An antimicrobial peptide with an asymmetric biphasic hydrophobic core and its preparation method and application

Abstract

The present invention discloses an antimicrobial peptide with an asymmetric biphasic hydrophobic core, a preparation method and an application thereof, and belongs to the field of bioengineering technology. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID No. 1. The antimicrobial peptide of the present invention was tested for antimicrobial activity, hemolytic activity, cytotoxicity and salt ion stability, and it was found that the antimicrobial peptide had extremely high cell selectivity (SI = 154.64), and did not show obvious cytotoxicity below the test concentration of 32 μM, and was able to maintain high stability in salt ions of different physiological concentrations. In summary, the antimicrobial peptide with an asymmetric biphasic hydrophobic core of the present invention has the potential to become a broad-spectrum antimicrobial drug for the treatment of Gram-positive and Gram-negative bacterial infections.

Description

Technical Field

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

[0002] Antimicrobial peptides (AMPs), as an important component of the innate immune system, have broad-spectrum antimicrobial activity and are less likely to induce pathogen resistance, and are considered to be ideal alternatives to traditional antibiotics. However, natural antimicrobial peptides face multiple challenges in clinical translation: for example, most natural antimicrobial peptides have high hemolytic activity and cytotoxicity against host cells (such as red blood cells and epithelial cells), resulting in a narrow therapeutic window; their antimicrobial activity is easily affected by high concentrations of salt ions (such as Na) in the physiological environment; + Mg 2+ , Ca 2+ ) inhibition, significantly reducing in vivo efficacy; some antimicrobial peptides lack selectivity for Gram-positive and Gram-negative bacteria, potentially disrupting the balance of the host's symbiotic flora. Therefore, optimizing the molecular characteristics of antimicrobial peptides through rational design to achieve a synergistic improvement in "high selectivity and ideal salt stability" has become a core goal of current antimicrobial peptide engineering.

[0003] In recent years, researchers have discovered through structure-activity relationship (SAR) analysis that the hemolytic toxicity of antimicrobial peptides is closely related to the ratio of hydrophobic residues and their amphipathic structure. For example, overexposure of the hydrophobic surface of α-helical antimicrobial peptides (such as LL-37 and magainin) can enhance hydrophobic interactions with eukaryotic cell membranes, leading to cell membrane dissolution. To address this issue, studies have attempted to manipulate the amphipathic balance by reducing the hydrophobicity index (e.g., replacing leucine and phenylalanine with serine or glycine) or introducing charged residues, but this may come at the expense of antimicrobial activity. Furthermore, the electrostatic interactions of cationic antimicrobial peptides are susceptible to interference from salt ion shielding, resulting in reduced binding to negatively charged bacterial membranes. Studies have shown that optimizing the net positive charge (+4 to +6) and introducing aromatic residues or rigid domains (such as proline turns) can enhance the salt tolerance of antimicrobial peptides, but their impact on selectivity requires systematic investigation. Summary of the Invention

[0004] Based on the above shortcomings, the purpose of the present invention is to provide an antimicrobial peptide with an asymmetric biphasic hydrophobic core to solve the problem that the antimicrobial peptide has high hemolytic activity, high cytotoxicity and instability in the presence of physiological salt conditions, making it difficult to use as an antimicrobial drug.

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

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

[0007]

[0008] The present invention also provides a method for preparing the antimicrobial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described above, comprising the following steps:

[0009] Step 1: Select aromatic amino acids phenylalanine and isoleucine as a double hydrophobic core to jointly provide hydrophobicity for the polypeptide, select arginine to form a positively charged cluster of the polypeptide, and place arginine at the C-terminal position of the polypeptide to improve the selectivity for bacterial membranes. Finally, the arginine at the two positions jointly provide a positive charge for the polypeptide; select proline-glycine and glutamine for transition, proline-glycine separates the positively charged cluster and the hydrophobic cluster, and glutamine can neutralize the strong positive charge of arginine to reduce electrostatic attraction to host cells, thereby reducing hemolytic activity and cytotoxicity; glycine is used to cap the N-terminus of the polypeptide to reduce the attraction of the positive charge to the host cell membrane and reduce hemolytic activity; the amino acid sequence of the obtained polypeptide is shown in SEQ ID No. 1, and -NH2 is used at the C-terminus of the polypeptide sequence for amidation to increase the net positive charge of the polypeptide, improve the selectivity for bacterial membranes, and enhance the antibacterial activity;

[0010] Step 2: The polypeptide was synthesized by solid-phase chemical synthesis, and after purification by reverse-phase high-performance liquid chromatography and identification by mass spectrometry, the polypeptide was tested for antibacterial activity, hemolytic activity, cytotoxicity, and salt ion stability, and finally named the antimicrobial peptide GRF3Q.

[0011] Another object of the present invention is to provide the use of the antimicrobial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described above in the preparation of drugs for treating Gram-positive and / or Gram-negative bacterial infections.

[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 and / or Gram-negative bacterial infections, wherein the drug contains the antimicrobial peptide GRF3Q with an asymmetric biphasic hydrophobic core as described above.

[0015] The present invention has the following advantages and beneficial effects: The antimicrobial peptide of the present invention significantly reduces hemolytic activity and cytotoxicity, potentially addressing the difficulties existing antimicrobial peptides face in maintaining the healthy development of animal husbandry. Testing of the antimicrobial peptide GRF3Q of the present invention for antimicrobial activity, hemolytic activity, cytotoxicity, and salt ion stability revealed high antimicrobial activity against several tested Gram-negative and Gram-positive bacteria, including Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis. Furthermore, the antimicrobial peptide GRF3Q exhibited high hemolytic activity, with no significant hemolysis at 256 μM, and no significant cytotoxicity at 32 μM. Furthermore, the antimicrobial peptide GRF3Q maintained a low MIC in various physiological salt ion concentrations and serum concentrations, demonstrating high salt ion stability and serum stability. In summary, the antimicrobial peptide GRF3Q of the present invention has the potential to become a broad-spectrum antimicrobial drug for treating Gram-positive and Gram-negative bacterial infections, thereby achieving the goal of maintaining the healthy development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 2 The mass spectrum of the antimicrobial peptide GRF3Q with an asymmetric biphasic hydrophobic core;

[0018] Figure 3 is the hemolytic activity graph of the antimicrobial peptide GRF3Q;

[0019] Figure 4 This is a graph showing the cytotoxicity of the antimicrobial peptide GRF3Q. DETAILED DESCRIPTION

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

[0021] Example 1

[0022] Design of antimicrobial peptides

[0023] 1. Because tryptophan, an aromatic amino acid, is hydrophobic and the largest amino acid, but aggregates within aromatic amino acids, it can easily lead to significant cytotoxicity. Therefore, the aromatic amino acid phenylalanine and the aliphatic amino acid isoleucine were selected as a dual hydrophobic core to provide hydrophobicity to the antimicrobial peptide. Arginine, due to its strong positive charge, was selected to form the antimicrobial peptide's positively charged cluster. Arginine was placed at the peptide's C-terminus to enhance selectivity for bacterial membranes. Ultimately, these two arginines together provide the antimicrobial peptide with a positive charge. Proline-glycine and glutamine were used to bridge the gap between the positively charged and hydrophobic amino acids. Proline-glycine separates the positively charged and hydrophobic clusters, while glutamine moderately neutralizes the strong positive charge of arginine, hopefully reducing electrostatic attraction to host cells and thereby reducing hemolytic activity and cytotoxicity. Glycine was added to the N-terminus of the peptide sequence to reduce the attraction of the positive charge to host cell membranes and reduce hemolytic activity. Amidation with -NH2 at the C-terminus of the peptide sequence increases the net positive charge of the peptide sequence, improving selectivity for bacterial membranes and thereby enhancing antimicrobial activity. The designed antimicrobial peptide maximizes antimicrobial activity while exhibiting low hemolytic activity and good stability. The sequence, molecular weight, and charge 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 peptides

[0028] The designed antimicrobial peptide was synthesized by Nanjing Synpeptide Co., Ltd. via 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 reversed-phase high performance liquid chromatogram of the antimicrobial peptide GRF3Q is attached. Figure 1 .

[0030] The mass spectrum of the antimicrobial peptide GRF3Q is attached. Figure 2 .

[0031] Example 3

[0032] Antibacterial activity of peptides

[0033] The minimum inhibitory concentration of several antimicrobial peptides was determined by the microbroth dilution method. The antibacterial activity of the peptides was understood by determining the minimum inhibitory concentration (MIC) of the peptides. The designed and successfully synthesized peptides were dissolved in sterile ultrapure water in a sterile operating table to a concentration of 2.56 mM to obtain a storage solution of AMPs for subsequent biological activity determination. The bacteria were cultured overnight at 37°C and 220 rpm on a shaker, and then transferred to a new MHB culture until the logarithmic phase of growth. The bacteria cultured to the logarithmic phase were adjusted to OD using a UV-visible spectrophotometer. 600nm =0.38~0.40 for later use. Dilute the above-adjusted bacteria 1000 times, and take 50μL of bacterial culture to 50μL of sterile 96-well plates containing BSA with different concentrations of antimicrobial peptides, and incubate at 37°C for 16-18h. Seal the 96-well plates tightly with sealing film on all sides to prevent bacterial contamination, and place them in a 37°C incubator for incubation for 16-18h. After the incubation, the negative control wells remained clear and transparent, indicating that there was no contamination during the test. The turbidity of each well was measured by visual observation and using an enzyme-labeled instrument at a wavelength of 492nm. The lowest peptide concentration at which the turbidity did not increase compared to the negative control was defined as the MIC of the peptide. The experiment was repeated three times independently, with two replicates for each replicate. The minimum inhibitory concentration of the peptides is shown in Table 2.

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

[0035]

[0036]

[0037] As can be seen from the above chart, the MIC values ​​of the antimicrobial peptide GRF3Q against all bacteria were 2-4 μM, showing good broad-spectrum antimicrobial activity.

[0038] Example 4

[0039] Hemolytic activity of antimicrobial peptides

[0040] To assess the safety of antimicrobial peptides, the destructive effects of peptides on human red blood cells (hRBCs) at concentrations ranging from 2 to 256 μM were investigated. One mL of fresh blood was collected from healthy volunteers and stored in sodium heparin tubes. The blood was centrifuged at 3000 r / min at 4°C for 10 minutes, the supernatant discarded, and the cells were washed two to three times with sterile PBS buffer before resuspending in 10 mL of PBS. Fifty μL of the RBC suspension was mixed with 50 μL of peptide solutions of varying concentrations, serially diluted with PBS, and incubated in a 37°C incubator for 1 hour. After incubation, the cells were centrifuged at 1000 g for 10 minutes at 4°C. Seventy μL of the supernatant was removed and transferred to a fresh 96-well plate. The absorbance was measured at 570 nm using a microplate reader. The average values ​​for each group were calculated and compared. A negative control consisted of 50 μL of RBCs plus 50 μL of PBS, and a positive control consisted of 50 μL of RBCs plus 50 μL of 0.1% Triton X-100. This experiment was repeated three times. The test results are shown in the instructions. Figure 3 The minimum hemolytic concentration is the concentration of antimicrobial peptide that causes 10% hemolysis. 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 that causes 10% hemolysis of human red blood cells. The therapeutic potential of the antimicrobial peptide was further evaluated by calculating the therapeutic index (SI), 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 (GM) of the antimicrobial peptides against the assayed bacteria was 128 μM, when no detectable antimicrobial activity was observed at 64 μM;

[0044] b MHC is the lowest concentration of antimicrobial peptide that causes 10% hemolysis of human red blood cells (hRBCs), and when no detectable hemolytic activity is observed at 256 μM, 512 μM is used to calculate the therapeutic index;

[0045] c SI is calculated as MHC / GM. A larger therapeutic value indicates a higher therapeutic potential.

[0046] Attached to the instruction manual Figure 3As can be seen, the antimicrobial peptide GRF3Q did not cause significant erythrocyte damage at the highest concentration tested. The minimum hemolytic concentration of the antimicrobial peptide GRF3Q was significantly greater than its minimum inhibitory concentration, demonstrating that the antimicrobial peptide GRF3Q exhibits both antimicrobial activity and high biosafety. Calculation of the therapeutic index (SI) for the antimicrobial peptide GRF3Q revealed a high SI value of 154.64. These results suggest that the antimicrobial peptide GRF3Q has strong therapeutic potential.

[0047] Example 5

[0048] Cytotoxicity of the antimicrobial peptide GRF3Q

[0049] The cytotoxicity of AMPs was determined by MTT assay. Human embryonic kidney RAW264.7 cells were selected as the test cells.

[0050] Prepare high-glucose RAMP-1640 complete medium containing 10% fetal bovine serum and 1% double-antibody antibodies and store at 4°C until ready. Heat the medium in a 37°C water bath before use. Resuspend cells frozen in liquid nitrogen in the corresponding complete medium after water bathing and culture in a CO2 incubator. Once cells have covered at least 80% of the bottom of the cell culture flask under a microscope, proceed to the next subculture. After completing the cell subculture, discard the complete medium from the cell culture flask and rinse the cells two to three times with sterile PBS filtered through a 0.22μM water filter. Add 1 mL of 0.25% trypsin solution to the cell culture flask and place in a CO2 incubator for 1 minute to digest the adherent cells. After digestion, observe under a microscope. If there are still cells attached to the wall, gently tap until they are completely detached. Then add complete culture medium and pipette to form a cell suspension. Add 50 μL of cell suspension to each well of the 1st to 11th columns of a sterile 96-well culture plate, and plate 3 to 5 × 10 cells per well. 5 cells. Finally, place the 96-well plate in a carbon dioxide incubator and culture it statically overnight. Take a new sterile 96-well plate and dilute the AMPs serially in series with complete culture medium to the 10th column. Then extract 50 μL of AMPs of various concentrations and add them to the corresponding columns 1 to 10 of the 96-well plate filled with cell suspension, of which 50 μL of complete culture medium is added to the 11th column as a positive control, and 100 μL of complete culture medium is added to the 12th column as a negative control. The 96-well plate continues to be placed in a carbon dioxide incubator and cultured statically for 4 hours. Add 5 mg / mL MTT solution to each well of the 96-well plate, 50 μL per well. Continue to culture statically in the carbon dioxide incubator for 3 hours, then aspirate and discard all the liquid in each well. Finally, add 100 μL DMSO to each well to fully dissolve the purple crystals, and measure the absorbance at 570 nm. This experiment was repeated three times independently. See the test results. Figure 4 .

[0051] Attached to the instruction manual Figure 4 It can be seen that the cell survival rate of the antimicrobial peptide GRF3Q in the concentration range of 1 to 32 μM was higher than 80%, and no obvious cytotoxicity was shown.

[0052] Example 6

[0053] Salt ion stability of antimicrobial peptides

[0054] E. coli 25922 and S. aureus 29213 were selected as representative Gram-negative bacteria, respectively, to determine the MIC values ​​of AMPs under different physiological salt concentrations. Different salt concentrations were dissolved in 0.2% BSA (containing 0.01% glacial acetic acid) solutions filtered through a 0.22μM water filter. The initial salt concentrations were 300mM NaCl, 9mM KCl, 5mM CaCl2, 2mM MgCl2, 12μM NH4Cl, 16μM ZnCl2, and 8μM FeCl3. The MICs of the antimicrobial peptides in these different salts were determined using the broth microdilution method using these different salt diluents. This experiment was repeated three times. The 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 against S. aureus 29213 and E. coli 25922 in the presence of physiological salts range from 4 μM to 16 μM, showing strong salt ion stability. 2+ , Ca 2+ and Na + While the presence of 2-hydroxy-1,4-dimethylthiazolinone (DH) produces a stronger charge interaction with the antimicrobial peptide, which has a slightly greater impact, the MIC values ​​exhibited in the presence of other salt ions range from 2μM to 8μM, also demonstrating strong salt ion stability. Overall, the antimicrobial peptide GRF3Q exhibits excellent performance.

Claims

1. An antimicrobial peptide GRF3Q with an asymmetric biphasic hydrophobic core, characterized by: Its amino acid sequence is shown in SEQ ID No. 1, its C-terminus is amidated with -NH2, and its molecular formula is shown in formula (I):

2. The method for preparing the antimicrobial short peptide GRF3Q with an asymmetric biphasic hydrophobic core as claimed in claim 1, comprising the following steps: Step 1: Select aromatic amino acids phenylalanine and isoleucine as a double hydrophobic core to jointly provide hydrophobicity for the polypeptide; select arginine to form a positively charged cluster of the polypeptide, and place arginine at the C-terminal position of the polypeptide to improve the selectivity for bacterial membranes. Finally, the arginine at the two positions jointly provide a positive charge for the polypeptide; select proline-glycine and glutamine for transition, proline-glycine separates the positively charged cluster and the hydrophobic cluster, and glutamine can neutralize the strong positive charge of arginine to reduce electrostatic attraction to host cells, thereby reducing hemolytic activity and cytotoxicity; glycine is used to cap the N-terminus of the polypeptide to reduce the attraction of the positive charge to the host cell membrane and reduce hemolytic activity; the amino acid sequence of the obtained polypeptide is shown in SEQ ID No. 1, and -NH2 is used at the C-terminus of the polypeptide sequence for amidation to increase the net positive charge of the polypeptide, improve the selectivity for bacterial membranes, and enhance the antibacterial activity; Step 2: The polypeptide is synthesized by solid-phase chemical synthesis, and after reverse-phase high-performance liquid chromatography purification and mass spectrometry identification, the polypeptide is subjected to antibacterial activity testing, hemolytic activity testing, cytotoxicity testing, and salt ion stability testing, and is finally named the antibacterial short peptide GRF3Q.

3. Use of the antibacterial peptide GRF3Q with an asymmetric biphasic hydrophobic core as claimed in claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis; and the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium or Salmonella pullorum.

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

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