Gemini surfactant-like antimicrobial peptide resistant to enzymatic hydrolysis and preparation method and application thereof

Abstract

The present invention discloses a Gemini surfactant-like antimicrobial peptide resistant to enzymolysis, a preparation method and an application thereof, and the amino acid sequence thereof is shown in SEQ ID NO.1. Two prolines are used as inherent rotation forming units to guide two "tails" to extend to the N-terminus and the C-terminus with two isoleucines and two tryptophans, and the tryptophan residues utilize π-π stacking to stabilize the peptide chain structure and provide hydrophobic forces; the "head group" comprises three arginines to provide a positive charge, and D-arginine is selected to reduce the protease cleavage rate, and the overall layout is a combination of a hydrophilic head and two hydrophobic tails, and the free carboxyl group is modified by C-terminal amidation. The antimicrobial peptide has an amphiphilic structure similar to a surfactant, effectively inhibits bacterial growth, no hemolysis is found within a safe range, has strong protease stability, can self-assemble into an ordered nanostructure, and has the potential for application as an antibiotic substitute.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a gemini surfactant-like antimicrobial peptide resistant to enzymolysis and a preparation method thereof. Background Art

[0002] Antimicrobial peptides have multiple biological activities and can kill or inhibit bacteria, fungi, viruses and parasites, etc. They are not easy to develop drug resistance. Antimicrobial peptides generally have an amphiphilic structure. The positive charge of some peptides makes them similar to cationic surfactants, which are water-soluble and have a strong bactericidal effect. The amphiphilicity of polypeptides is affected by orderly arranging amino acids, and the hydrophobic effect is used to drive the molecules to self-assemble into a compact nanostructure. This construction method is not only conducive to the penetration of antimicrobial peptides into bacterial membranes, but also can reduce the dosage and frequency of administration. However, most antimicrobial peptides are sensitive to gastrointestinal proteases. This natural characteristic hinders oral administration and limits their drug development and clinical application. How to enhance their ability to resist protease hydrolysis is the focus of the molecular improvement project of efficient antimicrobial peptides. Summary of the invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide an enzymatically resistant gemini surfactant-like antimicrobial peptide IPr, which has a surfactant-like amphiphilic structure, has a broad-spectrum antibacterial activity in vitro, and can self-assemble to form a nanostructure to resist protease cleavage, shows good in vivo therapeutic potential in an acute peritonitis-sepsis mouse model, and achieves high activity and protease stability of the nanopeptide.

[0004] The technical scheme adopted by the present invention is as follows: an enzymatically resistant gemini surfactant-like antimicrobial peptide IPr, whose amino acid sequence is shown in SEQ ID NO.1, wherein the C-terminus is amidated with -NH2, and the arginine at the 6th position is D-arginine.

[0005] Furthermore, the enzymatically resistant gemini surfactant-like antimicrobial peptide IPr described above has a molecular formula as shown in formula (I):

[0006]

[0007] Furthermore, the self-assembly method of the enzymatically resistant gemini surfactant-like antimicrobial peptide IPr as described above has the following self-assembly conditions: concentration of 26.75-256uM, incubation at 37°C for 24 hours.

[0008] Another object of the present invention is to provide a method for preparing a Gemini surfactant-like antimicrobial peptide IPr that is resistant to enzymatic hydrolysis, as follows: the overall layout of the polypeptide is a hydrophilic head and two hydrophobic tails, two prolines are used as inherent rotation forming units, the two hydrophobic tails of the polypeptide are guided to extend to the N-terminus and the C-terminus with two isoleucines and two tryptophans respectively, the tryptophan residues on the two hydrophobic tails stabilize the peptide chain structure by π-π stacking, and provide hydrophobic forces; the hydrophilic head contains three arginines for providing positive charges to electrostatically attract negatively charged components on bacterial cell membranes, one of which is D-arginine to reduce the protease cleavage rate; the amino acid sequence of the obtained polypeptide is as shown in SEQ ID As shown in NO.1, the C-terminus of the polypeptide is amidated with -NH2 to enhance metabolic stability; the polypeptide is separated and purified by solid phase chemical synthesis and reversed-phase high performance liquid chromatography, and then freeze-dried. The obtained polypeptide is subjected to minimum inhibitory activity determination, hemolytic activity determination, cytotoxicity determination, protease stability determination, nano-characterization analysis, and mouse peritonitis treatment test, and is finally named as anti-enzymatic gemini surfactant-like antimicrobial peptide IPr.

[0009] Another object of the present invention is to provide the use of the enzymatically resistant gemini surfactant-like antimicrobial peptide IPr as described above in the preparation of a drug for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

[0010] The present invention has the following advantages and beneficial effects: the raw materials of the present invention are basically natural amino acids, the production cost is low, the synthesis process is simple, the amphiphilic structure of the surfactant has a strong inhibitory effect on common pathogens, and is not easy to produce drug resistance, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella pullorum, Staphylococcus epidermidis, and Enterococcus faecalis; no hemolysis is found within the safe range, and it has strong protease stability; it self-assembles into an ordered nanostructure driven by hydrophobic forces, showing high antibacterial activity and stability; it shows a therapeutic effect similar to antibiotics on mouse peritonitis caused by Escherichia coli, and significantly reduces the bacterial load of mouse organs. In summary, the antimicrobial peptide IPr has the potential to become an alternative to antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the reverse phase high performance liquid chromatogram of antimicrobial peptide IPr;

[0012] Figure 2 is the mass spectrum of antimicrobial peptide IPr;

[0013] Figure 3 is the fluorescence spectrum of the antimicrobial peptide IPr;

[0014] Figure 4 This is the critical aggregation concentration determination diagram of the antimicrobial peptide IPr;

[0015] Figure 5 The nanostructure of the self-assembled antimicrobial peptide IPr under a transmission electron microscope;

[0016] Figure 6 Comparison of the antibacterial effect of antimicrobial peptide IPr in mice, (a) liver, (b) kidney, (c) lung, (d) spleen;

[0017] Figure 7 This is a graph showing the cytotoxicity assay of the antimicrobial peptide IPr on HEK 293T cells. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0019] Example 1

[0020] Design of antimicrobial peptide IPr

[0021] Two prolines act as inherent rotational forming units, guiding the two tails of the antimicrobial peptide to extend to the N-terminus and C-terminus with two isoleucines and two tryptophans, respectively. Isoleucine is less susceptible to protease cleavage than other hydrophobic amino acids, and tryptophan has a large aromatic ring. The tryptophan residues on the two hydrophobic tails stabilize the peptide chain structure by π-π stacking, providing hydrophobic forces; the hydrophilic head contains three arginines, providing positive charges for the antimicrobial peptide to electrostatically attract the negatively charged components on the bacterial cell membrane, and one of them is D-arginine to reduce the protease cleavage rate, forming a combination of a hydrophilic head and two hydrophobic tails, and the C-terminus of the peptide is modified by -NH2 amidation, which enhances the metabolic stability of the peptide. The sequence of the antimicrobial peptide is shown in Table 1.

[0022] The amino acid sequence of the antimicrobial peptide IPr is as follows:

[0023]

[0024] Table 1 Amino acid sequences of antimicrobial peptides

[0025]

[0026] The molecular formula is shown in formula (I):

[0027]

[0028] Example 2

[0029] The linear peptides were synthesized by solid phase chemical synthesis, and the peptides were synthesized from the C-terminus to the N-terminus.

[0030] 1. Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 ml reactor and soak it with 50 ml of dichloromethane (DCM).

[0031] 2. After 2 hours, wash the resin with nitrogen-dimethylformamide (DMF) 3 times the volume of the resin, and then drain it. Repeat this four times. Drain the resin and set it aside for later use.

[0032] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF 3 times the volume of the resin and then drain it.

[0033] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100°C for 1 minute). If the resin has color, it means that the deprotection is successful.

[0034] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxybenzotriazole (HOBT) into a 50 ml centrifuge tube, add 20 ml of DMF to dissolve them, then add 3 ml of N, N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30 ° C for reaction.

[0035] 6. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with DMF 3 times the volume of the resin, and drain it for later use.

[0036] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it.

[0037] 8. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100°C for 1 min). If the resin has color, it means that the deprotection is successful.

[0038] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50 ml centrifuge tube, add 25 ml of DMF to dissolve them, then add 2.5 ml of DIC and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0039] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, it means that the condensation is incomplete and the reaction should continue.

[0040] 11. After the reaction is complete, wash the resin four times with DMF, then drain, add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain to check whether the protection is removed.

[0041] 12. Follow steps 9-11 to connect the following amino acids.

[0042] 13. After the last amino acid is attached, remove the protection, wash with DMF four times, and then drain the resin with methanol. Then use 95 cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) to cut the polypeptide from the resin (add 10 ml of cutting solution per gram of resin), and centrifuge and precipitate four times with ice ether (cutting solution: ether = 1:9). Finally, use HPLC to separate and purify, and then freeze-dry to obtain a polypeptide with a purity of >95%, such as Figure 1-2 shown.

[0043] 14. Purification conditions:

[0044] (1) Stationary phase: C18;

[0045] (2) Configuration of mobile phase: Pump A: V(TFA) / V(water)=1 / 1000; Pump B: V(TFA) / V(acetonitrile)=1 / 1000; flow rate: 10 ml / min; retention time: between 20-30 min, prepared twice.

[0046] Example 3

[0047] Determination of antibacterial activity of antimicrobial peptide IPr

[0048] The experimental procedure of this experiment slightly changes the method for determining the minimum inhibitory concentration (MIC) recommended by the Clinical and Laboratory Standards Institute (CLSI). Take out 100 μL of frozen bacterial solution from the -40℃ refrigerator, inoculate it into 10mL MHB broth medium in a sterile operating table, and culture it at 37℃ overnight with shaking. The next day, transfer 200 μL of bacterial solution to a new 10mL MHB medium, continue to culture for 3-4 hours until it grows to the logarithmic phase, and then dilute the bacterial solution turbidity to OD using an enzyme reader. 600 nm =0.4.

[0049] 50 μL of peptides of different concentrations (final concentration of peptides was 1-128 μM) and an equal volume of bacterial suspension were added to the wells of the 96-well plate. The wells with only culture medium were used as negative controls and the wells with bacteria and culture medium were used as positive controls. The 96-well plate was then placed in a 37°C constant temperature incubator for 18-20 hours. The absorbance value was measured at an OD value of 492 nm using an ELISA reader. The turbidity at the bottom of the 96-well plate was observed by the naked eye to assist in the detection and determine the minimum inhibitory concentration. The test was repeated three times independently, with two parallels for each repeat. The results are shown in Table 2.

[0050] Table 2 Minimum inhibitory concentration of antimicrobial peptide IPr (μM)

[0051]

[0052] It can be seen from Table 2 that the gemini surfactant-like antimicrobial peptide IPr exhibits high antibacterial activity against both common Gram-negative bacteria and Gram-positive bacteria.

[0053] Example 4

[0054] Determination of hemolytic activity of antimicrobial peptides

[0055] 2 mL of blood was drawn from 3 healthy volunteers; 1 mL of fresh blood was centrifuged at 1000 g for 10 min, the supernatant was discarded, and red blood cells were collected; the red blood cells were washed with PBS solution (pH = 7.4), gently blown and centrifuged again for 5 min, and after 3 times, the red blood cells were resuspended in 10 mL of PBS solution (pH = 7.4) for use; 90 μL of PBS solution (pH = 7.4) filtered through a 0.22 μM water filter membrane was added to the first well of a 96-well plate, and 50 μL was added to wells 2-11; 50 μL of peptides of different concentrations (the final concentration of the peptide was 1-128 μM) and an equal volume of red blood cell suspension were added to the sample wells of the 96-well plate, the 11th well was a negative control (50 μL PBS + 50 μL red blood cells), and the 12th well was a positive control (50 μL 0.1% TritonX-100 + 50 μL red blood cells); incubate in a 37°C incubator for 1 h, then centrifuge the 96-well plate (1000 g, 5 min, 4°C), and use a pipette to transfer an equal amount of supernatant to another new sterile 96-well plate. 570 nm The absorbance was measured. The experiment was repeated three times independently, with two replicates for each. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = [(sample OD 570 - Negative control OD 570 ) / (positive control OD 570 - Negative control OD 570 )]×100%, the minimum hemolytic concentration is the concentration when the antimicrobial peptide causes a 10% hemolytic rate. The test results are shown in Table 3.

[0056] Table 3 Hemolytic activity and therapeutic index of antimicrobial peptide IPr

[0057]

[0058] Table 3 shows that the antimicrobial peptide IPr had no hemolytic activity within the safety range. The ratio of the geometric mean of the minimum hemolytic concentration and the minimum inhibitory concentration was used to calculate the therapeutic index, which was 21.12.

[0059] Example 5

[0060] Determination of the stability of antimicrobial peptide proteases

[0061] The common digestive enzyme trypsin was selected for the protease stability test. The antimicrobial peptide (1.28 mM) was incubated with an equal volume of trypsin (4 mg / mL) at 37°C for 1, 2, 4, and 8 hours, respectively, and the peptide not treated with the protease was used as a control. The incubation time was marked, and the subsequent operation was the same as the minimum inhibitory concentration experiment (as described in the steps of Example 3), and the test was repeated three times independently.

[0062] Table 4 Minimum inhibitory concentration (μM) of IPr after trypsin treatment for different time periods

[0063]

[0064] It can be seen from Table 4 that after 8 h of trypsin treatment, the antibacterial activity of the antimicrobial peptide IPr against Gram-negative bacteria E. coli 25922 remained basically unchanged, and the antibacterial activity against Gram-positive bacteria S. aureus 29213 decreased, but was not completely lost. Considering that the drug may work in a shorter time, the above results indicate that the gemini surfactant-like antimicrobial peptide IPr has strong protease stability.

[0065] Example 6

[0066] Antimicrobial Peptide Nanoparticle Characterization

[0067] Critical aggregation concentration determination: Dissolve the ANS dye in dimethylformamide (N,N-Dimethylformamide, DMF) to a final concentration of 2mM; dissolve the polypeptide powder in deionized water and add it to a 96-well plate. The final concentration of the polypeptide is 2-256μM. The total volume of each well is 100μL, and incubate in a 37°C incubator for 24h. Then add 1μL of ANS dye to make the final concentration of ANS 20μM and incubate for 15 minutes. Use an F-4500 fluorescence spectrophotometer (Hitachi, Japan) for testing, with an excitation wavelength of 369nm, and collect fluorescence spectra at 420-550nm. Then use Origin software to draw a fitting curve, with concentration as the horizontal axis and fluorescence intensity peak as the vertical axis to calculate the critical aggregation concentration of the polypeptide. The test results are shown in Figure 3-4 .

[0068] As the concentration of antimicrobial peptide IPr increases, the fluorescence intensity increases significantly, the curve has an obvious peak, and its emission peak intensity shows a blue shift trend. After fitting the curve, the critical aggregation concentration of IPr is 26.75 μM, which preliminarily indicates that when the concentration of antimicrobial peptide exceeds 26.75 μM, monomeric polypeptides can aggregate into macromolecules in aqueous solution.

[0069] Example 7

[0070] Microstructure observation of antimicrobial peptides

[0071] Transmission electron microscopy observation of nanostructures: The antimicrobial peptide IPr was diluted in sterile water to a final concentration of 128 μM and incubated at 37°C for 24 hours. The sample was deposited on a carbon-coated grid and observed using a Hitachi h-7800TEM (Hitachi, Japan) at 100 KV with 1% phosphotungstic acid negative staining for 10 seconds. The test results are shown in Figure 5 .

[0072] Gemini surfactant-like antimicrobial peptide IPr tends to form a nanofiber network structure, in which monomers aggregate to form long chains, which attract each other and coil to form a network, and combine into aggregates in the solution.

[0073] Example 8

[0074] In vivo antimicrobial effect of antimicrobial peptide IPr

[0075] Establishment of acute peritonitis-sepsis mouse model: 24 healthy female C57BL / 6 mice (6-8 weeks old) were randomly divided into 4 groups, 6 mice in each group. The first group of mice served as the healthy control group without treatment and were intraperitoneally injected with 100 μL of normal saline. The mice in the last three groups were intraperitoneally injected with 100 μL of E. coli ATCC 25922 bacterial suspension (OD 600nm=0.25). The mice were in a dying state 12 hours after intraperitoneal infection and were considered to have established an acute peritonitis-sepsis model. One hour after infection, the first group of mice were injected intraperitoneally with 100 μL of saline again, and the next three groups of mice were injected intraperitoneally with 100 μL of saline and a single dose of 10 mg kg - 1 IPr of BW, single dose of 1 mg kg -1 Colistin of BW. The animals were monitored for signs of pain and survival at different time points (1, 2, 4, 8, 12h), and the four groups of animals were killed and weighed after 12h. Blood was collected through the retro-orbital vein, and the blood samples were used to evaluate the levels of cytokines in the serum. Part of the liver, kidney, spleen and lung were used for histological examination, and the remaining organs were weighed, washed in normal saline and immediately homogenized with a homogenizer. Then, the tissue homogenate and peritoneal fluid were diluted to the appropriate concentration (100 or 1000 times), spread on the prepared MHA solid culture medium plates, incubated at 37°C overnight, and the number of colonies per gram of tissue was counted. The results of organ colony counts are shown in Figure 6 The results showed that compared with the untreated bacterial control group, the bacterial load in the liver, kidney, lung, and spleen was significantly reduced after treatment with antimicrobial peptide IPr and colistin, and there was no significant difference in the bacterial load in the liver, kidney, and lung of mice after treatment with antimicrobial peptide IPr and colistin, indicating that the effect of the antimicrobial peptide IPr group in treating mouse peritonitis is similar to that of the antibiotic group.

[0076] Example 9

[0077] Determination of cytotoxicity of antimicrobial peptide IPr

[0078] The survival and growth of human embryonic kidney epithelial cells (HEK 293T) after antimicrobial peptide treatment were detected by MTT colorimetry. The cells were cultured until they covered about 80% of the bottom of the cell bottle, and then the cells were digested with trypsin. The cell concentration was adjusted using a cell counter, and the final concentration was 2×10 4 Cells / well, 50 μL of cells per well were added to the first six wells of the 1st to 7th rows and the 8th row of the 96-well plate, and cell culture medium was added to the last six wells, and cultured in an environment of humidified air at 37°C and 5% CO2 for 24 hours. In the 1st to 7th rows of another 96-well plate, the antimicrobial peptides were diluted in multiples with blank culture medium without serum, and the antimicrobial peptide solutions of different concentrations were drawn out and injected into the 1st to 7th rows of the 96-well plate containing cells in turn. Each well of the 8th row was supplemented with 50 μL of blank culture medium without serum, and the first six wells were used as positive controls, and the last six wells were used as negative controls, and cultured at 37°C and 5% CO2 for 4 hours.

[0079] Prepare MTT solution: dissolve MTT powder in sterile PBS to a final concentration of 5 mg / mL, store in dark, add to the above 96-well plate, 50 μL per well, and culture for 3 hours. Centrifuge the 96-well plate at 1000g for 10 minutes, remove the supernatant, add 100 μL of dimethyl sulfoxide (DMSO) to each well, mix gently to completely dissolve the purple crystals. Use an ELISA reader to measure the absorbance at 570 nm. Set up two parallels for each test, and repeat the test three times independently. Cell viability (%) = [(sample OD 570 - Negative control OD 570 ) / (positive control OD 570 - Negative control OD 570 )]×100%.

[0080] like Figure 7 As shown, the survival rate of human embryonic kidney epithelial cells (HEK 293T) was close to 100% in the presence of antimicrobial peptide IPr at a concentration of 2-64 μM, indicating that the antimicrobial peptide IPr had no effect on the survival rate of cells at a concentration below 64 μM, indicating that it has good biocompatibility.

Claims

1. A Gemini surfactant-like antimicrobial peptide IPr resistant to enzymatic hydrolysis, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1, wherein the C-terminus is amidated with -NH2, the arginine at position 6 is D-arginine, and its molecular formula is shown in formula (I), 2. The self-assembly method of a Gemini surfactant-like antimicrobial peptide IPr resistant to enzymatic degradation according to claim 1, characterized in that: The self-assembly conditions are: concentration of 26.75-256 μM, incubation at 37° C. for 24 hours.

3. The method for preparing a Gemini surfactant-like antimicrobial peptide IPr resistant to enzymatic hydrolysis according to claim 1, characterized in that: The preparation method is as follows: the overall layout of the polypeptide is a hydrophilic head and two hydrophobic tails, two prolines are used as inherent rotation forming units, the two hydrophobic tails of the polypeptide are guided to extend to the N-terminus and the C-terminus with two isoleucines and two tryptophans respectively, and the tryptophan residues on the two hydrophobic tails stabilize the peptide chain structure by π-π stacking effect, providing hydrophobic force; The hydrophilic head contains three arginines for providing positive charge and electrostatic attraction with negatively charged components on the bacterial cell membrane, one of which is D-arginine to reduce the protease cleavage rate; the amino acid sequence of the obtained polypeptide is shown in SEQ ID NO.1, and the C-terminus of the polypeptide is amidated with -NH2 to enhance metabolic stability; the polypeptide is subjected to solid phase chemical synthesis and reverse phase high performance liquid chromatography separation and purification, and then freeze-dried, and the obtained polypeptide is subjected to minimum inhibitory activity determination, hemolytic activity determination, cytotoxicity determination, protease stability determination, nano-characterization analysis, and mouse peritonitis treatment test, and is finally named as anti-enzymatic gemini surfactant-like antimicrobial peptide IPr.

4. Use of an enzymolysis-resistant gemini surfactant-like antimicrobial peptide IPr according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria or / and Gram-positive bacteria, wherein the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa or Salmonella pullorum, and the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

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