Dendritic branched antimicrobial peptide rich in non-natural amino acids and preparation method and application thereof

Abstract

The present invention discloses a dendritic branched antimicrobial peptide rich in non-natural amino acids, a preparation method and an application thereof, and belongs to the field of bioengineering technology. The antimicrobial peptide comprises a Lys and two groups of α-helical structured heptapeptide groups, and the amino acid sequence of the heptapeptide group is: LW

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a dendritic branched antimicrobial peptide rich in non-natural amino acids and a preparation method and application thereof. Background Art

[0002] Initially, although the emergence of antibiotics provided effective protection for the treatment of various bacterial infections, the abuse of antibiotics led to the emergence of bacterial resistance. At present, the rate of antibiotic research and development is far less than the rate at which bacteria develop resistance, which puts people in a situation where no drugs are available. Therefore, people are eager to seek new drugs to replace traditional antibiotics. Compared with traditional antibiotics, antimicrobial peptides (AMPs) have a wide range of sources and have antibacterial activity against a variety of bacteria and multi-drug resistant bacteria. Moreover, their mechanism of action mainly acts on the bacterial cell membrane and is not easy to develop resistance. Therefore, antimicrobial peptides are considered to be an ideal substitute for antibiotics.

[0003] However, there are still many limitations in the development and application of antimicrobial peptides, such as the difficulty in extracting natural antimicrobial peptides from organisms, the instability of some natural antimicrobial peptides in vivo, and the problem of being easily degraded by various proteases in the digestive tract. This results in the fact that most of them can only be administered locally or intravenously, which greatly limits the scope of application of antimicrobial peptides. Studies have shown that the rational introduction of non-natural amino acids into polypeptide sequences is a common modification strategy to improve the resistance of antimicrobial peptides to various proteases. However, at the same time, the introduction of non-natural amino acids may cause increased toxicity in the body, resulting in the inability of polypeptides rich in non-natural amino acids to be truly applied in vivo. Therefore, we need to avoid unnecessary toxicity as much as possible. Dendritic branched antimicrobial peptides are a new antimicrobial peptide design strategy that introduces branch points to construct branch structures based on traditional linear antimicrobial peptides. Studies have shown that this strategy significantly reduces its hemolytic activity and toxicity by reducing the helicity of antimicrobial peptides and changing their secondary structure. Therefore, the rational design of a dendritic branched antimicrobial peptide rich in non-natural amino acids improves its stability in the digestive tract environment through non-natural amino acid modification, and reduces its toxicity to animals through branch design, which is expected to achieve oral administration of antimicrobial peptides. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a dendritic branched antimicrobial peptide KDabr rich in non-natural amino acids to solve the problem that the existing natural antimicrobial peptides have poor enzyme stability and are difficult to administer orally.

[0005] The technical scheme adopted by the present invention is as follows: a dendritic branched antimicrobial peptide KDabr rich in non-natural amino acids, comprising a Lys and two groups of α-helical structured heptapeptide groups, wherein the amino acid sequence of the heptapeptide group is: LW Dab L DabW D R , wherein Dab is 2,4-diaminobutyric acid, D R is D-Arg, the C-terminus of the D-Arg at the 7th position of the heptapeptide group described in the first group is connected to the ε-N-terminus of Lys, the N-terminus of the Leu at the 1st position of the heptapeptide group described in the second group is connected to the C-terminus of Lys, and the C-terminus of the antimicrobial peptide KDabr is amidated with -NH2.

[0006] Furthermore, the above-mentioned dendritic branched antimicrobial peptide KDabr rich in non-natural amino acids has a molecular formula as shown in formula (I),

[0007]

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned dendritic branched antimicrobial peptide KDabr rich in unnatural amino acids, comprising the following steps:

[0009] S1: Using the α-helical heptapeptide repeat sequence "abcdefg" as a template, Leu was selected and placed at positions a and d to form a Leu zipper that stabilized the α-helical structure; Trp was added at positions b and f to further improve the stability of the α-helical structure through Trp-Trp interaction, thereby improving the antibacterial activity; Dab, Dab, and D-type Arg were added at positions c, e, and g, respectively, to increase the number of positive charges, and the amino acid sequence of the heptapeptide group was obtained as follows: LW Dab L Dab W D R , connecting the C-terminus of the D-Arg at the 7th position of the first group of heptapeptide groups to the ε-N-terminus of Lys, connecting the N-terminus of the Leu at the 1st position of the second group of heptapeptide groups to the C-terminus of Lys, to obtain a polypeptide, and further amidating the C-terminus of the polypeptide with -NH2;

[0010] S2: The polypeptide is synthesized by solid phase chemical synthesis, and then purified by reverse phase high performance liquid chromatography and identified by mass spectrometry. The polypeptide is then tested for antibacterial activity, hemolytic activity and protease stability, and is finally named as the dendritic branched antimicrobial peptide KDabr.

[0011] Another object of the present invention is to provide the use of the above-mentioned dendritic branched antimicrobial peptide KDabr rich in non-natural amino acids in the preparation of drugs for treating Gram-positive and / or Gram-negative bacterial infectious diseases.

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

[0013] Furthermore, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.

[0014] The present invention has the following advantages and beneficial effects: the present invention significantly improves the stability of the polypeptide in trypsin and simulated intestinal fluid by rationally introducing non-natural amino acids and constructing a modification strategy of branched structure, while reducing the toxicity of the antimicrobial peptide, and is expected to achieve oral administration of the antimicrobial peptide. The obtained dendritic branched antimicrobial peptide KDabr rich in non-natural amino acids was tested for antibacterial and hemolytic activity and enzyme stability, and it was found that the dendritic branched antimicrobial peptide KDabr had high antibacterial activity against several Gram-negative bacteria and Gram-positive bacteria measured, such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, etc. At the same time, the dendritic branched antimicrobial peptide KDabr has the best cell selectivity, and its therapeutic index (TI) is 112.77. In addition, the dendritic branched antimicrobial peptide KDabr can still exert a stable and efficient antibacterial effect in artificial intestinal fluid (SIF). In summary, the dendritic branched antimicrobial peptide KDabr has the potential to become a broad-spectrum antibacterial drug for the treatment of Gram-positive and Gram-negative bacterial infections, maintains high antibacterial activity in the intestine, and is expected to be administered orally. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the reverse phase high performance liquid chromatogram of the linear antimicrobial peptide Dabr rich in unnatural amino acids;

[0016] Figure 2 It is a reverse phase high performance liquid chromatogram of a linear antimicrobial peptide 2Dabr rich in unnatural amino acids;

[0017] Figure 3 The reverse phase high performance liquid chromatogram of the antimicrobial peptide KDabr rich in unnatural amino acids dendritic branched chains;

[0018] Figure 4 The mass spectrum of the linear antimicrobial peptide Dabr rich in unnatural amino acids;

[0019] Figure 5 The mass spectrum of the linear antimicrobial peptide 2Dabr rich in unnatural amino acids;

[0020] Figure 6 The mass spectrum of the antimicrobial peptide KDabr rich in unnatural amino acids;

[0021] Figure 7 This is the hemolytic activity graph of antimicrobial peptides Dabr, 2Dabr and KDabr; DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with the embodiments and drawings.

[0023] Example 1

[0024] Design of antimicrobial peptides

[0025] 1. Using the α-helical heptapeptide repeat sequence "abcdefg" as a template, Leu was selected and placed at positions a and d to form a Leu zipper with a stable α-helical structure; Trp was added at positions b and f to further improve the stability of the α-helical structure through Trp-Trp interaction, thereby improving the antibacterial activity; Dab, Dab, and D-type Arg were added to the remaining c, e, and g positions to increase the number of positive charges, respectively, to obtain the antibacterial unit LW Dab L Dab W D R The C-terminus of the antibacterial unit was amidated with -NH2 to form a linear heptapeptide named Dabr.

[0026] 2. The above antibacterial unit LW Dab L Dab W D R This reaction was repeated twice and amidated with -NH2 at its C-terminus to form a linear tetradecapeptide named 2Dabr.

[0027] 3. The first group of antibacterial units LW Dab L Dab W D R The C-terminus of D-Arg at position 7 of the first peptide is connected to the ε-N-terminus of Lys, and the N-terminus of Leu at position 1 of the second group of heptapeptide groups is connected to the C-terminus of Lys. The C-terminus of the peptide is further amidated with -NH2, and finally a dendritic branched antimicrobial peptide rich in non-natural amino acids is obtained, named KDabr. The sequence, molecular weight and charge number of the antimicrobial peptide are shown in Table 1.

[0028] Table 1 Sequence, molecular weight and charge number of antimicrobial peptides Dabr, 2Dabr and KDabr

[0029]

[0030]

[0031] Example 2

[0032] Synthesis and identification of antimicrobial peptides

[0033] The designed antimicrobial peptide was synthesized by solid phase synthesis using a peptide synthesizer, identified by electrospray ionization mass spectrometry and purified by reversed-phase high performance liquid chromatography to obtain the target compound for subsequent antimicrobial and hemolytic activity and enzyme stability determination.

[0034] The reverse phase HPLC chromatograms of antimicrobial peptides Dabr, 2Dabr, and KDabr are attached. Figure 1 , 2 、3.

[0035] The mass spectra of antimicrobial peptides Dabr, 2Dabr, and KDabr are attached. Figure 4 , 5 、6.

[0036] Example 3

[0037] Antibacterial activity of antimicrobial peptides

[0038] The antibacterial activity of antimicrobial peptides was understood by measuring the minimum inhibitory concentration (MIC) of antimicrobial peptides. The designed and successfully synthesized antimicrobial peptides were formulated into a 5.12 mg / mL stock solution for biological activity determination. Take the bacterial solution frozen at -20°C, streak it on the MHA solid culture medium in a sterile operating table, and culture it overnight in a shaker at 37°C with a shaking speed of 220 rpm. Then pick a single colony of the strain and inoculate it in a sterile MHB liquid culture medium and culture it until the logarithmic phase of growth. Use a spectrophotometer to measure the OD of the bacterial solution. 600nm The value was adjusted to about 0.40 for standby use. Take a sterile 96-well culture plate, use 0.2% BSA (containing 0.01% acetic acid) filtered through a 0.22μM water filter as the diluent, and use the microbroth dilution method to determine the MIC of the antimicrobial peptide. Add the antimicrobial peptide stock solution with an initial concentration of 5.12mg / mL to the diluent of the 96-well culture plate, perform a 2-fold gradient dilution, and then add 50μL of the bacterial solution diluted 1000 times to each well. MHB containing bacteria was used as a positive control, and sterile MHB culture medium was used as a negative control. Seal the 96-well culture plate with a sealing film to prevent bacterial contamination and place it in a 37°C incubator for incubation for 16-18h. The negative control wells are clear and transparent, indicating that there is no contamination in the test process. Under naked eye observation, the lowest peptide concentration at which the turbidity does not increase compared with the negative control is defined as the MIC of the peptide. Perform three independent replicates, with two parallels for each replicate. The minimum inhibitory concentrations of antimicrobial peptides are shown in Table 2.

[0039] Table 2 Antibacterial activity of antimicrobial peptides Dabr, 2Dabr, and KDabr (μg / mL)

[0040]

[0041]

[0042] As can be seen from the table, the linear antimicrobial peptide Dabr rich in non-natural amino acids showed good antibacterial activity against the 6 tested Gram-negative bacteria, and only showed a certain inhibitory effect on the 5 tested Gram-positive bacteria. The linear antimicrobial peptide 2Dabr showed good antibacterial activity against all 11 strains tested, and its MIC value was between 2-32μg / mL, and its antibacterial activity was significantly better than Dabr. The dendritic branched antimicrobial peptide KDabr rich in non-natural amino acids had an MIC value of 2-4μg / mL against the 11 strains tested, showing even better antibacterial activity.

[0043] Example 4

[0044] Hemolytic activity of antimicrobial peptides

[0045] To evaluate the safety of antimicrobial peptides, the hemolytic behavior of peptides against human red blood cells (hRBC) was studied in the concentration range of 1-128 μg / mL.

[0046] 2 mL of fresh blood from a healthy person was collected and centrifuged at 4°C, 3000r for 10 min, then the supernatant was discarded and the red blood cells were collected. The collected red blood cells were then washed 3 times with PBS buffer under the same centrifugal conditions, and finally resuspended with 10 mL PBS for use. Take a sterile 96-well culture plate, use PBS buffer as the diluent, add the antimicrobial peptide stock solution with an initial concentration of 5.12 mg / mL to the diluent of the 96-well culture plate, perform a 2-fold gradient dilution, and then add 50 μL of red blood cell suspension to each well, and incubate in a 37°C incubator for 1 hour. Then the 96-well culture plate was centrifuged at 4°C, 1000×g for 10 min; 50 μL of the supernatant after centrifugation was aspirated and transferred to a new sterile 96-well culture plate, and the absorbance at 570 nm was measured using an ELISA reader. 50 μL of red blood cells plus 50 μL of 0.1% Triton X-100 was used as a positive control, and 50 μL of red blood cells plus 50 μL of PBS buffer was used as a negative control. Figure 7 The biocompatibility of antimicrobial peptides Dabr, 2Dabr, and KDabr was evaluated by the minimum concentration (MHC) of antimicrobial peptides when they caused 5% hemolysis of human red blood cells, and their cell selectivity was further evaluated by calculating the therapeutic index (TI) of Dabr, 2Dabr, and KDabr, as shown in Table 3.

[0047] Table 3 Biocompatibility of antimicrobial peptides Dabr, 2Dabr, and KDabr

[0048]

[0049]

[0050] a The geometric mean (GM) of the minimum inhibitory concentration of antimicrobial peptides against the tested bacteria, when no detectable antimicrobial activity was observed at 64 μg / mL, 128 μg / mL was used to calculate the therapeutic index;

[0051] b MHC is the lowest concentration of antimicrobial peptide that causes 5% hemolysis of human red blood cells (hRBCs), and when no detectable hemolytic activity is observed at 128 μg / mL, 256 μg / mL is used to calculate the therapeutic index;

[0052] c The therapeutic index (TI) is the ratio of MHC to GM.

[0053] Attached to the instruction manual Figure 7 It can be seen that 2Dabr caused 18% hemolysis at the highest concentration of 128μg / mL, while Dabr and KDabr did not cause hemolysis at the highest concentration. By measuring the minimum concentration (MHC) of the antimicrobial peptides that caused 5% hemolysis of human red blood cells (hRBC), it was found that the MHC values ​​of Dabr and KDabr (>128μg / mL) were much higher than the MHC value (32μg / mL), which indicated that Dabr and KDabr had higher biosafety. Combining the geometric mean (GM) of the minimum inhibitory concentration of antimicrobial peptides Dabr, 2Dabr, and KDabr for the measured bacteria, the therapeutic index (TI) of Dabr, 2Dabr, and KDabr was further calculated to evaluate the cell selectivity of the two. A higher TI value indicates that the antimicrobial peptide has a higher cell selectivity. Among them, the TI value of Dabr (16.00) is significantly higher than the TI value of 2Dabr (6.21), which indicates that when the heptapeptide sequence is repeated twice, although the antibacterial activity is significantly improved, its hemolytic activity is also increased accordingly, which will eventually lead to a decrease in cell selectivity. However, the TI value of the dendritic branched antimicrobial peptide KDabr (112.77) is much higher than that of other antimicrobial peptides. This indicates that the hemolytic activity of the peptide has been significantly improved after branching, which increases the cell selectivity. In summary, the dendritic branched antimicrobial peptide KDabr rich in unnatural amino acids has the best cell selectivity.

[0054] Example 5

[0055] Enzyme stability of antimicrobial peptides

[0056] E. coli 25922 was used as a typical Gram-negative bacterium, and S. aureus 29213 was used as a typical Gram-positive bacterium to determine the sensitivity of the antimicrobial peptides to simulated intestinal fluid (SIF) and trypsin at an initial concentration of 10 mg / ml.

[0057] The peptide was mixed with an equal volume of SIF or Trypsin with an initial concentration of 10 mg / ml, and incubated at 37°C for 8 h before the minimum inhibitory concentration test. The test results are shown in Table 4.

[0058] Table 4 Enzyme stability of antimicrobial peptides Dabr, 2Dabr, and KDabr

[0059]

[0060]

[0061] By measuring the protease sensitivity of antimicrobial peptides Dabr, 2Dabr, and KDabr to E.coli 25922 and S.aureus 29213, it was found that Dabr, 2Dabr, and KDabr were able to maintain stable antimicrobial activity after incubation with 10mg / ml Trypsin in equal volumes for 8 hours, but when incubated in SIF in equal volumes for 8 hours, which is closer to the complex environment of the intestine, Dabr and 2Dabr could not maintain their original antimicrobial activity, and their MIC values ​​for E.coli 25922 increased by 8 times. However, KDabr can still maintain relatively stable antimicrobial activity, and its MIC values ​​for E.coli 25922 and S.aureus 29213 only increased by 2-4 times, indicating that the dendritic branched antimicrobial peptides rich in non-natural amino acids are more stable in the SIF environment. In summary, the dendritic branched antimicrobial peptide KDabr rich in non-natural amino acids has excellent antimicrobial activity, good stability in the digestive tract environment, and low toxicity to animals, and is expected to truly realize the oral administration of antimicrobial peptides.

Claims

1. A dendritic branched antimicrobial peptide KDabr rich in unnatural amino acids, characterized in that: Its molecular formula is shown in formula (I), including a Lys and two groups of α-helical heptapeptide groups, and the amino acid sequence of the heptapeptide group is: LW Dab L Dab W D R , wherein Dab is 2,4-diaminobutyric acid, D R is D-type Arg, the C-terminus of the D-type Arg at the 7th position of the heptapeptide group of the first group is connected to the ε-N-terminus of Lys, the N-terminus of the Leu at the 1st position of the heptapeptide group of the second group is connected to the C-terminus of Lys, and the C-terminus of the antimicrobial peptide KDabr is amidated with -NH2, 2. The method for preparing a dendritic branched antimicrobial peptide KDabr rich in unnatural amino acids as claimed in claim 1, comprising the following steps: S1: Using the α-helical heptad repeat sequence "abcdefg" as a template, Leu was selected and placed at positions a and d to form a Leu zipper that stabilized the α-helical structure; Trp was added at positions b and f to further improve the stability of the α-helical structure through Trp-Trp interaction, thereby improving the antibacterial activity; Dab, Dab, and D-type Arg were added at positions c, e, and g, respectively, to increase the number of positive charges, and the amino acid sequence of the heptad group was obtained as follows: LW Dab L Dab W D R , connecting the C-terminus of the D-Arg at the 7th position of the first group of heptapeptide groups to the ε-N-terminus of Lys, connecting the N-terminus of the Leu at the 1st position of the second group of heptapeptide groups to the C-terminus of Lys, to obtain a polypeptide, and further amidating the C-terminus of the polypeptide with -NH2; S2: The polypeptide is synthesized by solid phase chemical synthesis, and then purified by reverse phase high performance liquid chromatography and identified by mass spectrometry. The polypeptide is then tested for antibacterial activity, hemolytic activity and protease stability, and is finally named as the dendritic branched antimicrobial peptide KDabr.

3. Use of a non-natural amino acid-rich dendritic branched antimicrobial peptide KDabr as claimed in claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria or / and Gram-negative bacteria, wherein the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis, and the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.

Images