Aromatic group-induced self-assembly of antimicrobial short peptides and uses thereof

Abstract

The application discloses a kind of self-assembly antibacterial short peptides based on aromatic group induction and application thereof, first with arginine to provide positive charge, f and Nal provide hydrophobicity, aromatic group X provides self-assembly driving force, and self-assembly antibacterial short peptides are obtained, and the structural general formula is X-NalRRRff-NH2, marked as X-f5f6, wherein R is Arg, f is D-Phe, and X is aromatic group.The antibacterial short peptides are dissolved in PBS or physiological saline under ultrasonic, and can form nanostructure.In vitro antibacterial experiment and toxicity test show that the self-assembly antibacterial short peptides have broad-spectrum antibacterial activity, low hemolytic activity and low cytotoxicity;Serum stability experiment shows that it has excellent stability in mouse serum;In acute toxicity test of mice, the self-assembly antibacterial short peptides show much higher safety than Polymyxin B.Therefore, the self-assembly antibacterial short peptides in the application have good application prospect in preparing clinical antibacterial drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemistry, and relates to a kind of self-assembly antibacterial short peptide based on aromatic group induction, and the present application also relates to the application of the self-assembly antibacterial short peptide in preparing clinical antibacterial drugs. BACKGROUND

[0002] With the misuse and abuse of antibiotics, bacteria began to develop drug resistance. With the increase of drug-resistant bacteria, the therapeutic effect of existing antibiotics is weakened, making it more difficult to control diseases that can be cured (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 the advantages of not being prone to drug resistance. Although AMPs have many advantages compared to traditional antibiotics, some inherent shortcomings of AMPs hinder the development of AMPs in clinical applications, such as high hemolytic activity, mammalian cytotoxicity, poor stability in serum and physiological salt conditions, easy decomposition by proteases, short half-life, high production cost, etc. (Drug Discovery Today, 2023, 28(8):103629.). In order to solve these problems, researchers try to enhance the activity and stability of peptides or reduce toxicity by introducing non-natural amino acids, cyclization, fatty acid modification, glycosylation, polymer design, etc. (Biotechnology Advances, 2022, 59:107968.).

[0003] Many studies have shown 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, etc. (Bioactive Materials, 2021, 28, 11:268-282). Self-assembling peptides have shown broad application prospects in drug carrier development and antibacterial fields due to their excellent biocompatibility, outstanding stability, flexible environmental responsiveness, long-acting release capability, and easy-to-synthesize characteristics (Acs Applied Bio Materials, 2019, 202(5):2208-2218). SUMMARY

[0004] One of the purposes of the present application is to provide a kind of self-assembly antibacterial short peptide based on aromatic group induction.

[0005] The second purpose of the present application is to provide a self-assembly nanostructure formed by the above antibacterial short peptide.

[0006] The third purpose of the present application is to provide the application of the above self-assembly antibacterial short peptide in preparing clinical antibacterial drugs.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] (I) Self-assembled antibacterial short peptides based on aromatic group induction

[0009] The self-assembled antibacterial short peptides based on aromatic group induction provided by the present application are obtained by providing positive charges with R (arginine), providing hydrophobicity with f and Nal, and providing self-assembly driving force with aromatic group X; the general structure 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 optimization of the technical solutions of the present application, the aromatic group is a group condensed with 2-naphthaleneoxyacetic acid and the N terminal of the peptide chain, and the obtained self-assembled antibacterial short peptide is Nap-NalRRRff-NH2, marked as Nap-f5f6, the amino acid sequence of which is Nap-Nal-Arg-Arg-Arg-D-Phe-D-Phe-NH2, and the structural formula is shown in formula (I);

[0011] or the aromatic group is a group condensed with the carboxyl of naproxen and the amino of the N terminal of the peptide chain, and the obtained self-assembled antibacterial short peptide is Npx-NalRRRff-NH2, marked as Npx-f5f6, the amino acid sequence of which is Npx-Nal-Arg-Arg-Arg-D-Phe-D-Phe-NH2, and the structural formula is shown in formula (II);

[0012]

[0013] The above self-assembled short peptides based on aromatic group induction are prepared by classical solid-phase synthesis.

[0014] (II) Self-assembled nanostructure of self-assembled antibacterial short peptides based on aromatic group induction

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

[0016] The self-assembled nanostructure can be obtained by ultrasonic dissolution of the self-assembled antibacterial short peptides based on aromatic group induction in the present application in PBS. Nile red is a hydrophobic fluorescent dye, and when the Nile red is combined with the hydrophobic site in the self-assembled body of the polypeptide, the fluorescence intensity of the Nile red will be significantly enhanced. Different concentrations of polypeptide solutions are prepared using sterile water, PBS buffer or physiological saline, respectively, and the polypeptide and the Nile red solution are mixed in equal volume, and the fluorescence is measured by an enzyme marker under excitation of 550 nm and emission of 600-750 nm. The log2C is taken as the abscissa, and the fluorescence intensity is taken as the ordinate to draw a graph (such as Figure 1). The peptide concentration corresponding to the intersection is the critical aggregation concentration (CAC), and the CAC of Nap-f5f6 in PBS is 9.51 μM and in physiological saline is 11.00 μM. The CAC of Npx-f5f6 in PBS is 5.17 μM and in physiological saline is 4.40 μM.

[0017] 2. Particle size of the peptide

[0018] The self-assembled antibacterial short peptides based on aromatic group induction in the present application were dissolved in water, PBS or physiological saline respectively, with a concentration of 256 μM. The particle size of the peptides in different solvents was determined by a nanoparticle size analyzer, and the results are shown in Table 3. Figure 2

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

[0020] (III) Application of the self-assembled antibacterial short peptide based on aromatic group induction in preparing clinical antibacterial drugs

[0021] In the following experiments, the self-assembled antibacterial short peptides were dissolved and administered according to the experimental requirements. In the in vitro antibacterial test and the cytotoxicity test, the self-assembled antibacterial short peptides were dissolved in broth medium and DMEM medium respectively. In the hemolysis test, the self-assembled antibacterial short peptides were dissolved in PBS. In the serum stability and in vivo acute toxicity test, the self-assembled antibacterial short peptides were dissolved in physiological saline to form nanostructures.

[0022] 1. In vitro antibacterial test

[0023] The minimum inhibitory concentration of the above-mentioned AMPs on gram-positive standard strains (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Enterococcus faecalis) and clinically drug-resistant bacteria (methicillin-resistant Staphylococcus aureus) and gram-negative standard strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and clinically drug-resistant bacteria (Pseudomonas aeruginosa clinically drug-resistant strain) was determined by the classic micro-dilution method. Polymyxin B was used as a positive control, 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 nanostructure antibacterial short peptide in the present application against standard strains

[0025]

[0026] ​

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

[0028]

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

[0030] 2. In vitro toxicity experiment

[0031] To investigate the toxicity of the self-assembled antibacterial short peptides based on aromatic group induction in the present application on mammalian cells, hemolysis of mouse red blood cells and cytotoxicity of HK-2 cells were determined for Nap-f5f6 and Npx-f5f6. The results are shown in Figure 3 and Figure 4 .

[0032] Figure 3 The hemolysis results show that the hemolysis rate of the antibacterial short peptides on mouse red blood cells is less than 10% at the highest concentration of 256 μM. Figure 4 The cytotoxicity results show that the survival rate of HK-2 cells is still higher than 80% at 128 μM. The experimental results show that these antibacterial short peptides do not show obvious toxicity on mammalian cells even under conditions far higher than their antibacterial concentration, which indicates that they have excellent biocompatibility.

[0033] 3. Serum stability experiment

[0034] To investigate the stability of the self-assembled antibacterial short peptides based on aromatic group induction synthesized in the present application in serum, Nap-f5f6 and Npx-f5f6 were incubated with mouse serum at 37°C, and the degradation of the peptides was analyzed by RP-HPLC at different time points to determine the serum half-life of the peptides. The results are shown in Figure 5 .

[0035] Figure 5 The results show that the half-life of Nap-f5f6 in mouse serum is 1103 min, and the half-life of Npx-f5f6 in mouse serum is 1010 min, indicating that these self-assembled antibacterial short peptides based on aromatic group induction have high stability in mouse serum.

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

[0037] The experiment selects BALB / C mice, male, 18-22g, and is fed according to the experimental animal ethics management method of Lanzhou University.

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

[0039] From the results Figure 6 It can be seen from the results that the in-vivo toxicity of Nap-f5f6 and Npx-f5f6 is much lower than that of Polymyxin B, and the median lethal dose (LD 50 ) of Nap-f5f6 and Npx-f5f6 is about 6 times and 11 times that of Polymyxin B respectively.

[0040] The present application provides positive charges with R (arginine), hydrophobicity with f and Nal, and self-assembly driving force with aromatic group X, and obtains a self-assembled antibacterial short peptide with a general structure of X-NalRRRff-NH2, which is marked as X-f5f6, wherein R is Arg, f is D-Phe, and X is an aromatic group. The antibacterial short peptide is dissolved in PBS or normal saline by ultrasonic, and the nanostructure thereof can be obtained. In-vitro bacteriostatic experiments and toxicity tests show that the self-assembled antibacterial short peptide has broad-spectrum antibacterial activity, low hemolytic activity and low cytotoxicity; serum stability experiments show that the self-assembled antibacterial short peptide has excellent stability in mouse serum; in the acute toxicity experiment of mice, the self-assembled antibacterial short peptide shows much higher safety than Polymyxin B. Therefore, the self-assembled antibacterial short peptide in the present application has a good application prospect in the preparation of clinical antibacterial drugs. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The critical aggregation concentration of the antibacterial peptide in the present application is

[0042] Figure 2 The particle size of the antibacterial peptide in the present application in water or PBS is

[0043] Figure 3 The hemolytic activity of the antibacterial peptide in the present application to red blood cells after incubation with mouse red blood cells for 1h is

[0044] Figure 4 Toxicity of the antibacterial peptide of the present application to HK-2 cells after 3h incubation;

[0045] Figure 5 Serum stability of the antibacterial peptide of the present application after co-incubation with mouse serum;

[0046] Figure 6 Acute toxicity of the antibacterial peptide of the present application and Polymyxin B;

[0047] Figure 7 Mass spectrum of Nap-f5f6;

[0048] Figure 8 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 application is further described below through specific examples.

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

[0051] (1) Activation and pretreatment of the resin

[0052] 0.42 g of MBHA resin (0.48 mmol / g) was weighed and added to a polypeptide solid-phase synthesizer, swelled in DCM for 30 min, washed with DMF, and then identified by ninhydrin coloration. No color indicated that the resin was normal and could be used.

[0053] (2) Synthesis of Nap-f5f6

[0054] The above swelled resin was washed with a DMF solution containing 20% piperidine to remove the Fmoc protecting group. After washing with DMF, ninhydrin test showed blue-purple color, indicating successful removal. A 3-fold excess of D-Phe, 3-fold excess of HOBt, HBTU, and 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer and stirred for 1 h. After the reaction time reached, ninhydrin test showed that the resin was colorless and transparent, indicating successful condensation, and Fmoc-D-Phe-MBHA was obtained.

[0055] Fmoc-D-Phe-D-Phe. The Fmoc group was removed by washing the resin with 20% piperidine in DMF, and the resin was washed with DMF. A 3-fold excess of 2-naphthoxyacetic acid, 3-fold excess of HOBt, HBTU, and 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer and stirred for 3 h. After the reaction time had elapsed, the resin was tested with ninhydrin and was found to be colorless and transparent, indicating that the condensation was successful, and Nap-D-Phe-D-Phe-MBHA was obtained.

[0056] (3) Cleavage of the polypeptide

[0057] The resulting Nap-D-Phe-D-Phe-MBHA was washed with DCM and MeOH and was dried to a sand-like consistency. Ten mL of cleavage reagent (TFA: Tris: H2O = 9.5:0.25:0.25) was added and the reaction was allowed to proceed for 3 h. After extraction with ether and water, the aqueous phase was lyophilized to obtain a crude peptide lyophilized powder.

[0058] (4) Purification of the polypeptide

[0059] The RP-HPLC purification conditions were A: 0.1% TFA / ACN, mobile phase B: 0.1% TFA / H2O. Gradient elution was performed, and the target peak was collected and lyophilized to obtain Nap-f5f6, which had a mass spectrum as shown in Figure 7 Example 2: Synthesis of the antibacterial peptide Npx-f5f6

[0060] (1) Activation and pretreatment of the resin

[0061] As in Example 1.

[0062] (2) Synthesis of Npx-f5f6

[0063] The Fmoc group was removed by washing the resin with 20% piperidine in DMF, and the resin was washed with DMF. A 3-fold excess of 2-naphthoxyacetic acid, 3-fold excess of HOBt, HBTU, and 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer and stirred for 3 h. After the reaction time had elapsed, the resin was tested with ninhydrin and was found to be colorless and transparent, indicating that the condensation was successful, and Nap-D-Phe-D-Phe-MBHA was obtained.

[0064] Fmoc-Nal-Arg-Arg-Arg-D-Phe-D-Phe. Fomc was removed with 20% piperidine in DMF, washed with DMF, and 3 times the amount of Npx, 3 times excess HOBt, HBTU, 6 times excess DIEA were dissolved in DMF and added to the synthesizer and the reaction was stirred for 3 h. After the reaction time was up, a ninhydrin test of the resin was clear indicating a successful coupling, Npx-Nal-Arg-Arg-Arg-D-Phe-D-Phe-MBHA.

[0065] (3) Polypeptide Cleavage

[0066] Example 1.

[0067] (4) Polypeptide Purification

[0068] Example 1. The antibacterial peptide Npx-f5f6 was obtained and had a mass spectrum as shown in Figure 2. Figure 8 Example 1. The antibacterial peptide Npx-f5f6 was obtained and had a mass spectrum as shown in Figure 2.

Claims

1. A class of aromatic group-induced self-assembly antibacterial short peptides, characterized in that, The antibacterial short peptide structure general formula is X-NalRRRff-NH2, marked as X-f5f6, wherein R is Arg, f is D-Phe, and X is an aromatic group; the antibacterial short peptide is obtained by providing a positive charge with R, providing hydrophobicity with f and Nal, and providing self-assembly driving force with the aromatic group X; The aromatic group is a group obtained by condensing 2-naphthaleneoxyacetic acid with the N-terminal of the peptide chain, and the obtained self-assembly antibacterial short peptide is Nap-NalRRRff-NH2, marked as Nap-f5f6, and the structural formula is shown as formula (I); Or the aromatic group is a group obtained by condensing the carboxyl of naproxen with the N-terminal amino of the peptide chain, and the obtained self-assembly antibacterial short peptide is Npx-NalRRRff-NH2, marked as Npx-f5f6, and the structural formula is shown as formula (II); 。 2. Use of the self-assembling antimicrobial short peptide according to claim 1 for the preparation of a clinical antimicrobial drug, characterized in that, The bacteria inhibited by the antibacterial drug are gram-positive bacteria or gram-negative bacteria; The gram-positive bacteria are Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Enterococcus faecalis or methicillin-resistant Staphylococcus aureus; and the gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa.

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