A branched antibacterial peptide KW, its preparation method and application

Abstract

The present invention discloses a preparation method and application of a branched antibacterial peptide KW, belonging to the field of biotechnology. The antibacterial peptide KW includes a Lys and two heptapeptide groups with two α-helix structures. The amino acid sequence of the heptapeptide group is shown in SEQ ID No.1. The C-terminus of Arg at the 7th position of the first heptapeptide group is connected to the ε-N terminus of Lys, and the N-terminus of Leu at the 1st position of the second heptapeptide group is connected to the C-terminus of Lys. Moreover, the C-terminus of the antibacterial peptide KW is amidated with an amino group. The branched antibacterial peptide can significantly improve the problem of high toxicity caused by linear polypeptides and further enhance cell selectivity on the basis of ensuring a certain antibacterial activity. In addition, the branched antibacterial peptide KW has the potential to become a broad-spectrum antibacterial drug for treating Gram-positive and Gram-negative bacterial infections and has high application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a preparation method and application of a branched structure antimicrobial peptide KW. Background Art

[0002] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides widely present in the natural immune defense system of organisms. They have broad-spectrum antibacterial activity and act rapidly. Due to their unique antibacterial mechanism of action, they are regarded as ideal alternative drugs to traditional antibiotics. In recent years, researchers have discovered a new type of antimicrobial peptide, namely branched peptide, also known as multi-peptide or dendritic polypeptide, which has good application prospects in the fields of drug delivery, disease treatment, gene carriers, etc. due to its high biological activity and high stability. Since natural antimicrobial peptides or traditional newly designed linear antimicrobial peptides often have disadvantages such as weak activity, unstable metabolism, and high toxicity, their development and application in animal husbandry are restricted. Summary of the Invention

[0003] Based on the above deficiencies, the purpose of the present invention is to provide a preparation method of a branched structure antimicrobial peptide KW. By utilizing the characteristics of Lys having two N-terminals and one C-terminal, a branched structure is constructed. Compared with linear polypeptides with the same amino acid composition, the branched antimicrobial peptide can still maintain certain antibacterial activity and significantly reduce toxicity, solving the problems of low activity and high toxicity of existing natural antimicrobial peptides or newly designed traditional linear antimicrobial peptides.

[0004] The technical solution adopted by the present invention is as follows: A branched structure antimicrobial peptide KW includes one Lys and two heptapeptide groups with α-helix structures. The amino acid sequence of the heptapeptide group is as shown in SEQ ID No.1. The C-terminal of Arg at the 7th position of the first heptapeptide group is connected to the ε-N-terminal of Lys, and the N-terminal of Leu at the 1st position of the second heptapeptide group is connected to the C-terminal of Lys. And the C-terminal of the antimicrobial peptide KW is amidated with an amino group.

[0005] Further, for a branched structure antimicrobial peptide KW as described above, its molecular formula is as shown in formula (I).

[0006]

[0007] Another object of the present invention is to provide a preparation method of a branched structure antimicrobial peptide KW as described above. The method steps are as follows:

[0008] Step 1: Using the α-helical heptapeptide repeat sequence "abcdefg" as a basis, place Leu at positions a and d to form a Leu zipper structure to provide a certain degree of hydrophobicity; add Trp at positions c and f to enable Trp-Trp interaction to improve the stability of the α-helical structure, thereby enhancing the antibacterial activity, and add Arg at the remaining positions to increase the number of positive charges, obtaining a heptapeptide group sequence as shown in SEQ ID No.1. Connect the C-terminus of Arg at the 7th position of the first group of heptapeptide groups to the ε-N terminus of Lys, and connect the N-terminus of Leu at the 1st position of the second group of heptapeptide groups to the C-terminus of Lys to obtain a polypeptide, and amidate the C-terminus of the polypeptide with an amino group to provide sufficient positive charges to ensure antibacterial activity;

[0009] Step 2: Synthesize the polypeptide by solid-phase chemical synthesis method, and after purification by reverse-phase high-performance liquid chromatography and identification by mass spectrometry, further perform antibacterial activity detection and hemolytic activity detection on the polypeptide, and finally name it antibacterial peptide KW.

[0010] Another object of the present invention is to provide an application of the branched structure antibacterial peptide KW as described above in the preparation of a drug for treating Gram-positive bacteria and / or Gram-negative bacteria infectious diseases.

[0011] Further, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or / and Salmonella typhimurium.

[0012] Further, the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis or / and Enterococcus faecalis.

[0013] The present invention has the following advantages and effects: The present invention can achieve good antibacterial effects with only 15 amino acids, effectively reducing the synthesis cost and significantly reducing the toxicity of antibacterial peptides. The antibacterial and hemolytic activities of the obtained branched antibacterial peptide KW were detected, and it was found that the branched antibacterial peptide KW had high antibacterial activities against several tested Gram-positive and Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis. Although the antibacterial activity of the branched antibacterial peptide KW was slightly lower than that of the linear antibacterial peptide W, the minimum inhibitory concentration of the branched antibacterial peptide against all tested strains still remained within the range of 8-16 μM. The therapeutic index of the branched antibacterial peptide KW was 30.00, showing good cell selectivity, and its TI value was increased by 3 times compared with the therapeutic index of the linear antibacterial peptide W (TI = 10.56). In summary, it shows that the strategy of transforming linear antibacterial peptides into branched antibacterial peptides can significantly improve the problem of high toxicity caused by linear polypeptides and further improve cell selectivity on the basis of ensuring a certain antibacterial activity. In addition, the branched antibacterial peptide KW has the potential to become a broad-spectrum antibacterial drug for the treatment of Gram-positive and Gram-negative bacterial infections and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the reverse-phase high-performance liquid chromatography chart of the linear antibacterial peptide W;

[0015] Figure 2 is the reverse-phase high-performance liquid chromatography chart of the branched antibacterial peptide KW;

[0016] Figure 3 is the mass spectrometry chart of the linear antibacterial peptide W;

[0017] Figure 4 is the mass spectrometry chart of the branched antibacterial peptide KW;

[0018] Figure 5 is the hemolytic activity chart of the linear antibacterial peptide W and the branched antibacterial peptide KW; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The present invention will be further described in detail below with reference to the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0020] Example 1

[0021] Design of Antibacterial Peptides

[0022] 1. Using the α-helical heptapeptide repeat sequence "abcdefg" as a basis, Leu is selected and placed at positions a and d to form a Leu zipper structure to provide a certain degree of hydrophobicity; Trp is added at positions b and f to enable Trp-Trp interaction to enhance the stability of the α-helical structure, thereby improving antibacterial activity. Arg is added at the remaining positions to increase the number of positive charges, and such a heptapeptide repeat sequence is repeated twice and amidated at the C-terminus to provide sufficient positive charges to ensure antibacterial activity. Finally, the linear antibacterial peptide W is obtained, and its amino acid sequence is LWRLRWRLWRLRWR-NH 2 。

[0023] 2. Using the α-helical heptapeptide repeat sequence "abcdefg" as a basis, Leu is selected and placed at positions a and d to form a Leu zipper structure to provide a certain degree of hydrophobicity; Trp is added at positions c and f to enable Trp-Trp interaction to enhance the stability of the α-helical structure, thereby improving antibacterial activity. Arg is added at the remaining positions to increase the number of positive charges, and the heptapeptide group sequence is as shown in SEQ ID No.1. The C-terminus of Arg at the 7th position of the first group of heptapeptide groups is connected to the ε-N terminus of Lys, and the N-terminus of Leu at the 1st position of the second group of heptapeptide groups is connected to the C-terminus of Lys to obtain a polypeptide, and the C-terminus of the polypeptide is amidated with an amino group to provide sufficient positive charges to ensure antibacterial activity; a branched antibacterial peptide is obtained, named KW, and its amino acid sequence is K(LWRLRWR)LWRLRWR-NH 2 。

[0024] Table 1 shows the sequences, molecular weights and charge numbers of the linear antibacterial peptide W and the branched antibacterial peptide KW

[0025]

[0026] Example 2

[0027] Synthesis and identification of antibacterial peptides

[0028] The designed antibacterial peptides were synthesized by solid-phase synthesis using a peptide synthesizer, identified by electrospray mass spectrometry and purified by reverse-phase high-performance liquid chromatography to obtain the target compounds for subsequent antibacterial and hemolytic activity assays.

[0029] The reverse-phase high-performance liquid chromatography diagram of the linear antibacterial peptide W is as Figure 1 shown, and the mass spectrometry diagram is as Figure 3 shown.

[0030] The reverse-phase high-performance liquid chromatography diagram of the branched antibacterial peptide KW is as Figure 2 shown, and the mass spectrometry diagram is as Figure 4 shown.

[0031] Example 3

[0032] Bacteriostatic Activity of Antimicrobial Peptides

[0033] The bacteriostatic activity of antimicrobial peptides was understood by measuring the minimum inhibitory concentration (MIC) of antimicrobial peptides. The designed and successfully synthesized antimicrobial peptides were prepared into a 2.56 mM stock solution for bioactivity determination. 100 μL of the frozen test bacterial strain was inoculated into fresh MHB broth medium in a sterile operating table, cultured overnight in a shaker at 37 °C with a shaking speed of 220 rpm, and then transferred to a new, sterile MHB broth medium and cultured until the logarithmic phase of cell growth. The turbidity of the test bacterial solution was adjusted to OD 600nm = 0.37 - 0.4 for standby. A sterile 96-well culture plate was taken, and 0.2% BSA aqueous solution (containing 0.01% acetic acid) filtered through a 0.22 μM aqueous filter membrane was used as the diluent, and the MIC of antimicrobial peptides was determined by the microbroth dilution method. The 2.56 mM antimicrobial peptide stock solution was added to the diluent in the 96-well culture plate for 2-fold serial dilution, and then 50 μL of the bacterial suspension diluted 1000-fold was added to each well. Among them, MHB containing bacteria was used as the positive control, and sterile MHB medium was used as the negative control. The culture plate was incubated in an incubator at 37 °C for 16 - 18 h. The clear negative control wells indicated that the test was not contaminated; the lowest peptide concentration without increased turbidity compared with the negative control under naked eye observation was the MIC of the tested polypeptide against the test bacteria. Three independent repeated tests were carried out, with two parallels for each repetition. The minimum inhibitory concentrations of antimicrobial peptides are shown in Table 2.

[0034] Table 2 shows the bacteriostatic activities (μM) of linear antimicrobial peptide W and branched antimicrobial peptide KW

[0035]

[0036] As can be seen from the table, both linear antimicrobial peptide W and branched antimicrobial peptide KW showed good antibacterial activities against the 10 tested strains. Although the bacteriostatic activity of branched antimicrobial peptide KW was slightly lower than that of linear antimicrobial peptide W, the minimum inhibitory concentrations of branched antimicrobial peptide against all tested strains still remained in the range of 8 - 16 μM.

[0037] Example 5

[0038] Hemolytic Activity of Antimicrobial Peptides

[0039] To evaluate the safety of antimicrobial peptides, the hemolytic behaviors of linear antimicrobial peptide W and branched antimicrobial peptide KW on human red blood cells (hRBC) were studied in the concentration range of 1 - 128 μM.

[0040] Randomly collect 2 mL of fresh blood from 3 healthy volunteers each. Centrifuge at 1000×g for 5 min, discard the supernatant, and collect the red blood cells. Wash the collected red blood cells 3 times with PBS buffer, and finally resuspend them in 10 mL of PBS buffer for standby. Add PBS buffer to the red blood cells in proportion to make the light absorption value measured at 570 nm of the positive control around 1. Take a sterile 96-well culture plate, use PBS buffer as the diluent, add the antimicrobial peptide stock solution to the first well of the 96-well culture plate, perform 2-fold serial dilution, then add 50 μL of the red blood cell suspension to each well, place it in an incubator at 37 °C for constant incubation for 1 h, and then centrifuge at 4 °C and 1000×g for 10 min; use a pipette to aspirate 50 μL of the supernatant from each well and transfer it to a new sterile 96-well culture plate, and measure the light absorption value at 570 nm using an enzyme-linked immunosorbent assay reader. Among them, 50 μL of red blood cells plus 50 μL of 0.1% Triton X-100 is used as the positive control, and 50 μL of red blood cells plus 50 μL of PBS buffer is used as the negative control. The test results are shown in the appendix of the specification Figure 5 The minimum hemolytic concentration (MHC) of the antimicrobial peptide when causing 5% hemolysis of human red blood cells was determined for the linear antimicrobial peptide W and the branched antimicrobial peptide KW to evaluate their biocompatibility, and further the therapeutic index (TI) of the linear antimicrobial peptide W and the branched antimicrobial peptide KW was calculated to evaluate their cell selectivity, as shown in Table 3.

[0041] Table 3 shows the biocompatibility of the linear antimicrobial peptide W and the branched antimicrobial peptide KW

[0042]

[0043] a The geometric mean (GM) of the minimum inhibitory concentration of the antimicrobial peptide against the tested bacteria;

[0044] b MHC is the minimum concentration of the antimicrobial peptide when causing 5% hemolysis of human red blood cells (hRBC). When no detectable hemolytic activity is observed at 128 μM, 256 μM is used to calculate the therapeutic index;

[0045] c The therapeutic index (TI) is the ratio of MHC to GM.

[0046] To evaluate the safety of the linear antimicrobial peptide W and the branched antimicrobial peptide KW, their hemolytic activities against human red blood cells were determined in the concentration range of 1 - 128 μM. As shown in the appendix of the specification Figure 5It can be seen that although there is no hemolysis phenomenon in both of them within the MIC range, with the increase of the concentration of the linear antimicrobial peptide W, its hemolysis increases accordingly, and it can cause 20% hemolysis at a concentration of 128 μM; while the branched antimicrobial peptide KW will not cause hemolysis even at the highest measured concentration. By measuring the minimum concentration (MHC) of the antimicrobial peptide when both of them cause 5% hemolysis of human red blood cells (hRBC), it is found that the MHC value of KW (>128 μM) is much higher than that of W (32 μM), which indicates that KW has higher biocompatibility. Further calculating the therapeutic index (TI) of the linear antimicrobial peptide W and the branched antimicrobial peptide KW to evaluate the cell selectivity of the two, a higher TI value indicates that the antimicrobial peptide has higher cell selectivity. Compared with the TI value of the linear antimicrobial peptide W (10.56), the TI value of the branched antimicrobial peptide KW (32.00) has increased by 3 times, indicating that although the antibacterial activity of the antimicrobial peptide decreases to a certain extent after forming branches, it has better cell selectivity.

Claims

1. A branched - structure antimicrobial peptide KW, Characterized in that: Its molecular formula is shown in formula (I), including one Lys and two heptapeptide groups with α - helix structures. The amino acid sequence of the heptapeptide group is shown in SEQ ID No.

1. The C - terminus of Arg at the 7th position of the first heptapeptide group is connected to the ε - N - terminus of Lys, and the N - terminus of Leu at the 1st position of the second heptapeptide group is connected to the C - terminus of Lys. And the C - terminus of the antimicrobial peptide KW is amidated with an amino group.

2. A preparation method of the branched - structure antimicrobial peptide KW according to claim 1, Characterized in that, The method steps are as follows: Step 1: Using the α - helix heptapeptide repeat sequence "abcdefg" as a basis, place Leu at positions a and d to form a Leu - zipper structure to provide a certain hydrophobicity; add Trp at positions c and f so that Trp - Trp interaction improves the stability of the α - helix structure, thereby enhancing the antibacterial activity, and add Arg at the remaining positions to increase the number of positive charges, obtaining a heptapeptide group sequence shown in SEQ ID No.

1. Connect the C - terminus of Arg at the 7th position of the first heptapeptide group to the ε - N - terminus of Lys, and connect the N - terminus of Leu at the 1st position of the second heptapeptide group to the C - terminus of Lys to obtain a polypeptide, and amidate the C - terminus of the polypeptide with an amino group to provide sufficient positive charges to ensure antibacterial activity; Step 2: Synthesize the polypeptide by solid - phase chemical synthesis method, then purify it by reverse - phase high - performance liquid chromatography and identify it by mass spectrometry, further detect the antibacterial activity and hemolytic activity of the polypeptide, and finally name it antimicrobial peptide KW.

3. Use of the branched - structure antimicrobial peptide KW according to claim 1 in the preparation of a drug for treating Gram - positive bacteria and / or Gram - negative bacteria infectious diseases, 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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