A PG-centered asymmetric antimicrobial peptide and its preparation method and application

Abstract

The present invention discloses an asymmetric antimicrobial peptide centered on PG, a preparation method thereof, and an application thereof, belonging to the field of bioengineering technology. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID No. 1, and its C-terminus is amidated with ‑NH2; the antimicrobial peptide of the present invention is tested for antimicrobial activity, hemolytic activity, cytotoxicity, and salt ion stability, and it is found that it has the best cell selectivity (SI=72.60), and does not show obvious cytotoxicity below the test concentration of 32 μM, and can maintain relatively ideal stability in salt ions of different physiological concentrations. In summary, the antimicrobial peptide 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 specifically relates to an asymmetric antimicrobial peptide GPRW3Q with PG as the center, and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of small peptides widely found in nature, typically composed of 10-60 amino acids. They are important components of the innate immune system and exhibit a wide range of inhibitory effects against bacteria, fungi, parasites, and viruses. Due to their unique bactericidal mechanisms and multiple biological activities, they are less susceptible to drug resistance and are considered a strategic solution in the post-antibiotic era. However, existing natural AMPs face significant bottlenecks in livestock applications. For example, some natural AMPs exhibit high hemolytic and toxic properties, and the high number of amino acids in their peptide composition leads to high synthetic costs, hindering their further application in livestock production. This significantly limits the application of AMPs in livestock production. While numerous design strategies have made progress in improving the biocompatibility of AMPs, some of these strategies have negatively impacted AMPs. For example, improving biocompatibility comes at the expense of antimicrobial activity, and the use of D-amino acids and other unnatural amino acids to maintain antimicrobial activity potentially increases synthetic costs and subsequent expression barriers, hindering their application as antimicrobial agents. Summary of the Invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide an asymmetric antimicrobial peptide GPRW3Q centered on PG, which solves the problems of high hemolytic activity and high cytotoxicity caused by antimicrobial peptides during application and polypeptide instability in the presence of physiological salt conditions. It can be used as an antimicrobial drug, thereby ensuring the health of livestock and poultry and improving the quality of animal husbandry production.

[0004] The technical solution adopted by the present invention is as follows: an asymmetric antibacterial short peptide GPRW3Q with PG as the center, the amino acid sequence of which is shown in SEQ ID No.1, and the C-terminus is amidated with -NH2.

[0005] Furthermore, the molecular formula of the antibacterial short peptide GPRW3Q is shown in formula (I):

[0006]

[0007] The present invention also provides a method for preparing the above-mentioned asymmetric antimicrobial peptide GPRW3Q with PG as the center, comprising the following steps:

[0008] Step 1: Select arginine Arg as a positively charged amino acid and place it at positions 2, 3, 4 and 13 of the polypeptide amino acid sequence. The 13th arginine is placed at the C-terminus to enhance the positive charge effect of the polypeptide and provide a positive charge to the polypeptide. Select tryptophan Trp and isoleucine Ile as hydrophobic amino acids to provide hydrophobicity to the polypeptide. Trp is placed at positions 7, 8 and 9 of the polypeptide amino acid sequence. Ile is placed at positions 10 and 11 of the polypeptide amino acid sequence to provide hydrophobicity as an auxiliary tryptophan. Select uncharged glutamine Gln to be placed at positions 10 and 11 of the polypeptide amino acid sequence. The 12th position of the peptide sequence is selected to achieve a flexible balance between the positive charge and hydrophobicity of the peptide; proline Phe and glycine Gly are selected to be placed at positions 5 and 6 of the peptide amino acid sequence, respectively, with Phe expected to enhance the stability of Arg at position 4, and the PG-containing structure has enhanced antibacterial properties and low cytotoxicity; the N-terminus of the peptide amino acid sequence is capped with glycine Gly to prevent the R group at the N-terminus from being directly exposed on the surface, thereby reducing electrostatic attraction to the neutral surface structure of human hemoglobin; finally, the C-terminus of the peptide is amidated to construct a peptide with an amino acid sequence of +5 as shown in SEQ ID No. 1;

[0009] 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 GPRW3Q.

[0010] Another object of the present invention is to provide the use of the above-mentioned asymmetric antimicrobial peptide GPRW3Q centered around PG in the preparation of a drug for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

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

[0012] Furthermore, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium or Salmonella pullorum.

[0013] 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 asymmetric antimicrobial peptide GPRW3Q centered around PG as described above.

[0014] The present invention has the following advantages and beneficial effects: Through a flexible balance of neutral amino acids and an N-terminal glycine cap, the present invention significantly reduces hemolytic activity and cytotoxicity without compromising antimicrobial activity, potentially resolving the difficulties currently faced in applying existing antimicrobial peptides to maintain the healthy development of animal husbandry. Testing of the present invention's asymmetric antimicrobial peptide GPRW3Q, centered around the "PG" moiety, revealed high antimicrobial activity against several Gram-negative and Gram-positive bacteria tested, including Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis. Furthermore, GPRW3Q exhibited high hemolytic activity, with no significant hemolysis at 256 μM, and no significant cytotoxicity at 32 μM. Furthermore, GPRW3Q maintained low MICs in various physiological salt ion concentrations and serum concentrations, demonstrating high salt ion and serum stability. In summary, the asymmetric antimicrobial peptide GPRW3Q centered on "PG" has the potential to become a broad-spectrum antimicrobial drug for the treatment of Gram-positive and Gram-negative bacterial infections, thereby achieving the goal of maintaining the healthy development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the reverse phase high performance liquid chromatogram of the antimicrobial peptide GPRW3Q;

[0016] Figure 2 is the mass spectrum of the antimicrobial peptide GPRW3Q;

[0017] Figure 3 This is a comparison of the hemolytic activities of the antimicrobial peptides PRW3 and GPRW3Q;

[0018] Figure 4 This is a comparison of the cytotoxicity of antimicrobial peptides PRW3 and GPRW3Q. DETAILED DESCRIPTION

[0019] The present invention will be described in further detail below with reference to the embodiments and accompanying drawings.

[0020] Example 1

[0021] Design of antimicrobial peptides

[0022] 1. Arginine (Arg) is selected as the positively charged amino acid and is placed at positions 2, 3, 4, and 13 of the peptide's amino acid sequence. Arginine at positions 2, 3, and 4 forms an arginine cluster at the N-terminus. Arginine at position 13 is placed at the C-terminus to enhance the peptide's positive charge, ultimately providing the peptide with a positive charge. Tryptophan (Trp) and isoleucine (Ile) are selected as hydrophobic amino acids to provide hydrophobicity to the peptide. Trp is placed at positions 7, 8, and 9 to form a polytryptophan, and Ile is placed at positions 10 and 11 to assist the tryptophan in providing hydrophobicity. The uncharged neutral amino acid glutamine (Gln) is selected and placed at position 12 to achieve a reasonable and flexible balance between the peptide's positive charge and hydrophobicity. Proline (Phe) and glycine (Gly) were selected and placed at positions 5 and 6, respectively. Phe was used to enhance the stability of the Arg residue at position 4, thereby enhancing antimicrobial properties and reducing cytotoxicity. The N-terminus was capped with glycine (Gly). This arrangement prevented the N-terminal R from being directly exposed to the surface, thereby reducing electrostatic attraction to the neutral surface structure of human hemoglobin. Finally, the C-terminus of the peptide was amidated to obtain the antimicrobial peptide GPRW3Q, which has a net charge of +5. Its amino acid sequence is shown in SEQ ID No. 1. The sequence, molecular weight, and charge of the antimicrobial peptide are shown in Table 1.

[0023] Table 1 Sequence, molecular weight and charge number of peptides PRW3 and GPRW3Q

[0024]

[0025] Example 2

[0026] Synthesis and identification of antimicrobial peptides

[0027] 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.

[0028] The reversed-phase high performance liquid chromatogram of the antimicrobial peptide GPRW3Q is attached. Figure 1 .

[0029] The mass spectrum of the antimicrobial peptide GPRW3Q is attached. Figure 2 .

[0030] Example 3

[0031] Antibacterial activity of peptides

[0032] 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 process. 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 test was repeated three times independently, with two replicates for each replicate. The minimum inhibitory concentration is shown in Table 2.

[0033] Table 2 Antibacterial activity of peptide PRW3 and antimicrobial peptide GPRW3Q (μM)

[0034]

[0035] As can be seen from the above table, the MIC value of the peptide PRW3 against Gram-negative bacteria is 2-16 μM, and the MIC value against Gram-negative bacteria is 2-4 μM, while the MIC value of the antimicrobial peptide GPRW3Q against all bacteria is 2-4 μM, both showing good antibacterial activity, and the activity is better than that of the peptide PRW3.

[0036] Example 4

[0037] Hemolytic activity of antimicrobial peptides

[0038] To assess the safety of antimicrobial peptides, the destructive effects of peptides on human red blood cells (hRBCs) at concentrations ranging from 2 to 128 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 HRCB 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 HRCB supplemented with 50 μL of PBS, and a positive control consisted of 50 μL of HRCB supplemented with 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.

[0039] Table 3 Hemolytic activity of peptide PRW3 and antimicrobial peptide GPRW3Q

[0040]

[0041] 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;

[0042] 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 128 μM, 256 μM is used to calculate the therapeutic index;

[0043] c SI is calculated as MHC / GM. A larger therapeutic value indicates a higher therapeutic potential.

[0044] Attached to the instruction manual Figure 3As can be seen, the antimicrobial peptide GPRW3Q did not cause hemolysis at the highest concentration tested. The minimum hemolytic concentration of the antimicrobial peptide GPRW3Q was significantly greater than its minimum inhibitory concentration, demonstrating that the antimicrobial peptide GPRW3Q exhibits both antimicrobial activity and high biosafety. Calculation of the therapeutic index (SI) for PRW3 and GPRW3Q revealed that the SI value for GPRW3Q (72.60) was significantly higher than that for PRW3 (2.42). These results suggest that the antimicrobial peptide GPRW3Q possesses the greatest therapeutic potential.

[0045] Example 5

[0046] Cytotoxicity of antimicrobial peptides

[0047] The cytotoxicity of AMPs was determined by MTT assay. Human embryonic kidney RAW264.7 cells were selected as the test cells.

[0048] 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 .

[0049] Attached to the instruction manual Figure 4 It can be seen that the cell survival rates of the polypeptide PRW3 and the antimicrobial peptide GPRW3Q were both higher than 80% in the concentration range of 1 to 32 μM, and no obvious cytotoxicity was shown.

[0050] Example 6

[0051] Salt ion stability of antimicrobial peptides

[0052] E. coli 25922 and S. aureus 29213 were selected as representative Gram-negative bacteria, respectively, to determine the MICs of antimicrobial peptides under different physiological salt concentrations. Different salt concentrations were dissolved in a 0.2% BSA solution (containing 0.01% glacial acetic acid) filtered through a 0.22μM aqueous 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.

[0053] Table 4 Salt ion stability of antimicrobial peptide GPRW3Q

[0054]

[0055] As can be seen from Table 4, the MIC value of the antimicrobial peptide GPRW3Q against S. aureus in the presence of physiological salt varies from 4μM to 32μM, showing strong salt ion stability. 2+ and Ca 2+ While the presence of 2-hydroxy-2-methyl-1-oxo-2-nitropropene (NH4-2-nitropropene) produces a stronger charge interaction with the antimicrobial peptide, the MIC values ​​in the presence of other salt ions range from 4 μM to 8 μM, demonstrating strong salt ion stability. Overall, the antimicrobial peptide GPRW3Q exhibits superior performance.

Claims

1. An asymmetric antimicrobial peptide GPRW3Q centered around PG, 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 asymmetric antimicrobial peptide GPRW3Q centered on PG according to claim 1, wherein: Here are the steps: Step 1: Select arginine Arg as a positively charged amino acid and place it at positions 2, 3, 4 and 13 of the polypeptide amino acid sequence. The 13th arginine is placed at the C-terminus to enhance the positive charge effect of the polypeptide and provide a positive charge to the polypeptide. Select tryptophan Trp and isoleucine Ile as hydrophobic amino acids to provide hydrophobicity to the polypeptide. Trp is placed at positions 7, 8 and 9 of the polypeptide amino acid sequence, and Ile is placed at positions 10 and 11 of the polypeptide amino acid sequence as auxiliary tryptophan to provide hydrophobicity to the polypeptide. Select uncharged glutamine Gln and place it at positions 10 and 11 of the polypeptide amino acid sequence. The peptide is positioned at position 12 to achieve a flexible balance between the positive charge and hydrophobicity of the peptide; proline (Phe) and glycine (Gly) are selected and placed at positions 5 and 6, respectively, of the peptide's amino acid sequence. Phe is expected to enhance the stability of Arg at position 4, and the PG-containing structure has enhanced antibacterial properties and low cytotoxicity; the N-terminus of the peptide's amino acid sequence is capped with glycine (Gly) to prevent the N-terminal R group from being directly exposed on the surface, thereby reducing electrostatic attraction to the neutral surface structure of human hemoglobin; finally, the C-terminus of the peptide is amidated with -NH2 to construct a peptide with a net charge of +5, as shown in SEQ ID No.

1. 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 GPRW3Q.

3. Use of the PG-centered asymmetric antimicrobial peptide GPRW3Q as claimed in claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive 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 asymmetric antimicrobial peptide GPRW3Q centered on PG as claimed in claim 1.

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