Self-assembled antibacterial oligopeptide based on aromatic group induction and application thereof

Abstract

The invention discloses a self-assembly antibacterial oligopeptide based on aromatic group induction and application thereof.The self-assembly antibacterial oligopeptide is obtained through arginine providing positive charges, f and Nal providing hydrophobicity and aromatic group X providing self-assembly driving force, the structural general formula of the self-assembly antibacterial oligopeptide is X-NalRRRff-NH2, the self-assembly antibacterial oligopeptide is marked as X-f5f6, R is Arg, f is D-Phe, and X is aromatic group. The antibacterial oligopeptide is ultrasonically dissolved in PBS or normal saline to form a nano structure. An in-vitro antibacterial experiment and a toxicity test show that the self-assembled antibacterial oligopeptide has broad-spectrum antibacterial activity, low hemolytic activity and low cytotoxicity; serum stability experiments show that the compound has excellent stability in mouse serum; in an acute toxicity experiment of a mouse, the safety of the self-assembled antibacterial oligopeptide is far higher than that of Polymyxin B. Therefore, the self-assembled antibacterial oligopeptide provided by the invention has a good application prospect in preparation of clinical antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and relates to a self-assembling antibacterial short peptide based on aromatic group induction. The invention also relates to the application of the self-assembling antibacterial short peptide in the preparation of clinical antibacterial drugs. Background Art

[0002] With the misuse and abuse of antibiotics, bacteria begin to develop drug resistance. With the increase of drug-resistant bacteria, the effectiveness of existing antibiotic treatments has weakened, making diseases that could have been cured more difficult to control (Embo Reports, 2020, 21(12): e51034.). Antimicrobial peptides (AMPs) have attracted the attention of researchers due to their high antibacterial efficiency and broad-spectrum antibacterial activity, and their resistance to drug resistance. Although AMPs have many advantages over traditional antibiotics, some inherent disadvantages of AMPs have hindered the development of AMPs in clinical applications, such as high hemolytic activity, mammalian cell toxicity, poor stability under serum and physiological salt conditions, easy to be decomposed by proteases, short half-life, and high production costs (Drug Discovery Today, 2023, 28(8): 103629.). To address these problems, researchers have tried to enhance the activity and stability of peptides or reduce their toxicity by introducing non-natural amino acids, cyclization, fatty acid modification, glycosylation, polymer design, etc. (Biotechnology Advances, 2022, 59: 107968.).

[0003] Many studies have pointed out that the self-assembly process of peptides is mainly dominated by a series of non-covalent forces, including hydrogen bonds, hydrophobic interactions, π-π stacking, electrostatic attraction, and van der Waals forces (Bioactive Materials, 2021, 28, 11: 268-282). Self-assembling peptides have shown broad application prospects in the fields of drug carrier development and antibacterial due to their excellent biocompatibility, outstanding stability, flexible environmental responsiveness, long-term release ability, and easy synthesis (Acs Applied Bio Materials, 2019, 202(5): 2208-2218). Summary of the invention

[0004] One of the purposes of the present invention is to provide a class of self-assembling antibacterial short peptides induced by aromatic groups.

[0005] The second purpose of the present invention is to provide a self-assembled nanostructure formed by the above-mentioned antibacterial short peptides.

[0006] The third object of the present invention is to provide the use of the self-assembling antibacterial short peptide in the preparation of clinical antibacterial drugs.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] (I) Self-assembly of antibacterial short peptides induced by aromatic groups

[0009] The present invention provides a type of self-assembling antibacterial short peptide based on aromatic group induction, which is obtained by providing positive charge with R (arginine), hydrophobicity with f and Nal, and self-assembly driving force with aromatic group X; its general structural formula is X-NalRRRff-NH2, marked as X-f5f6, wherein R is Arg, f is D-Phe, and X is an aromatic group.

[0010] As a further preferred embodiment of the technical solution of the present invention, the aromatic group is a group formed by condensation of 2-naphthyloxyacetic acid and the N-terminus of the peptide chain, and the obtained self-assembling antibacterial short peptide is Nap-NalRRRRff-NH2, marked as Nap-f5f6, and its amino acid sequence is Nap-Nal-Arg-Arg-Arg-D-Phe-D-Phe-NH2, and its structural formula is shown in formula (I);

[0011] Or the aromatic group is the group formed by condensation of the carboxyl group of naproxen and the amino group at the N-terminus of the peptide chain, and the obtained self-assembling antibacterial short peptide is Npx-NalRRRRW-NH2, marked as Npx-f5f6, and its amino acid sequence is Npx-Nal-Arg-Arg-Arg-D-Phe-D-Phe-NH2, and its structural formula is shown in formula (II);

[0012]

[0013] The above-mentioned self-assembling short peptides induced by aromatic groups were all prepared by classical solid-phase synthesis method.

[0014] (II) Self-assembled nanostructures based on aromatic group-induced self-assembly of antibacterial short peptides

[0015] 1. Nile red determination of critical aggregation concentration

[0016] The self-assembled antibacterial short peptide induced by aromatic groups in the present invention is ultrasonically dissolved in PBS to obtain a self-assembled nanostructure. Nile red is a hydrophobic fluorescent dye. When Nile red binds to the hydrophobic sites in the polypeptide self-assembly, the fluorescence intensity of Nile red will be significantly enhanced. Different concentrations of polypeptide solutions are prepared using sterile water, PBS buffer or physiological saline, and the polypeptide and Nile red solution are mixed in equal volumes. The fluorescence is measured using an enzyme marker at an excitation of 550nm and an emission of 600-750nm. Plot the graph with log2C as the horizontal axis and fluorescence intensity as the vertical axis (such as Figure 1). The peptide concentration corresponding to the intersection is the critical aggregation concentration (CAC). It can be seen that the CAC of Nap-f5f6 in PBS is 9.51μM, and the CAC in normal saline is 11.00μM. The CAC of Npx-f5f6 in PBS is 5.17μM, and the CAC in normal saline is 4.40μM.

[0017] 2. Peptide particle size

[0018] The aromatic group-induced self-assembly antibacterial short peptide of the present invention was dissolved in water, PBS or saline at a concentration of 256 μM. The particle size of the peptide in different solvents was measured using a nanoparticle size analyzer. The results are as follows: Figure 2 shown.

[0019] Figure 2 The results show that the particle size of Nap-f5f6 in water is 226nm, the particle size in PBS is 896nm, and the particle size in saline is 643nm. The particle size of Npx-f5f6 in water is 147nm, the particle size in PBS is 310nm, and the particle size in saline is 218nm, indicating that the particle size of the peptide increases after self-assembly in PBS or saline.

[0020] (III) Application of aromatic group-induced self-assembly of antibacterial peptides in the preparation of clinical antibacterial drugs

[0021] In the following experiments, the self-assembled antimicrobial peptides were dissolved and administered according to the experimental needs. In the in vitro antibacterial test and cytotoxicity test, the self-assembled antimicrobial peptides were dissolved in broth culture medium and DMEM culture medium respectively and administered, the hemolysis experiment was conducted by dissolving the self-assembled antimicrobial peptides in PBS and administered, and the serum stability and in vivo acute toxicity were conducted by dissolving the self-assembled antimicrobial peptides in physiological saline to form nanostructures and administering them.

[0022] 1. In vitro antibacterial test

[0023] The minimum inhibitory concentration of the above AMPs against Gram-positive standard strains (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Enterococcus faecalis) and clinical drug-resistant bacteria (methicillin-resistant Staphylococcus aureus) and Gram-negative standard strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and clinical drug-resistant bacteria (clinical drug-resistant strains of Pseudomonas aeruginosa) was determined by the classic micro-two-fold dilution method. Polymyxin B was used as a positive control in the experiment, and the experiment was repeated three times in parallel. The results are shown in Tables 1 and 2.

[0024] Table 1 Minimum inhibitory concentration of the self-assembled nanostructured antibacterial peptides against standard strains

[0025]

[0026]

[0027] Table 2 Minimum inhibitory concentration of the self-assembled antibacterial peptides of the present invention against clinical drug-resistant strains

[0028]

[0029] The results in Table 1 and Table 2 show that the self-assembling antibacterial short peptides of the present invention have a strong inhibitory effect on standard strains of Gram-positive bacteria and clinical drug-resistant bacteria and standard strains of Gram-negative bacteria and clinical drug-resistant bacteria, showing a broad-spectrum antibacterial activity, especially a stronger inhibitory effect on standard strains of Gram-positive bacteria and clinical drug-resistant bacteria, and show better antibacterial activity than the control drug Polymyxin B.

[0030] 2. In vitro toxicity test

[0031] In order to investigate the toxicity of the aromatic group-induced self-assembly antibacterial short peptides to mammalian cells, the hemolysis of Nap-f5f6 and Npx-f5f6 on mouse erythrocytes and the cytotoxicity to HK-2 cells were determined. Figure 3 and Figure 4 .

[0032] Figure 3 The hemolysis results showed that at the highest tested concentration of 256 μM, the hemolysis rate of the antibacterial peptide on mouse erythrocytes was less than 10%. Figure 4 Cytotoxicity results showed that at 128 μM, the survival rate of HK-2 cells was still above 80%. The experimental results showed that even at conditions far higher than their antibacterial concentrations, these antibacterial short peptides did not show obvious toxicity to mammalian cells, indicating that they have excellent biocompatibility.

[0033] 3. Serum stability test

[0034] In order to investigate the stability of the aromatic group-induced self-assembly antibacterial short peptides synthesized in the present invention in serum, Nap-f5f6 and Npx-f5f6 were co-incubated with mouse serum at 37°C, and samples were taken at different time points to analyze the degradation of the peptides by RP-HPLC, and the serum half-life of the peptides was determined. Figure 5 .

[0035] Figure 5 The results showed that the half-life of Nap-f5f6 in mouse serum was 1103 min, and the half-life of Npx-f5f6 in mouse serum was 1010 min, indicating that these self-assembled antibacterial short peptides induced by aromatic groups have high stability in mouse serum.

[0036] 4. In vivo acute toxicity experiment of antimicrobial peptides

[0037] The experiment used BALB / C mice, male, 18-22 g, and fed according to the Lanzhou University Experimental Animal Ethics Management Methods.

[0038] Nap-f5f6, Npx-f5f6 and Polymyxin B were divided into three groups of high, medium and low doses, with 8 mice in each group. The high, medium and low doses of Nap-f5f6 were 150mg / kg, 104mg / kg and 72mg / kg respectively; the high, medium and low doses of Nx-f5f6 were 300mg / kg, 245mg / kg and 200mg / kg respectively; the high, medium and low doses of the control drug Polymyxin B were 23.8mg / kg, 18.9mg / kg and 15mg / kg respectively. After a single intraperitoneal injection, the mice were observed for death for 7 days. The survival curve is shown in Figure 6 .

[0039] from Figure 6 The results show that the in vivo toxicity of Nap-f5f6 and Npx-f5f6 is much lower than that of Polymyxin B. The median lethal dose (LD50) of Nap-f5f6 and Npx-f5f6 50 ) are approximately 6 and 11 times that of Polymyxin B, respectively.

[0040] The present invention provides positive charge with R (arginine), hydrophobicity with f and Nal, and self-assembly driving force with aromatic group X, thereby obtaining a self-assembly antibacterial short peptide, the general structural formula of which is X-NalRRRRW-NH2, labeled as X-f5f6, wherein R is Arg, f is D-Phe, and X is an aromatic group. The antibacterial short peptide is ultrasonically dissolved in PBS or saline to obtain its nanostructure. In vitro antibacterial experiments and toxicity tests show that the self-assembly antibacterial short peptide has broad-spectrum antibacterial activity, low hemolytic activity and low cytotoxicity; serum stability experiments show that the self-assembly antibacterial short peptide has excellent stability in mouse serum; in acute toxicity experiments of mice, the self-assembly antibacterial short peptide shows much higher safety than Polymyxin B. Therefore, the self-assembly antibacterial short peptide in the present invention has a good application prospect in the preparation of clinical antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the critical aggregation concentration of the antimicrobial peptide of the present invention;

[0042] Figure 2 is the particle size of the antimicrobial peptide of the present invention in water or PBS;

[0043] Figure 3 The results of the hemolytic activity of the antimicrobial peptide of the present invention on mouse erythrocytes after incubation with erythrocytes for 1 hour;

[0044] Figure 4 The toxicity results of the antimicrobial peptide of the present invention on HK-2 cells after incubation for 3 hours;

[0045] Figure 5 The serum stability results after the antimicrobial peptide of the present invention was co-incubated with mouse serum;

[0046] Figure 6 The acute toxicity results of the antimicrobial peptides and Polymyxin B of the present invention;

[0047] Figure 7 is the mass spectrum of Nap-f5f6;

[0048] Figure 8 This is the mass spectrum of Npx-f5f6. DETAILED DESCRIPTION

[0049] The synthesis of the self-assembling peptide with broad-spectrum antibacterial activity and low toxicity of the present invention is further illustrated by specific examples below.

[0050] Example 1: Synthesis of antimicrobial peptide Nap-f5f6

[0051] (1) Activation and pretreatment of resin

[0052] Weigh 0.42 g of MBHA resin (0.48 mmol / g), add it to a peptide solid phase synthesizer, swell it with DCM for 30 min, wash it with DMF, and identify the resin with the ninhydrin colorimetric method. If it is colorless, it means that the resin is normal and can be used.

[0053] (2) Synthesis of Nap-f5f6

[0054] The swollen resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc protecting group. After washing with DMF, the ninhydrin test showed a blue-purple color, indicating successful removal. Dissolve 3 times excess D-Phe, 3 times excess HOBt, HBTU, and 6 times excess DIEA in DMF, add them to the synthesizer and stir for 1 hour. After the reaction time is reached, the ninhydrin test resin is colorless and transparent, indicating that the condensation is successful, and Fmoc-D-Phe-MBHA is obtained.

[0055] According to the above method, D-Phe, Arg, Arg, Arg, Nal are condensed in sequence to obtain Fmoc-Nal-Arg-Arg-Arg-D-Phe-D-Phe. Fmoc is removed with a DMF solution containing 20% ​​piperidine, washed with DMF, and 3 times the amount of 2-naphthoxyacetic acid, 3 times the excess of HOBt, HBTU, and 6 times the excess of DIEA are dissolved in DMF, added to the synthesizer and stirred for 3 hours. After the reaction time is reached, the ninhydrin test resin is colorless and transparent, indicating that the condensation is successful, Nap-Nal-Arg-Arg-Arg-D-Phe-D-Phe-MBHA.

[0056] (3) Peptide cleavage

[0057] The obtained Nap-Nal-Arg-Arg-Arg-D-Phe-D-Phe-MBHA was washed with DCM and MeOH in turn, and dried to a sandy state. 10 mL of a cutting agent (TFA:Tris:H2O=9.5:0.25:0.25) was added to react for 3 h, and after extraction with ether and water, the aqueous phase was freeze-dried to obtain a crude peptide freeze-dried powder.

[0058] (4) Peptide purification

[0059] The RP-HPLC purification conditions were A: 0.1% TFA / ACN, mobile phase B: 0.1% TFA / H2O. The target peak effluent was collected by gradient elution and freeze-dried to obtain Nap-f5f6. The mass spectrometry results were as follows: Figure 7 Example 2: Synthesis of antimicrobial peptide Npx-f5f6

[0060] (1) Activation and pretreatment of resin

[0061] Same as Example 1.

[0062] (2) Synthesis of Npx-f5f6

[0063] The swollen resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc protecting group. After washing with DMF, the ninhydrin test showed a blue-purple color, indicating successful removal. Dissolve 3 times the amount of D-Phe, 3 times the excess of HOBt and HBTU, and 6 times the excess of DIEA in DMF, add them to the synthesizer and stir for 1 hour. After the reaction time is reached, the ninhydrin test resin is colorless and transparent, indicating that the condensation is successful, and Fmoc-D-Phe-MBHA is obtained.

[0064] According to the above method, D-Phe, Arg, Arg, Arg, Nal are condensed in sequence to obtain Fmoc-Nal-Arg-Arg-Arg-D-Phe-D-Phe. Use DMF solution containing 20% ​​piperidine to remove Fomc, wash with DMF, dissolve 3 times of Npx, 3 times of excess HOBt, HBTU, 6 times of excess DIEA in DMF, add to the synthesizer and stir to react for 3 hours. After the reaction time is reached, the ninhydrin test resin is colorless and transparent, indicating that the condensation is successful, Npx-Nal-Arg-Arg-Arg-D-Phe-D-Phe-MBHA.

[0065] (3) Peptide cleavage

[0066] Same as Example 1.

[0067] (4) Peptide purification

[0068] Same as Example 1. Antimicrobial peptide Npx-f5f6 was obtained, and its mass spectrometry results are as follows Figure 8 shown.

Claims

1. A type of self-assembling antibacterial short peptide induced by aromatic groups, characterized in that: The antibacterial short peptide is obtained by providing positive charge with R, hydrophobicity with f and Nal, and self-assembly driving force with the aromatic group X; Its general structural formula is X-NalRRRff-NH2, marked as X-f5f6, where R is Arg, f is D-Phe, and X is an aromatic group.

2. A type of self-assembling antibacterial short peptide based on aromatic group induction as claimed in claim 1, characterized in that: The aromatic group is a group formed by condensation of 2-naphthyloxyacetic acid and the N-terminal of the peptide chain, and the obtained self-assembling antibacterial short peptide is Nap-NalRRRff-NH2, marked as Nap-f5f6, and its structural formula is shown in formula (I); Or the aromatic group is a group formed by condensation of the carboxyl group of naproxen and the amino group at the N-terminus of the peptide chain, and the obtained self-assembling antibacterial short peptide is Npx-NalRRRRW-NH2, marked as Npx-f5f6, and its structural formula is shown in formula (II); 3. The use of the self-assembling antibacterial short peptide according to claim 1 or 2 in the preparation of clinical antibacterial drugs, characterized in that: The bacteria inhibited by the antibacterial drug are Gram-positive bacteria or Gram-negative bacteria.

4. The use of the self-assembling antibacterial short peptide according to claim 3 in the preparation of clinical antibacterial drugs, characterized in that: The antibacterial drug is a drug for treating infectious diseases caused by Gram-positive bacteria and Gram-negative bacteria.

5. The use of the self-assembling antibacterial short peptide according to claim 4 in the preparation of clinical antibacterial drugs, characterized in that: The Gram-positive bacteria and Gram-negative bacteria include standard bacteria and clinical drug-resistant bacteria.

6. The use of the self-assembling antibacterial short peptide according to claim 5 in the preparation of clinical antibacterial drugs, characterized in that: The Gram-positive standard bacteria are Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis or Enterococcus faecalis, and the drug-resistant bacteria are methicillin-resistant Staphylococcus aureus; the Gram-negative standard bacteria are Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa, and the drug-resistant bacteria are clinical drug-resistant bacteria of Pseudomonas aeruginosa.

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