A PEG-conjugated dendritic branched antimicrobial peptide, its preparation method and application

Abstract

The present invention provides a method for preparing and an application of a PEG-conjugated dendritic branched antimicrobial peptide, belonging to the field of biotechnology, the antimicrobial peptide PEG 1000 -K3 comprises two Lys, a heptapeptide group with three groups of α-helix structures and a PEG macromolecule, and the amino acid sequence of the heptapeptide group is shown as SEQ ID No.1. The present invention significantly improves the serum stability of the polypeptide through PEG conjugation, and simultaneously inhibits the toxicity of the antimicrobial peptide. And it further discloses the application of the antimicrobial peptide PEG 1000 -K3 in the preparation of drugs for treating Gram-positive and Gram-negative bacterial infectious diseases, and the antimicrobial peptide PEG 1000 -K3 has higher serum stability. In summary, the PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K3 has the potential to become a broad-spectrum antimicrobial 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 PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 preparation method and application. Background Art

[0002] Antimicrobial peptides (AMPs) have been proven to be ideal alternatives to traditional antibiotics, with advantages such as wide sources, safety, no residue, rapid action, and being less affected by bacterial resistance mechanisms. However, in the production application of natural antimicrobial peptides in animal husbandry, there are still certain limitations, including: being easily degraded by serum and gastrointestinal proteases, non-specific toxicity to host cells, and a decrease in activity at physiological pH and salt concentrations. In recent years, in order to overcome some limitations of natural antimicrobial peptides, the conjugation of antimicrobial peptides with other biomolecules has been widely regarded as an effective strategy. Therefore, a polypeptide conjugated with an antimicrobial peptide and other biomolecules is needed to improve biocompatibility and stability. Summary of the Invention

[0003] In view of the deficiencies of the above background art, the purpose of the present invention is to provide a PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 to solve the problems of poor biocompatibility and poor stability of existing natural antimicrobial peptides, and inhibit the toxicity of antimicrobial peptides.

[0004] The technical solution adopted by the present invention is as follows: A PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 , comprising two Lys, three heptapeptide groups with an α-helix structure, and polyethylene glycol with a molecular weight of 1000. The amino acid sequence of the heptapeptide group is 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 the first Lys, the C-terminal of Arg at the 7th position of the second heptapeptide group is connected to the ε-N terminal of the second Lys, the N-terminal of Leu at the 1st position of the third heptapeptide group is connected to the C-terminal of the second Lys, the C-terminal of the first Lys is connected to the α-N terminal of the second Lys by an amide bond, a Cys is connected to the α-N terminal of the first Lys, and polyethylene glycol is conjugated to the Cys; the C-terminal of the antimicrobial peptide PEG 1000 -K 3 is amidated with an amino group.

[0005] Further, for a PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 as described above, its molecular formula is shown in formula (I),

[0006]

[0007] Another object of the present invention is to provide a PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 The preparation method is as follows:

[0008] Step 1: Using the α-helical heptapeptide repeat sequence "abcdefg" as a template, Leu is selected and placed at positions a and d to form a Leu zipper with a stable α-helical structure; Trp is added at positions b and f, and the Trp-Trp interaction is used to further improve the stability of the α-helical structure, thereby enhancing the antibacterial activity, and Arg is added at the remaining positions to increase the number of positive charges, obtaining a heptapeptide group sequence as shown in SEQ ID No.1. The C-terminus of the Arg at the 7th position of the first group of antibacterial units LWRLRWR is connected to the ε-N terminus of the first Lys, the C-terminus of the Arg at the 7th position of the second group of heptapeptide groups is connected to the ε-N terminus of the second Lys, the N-terminus of the Leu at the 1st position of the third group of heptapeptide groups is connected to the C-terminus of the second Lys, and the C-terminus of the first Lys is connected to the α-N terminus of the second Lys by an amide bond; for convenient PEG conjugation, a Cys is added to the α-N terminus of the first Lys, and polyethylene glycol is conjugated to the Cys, finally obtaining a polyethylene glycol-conjugated dendritic branched polypeptide sequence, and its C-terminus is amidated with an amino group;

[0009] Step 2: The designed polypeptide is synthesized by solid-phase chemical synthesis, further purified by reverse-phase high-performance liquid chromatography and identified by mass spectrometry, then the polypeptide is conjugated with polyethylene glycol to prepare a polyethylene glycol-conjugated dendritic branched polypeptide, and finally, antibacterial activity detection, hemolytic activity detection and serum stability detection are carried out, and finally it is named antimicrobial peptide PEG 1000 -K 3 。

[0010] Another object of the present invention is to provide an application of the above-mentioned PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 in the preparation of drugs for treating Gram-positive bacteria or / and Gram-negative bacteria infectious diseases.

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

[0012] Further, the Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium.

[0013] The present invention has the following advantages: By PEG conjugation, the serum stability of the polypeptide is significantly improved, and the toxicity of the antimicrobial peptide is inhibited. For the obtained PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 antibacterial and hemolytic activities and serum stability were detected, and it was found that the antimicrobial peptide PEG 1000 -K 3 has good antibacterial activity against several tested Gram-negative and Gram-positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis, and has excellent biocompatibility and serum stability. By measuring the minimum concentration (MHC) of the antimicrobial peptide when the dendritic branched antimicrobial peptide K 3 without PEG conjugation, PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 causes 5% hemolysis of human red blood cells (hRBC), it was found that the MHC value of the antimicrobial peptide PEG 1000 -K 3 (>256 μg / mL) is much higher than the HC 3 value (32 μg / mL) of the antimicrobial peptide K 50 , which indicates that the antimicrobial peptide PEG 1000 -K 3 has high biosafety. Further calculating the therapeutic index (TI) of the antimicrobial peptide K 3 and the antimicrobial peptide PEG 1000 -K 3 to evaluate the cell selectivity of the two, the TI value of the PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 is much higher than the TI value (4.00) of the dendritic branched antimicrobial peptide K 3 , and the TI value increases by 4 times after PEG modification. In summary, the PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 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 (RP-HPLC) chromatogram of the dendritic branched antimicrobial peptide K 3 ;

[0015] Figure 2 is the RP-HPLC chromatogram of the dendritic branched antimicrobial peptide K 3 modified by Cys (i.e., the naked peptide before conjugation);

[0016] Figure 3 is the dendritic branched antimicrobial peptide K3 Mass spectrum;

[0017] Figure 4 is the dendritic branched antimicrobial peptide K modified by Cys 3 (i.e., the naked peptide before conjugation) mass spectrum;

[0018] Figure 5 is PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI ToF) spectrum;

[0019] Figure 6 is dendritic branched antimicrobial peptide K 3 , PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 hemolytic activity diagram; Detailed implementation mode

[0020] The present invention will be further described in detail below in conjunction with examples and drawings.

[0021] Example 1

[0022] Design of antimicrobial peptide

[0023] 1. Using the α-helical heptapeptide repeat sequence "abcdefg" as a template, Leu is selected and placed at positions a and d to form a Leu zipper with a stable α-helical structure; pairs of Trp are added at positions b and f, and the Trp-Trp interaction is used to further improve the stability of the α-helical structure, thereby enhancing the antibacterial activity, and Arg is added at the remaining positions to increase the number of positive charges. The C-terminus of the Arg at the 7th position of the first group of antibacterial units LWRLRWR is connected to the ε-N terminus of the first Lys, the C-terminus of the Arg at the 7th position of the second group of heptapeptide groups is connected to the ε-N terminus of the second Lys, the N-terminus of the Leu at the 1st position of the third group of heptapeptide groups is connected to the C-terminus of the second Lys, the C-terminus of the first Lys is connected to the α-N terminus of the second Lys by an amide bond, and amidation is performed at the C-terminus of the polypeptide to form a dendritic branched antimicrobial peptide with 3 branches, named K 3 .

[0024] 2. On the basis of the above dendritic branched antimicrobial peptide K 3 , a Cys is added to the α-N terminus of its first Lys scaffold for the purpose of facilitating the conjugation of PEG. Further, PEG 1000 is conjugated to Cys, and finally, the PEG-conjugated dendritic branched antimicrobial peptide is named PEG 1000 -K 3 . The sequences of the antimicrobial peptides are shown in Table 1.

[0025] Table 1 Dendritic branched antimicrobial peptide K 3 and PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 Sequence

[0026]

[0027] Example 2

[0028] Synthesis and identification of antimicrobial peptides

[0029] 1. Synthesis of dendritic branched antimicrobial peptide K by solid-phase chemical synthesis method 3 and dendritic branched antimicrobial peptide K modified by Cys 3 (i.e., the naked peptide before conjugation)

[0030] First, Fmoc-Arg(pbf)-OH was coupled to Rink resin. After 30 min of piperidine deprotection reaction, piperidine was removed and washed with dimethylformamide (DMF), and the deprotection color was detected by ninhydrin. Then the subsequent linear amino acids were sequentially linked until Cys at the N-terminus was reached. After removing the Fmoc at the N-terminus, Boc was attached to obtain the following substance:

[0031] Boc-C(trt)K(dde)K(dde)LW(boc)R(pbf)LR(pbf)W(boc)R(pbf)-Rink Resin

[0032] The above resin was treated with 2% hydrazine hydrate in DMF solution for 15 min to remove hydrazine hydrate and washed 5 times with DMF. At this time, the dde protecting group of the Lys side chain was removed and the amino group was exposed. Then, in the previous condensation method, these amino acids were sequentially linked to the amino group of the Lys side chain in the order of R, W, R, L, R, W, L to obtain the resin of the target peptide.

[0033] The target peptide was cleaved from the resin with 95% TFA, and all side chain protecting groups of the sequence were cut off simultaneously. The crude product of the target peptide was obtained. After liquid-phase purification and lyophilization, the pure target peptide was obtained.

[0034] 2. PEG conjugation of dendritic branched antimicrobial peptide

[0035] The target peptide and mPEG1000-Mal were each dissolved in PBS with pH = 7.5, mixed and reacted at room temperature for 4 h. Then the reaction solution was dialyzed with an MD4000 dialysis bag, and the pure product solution of PEG 1000 -CK(LWRLRWR)K(LWRLRWR)LWRLRWR-NH 2 was obtained after dialysis and lyophilized.

[0036] Dendritic antimicrobial peptide K 3 , Cys-modified dendritic antimicrobial peptide K 3 The reversed-phase HPLC chromatogram of the uncoupled peptide is shown in the attached Figure 1 , 2 .

[0037] Dendritic antimicrobial peptide K 3 , Cys-modified dendritic antimicrobial peptide K 3 The mass spectrum of the uncoupled peptide is shown in the attached Figure 3 , 4 .

[0038] PEG-coupled dendritic branched antimicrobial peptide PEG 1000 -K 3 The mass spectrum of Figure 5 .

[0039] Example 3

[0040] Antibacterial activity of antimicrobial peptides

[0041] The antibacterial activity of antimicrobial peptides was determined by measuring the minimum inhibitory concentration (MIC) of antimicrobial peptides. The designed and successfully synthesized antimicrobial peptides were formulated into a 5120 μg / mL stock solution for biological activity determination. The bacterial solution frozen at -20°C was streaked and inoculated on MHA solid culture medium, and cultured overnight at 37°C with a shaking speed of 220 rpm. Subsequently, a single colony of the overnight culture was inoculated in fresh, sterile MHB and cultured until the logarithmic phase of growth. The OD of the bacterial solution was measured using a spectrophotometer. 600nm The value was adjusted to 0.38-0.40 for standby use. A sterile 96-well culture plate was taken, and 0.2% BSA (containing 0.01% acetic acid) filtered through a 0.22μM water filter was used as the diluent, and the MIC of the antimicrobial peptide was determined by the microbroth dilution method. The antimicrobial peptide stock solution was added to the diluent of the 96-well culture plate, and a 2-fold gradient dilution was performed, and then 50μL of the bacterial solution diluted 1000 times was added to each well. MHB containing bacteria was used as a positive control, and sterile MHB culture medium was used as a negative control. The 96-well culture plate was sealed with a sealing film to prevent bacterial contamination and placed in a 37°C incubator for incubation for 16-18h. The negative control well remained clear, indicating that the test process was pollution-free. Under naked eye observation, the lowest peptide concentration at which the turbidity did not increase compared with the negative control was defined as the MIC of the peptide. Three independent repeated experiments were performed, with two parallels for each repeat. The minimum inhibitory concentration of antimicrobial peptides is shown in Table 2.

[0042] Table 2 Dendritic branched antimicrobial peptide K 3 PEG-coupled dendritic antimicrobial peptide PEG1000 -K 3 Antibacterial activity (μg / mL)

[0043]

[0044]

[0045] As can be seen from the table, the dendritic branched antimicrobial peptide K 3 , the PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 showed excellent antibacterial activity against all 8 tested strains. Although the antibacterial activity of PEG 1000 -K 3 was less than that of K without modified PEG 3 , its MIC value was between 16 - 32 μg / mL, indicating that PEG 1000 -K 3 still had a certain antibacterial activity.

[0046] Example 4

[0047] Hemolytic activity of antimicrobial peptides

[0048] To evaluate the safety of antimicrobial peptides, the hemolytic behavior of peptides in the concentration range of 1 - 256 μg / mL against human red blood cells (hRBC) was studied.

[0049] Collect 1 mL of fresh blood from healthy people and discard the supernatant after centrifugation at 4°C, 1000×g for 5 min, and collect the red blood cells. Subsequently, wash the collected red blood cells 3 times with PBS buffer under the same centrifugation conditions, and finally resuspend them with 10 mL of PBS for standby. Take a sterile 96-well culture plate, use PBS buffer as the diluent, add the antimicrobial peptide stock solution to the diluent in the 96-well culture plate, perform a 2-fold serial dilution, and then add 50 μL of the red blood cell suspension to each well. Incubate at a constant temperature of 37°C in an incubator for 1 h. After incubation, centrifuge the 96-well culture plate at 4°C, 1000×g for 10 min; aspirate 50 μL of the supernatant after centrifugation and transfer it to a new 96-well culture plate, and measure the absorbance at 570 nm using an enzyme-linked immunosorbent assay reader. Use 50 μL of red blood cells plus 50 μL of 0.1% Triton X-100 as the positive control and 50 μL of red blood cells plus 50 μL of PBS buffer as the negative control. The test results are shown in the appendix of the specification Figure 6 . Use the minimum concentration (MHC) of the antimicrobial peptide when causing 5% hemolysis of human red blood cells by the dendritic branched antimicrobial peptide K 3 , the PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 to evaluate the biocompatibility of the two, and further calculate K 3 , PEG1000 -K 3 The therapeutic index (TI) was used to evaluate the cell selectivity of the two, as shown in Table 3.

[0050] Table 3 Dendritic branched antimicrobial peptide K 3 , PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 biocompatibility

[0051]

[0052] a Geometric mean (GM) of the minimum inhibitory concentration of the antimicrobial peptide against the tested bacteria;

[0053] b MHC is the lowest concentration of the antimicrobial peptide when 5% hemolysis of human red blood cells (hRBC) occurs. When no detectable hemolytic activity was observed at 256 μg / mL, 512 μg / mL was used to calculate the therapeutic index;

[0054] c The therapeutic index (TI) is the ratio of MHC to GM.

[0055] As shown in the attached Figure 6 It can be seen that dendritic branched antimicrobial peptide K 3 could cause hemolysis within the MIC range (4 - 32 μg / mL) and nearly 40% hemolysis at the highest concentration tested, while PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 did not cause hemolysis even at the highest concentration. By measuring the lowest concentration (MHC) of dendritic branched antimicrobial peptide K 3 , PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 when it caused 5% hemolysis of human red blood cells (hRBC), it was found that the MHC value of PEG 1000 -K 3 (>256 μg / mL) was much higher than that of K 3 's HC 50 value (32 μg / mL), indicating that PEG 1000 -K 3 had higher biosafety. Further calculation of the therapeutic index (TI) of K 3 , PEG 1000 -K 3 was used to evaluate the cell selectivity of the two. A higher TI value indicates higher cell selectivity of the antimicrobial peptide. Among them, the TI value of PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 (21.11) was much higher than that of dendritic branched antimicrobial peptide K3 The TI value (4.00) increased by 4 times after PEG modification. This indicates that although the antibacterial activity of the polypeptide decreased to a certain extent after PEG modification, its hemolytic activity was significantly improved, enhancing cell selectivity. PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 has better cell selectivity.

[0056] Example 5

[0057] Serum stability of antimicrobial peptides

[0058] Using E. coli 25922 as a typical Gram-negative bacterium and S. aureus 29213 as a typical Gram-positive bacterium, the sensitivity of antimicrobial peptides to serum was determined. The polypeptide was mixed with an equal volume of serum at different concentrations (25%, 50%, 100%) and incubated at 37°C for 3 h, followed by a minimum inhibitory concentration experiment. The test results are shown in Table 4.

[0059] Table 4 Dendritic branched antimicrobial peptide K 3 and PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 serum stability

[0060]

[0061] By measuring the serum sensitivity of dendritic branched antimicrobial peptide K 3 and PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 against E. coli 25922 and S. aureus 29213, it was found that K 3 could not maintain its original antibacterial activity in serum environments at all concentrations. After PEG modification, PEG 1000 -K 3 could maintain its original antibacterial activity against both bacteria in a 25% serum environment. In a 50% serum environment, it could maintain its original antibacterial activity against E. coli 25922, while the MIC value against S. aureus 29213 increased from the original 32 μg / mL to 64 μg / mL. This indicates that PEG 1000 -K 3 has higher serum stability, and the PEG modification strategy of polypeptides can significantly improve the serum stability of polypeptides.

Claims

1. A PEG-conjugated dendritic branched antimicrobial peptide PEG 1000 -K 3 , Characterized in that: Its molecular formula is shown in Formula (I), including two Lys, a heptapeptide group with three groups of α-helix structures, and polyethylene glycol with a molecular weight of 1000. 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 group of heptapeptide groups is connected to the ε-N terminus of the first Lys. The C-terminus of Arg at the 7th position of the second group of heptapeptide groups is connected to the ε-N terminus of the second Lys. The N-terminus of Leu at the 1st position of the third group of heptapeptide groups is connected to the C-terminus of the second Lys. The C-terminus of the first Lys and the α-N terminus of the second Lys are connected by an amide bond. A Cys is connected to the α-N terminus of the first Lys, and polyethylene glycol is conjugated to the Cys; the antimicrobial peptide PEG 1000 -K 3 The C-terminus of is amidated with an amino group, ， Formula (I).

2. A preparation method of a PEG-coupled dendritic branched antibacterial peptide PEG according to claim 1 1000 -K 3 ​ Characterized in that The method steps are as follows: Step 1: Using the α-helical heptapeptide repeat sequence "abcdefg" as a template, place Leu at positions a and d to form a Leu zipper with a stable α-helical structure; add Trp at positions b and f to further enhance the stability of the α-helical structure through Trp-Trp interaction, thereby enhancing 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 the Arg at the 7th position of the first group of antibacterial unit LWRLRWR to the ε-N terminus of the first Lys, connect the C-terminus of the Arg at the 7th position of the second group of heptapeptide groups to the ε-N terminus of the second Lys, connect the N-terminus of the Leu at the 1st position of the third group of heptapeptide groups to the C-terminus of the second Lys, and connect the C-terminus of the first Lys to the α-N terminus of the second Lys with an amide bond; for convenient PEG conjugation, add a Cys to the α-N terminus of the first Lys and conjugate polyethylene glycol to the Cys, finally obtaining a polypeptide sequence with polyethylene glycol-conjugated dendritic branches, and its C-terminus is amidated with an amino group; Step 2: Synthesize the designed polypeptide by solid-phase chemical synthesis, further purify it by reverse-phase high-performance liquid chromatography and identify it by mass spectrometry, then couple the polypeptide with polyethylene glycol to prepare a polypeptide with polyethylene glycol-conjugated dendritic branches, and finally perform antibacterial activity detection, hemolytic activity detection and serum stability detection, and finally name it antibacterial peptide PEG 1000 -K 3 。 3. Use of a PEG-conjugated dendritic branched antimicrobial peptide PEG according to claim 1 1000 -K 3 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria, wherein the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis and Staphylococcus epidermidis, and the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium.

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