Antibacterial peptide with asymmetric biphase hydrophobic core as well as preparation method and application of antibacterial peptide

Abstract

The invention discloses an antibacterial peptide with an asymmetric biphase hydrophobic core as well as a preparation method and application of the antibacterial peptide, and belongs to the technical field of bioengineering. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID No. 1. Detection on antibacterial activity, hemolytic activity, cytotoxicity and salt ion stability of the antibacterial peptide shows that the antibacterial peptide has extremely high cell selectivity (SI = 154.64), does not show obvious cytotoxicity when the test concentration is 32 mu M or below, and can keep high stability in salt ions with different physiological concentrations. In conclusion, the antibacterial peptide with the asymmetric biphasic hydrophobic core has the potential to become a broad-spectrum antibacterial drug for treating gram-positive bacterium infection and gram-negative bacterium infection.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to an antimicrobial peptide GRF3Q with an asymmetric biphasic hydrophobic core, a preparation method thereof, and an application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), as an important part of the natural immune system, have broad-spectrum antibacterial activity and are not easily induced to produce pathogen drug resistance, and are considered ideal alternative candidates for traditional antibiotics. However, natural antimicrobial peptides face multiple challenges in clinical translation: for example, most natural antimicrobial peptides have high hemolytic activity and cytotoxicity to host cells (such as red blood cells, epithelial cells), resulting in a narrow therapeutic window; their antibacterial activity is easily inhibited by high-concentration salt ions (such as Na + , Mg 2+ , Ca 2+ ) in the physiological environment, significantly reducing the in vivo efficacy; some antimicrobial peptides have insufficient selectivity for Gram-positive and Gram-negative bacteria, which may disrupt the balance of the host commensal flora. Therefore, by rationally designing and optimizing the molecular characteristics of antimicrobial peptides to achieve a synergistic improvement of "high selectivity and ideal salt stability" has become the core goal of the current engineering transformation of antimicrobial peptides.

[0003] In recent years, through structure-activity relationship (SAR) analysis, researchers have found that the hemolytic toxicity of antimicrobial peptides is closely related to the proportion of hydrophobic residues and the amphiphilic structure. For example, the excessive exposure of the hydrophobic surface of α-helical antimicrobial peptides (such as LL-37, magainin) will enhance the hydrophobic interaction with eukaryotic cell membranes, resulting in cell membrane lysis. To address this problem, some studies have attempted to regulate the amphiphilic balance by reducing the hydrophobicity index (such as replacing leucine and phenylalanine with serine and glycine) or introducing charged residues, but this may be at the cost of sacrificing antibacterial activity. In addition, the electrostatic interaction of cationic antimicrobial peptides is easily interfered by the salt ion shielding effect, resulting in a decrease in their binding ability to negatively charged bacterial membranes. Studies have shown that by optimizing the net positive charge number (+4 to +6), introducing aromatic residues or rigid domains (such as proline turns), the salt tolerance of antimicrobial peptides can be enhanced, but the impact on selectivity still needs to be systematically explored. Summary of the Invention

[0004] Based on the above deficiencies, the purpose of the present invention is to provide an antimicrobial peptide with an asymmetric biphasic hydrophobic core, so as to solve the problems of high hemolytic activity, high cytotoxicity of antimicrobial peptides and instability under physiological salt conditions, thus making it difficult to be used as an antibacterial drug.

[0005] The technical solution adopted by the present invention is as follows: An antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core, whose amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2.

[0006] Furthermore, its molecular formula is shown in formula (I):

[0007]

[0008] The present invention also provides a preparation method of an antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described above, and the steps are as follows:

[0009] Step 1: Select aromatic amino acid phenylalanine and isoleucine as the double hydrophobic cores to jointly provide hydrophobicity for the polypeptide. Select arginine to form the positive charge cluster of the polypeptide, and place arginine at the C-terminal position of the polypeptide to improve the selectivity for the bacterial membrane. Finally, the arginines at the two positions jointly provide positive charges for the polypeptide. Select proline-glycine and glutamine for transition. Proline-glycine separates the positive charge cluster and the hydrophobic cluster, and glutamine can neutralize the strong positive charge of arginine to reduce the electrostatic attraction to host cells, thereby reducing hemolytic activity and cytotoxicity. Use glycine to cap at the N-terminus of the polypeptide in order to reduce the attraction of positive charges to the host cell membrane and reduce hemolytic activity. The obtained polypeptide has an amino acid sequence shown in SEQ ID No.1, and the C-terminus of the polypeptide sequence is amidated with -NH2 to increase the net positive charge of the polypeptide, improve the selectivity for the bacterial membrane, and enhance antibacterial activity.

[0010] Step 2: Synthesize the polypeptide by solid-phase chemical synthesis method, and after purification by reverse-phase high-performance liquid chromatography and mass spectrometry identification, perform antibacterial activity detection, hemolytic activity detection, cytotoxicity detection, and salt ion stability detection on the polypeptide, and finally name it antibacterial peptide GRF3Q.

[0011] Another object of the present invention is to provide the application of an antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described above in the preparation of drugs for treating Gram-positive bacteria and / or Gram-negative bacteria 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, Salmonella typhimurium or Salmonella pullorum.

[0014] 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 infections, and the drug contains an antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described above.

[0015] The present invention has the following advantages and beneficial effects: The antibacterial peptide of the present invention significantly reduces hemolytic activity and cytotoxicity, and is expected to solve the problem that existing antibacterial peptides are difficult to be applied to maintaining the healthy development of animal husbandry. By detecting the antibacterial activity, hemolytic activity, cytotoxicity, and salt ion stability of the antibacterial peptide GRF3Q of the present invention, it is found that the antibacterial peptide GRF3Q has high antibacterial activity against several measured Gram-negative and Gram-positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis. At the same time, the antibacterial peptide GRF3Q has a high hemolysis value, does not produce obvious hemolysis at 256 μM, and does not show obvious cytotoxicity at 32 μM. In addition, the antibacterial peptide GRF3Q still maintains a low MIC in different physiological concentrations of salt ions and different concentrations of serum environments, and has high salt ion stability and serum stability. In summary, the antibacterial peptide GRF3Q of the present invention has the potential to become a broad-spectrum antibacterial drug for treating Gram-positive and Gram-negative bacterial infections, so as to achieve the purpose of maintaining the healthy development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Reverse-phase high performance liquid chromatography diagram of the antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core;

[0017] Figure 2 Mass spectrometry diagram of the antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core;

[0018] Figure 3 Hemolytic activity diagram of the antibacterial peptide GRF3Q;

[0019] Figure 4 Cytotoxicity diagram of the antibacterial peptide GRF3Q. DETAILED DESCRIPTION OF THE EMBODIMENTS

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

[0021] Example 1

[0022] Design of the antibacterial peptide

[0023] 1. Since tryptophan in aromatic amino acids is hydrophobic and the largest in volume among amino acids, but it is prone to form significant cytotoxicity after aggregation in aromatic amino acids, phenylalanine, an aromatic amino acid, and isoleucine, an aliphatic amino acid, are selected as the dual hydrophobic cores to jointly provide hydrophobicity for the antimicrobial peptide. Since arginine has a strong positive charge, arginine is selected to form the positive charge cluster of the antimicrobial peptide, and arginine is placed at the C-terminal position of the polypeptide to improve the selectivity for bacterial membranes. Eventually, the arginines at both positions jointly provide positive charges for the antimicrobial peptide. Between the positively charged amino acids and the hydrophobic amino acids, proline-glycine and glutamine are selected for transition. Proline-glycine separates the positive charge cluster and the hydrophobic cluster, and glutamine can moderately neutralize the strong positive charge of arginine, in order to reduce the electrostatic attraction to host cells, thereby reducing hemolytic activity and cytotoxicity. Glycine capping is carried out at the N-terminal of the polypeptide sequence to reduce the attraction of positive charges to the host cell membrane and lower hemolytic activity. Amidation with -NH2 is carried out at the C-terminal of the polypeptide sequence to increase the net positive charge for the overall peptide sequence, improve the selectivity for bacterial membranes, and thus enhance antibacterial activity. The designed antimicrobial peptide maximally improves antibacterial activity while having low hemolytic activity and good stability. The sequence, molecular weight, and charge number of the antimicrobial peptide are shown in Table 1.

[0024] Table 1 Sequence, molecular weight, and charge number of polypeptide GRF3Q

[0025]

[0026] Example 2

[0027] Synthesis and identification of antimicrobial peptide

[0028] The designed antimicrobial peptide was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. (Synpeptide Co Ltd.) through solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain the target compound for subsequent determination of antibacterial activity, hemolytic activity, cytotoxicity, and salt ion stability.

[0029] The reverse-phase high-performance liquid chromatography chart of antimicrobial peptide GRF3Q is shown in the appendix Figure 1 .

[0030] The mass spectrometry chart of antimicrobial peptide GRF3Q is shown in the appendix Figure 2 .

[0031] Example 3

[0032] Antibacterial activity of polypeptide

[0033] Determine the minimum inhibitory concentration of several antimicrobial peptides by the microbroth dilution method. Understand the antibacterial activity of the polypeptide by measuring the minimum inhibitory concentration (MIC) of the polypeptide. Dissolve the designed and successfully synthesized polypeptide in sterile ultrapure water in a sterile operating bench to a concentration of 2.56 mM to obtain a stock solution of AMPs for subsequent bioactivity assays. The bacteria are cultured overnight in a shaker at 37 °C and 220 rpm, and then transferred to fresh MHB for culturing until the logarithmic growth phase. For the bacteria cultured to the logarithmic growth phase, use an ultraviolet-visible spectrophotometer to adjust the bacteria to OD 600nm = 0.38 - 0.40 for standby. Dilute the above-prepared bacteria by 1000 times, and add 50 μL of the bacterial culture to 50 μL of a sterile 96-well plate containing BSA with different concentrations of antimicrobial peptides, and incubate at 37 °C for 16 - 18 h. Seal the 96-well plate tightly with sealing film around the perimeter to prevent bacterial contamination, and place it in an incubator at 37 °C for 16 - 18 h. After incubation, the negative control wells remain clear and transparent, indicating that the test process is free of contamination. Observe visually and measure the turbidity of each well using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 492 nm. The lowest peptide concentration at which the turbidity does not increase compared to the negative control is defined as the MIC of the polypeptide. Conduct 3 independent replicate experiments, with two parallels for each replicate. The minimum inhibitory concentrations of the polypeptides are shown in Table 2.

[0034] Table 2 Antibacterial activity of antimicrobial peptide GRF3Q (μM)

[0035]

[0036]

[0037] As can be seen from the above charts, the MIC values of antimicrobial peptide GRF3Q for all tested bacteria are 2 - 4 μM, all showing good broad-spectrum antibacterial activity.

[0038] Example 4

[0039] Hemolytic activity of antimicrobial peptide

[0040] To evaluate the safety of antimicrobial peptides, the disruptive behavior of the polypeptides in the concentration range of 2 - 256 μM on human red blood cells (hRBC) was studied. 1 mL of fresh blood from healthy volunteers was collected and stored in a heparin sodium anticoagulant tube. After centrifugation at 3000 r for 10 min at 4°C, the supernatant was discarded, and the cells were washed 2 - 3 times with sterile PBS buffer and resuspended in 10 mL of PBS for later use. 50 μL of the red blood cell suspension was mixed evenly with 50 μL of polypeptide solutions at different concentrations serially diluted with PBS in a 96-well plate, and incubated at a constant temperature of 37°C for 1 h. After incubation, the mixture was centrifuged at 1000 g for 10 min at 4°C. 70 μL of the supernatant was taken and transferred to a new 96-well plate, and the absorbance was measured at 570 nm using a microplate reader. The average value of each group was taken for comparative analysis. Among them, 50 μL of red blood cells plus 50 μL of PBS served as the negative control; 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 served as the positive control. This experiment was repeated three times. The test results are shown in the appendix of the specification Figure 3 The minimum hemolytic concentration is the concentration of the antimicrobial peptide when it causes a 10% hemolysis rate. The test results are shown in Table 3. The biocompatibility of the antimicrobial peptide was evaluated by the minimum concentration (MHC) of the antimicrobial peptide when it causes 10% hemolysis of human red blood cells, and its therapeutic potential was further evaluated by calculating the therapeutic index (SI) of the antimicrobial peptide, as shown in Table 3.

[0041] Table 3 Hemolytic activity of antimicrobial peptide GRF3Q

[0042]

[0043] a The geometric mean (GM) of the minimum inhibitory concentration of the antimicrobial peptide against the tested bacteria. When no detectable antimicrobial activity was observed at 64 μM, 128 μM was used to calculate the therapeutic index;

[0044] b MHC is the minimum concentration of the antimicrobial peptide when it causes 10% hemolysis of human red blood cells (hRBC). When no detectable hemolytic activity was observed at 256 μM, 512 μM was used to calculate the therapeutic index;

[0045] c The calculation method of SI is MHC / GM. The larger the therapeutic value, the higher the therapeutic potential.

[0046] As shown in the appendix of the specification Figure 3It can be seen that the antimicrobial peptide GRF3Q did not cause obvious red blood cell damage at the highest concentration tested. The minimum hemolytic concentration of the antimicrobial peptide GRF3Q was much higher than its minimum inhibitory concentration, indicating that the antimicrobial peptide GRF3Q has high biosafety while exerting its antibacterial activity. By calculating the therapeutic index (SI) of the antimicrobial peptide GRF3Q, it was found that the SI value of the antimicrobial peptide GRF3Q was as high as 154.64. Based on the above results, the antimicrobial peptide GRF3Q has strong therapeutic potential.

[0047] Example 5

[0048] Cytotoxicity of Antimicrobial Peptide GRF3Q

[0049] The MTT method was used to determine the cytotoxicity of AMPs. The cells selected for determination were human embryonic kidney cells RAW264.7.

[0050] Prepare high-glucose RAMP-1640 complete medium containing 10% fetal bovine serum and 1% double antibody, place it at 4°C for later use, and place it in a 37°C water bath until room temperature before use. Inoculate the cells frozen in liquid nitrogen into the corresponding water-bathed complete medium for resuscitation, and place them in a carbon dioxide incubator for culture. Observe under a microscope, and the next subculture can be carried out when the cells cover more than 80% of the bottom of the cell culture flask. After the cells are subcultured well, pour out the complete medium in the cell culture flask, and wash the cells 2-3 times with sterile PBS filtered through a 0.22 μM aqueous filter membrane. Add 1 mL of 0.25% trypsin solution to the cell culture flask and place it in a carbon dioxide incubator for 1 min to digest the adherent cells. After digestion, observe under a microscope. If there are still adherent cells, gently tap until they completely fall off, then add complete medium and pipette to form a cell suspension, and add 50 μL of the cell suspension to each well of columns 1-11 of a sterile 96-well culture plate, with 3-5×10 5 cells per well. Finally, place the 96-well plate in a carbon dioxide incubator and incubate overnight. Take a new sterile 96-well plate, serially dilute AMPs with complete medium to the 10th column. Then aspirate 50 μL of each concentration of AMPs and add them to the corresponding columns 1-10 of the 96-well plate filled with cell suspension. Among them, 50 μL of complete medium is added to column 11 as a positive control, and 100 μL of complete medium is added to column 12 as a negative control. The 96-well plate is continued to be placed in a carbon dioxide incubator and incubated for 4 h. Add 50 μL of MTT solution with a concentration of 5 mg / mL to each well of the 96-well plate. Continue to incubate in a carbon dioxide incubator for 3 h, and then aspirate and discard all the liquid in each well. Finally, add 100 μL of DMSO to each well to fully dissolve the purple crystals, and measure the absorbance at 570 nm. This experiment was independently repeated three times. The test results are shown in Figure 4 .

[0051] Attached to the specification Figure 4 It can be seen that the cell viability of the antimicrobial peptide GRF3Q is higher than 80% within the concentration range of 1 - 32 μM, and no obvious cytotoxicity is exhibited.

[0052] Example 6

[0053] Salt ion stability of antimicrobial peptides

[0054] E. coli 25922 and S. aureus 29213 were selected as typical Gram-negative bacteria and typical Gram-negative bacteria respectively to determine the MIC values of AMPs under different physiological concentrations of salt ions. Different concentrations of salt ions were dissolved in a 0.2% BSA (containing 0.01% glacial acetic acid) solution filtered through a 0.22 μM aqueous filter membrane, and the initial concentrations of the configured salt ions were 300 mM NaCl, 9 mM KCl, 5 mM CaCl2, 2 mM MgCl2, 12 μM NH4Cl, 16 μM ZnCl2, and 8 μM FeCl3. Using the above different salt ions as diluents, the MIC of the antimicrobial peptide in different salt ions was determined by the microbroth dilution method. This experiment was independently repeated three times. The test results are shown in Table 4.

[0055] Table 4 Salt ion stability of antimicrobial peptide GRF3Q

[0056]

[0057] As can be seen from Table 4, the MIC values of the antimicrobial peptide GRF3Q for S. aureus 29213 and E. coli 25922 vary in the range of 4 μM - 16 μM in the presence of physiological salts, showing strong salt ion stability. In the presence of physiological salts, for the antimicrobial peptide GRF3Q, except for the relatively large influence of the strong charge interaction with the antimicrobial peptide in the presence of Mg 2+ , Ca 2+ and Na + , the MIC value ranges from 2 μM to 8 μM in the presence of the remaining salt ions, also showing strong salt ion stability. Considering the above results, the antimicrobial peptide GRF3Q exhibits excellent performance.

Claims

1. An antibacterial short peptide GRF3Q with an asymmetric biphasic hydrophobic core, characterized in that: Its amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2.

2. The antibacterial short peptide GRF3Q with an asymmetric biphasic hydrophobic core as described in claim 1, characterized in that, The molecular formula is shown in formula (I):

3. The preparation method of an antibacterial short peptide GRF3Q with an asymmetric biphasic hydrophobic core as described in claim 1 is as follows: Step 1: Select the aromatic amino acid phenylalanine and isoleucine as the double hydrophobic core to jointly provide hydrophobicity for the polypeptide. Select arginine to form the positive charge cluster of the polypeptide, and place arginine at the C-terminal position of the polypeptide to improve the selectivity for the bacterial membrane. Eventually, the arginines at both positions jointly provide positive charges for the polypeptide. Select proline-glycine and glutamine for transition. Proline-glycine separates the positive charge cluster and the hydrophobic cluster, and glutamine can neutralize the strong positive charge of arginine to reduce the electrostatic attraction to host cells, thereby reducing hemolytic activity and cytotoxicity. Cap the N-terminus of the polypeptide with glycine to reduce the attraction of positive charges to the host cell membrane and lower hemolytic activity. The amino acid sequence of the obtained polypeptide is shown in SEQ ID No.1, and the C-terminus of the polypeptide sequence is amidated with -NH2 to increase the net positive charge of the polypeptide, improve the selectivity for the bacterial membrane, and enhance antibacterial activity. Step 2: Synthesize the polypeptide by solid-phase chemical synthesis method. After purification by reverse-phase high-performance liquid chromatography and identification by mass spectrometry, perform antibacterial activity detection, hemolytic activity detection, cytotoxicity detection, and salt ion stability detection on the polypeptide, and finally name it antibacterial peptide GRF3Q.

4. The application of an antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described in claim 1 in the preparation of a drug for treating infectious diseases caused by Gram-positive bacteria or / and Gram-negative bacteria.

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

6. The application according to claim 4, characterized in that: The Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, or Salmonella pullorum.

7. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that, The drug contains an antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described in claim 1.

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