An α-helical antimicrobial peptide W37 and its preparation method and application

Abstract

The present invention discloses an α-helical antimicrobial peptide W37 and a preparation method and application thereof, belonging to the field of bioengineering technology. The sequence of the antimicrobial peptide W37 is shown in SEQ ID No. 1. The application of the antimicrobial peptide W37 in the preparation of drugs for treating and / or preventing infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria, the antimicrobial peptide W37 can effectively inhibit a variety of common bacteria including Staphylococcus aureus and Escherichia coli, while inhibiting bacterial growth without causing hemolysis of red blood cells, and the antimicrobial peptide W37 has high serum stability. In summary, the antimicrobial peptide W37 has the potential to become a broad-spectrum anti-infective drug.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to an alpha-helical antimicrobial peptide W37 and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides are a type of effector molecules in the innate immune system of animals. They have natural antimicrobial activity and are a type of "endogenous antibiotics" encoded by genes. Compared with traditional antibiotics, antimicrobial peptides have a wide range of sources and types. Moreover, most antimicrobial peptides carry positive charges and can non-specifically electrostatically adsorb to negatively charged groups on the surface of bacteria, inserting their hydrophobic ends into the bacterial membrane and disintegrating the bacterial cell membrane. This non-specific physical membrane-breaking mechanism greatly reduces the occurrence and development of bacterial resistance. Therefore, the development of antimicrobial peptides to replace traditional antibiotics has brought hope for curbing the development of antibiotic resistance.

[0003] There are many types of antimicrobial peptides, which can be divided into α-helical antimicrobial peptides, β-fold antimicrobial peptides, extended structure antimicrobial peptides and cyclic antimicrobial peptides according to their secondary structure. Among them, α-helical and β-fold antimicrobial peptides are widely studied due to their typical secondary structures. However, most β-fold antimicrobial peptides contain disulfide bonds and have high synthesis costs. In contrast, α-helical peptides have a simple structure, and perfect amphipathic α-helical peptides usually have excellent antimicrobial activity. Antimicrobial peptide sequences that are too long are prone to produce greater biological toxicity and also increase production costs. However, shortening the sequence length is likely to lead to reduced antimicrobial activity. Summary of the invention

[0004] Based on the above shortcomings, the purpose of the present invention is to provide an α-helical antimicrobial peptide W37, which contains only 7 amino acid residues, has low synthesis cost, and has high biological activity, cell selectivity and serum stability.

[0005] The technical scheme adopted by the present invention is as follows: an α-helical antimicrobial peptide W37, whose amino acid sequence is shown in SEQ ID No. 1, and whose C-terminus is amidated with -NH2.

[0006] Another object of the present invention is to provide a method for preparing an α-helical antimicrobial peptide W37, comprising the following steps:

[0007] S1: Leu is placed at positions 1 and 4 of the α-helical polypeptide to stabilize the α-helical structure; Arg is selected to provide positive charge to the polypeptide, and Trp is selected to provide hydrophobicity to the polypeptide; Arg is placed at positions 2, 5, and 6 of the polypeptide, and Trp is placed at positions 3 and 7 to obtain a perfect amphipathic α-helical structure; the resulting polypeptide sequence is shown in SEQ ID No. 1, and -NH2 is used at the C-terminus of the polypeptide for amidation;

[0008] S2: A peptide resin is obtained by solid phase chemical synthesis, and the obtained peptide resin is cut by TFA, and then purified by reverse phase high performance liquid chromatography and identified by mass spectrometry to obtain a polypeptide;

[0009] S3: The polypeptide was then subjected to antibacterial activity testing, hemolytic activity testing, and serum stability testing, and was finally named antimicrobial peptide W37.

[0010] Another object of the present invention is to provide an α-helical antimicrobial peptide W37 for use in the preparation of a drug for treating and / or preventing Gram-positive and / or Gram-negative bacterial infectious diseases.

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

[0012] Furthermore, the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium or Pseudomonas aeruginosa.

[0013] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive bacteria and / or Gram-negative bacteria infection, wherein the drug contains the α-helical antimicrobial peptide W37 as described above.

[0014] The present invention has the following advantages and beneficial effects: the present invention successfully designs an amphipathic α-helical antimicrobial peptide W37 through reasonable amino acid selection and arrangement, thereby reducing the synthesis cost of the antimicrobial peptide. The antibacterial mechanism of the antimicrobial peptide W37 is to induce increased bacterial outer membrane permeability and plasma membrane depolarization, thereby increasing the permeability of the plasma membrane and destroying its integrity, leading to bacterial cytoplasm leakage, and ultimately causing bacterial death. The obtained antimicrobial peptide W37 was tested for antimicrobial activity, hemolytic activity and serum stability, and it was found that the antimicrobial peptide W37 had high antibacterial activity against Gram-positive bacteria and Gram-negative bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Salmonella typhimurium. At the same time, the antimicrobial peptide W37 did not cause hemolysis of red blood cells. In addition, the short perfect amphipathic α-helical antimicrobial heptapeptide W37 can still exert a highly effective antibacterial effect in serum. In summary, the short perfect amphipathic α-helical antibacterial heptapeptide W37 has the potential to become a broad-spectrum antibacterial drug for the treatment of Gram-positive and Gram-negative bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the helical wheel projection diagram of the antimicrobial peptide W37;

[0016] Figure 2 This is the spiral wheel projection diagram of the antimicrobial peptide 2W37;

[0017] Figure 3 is the chromatogram of antimicrobial peptide W37;

[0018] Figure 4is the mass spectrum of antimicrobial peptide W37;

[0019] Figure 5 This is a comparative chart of the hemolytic activity of the antimicrobial peptides W37 and 2W37. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the embodiments and drawings.

[0021] Example 1

[0022] Design of antimicrobial peptides

[0023] 1. Choose to place it at positions 1 and 4 of the α-helical polypeptide to stabilize the α-helical structure; the excellent antibacterial effect of antimicrobial peptides mainly depends on sufficient positive charge and hydrophobicity. Since the side chain guanidine group of Arg can combine with carboxylates and phosphates on the bacterial surface to form strong hydrogen bonds and enhance antibacterial activity, Arg is selected as a hydrophilic amino acid to provide positive charge for the polypeptide; Trp is easier to insert into the bacterial membrane due to its indole ring; and Arg and Trp can participate in cation-π interactions, allowing the polypeptide to further penetrate into the bacterial cell membrane and destroy the bacterial cell membrane. Place Arg at positions 2, 5, and 6 of the polypeptide, and Trp at positions 3 and 7 to ensure a perfect amphipathic α-helical structure, such as Figure 1 The positive charge of the peptide is between +4 and +9, and the antibacterial activity is the best, so the C-terminus of the peptide is amidated to increase the positive charge, and the antibacterial peptide W37 with a positive charge of +4 is obtained.

[0024] 2. Double the above spiral, e.g. Figure 2 As shown, the antimicrobial peptide 2W37 with a positive charge number of +7 was obtained. The sequences and physicochemical parameters of the two antimicrobial peptides are shown in Table 1.

[0025] Table 1 Sequences and physicochemical parameters of antimicrobial peptides W37 and 2W37

[0026]

[0027] Superscripts: a Average hydrophobicity calculated by HeliQuest analysis; b Relative hydrophobic moment calculated by HeliQuest analysis.

[0028] The above sequences were synthesized by solid phase chemical synthesis to obtain peptide resins. The obtained peptide resins were cleaved by TFA, purified by reverse phase high performance liquid chromatography and identified by mass spectrometry to obtain antimicrobial peptides W37 and 2W37, respectively. The chromatogram and mass spectrum of antimicrobial peptide W37 are shown in Figures 1 and 2, respectively. Figure 3-4 shown.

[0029] Example 2

[0030] Antibacterial activity test

[0031] The antimicrobial activity of antimicrobial peptides was determined by measuring the minimum inhibitory concentration (MIC) of antimicrobial peptides by microbroth dilution method. Single colonies grown on MHA solid medium were picked and inoculated into 10 mL sterile broth medium, cultured at 37 ° C and 220 rpm, and the strains in the logarithmic growth phase were centrifuged and resuspended, and the concentration was adjusted to 105 CFU / mL. The above bacterial solution was added to a 96-well plate at a volume of 50 μL / well. Then 50 μL of BSA solution containing different concentrations of antimicrobial peptides was added to each well, and the final concentration of antimicrobial peptides in the 96-well plate was 0.125 to 64 μM. The 96-well plate was placed in a 37 ° C constant temperature incubator for 16-18 hours. The negative control wells were clear and transparent, indicating that the experimental process was pollution-free. The lowest antimicrobial peptide concentration corresponding to the wells without visible turbid bacterial solution was the MIC of the peptide against the detected bacteria. Three independent repeated experiments were performed, with two parallels for each repeat. The minimum inhibitory concentration of antimicrobial peptides is shown in Table 2.

[0032] Table 2 Antibacterial activity of antimicrobial peptides W37 and 2W37 (μM)

[0033]

[0034]

[0035] As can be seen from Table 2, the antimicrobial peptide W37 showed good antibacterial activity against the 6 tested Gram-negative bacteria and 5 tested Gram-positive bacteria, with MIC values ​​ranging from 4 to 32 μM. The MIC values ​​of peptide 2W37 against the 11 tested strains were between 2 and 16 μM, showing a more excellent antibacterial activity.

[0036] Example 3

[0037] Hemolytic activity test

[0038] The safety of antimicrobial peptides was evaluated by determining whether antimicrobial peptides in the concentration range of 1-128 μM cause hemolysis of human red blood cells. 2 mL of fresh blood was collected from healthy volunteers, centrifuged at 4°C and 1000 g for 10 min, red blood cells were collected, washed 3 times with PBS, and resuspended in 10 mL of PBS to obtain a red blood cell suspension. The above red blood cell suspension was added to a 96-well plate at a volume of 50 μL / well. 50 μL of PBS solution containing different concentrations of antimicrobial peptides was then added to each well. 0.1% Triton X-100 was added to the red blood cell suspension as a positive control, and PBS buffer was added to the red blood cell suspension as a stealth control. The 96-well plate was placed in a 37°C constant temperature incubator for 1 hour. The incubated 96-well plate was centrifuged at 4°C and 1000 g for 10 min; 50 μL of supernatant was aspirated from each well and transferred to a new 96-well plate. The absorbance was measured at 570 nm.

[0039] like Figure 5As shown in the figure, the antimicrobial peptide W37 did not cause hemolysis of erythrocytes at the highest detection concentration of 128 μM, while the antimicrobial peptide 2W37 caused hemolysis of 10% erythrocytes at 4 μM, indicating that when the antimicrobial peptide W37 sequence was repeated twice, although the antimicrobial activity was significantly improved, its hemolytic activity also increased, resulting in a decrease in cell selectivity, that is, a decrease in safety. In summary, the antimicrobial peptide W37 has the best cell selectivity.

[0040] Example 4

[0041] Serum stability test

[0042] E. coli 25922 was used as a typical Gram-negative bacteria and S. aureus 29213 was used as a typical Gram-positive bacteria to determine the stability of antimicrobial peptides in serum. Antimicrobial peptide W37 was mixed with equal volumes of serum of different concentrations (25%, 50%, 100%) and incubated in a 37°C incubator for 3 hours. The minimum inhibitory concentration of antimicrobial peptide W37 was detected. The serum stability of antimicrobial peptide W37 is shown in Table 3.

[0043] Table 3 Serum stability of antimicrobial peptide W37

[0044]

[0045] As can be seen from Table 3, after co-incubation with 25% serum concentration, the inhibitory concentration of antimicrobial peptide W37 against E. coli 25922 and S. aureus 29213 was not affected. After co-incubation with 50% and 100% serum concentrations, the antibacterial concentration of antimicrobial peptide W37 against E. coli 25922 increased from 8 μM to 16 μM, and the antibacterial concentration against S. aureus 29213 did not change, which indicates that the antimicrobial peptide W37 has good serum stability.

Claims

1. An α-helical antimicrobial peptide W37, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

2. The method for preparing an α-helical antimicrobial peptide W37 according to claim 1, characterized in that: The steps are as follows: S1: Leu is placed at positions 1 and 4 of the α-helical polypeptide to stabilize the α-helical structure; Arg is selected to provide positive charge to the polypeptide, and Trp is selected to provide hydrophobicity to the polypeptide; Arg is placed at positions 2, 5, and 6 of the polypeptide, and Trp is placed at positions 3 and 7 to obtain a perfect amphipathic α-helical structure; the resulting polypeptide sequence is shown in SEQ ID No. 1, and -NH2 is used at the C-terminus of the polypeptide for amidation; S2: A peptide resin is obtained by solid phase chemical synthesis, and the obtained peptide resin is cut by TFA, and then purified by reverse phase high performance liquid chromatography and identified by mass spectrometry to obtain a polypeptide; S3: The polypeptide was then subjected to antibacterial activity testing, hemolytic activity testing, and serum stability testing, and was finally named antimicrobial peptide W37.

3. Use of an α-helical antimicrobial peptide W37 according to claim 1 in the preparation of a medicament for treating and / or preventing Gram-positive and / or Gram-negative bacterial infectious diseases.

4. The use according to claim 3, characterized in that: The Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

5. The use according to claim 3, characterized in that: The Gram-negative bacteria are Escherichia coli, Salmonella typhimurium or Pseudomonas aeruginosa.

6. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The medicine contains the α-helical antimicrobial peptide W37 as claimed in claim 1.

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