembly process of short peptides. The obtained amino acid sequence is Pba-WWDabDabDab-NH2, and its C-terminus is amidated with amino groups; the polypeptide is synthesized by solid phase chemical synthesis; and after the minimum aggregation concentration, antibacterial activity, hemolytic activity and protease stability are determined, it is finally named as the anti-enzymatic peptide Pba-Dab.

4. Use of the nanostructured anti-enzymatic antimicrobial peptide Pba-Dab according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria, wherein the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium, and the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis or Enterococcus faecalis.

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