A β-hairpin antimicrobial peptide with Trp-cation cross-chain interaction and its preparation method and application

Abstract

The present invention provides a β-hairpin antimicrobial peptide with a Trp-cation cross-chain interaction and its preparation method and application. Based on the β-hairpin antimicrobial peptide WRFPG, whose amino acid sequence is shown in SEQ ID No.2, five polypeptides are obtained, which are then synthesized by solid-phase chemical synthesis. Antimicrobial peptides with excellent antimicrobial activity and low toxicity are screened out by measuring antimicrobial activity, hemolytic activity and cytotoxicity. Its amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with NH2, and is finally named antimicrobial peptide KKRRPG. The antimicrobial peptide KKRRPG of the present invention not only ensures antimicrobial activity, but also reduces hemolytic activity and improves the selection index for human embryonic kidney cells HEK293t.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a Trp-cation cross-chain interactive beta-hairpin antimicrobial peptide and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs) are a class of small polypeptides widely distributed in animals, insects, and plants. As an important component of innate host defense, most AMPs can bind to negatively charged cell membranes, thereby disrupting the integrity of bacterial cell membranes. This unique membrane-breaking mechanism, distinct from antibiotics, makes it difficult for bacteria to develop resistance to AMPs. β-pleated antimicrobial peptides are often stabilized by disulfide bonds, resulting in an antiparallel folded structure. However, the chemical synthesis of these disulfide bonds is difficult and expensive. The tryptophan-cation, which forms a cross-chain interaction within the polypeptide molecule by utilizing the π bond of the tryptophan benzene ring with positively charged amino acids, provides cross-chain interactions within the β-hairpin structure, avoiding the negative antimicrobial activity associated with the introduction of complex and lengthy polypeptide sequences and disulfide bonds. However, the in vivo application of AMPs is hampered by the common problems of high toxicity and high hemolytic activity. Summary of the Invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide a Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG, which is highly effective and low-toxic, and solves the problems of high hemolytic activity and large toxic and side effects of existing antimicrobial peptides.

[0004] The technical solution adopted by the present invention is as follows: a Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG, wherein the antimicrobial peptide uses PG as a turn unit and forms a β-hairpin structure through Tryptophan-cation cross-chain interaction. Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

[0005] Another object of the present invention is to provide a method for preparing the Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG as described above, as follows: based on the β-hairpin antimicrobial peptide WRFPG, whose amino acid sequence is shown in SEQ ID No. 2, the four positively charged amino acids therein are positionally exchanged to obtain five polypeptides, which are then synthesized by solid-phase chemical synthesis. Antimicrobial activity, hemolytic activity, and cytotoxicity assays are performed to screen out an antimicrobial peptide with excellent antimicrobial activity and low toxicity, whose amino acid sequence is shown in SEQ ID No. 1, and whose C-terminus is amidated with -NH2, and finally named antimicrobial peptide KKRRPG.

[0006] Another object of the present invention is to provide a use of the Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG as described above in the preparation of a drug for treating and / or preventing infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

[0007] Furthermore, in the above application, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.

[0008] Furthermore, in the application described above, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

[0009] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, wherein the drug contains the antimicrobial peptide KKRRPG as described above.

[0010] The beneficial effects and advantages of the present invention are as follows: The antimicrobial peptide KKRRPG, which forms a stable β-hairpin structure without disulfide bonds formed by cysteine ​​pairs, has a sequence length of only 12 amino acids and reduces hemolytic activity and cytotoxicity while maintaining good antibacterial activity. In summary, the antimicrobial peptide KKRRPG is an antimicrobial peptide with high application value, exhibiting the advantages of high efficacy and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the HPLC chromatogram of the antimicrobial peptide KKRRPG;

[0012] Figure 2 This is the mass spectrum of the antimicrobial peptide KKRRPG. DETAILED DESCRIPTION

[0013] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1

[0015] Design of the antimicrobial peptide KKRRPG

[0016] Its amino acid sequence is:

[0017]

[0018] Based on the β-hairpin amphiphilic peptide arrangement, the Trp-cation cross-chain interaction serves as a force to assist the PG turn unit in forming the β-hairpin structure, and is named KKRRPG. The sequence is shown in Table 1.

[0019] 1 Amino acid sequence of the derived peptide

[0020]

[0021] By exchanging the positions of positively charged amino acids in AMPs, an antimicrobial peptide KKRRPG with high antibacterial activity and low hemolytic activity was obtained. Its sequence length is 12, the turn unit is a PG unit, and the β-hairpin structure is formed through the Trp-cation cross-chain interaction.

[0022] Example 2

[0023] Antimicrobial peptide KKRRPG synthesized by solid-phase chemical synthesis (Synthesized by Shanghai Sangon Biotechnology Co., Ltd.)

[0024] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); follow this procedure from the C-terminus to the N-terminus until the synthesis is complete, obtaining a resin with the side chain protection of the Fmoc group removed;

[0025] 2. Add a cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with TFA (trifluoroacetic acid) to precipitate, mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, and then add about 10 times the volume of pre-cooled anhydrous ether. Precipitate at -20°C for 3 hours to precipitate a white powder. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain the polypeptide, wherein the cleavage reagent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0026] 3. Use 0.2 mol / L sodium sulfate (adjusted to pH 7.4 with phosphoric acid) to equilibrate the column for 30 min, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and apply gradient elution (eluent: methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30) on a C18 reverse-phase atmospheric pressure column at a flow rate of 1 mL / min and a detection wave of 220 nm to collect the main peak and freeze-dry; further purification is performed on a reverse-phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, the elution concentration is 25% B to 40% B, the elution time is 12 min, the flow rate is 1 mL / min, and the main peak is collected as above and freeze-dried;

[0027] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray mass spectrometry. The molecular weights (e.g. Figure 1 、 2The molecular weight of the antimicrobial peptide was basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide was greater than 95%.

[0028] Example 3

[0029] 1. Determination of antimicrobial activity: The minimum inhibitory concentration of several antimicrobial peptides was determined by the broth microdilution method. Bacteria were incubated at 37°C overnight with constant shaking at 220 rpm and then transferred to a new MHB until the logarithmic phase of growth was reached. 50 μL of bacterial culture (~10 6 CFU / mL) were added to 50 μL of BSA containing various concentrations of antimicrobial peptides and incubated at 37°C for 18 hours. At the end of the 18-hour incubation, the absorbance at 492 nm was measured. The minimum inhibitory concentration (MIC) was defined as the concentration of antimicrobial peptide at which no microbial growth was observed visually or spectrophotometrically. Broth containing microbial cells served as a negative control, and uninoculated broth served as a positive control. The test was performed in triplicate, with two replicates per replicate.

[0030] The test results are shown in Table 2.

[0031] Table 2 Antibacterial activity of antimicrobial peptides (μM)

[0032]

[0033]

[0034] As can be seen from Table 2, the polypeptide KKRRPG exhibits high antibacterial activity against both Gram-negative and Gram-positive bacteria.

[0035] 2. Hemolytic Activity Assay: 1 mL of fresh blood from a healthy individual was centrifuged at 1000 g for 5 minutes to collect red blood cells (RBCs). The cells were washed three times with PBS and resuspended in 10 mL of PBS. 50 μL of the RBC suspension was mixed evenly with 50 μL of antimicrobial peptide solutions of varying concentrations dissolved in PBS and incubated in a 37°C incubator for 1 hour. After 1 hour, the cells were removed and centrifuged at 1000 g for 5 minutes at 4°C. The supernatant was collected and absorbance was measured at 570 nm using a microplate reader. The average values ​​for each group were calculated and compared. A negative control (50 μL of RBCs plus 50 μL of PBS) was used; a positive control (50 μL of RBCs plus 50 μL of 0.1% Triton X-100) was used. The minimum hemolytic concentration was the antimicrobial peptide concentration that caused 10% hemolysis. The test results are shown in Table 3.

[0036] Table 3 Determination of hemolytic activity and therapeutic index of antimicrobial peptides

[0037]

[0038] As can be seen from Table 3, compared with the known antimicrobial peptide WKFPG, the antimicrobial peptide KKRRPG reduced the hemolytic activity and increased the therapeutic index, with the therapeutic index reaching 140.11.

[0039] 3. Cytotoxicity assay: (1) Preparation of cell suspension: Porcine intestinal epithelial cells IPEC-J2 frozen in liquid nitrogen were revived and then transferred to 5 mL of DMEM medium containing 10% fetal bovine serum and cultured at 37°C. When the cells were confluent with 25 cm 2 When the bottom of the culture flask is about 70%, it means that the cells have entered the rapid growth phase and are passaged. Rinse the cells twice with sterile PBS and add 2mL of 0.25% trypsin to digest the cells. At the same time, observe the cell morphology during digestion. When the intercellular gap increases and most of them become round, aspirate the digestion solution, add 5mL of DMEM culture medium (containing 10% calf serum), blow gently, mix well, and form a single cell suspension. Adjust the cell concentration, and then add 50μL of cell suspension to wells 1 to 11 in each row of the 96-well plate. The final concentration is about 2×10 4 cells / well, add 50 μL of culture medium to well 12;

[0040] (2) Antimicrobial peptide treatment: 50 μL of antimicrobial peptide diluted in culture medium was added to wells 1 to 10 of a 96-well plate and incubated at 37°C for 16-18 h. Well 12 contained only culture medium as a negative control, well 11 contained cells but no antimicrobial peptide as a positive control, and wells 1 to 10 were assay wells.

[0041] (3) Result evaluation: After the incubation, 50 μL of 5 mg / mL MTT was added to each well of a 96-well plate and incubated at 37°C for 4 h. Then, 150 μL of DMSO was added and shaken for 10 min to dissolve the crystals. The OD value was measured using a microplate reader. 492 absorbance.

[0042] (4) Each experiment was repeated more than 3 times, and the cell survival rate was calculated according to the following formula:

[0043] Cell viability (100%) = (OD 测定值 / OD 阳性对照 )×100%

[0044] Table 4 Determination of the selection index of antimicrobial peptides HEK293t cells

[0045]

[0046] The above results show that by changing the position of positively charged amino acids to enable Trp-cation cross-chain interactions, the antimicrobial peptide KKRRPG designed with PG as the turn unit has good antibacterial activity and lower hemolytic activity, as well as an improved selection index for human embryonic kidney cells HEK293t, indicating that it has strong potential to replace antibiotics.

Claims

1. A β-hairpin antimicrobial peptide KKRRPG with Trp-cation cross-chain interaction, characterized in that: The antimicrobial peptide uses PG as a turn unit and forms a β-hairpin structure through tryptophan-cation cross-chain interaction. Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

2. The method for preparing a Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG according to claim 1, characterized in that: The method is as follows: based on the β-hairpin antimicrobial peptide WRFPG, whose amino acid sequence is shown in SEQ ID No. 2, the four positively charged amino acids therein are exchanged to obtain five polypeptides, which are then synthesized by solid-phase chemical synthesis. Antimicrobial activity, hemolytic activity and cytotoxicity assays are performed to screen out an antimicrobial peptide with excellent antimicrobial activity and low toxicity, whose amino acid sequence is shown in SEQ ID No. 1, and whose C-terminus is amidated with -NH2. The peptide is finally named antimicrobial peptide KKRRPG.

3. Use of a Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG according to claim 1 in the preparation of a medicament for treating and / or preventing infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria, wherein the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, or Salmonella typhimurium, and the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

4. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The drug contains the antimicrobial peptide KKRRPG according to claim 1.

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