D-Arg-rich enzymatically resistant tetradecapeptide 2rrr and its preparation method and application

Abstract

The present invention discloses an anti - enzymatic hydrolysis tetrapeptide 2rrr rich in D - Arg, its preparation method and application, belonging to the field of bioengineering technology. Its amino acid sequence is as shown in SEQ ID No.1, its C - terminus is amidated with - NH2, and the 3rd, 5th, and 7th positions of the sequence are D - type arginine. And the application of the anti - enzymatic hydrolysis tetrapeptide 2rrr rich in D - Arg of the present invention in the preparation of drugs for treating infectious diseases caused by Gram - positive bacteria or / and Gram - negative bacteria is disclosed. The anti - enzymatic hydrolysis tetrapeptide 2rr rich in D - Arg of the present invention has high cell selectivity and can maintain high stability in salt ions at different physiological concentrations and in high concentrations of pepsin and trypsin. In summary, the anti - enzymatic hydrolysis tetrapeptide 2rrr rich in D - Arg has the potential to become a broad - spectrum antibacterial drug for treating Gram - positive and Gram - negative bacterial infections and maintain high antibacterial activity in the digestive tract.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a D-Arg-rich enzymatically resistant tetradecapeptide 2rrr and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of small molecule peptides widely found in nature. They are usually composed of 10 to 60 amino acids and are an important part of the body's innate immune system. They have a wide range of inhibitory effects on bacteria, fungi, parasites and viruses. AMPs are considered to be an ideal substitute for antibiotics because they are not easy to induce drug resistance. However, AMPs have the defect of being easily degraded by various proteases in the digestive tract, which greatly limits the application of AMPs. Among them, trypsin is the biggest threat, which can specifically cleave the C-terminal peptide bond of the positively charged amino acid arginine (Arginine, Arg) and lysine (Lys). Although many design strategies have made progress in reducing the protease sensitivity of AMPs, some anti-enzyme hydrolysis strategies have had some negative effects on AMPs to varying degrees, such as increasing toxicity or achieving the purpose of anti-enzyme hydrolysis at the expense of certain antibacterial activity. Summary of the invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide a D-Arg-rich resistant tetradecapeptide 2rrr to solve the problem of poor protease stability of existing antimicrobial peptides. The resistant tetradecapeptide 2rrr of the present invention can resist degradation by high concentrations of trypsin.

[0004] The technical scheme adopted by the present invention is as follows: a D-Arg-rich enzymatically resistant fourteen peptide 2rrr, whose amino acid sequence is shown in SEQ ID No.1, whose C-terminus is amidated with -NH2, and the 3rd, 5th and 7th positions of the sequence are D-arginine.

[0005] Furthermore, the D-Arg-rich, enzymatically resistant tetradecapeptide 2rrr as described above has a molecular formula as shown in formula (I).

[0006]

[0007] Another object of the present invention is to provide a method for preparing the D-Arg-rich enzymatically resistant tetradecapeptide 2rrr as described above, the steps being as follows:

[0008] Step 1: Select leucine and tryptophan as hydrophobic amino acids to provide hydrophobicity for the polypeptide, place leucine at the 1st and 4th positions of the sequence to form a leucine zipper, and place tryptophan at the 2nd and 6th positions of the sequence to form a tryptophan zipper to stabilize the α-helical structure and improve the antibacterial activity; place D-arginine at the 3rd, 5th and 7th positions of the sequence to provide a positive charge and enhance the ability to resist protease degradation; on this basis, repeat the sequence twice to increase the hydrophobicity and net charge number, and the obtained polypeptide sequence is shown in SEQ ID No. 1, and amidate is performed at the C-terminus of the polypeptide so that the net charge number of the polypeptide is +7;

[0009] Step 2: synthesizing the polypeptide by solid phase chemical synthesis, and purifying it by reverse phase high performance liquid chromatography and identifying it by mass spectrometry;

[0010] Step 3: The polypeptide is subjected to antibacterial activity test, hemolytic activity test, salt ion stability test and protease stability test, and finally named as the enzymatically resistant tetradecapeptide 2rrr.

[0011] Another object of the present invention is to provide the use of the D-Arg-rich enzymatically resistant tetradecapeptide 2rrr as described above in the preparation of a drug for treating Gram-positive and / or Gram-negative bacterial infectious diseases.

[0012] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis and Staphylococcus epidermidis.

[0013] Furthermore, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium.

[0014] The present invention also provides a drug suitable for treating and / or preventing Gram-positive bacteria and / or Gram-negative bacteria infection, wherein the drug contains the D-Arg-rich enzymatically resistant tetradecapeptide 2rrr as claimed in claim 1.

[0015] The present invention has the following advantages and beneficial effects: the D-Arg-rich enzymatic resistant fourteen peptide 2rrr of the present invention has high antibacterial activity against several tested Gram-negative bacteria and Gram-positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, etc., and does not cause hemolysis at the highest test concentration (128 μg / mL), has high biosafety, and a therapeutic index of 60.10. In addition, it still maintains a low MIC in salt ions and pepsin and trypsin at different physiological concentrations, and has high salt ion stability and protease stability. In summary, the D-Arg-rich enzymatic resistant fourteen peptide 2rrr of the present invention has the potential to become a broad-spectrum antibacterial drug for treating Gram-positive and Gram-negative bacterial infections, and maintains high antibacterial activity in the digestive tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the reverse phase high performance liquid chromatogram of the D-Arg-rich enzymatic resistant tetradecapeptide 2rrr;

[0017] Figure 2 This is the mass spectrum of the D-Arg-rich enzymatic-resistant tetradecapeptide 2rrr. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the embodiments and drawings.

[0019] Example 1

[0020] Peptide design

[0021] Leu and Trp were selected as hydrophobic amino acids to provide hydrophobicity to the polypeptide. Leu was placed at positions 1 and 4 to form a Leu zipper, and Trp was placed at positions 2 and 6 to form a Trp zipper to stabilize the α-helical structure and improve the antibacterial activity. D-Arg was placed at positions 3, 5, and 7 to provide a positive charge to the polypeptide and enhance its ability to resist protease degradation. On this basis, the hydrophobicity and charge number were further increased, the above sequence was repeated twice, and the C-terminus of the antimicrobial peptide was amidated with -NH2 to obtain a D-Arg-rich, enzymatically resistant 14-peptide named 2rrr, with a charge number of +7 (D-Arg in the sequence is used D The sequence, molecular weight and charge number of the polypeptide are shown in Table 1.

[0022] Table 1 Sequence, molecular weight and charge number of the enzymatically resistant peptide 2rrr

[0023]

[0024] Example 2

[0025] Synthesis and identification of peptides

[0026] The designed peptide was synthesized by Nanjing 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 antibacterial activity, hemolytic activity, salt ion stability and protease stability determination.

[0027] The reverse phase HPLC chromatogram of the enzymatic resistant peptide 2rrr is attached. Figure 1 .

[0028] The mass spectrum of the enzymatically resistant peptide 2rrr is attached. Figure 2 .

[0029] Example 3

[0030] Antibacterial activity of anti-enzymatic peptides

[0031] The antibacterial activity of antimicrobial peptides was evaluated by measuring the minimum inhibitory concentration (MIC) of antimicrobial peptides. The MIC of antimicrobial peptides in this test was measured based on the test method recommended by the Clinical and laboratory standards institute (CLSI) of the United States with a slight improvement.

[0032] Dissolve the powdered polypeptide into a storage solution with sterile ultrapure water in a sterile operating table at a concentration of 5.12 mg / mL for biological activity determination. Inoculate 100 μL of the bacterial solution frozen at -40 into sterile MHB liquid medium and place it in a 37, 220 rpm shaker overnight. Then transfer the bacterial solution to a new sterile MHB liquid medium and culture it to its logarithmic growth phase. Use a UV-visible spectrophotometer to adjust the bacterial solution concentration to OD 600 nm =0.38~0.40 for standby use. Use 0.2% BSA (containing 0.01% acetic acid) filtered through a 0.22μm water filter as the diluent, add the above polypeptide stock solution to the diluent of the 96-well plate, perform a 2-fold gradient dilution, and then add 50μL of the bacterial solution diluted 1000 times to each well. Use MHB containing bacteria as the positive control, and sterile MHB medium as the negative control. Place the 96-well plate in a 37 incubator and incubate for 16~18h. After the incubation, the negative control wells remain clear and transparent, indicating that the test process is pollution-free. The lowest peptide concentration at which the turbidity does not increase compared with the negative control by naked eye observation is defined as the MIC of the polypeptide. Perform three independent repeated tests. The minimum inhibitory concentration of anti-enzymatic peptides is shown in Table 2.

[0033] Table 2 Antibacterial activity of anti-enzymatic peptide 2rrr (μg / mL)

[0034]

[0035] As can be seen from the table, the D-Arg-rich enzymatic-resistant tetradecapeptide 2rrr exhibited good antibacterial activity against all bacteria tested, with MIC values ​​ranging from 2 to 16 μg / mL.

[0036] Example 4

[0037] Hemolytic activity of enzymatically resistant peptides

[0038] To evaluate the safety of the anti-enzymatic peptides, the hemolytic behavior of the anti-enzymatic peptides on human red blood cells (hRBC) was measured in the concentration range of 1 to 128 μg / mL.

[0039] 2mL of fresh blood from healthy volunteers was collected and stored in a sodium heparin anticoagulant tube. After centrifugation at 4, 3000r for 10min, the supernatant was discarded, and the sample was washed 3 times with sterile PBS buffer. Finally, 10mL of PBS was resuspended for use. Using PBS buffer as the diluent, the peptide storage solution with an initial concentration of 5.12mg / mL was added to the diluent of the 96-well plate, and a 2-fold gradient dilution was performed. Then 50μL of red blood cell suspension was added to each well (50μL of red blood cells + 50μL of 0.1% Triton X-100 for the positive control and 50μL of red blood cells + 50μL of PBS buffer for the negative control), and incubated at 37 for 1h. After the incubation, the 96-well plate was placed at 4, 1000×g for 10min. After the centrifugation, 50μL of supernatant was aspirated from each well, transferred to a new 96-well plate, and the absorbance at 570nm was measured. This experiment was repeated three times. The lowest concentration of the anti-enzymatic peptide that causes 5% hemolysis of human red blood cells is defined as the minimum hemolytic concentration (MHC), and the MHC is used to evaluate the biocompatibility of the peptide. The GM value is the geometric mean of the MIC of the peptide against the tested bacteria, and the ratio of the MHC to the GM of AMPs is defined as the therapeutic index (TI). The cell selectivity of the peptide is evaluated by calculating the TI value of the peptide. The larger the TI value, the higher the cell selectivity of the AMPs. The calculation results are shown in Table 3.

[0040] Table 3 Therapeutic index of the resistant peptide 2rrr

[0041]

[0042] As shown in Table 3, 2rrr did not cause hemolysis at all tested concentrations, and its MHC was greater than 128 μg / mL, indicating that it has high biosafety, and the calculated TI value is 60.10, which has high cell selectivity.

[0043] Example 5

[0044] Salt ion stability of enzymatically resistant peptides

[0045] E. coli 25922 and S. aureus 29213 were selected as typical Gram-negative bacteria, and the MIC values ​​of the anti-enzymatic peptides under different physiological concentrations of salt ions were determined.

[0046] 0.2% BSA (containing 0.01% glacial acetic acid) filtered through a 0.22μm water filter membrane was used as a diluent to prepare salt ion solutions of different physiological concentrations. The initial concentrations of the salt ions were NaCl 300mM, KCl 9mM, CaCl2 5mM, MgCl22mM, NH4Cl 12μM, ZnCl2 16μM and FeCl3 8μM. Using the above-mentioned different salt ions as diluents, the determination method in Example 3 was used to determine the MIC of the anti-enzymatic peptide in different salt ions (with the MIC value of AMPs not treated with salt ions as a control). According to the changes in its MIC value, the stability of the anti-enzymatic peptide in salt ions of different physiological concentrations was determined. This experiment was repeated three times independently. The results are shown in Table 4.

[0047] Table 4 MIC values ​​of anti-enzymatic peptides in the presence of physiological concentrations of salt ions (μg / mL)

[0048]

[0049] 2rrr in Ca 2+ In the presence of 2, the MIC of E. coli increased from 4 μg / mL to 8 μg / mL, which was only 2 times higher, and the MIC of S. aureus increased from 4 μg / mL to 32 μg / mL. In summary, 2rrr showed high salt ion stability.

[0050] Example 6

[0051] Protease stability of resistant peptides

[0052] E. coli 25922 was used as a typical Gram-negative bacterium and S. aureus 29213 was used as a typical Gram-positive bacterium to determine the sensitivity of the resistant peptides to pepsin and trypsin at an initial concentration of 10 mg / ml.

[0053] Prepare pepsin solution and trypsin solution with an initial concentration of 10 mg / mL with sterile ultrapure water. Incubate the storage solution of the anti-enzymatic peptide to be tested with the pepsin and trypsin solutions at 37°C for 8 hours, boil for 15 minutes, and cool to room temperature. The solution after the pepsin and anti-enzymatic peptide are mixed with concentrated hydrochloric acid to pH = 2. The anti-enzymatic peptide is mixed with an equal volume of pepsin or trypsin with an initial concentration of 10 mg / ml, and after incubation at 37 for 8 hours, the MIC of the anti-enzymatic peptide in different proteases is determined by the determination method in Example 3 (the antibacterial activity of the polypeptide not treated with protease is used as a control). The stability of AMPs in different proteases is judged according to the changes in its MIC value. This experiment was repeated three times independently. The test results are shown in Table 5.

[0054] Table 5 MIC values ​​(μg / mL) of resistant peptides after incubation with different proteases for 8 h

[0055]

[0056] By measuring the protease sensitivity of 2rrr to E. coli 25922 and S. aureus 29213, it was found that 2rrr has good protease stability. After incubation with 10 mg / ml pepsin and trypsin for 8 hours, its MIC value was 4-8 μg / mL, and it still has high antibacterial activity. In summary, the D-Arg-rich enzymatic resistant 14-peptide 2rrr has excellent antibacterial activity and biocompatibility, and it has good stability in physiological concentration salt ion environment and high concentration protease environment.

Claims

1. A D-Arg-rich enzymatically resistant tetradecapeptide 2rrr, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, its molecular formula is shown in formula (I), its C-terminus is amidated with -NH2, and the 3rd, 5th and 7th positions of the sequence are D-arginine, 2. The method for preparing a D-Arg-rich enzymatically resistant tetradecapeptide 2rrr according to claim 1, comprising the following steps: Step 1: Select leucine and tryptophan as hydrophobic amino acids to provide hydrophobicity for the polypeptide, place leucine at the 1st and 4th positions of the sequence to form a leucine zipper, and place tryptophan at the 2nd and 6th positions of the sequence to form a tryptophan zipper to stabilize the α-helical structure and improve the antibacterial activity; place D-arginine at the 3rd, 5th and 7th positions of the sequence to provide a positive charge and enhance the ability to resist protease degradation; on this basis, repeat the sequence twice to increase the hydrophobicity and net charge number, and the obtained polypeptide sequence is shown in SEQ ID No. 1, and amidate is performed at the C-terminus of the polypeptide so that the net charge number of the polypeptide is +7; Step 2: synthesizing the polypeptide by solid phase chemical synthesis, and purifying it by reverse phase high performance liquid chromatography and identifying it by mass spectrometry; Step 3: The polypeptide is subjected to antibacterial activity test, hemolytic activity test, salt ion stability test, and protease stability test, and is finally named as the enzymatically resistant tetradecapeptide 2rrr.

3. Use of the D-Arg-rich enzymatically resistant tetradecapeptide 2rrr according to 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; the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.

4. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The drug contains the D-Arg-rich, enzymatically resistant tetradecapeptide 2rrr as claimed in claim 1.

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