Low-toxic broad-spectrum antibacterial peptide with WW as symmetry center and application thereof

Abstract

The application discloses a kind of low-toxicity broad-spectrum antibacterial peptide with WW as symmetry center, the antibacterial peptide is that WK is repeated twice and symmetrically distributed in two sides, and WW is used as symmetry center, obtain antibacterial peptide WKWKWWKWKW-NH2 (N2W2), then the tryptophan of C terminal and N terminal of N2W2 is replaced by leucine and / or isoleucine;The antibacterial peptide can basically maintain the excellent antibacterial activity of parent peptide, and their hemolytic activity is greatly reduced, and the safety is improved. The antibacterial activity of IL-N2W2 is highest, but the stability needs to be improved, so D-type amino acid replacement is carried out on IL-N2W2 to obtain D-IL-N2W2. D-IL-N2W2 maintains the high antibacterial activity and low toxicity of IL-N2W2, and can still completely inhibit bacterial growth after incubation with high-concentration trypsin or chymotrypsin, has very great potential in relieving antibiotic resistance, and has good application prospect in preparing clinical antibacterial drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry, and relates to a kind of low-toxicity broad-spectrum antibacterial peptide with KWWK as the symmetry center, and the application also relates to the application of the antibacterial peptide in the preparation of clinical antibacterial drugs. BACKGROUND

[0002] Antibiotic resistance poses a serious threat to world public health, but few new antibiotics have been developed and approved by the US Food and Drug Administration. It is estimated that the cost of developing a new antibiotic is about 1.5 billion US dollars, and it takes more than 10 years from basic research to FDA approval. Antimicrobial peptides (AMPs) are a potential antibiotic due to their unique mechanism of action on bacterial membranes, but they have some inherent defects, such as difficulty in combining high antibacterial activity, low toxicity, and high stability, and high cost. Therefore, there is an urgent need to develop new antibacterial peptides with simple sequences and easy synthesis.

[0003] Hydrophilicity and hydrophobicity are key factors affecting the antibacterial activity and toxicity of cationic AMPs. Cations help AMPs bind to negatively charged regions of bacterial membranes, and hydrophobic portions promote the insertion of AMPs into the hydrophobic phospholipid layer of bacterial membranes. The alternating arrangement of hydrophobic amino acids and cationic amino acids can promote the formation of an amphiphilic surface with one hydrophilic and one hydrophobic side, and "K" and "W" are used as a golden combination in the design of many AMPs. Some studies have shown that AMPs with repeating sequences and symmetry have high antibacterial activity and low toxicity. Therefore, the initially designed peptide is to repeat "WK" twice and symmetrically distribute it on both sides. In order to improve the antibacterial activity of the peptide, the tryptophan that can interact strongly with the bacterial membrane is repeated twice as the symmetry center of the peptide, resulting in the antibacterial peptide WKWKWWKWKW-NH2 (N2W2). N2W2 has high and broad-spectrum antibacterial activity, but its hemolytic activity is high. Studies have shown that excessive hydrophobicity can lead to an increase in hemolytic activity, and the hydrophobicity of N2W2 is as high as 0.954, so reducing its hydrophobicity appropriately may improve its toxicity. SUMMARY

[0004] One of the purposes of the present application is to reduce the toxicity of N2W2 (WKWKWWKWKW-NH2) with "WW" as the symmetry center and "WK" sequence repeated twice and symmetrically distributed on both sides, and to provide a class of antibacterial peptides derived from N2W2 as the structural basis.

[0005] The second purpose of the present application is to provide the application of the above-mentioned antibacterial peptide in the preparation of clinical antibacterial drugs.

[0006] To achieve its purpose, the present application adopts the following technical solutions:

[0007] One kind of low-toxic broad-spectrum antibacterial peptide with WW as the symmetry center

[0008] The low-toxic broad-spectrum antibacterial peptide with WW as the symmetry center provided by the application is obtained by repeating WK twice and symmetrically distributing on both sides, and taking WW as the symmetry center

[0009] WKWKWWKWKW-NH2 (N2W2), the amino acid sequence of which is shown in SEQ ID No. 1; then replacing the tryptophan at the C-terminal and N-terminal of N2W2 with leucine and / or isoleucine to obtain:

[0010] That is, the antibacterial peptide is LKWKWWKWKL-NH2, marked as LL-N2W2, the amino acid sequence of which is shown in SEQ ID No. 2;

[0011] Or: IKWKWWKWKL-NH2, marked as IL-N2W2, the amino acid sequence of which is shown in SEQ ID No. 3;

[0012] Or: IKWKWWKWKI-NH2, marked as II-N2W2, the amino acid sequence of which is shown in SEQ ID No. 4;

[0013] Or: LKWKWWKWKI-NH2, marked as LI-N2W2, the amino acid sequence of which is shown in SEQ ID No. 5.

[0014] As a further preferred technical solution of the application, the antibacterial peptide is IKWKWWKWKL-NH2, marked as IL-N2W2, the amino acid sequence of which is shown in SEQ ID No. 3.

[0015] Because IL-N2W2 has the highest antibacterial activity and low hemolytic activity, in order to improve the stability, further, all the amino acids in the sequence of IL-N2W2 are replaced with D-type amino acids to obtain a new antibacterial peptide, the structural formula of which is D-Ile-D-Lys-D-Trp-D-Lys-D-Trp-D-Trp-D-Lys-D-Trp-D-Lys-D-Leu-NH2, marked as D-IL-N2W2.

[0016] The above-mentioned newly designed antibacterial peptides are all prepared by using the classical solid-phase synthesis method.

[0017] Two, the application of a kind of low-toxic broad-spectrum antibacterial peptide with WW as the symmetry center in preparing clinical antibacterial drugs

[0018] 1. In vitro antibacterial experiment

[0019] To evaluate the in vitro antibacterial activity of the newly designed antibacterial peptides, the minimum inhibitory concentration of the peptides was tested by micro-broth dilution method. The peptides were prepared in a gradient concentration of 256 μM to 2 μM by two-fold dilution method, and the bacteria growing in logarithmic phase were diluted to 1 x 10 5 CFU / mL with MH broth, and equal volume of the peptide solution and the bacterial solution were added into a 96-well plate and incubated at 37°C for 18 h. The minimum concentration of the peptide without bacterial growth was observed by naked eyes, which was the minimum inhibitory concentration of the peptide to the bacteria. The experiment was repeated three times independently, with three parallel tests each time, and polymyxin B, ciprofloxacin and streptomycin were used as positive controls. The test results are shown in Table 1.

[0020] Table 1 Minimum inhibitory concentration (μM) of the antibacterial peptides of the present application to common standard strains

[0021]

[0022] The results in Table 1 show that the antibacterial peptides of the present application have good antibacterial effect on gram-positive bacteria represented by Staphylococcus aureus, Bacillus subtilis and Staphylococcus epidermidis, and on gram-negative bacteria represented by Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa, showing broad-spectrum antibacterial activity, in which the antibacterial activity of IL-N2W3 is the highest.

[0023] 2. Hemolytic activity experiment

[0024] To evaluate the toxicity of the newly designed antibacterial peptides of the present application to mammalian red blood cells, the antibacterial peptides were diluted twice with PBS and incubated with mouse red blood cells (8% v / v) at 37°C for 1 h, and the absorbance of the supernatant was detected. 1% Triton X-100 was used as a positive control, and PBS was used as a negative control. The hemolysis rate is shown in Figure 1 .

[0025] Figure 1 The results show that the parent peptide N2W2 causes more than 10% hemolysis at a concentration of 128 μM, while the hemolytic activity of the analogues with terminal amino acid substitution is significantly reduced and still less than 10% at 1024 μM, indicating that the antibacterial peptides provided by the present application have good biocompatibility.

[0026] 3. Enzymatic stability determination

[0027] To evaluate the stability of the antibacterial peptides of the present application, the antibacterial peptides with a final concentration of 2 x MIC were incubated with different concentrations of trypsin or chymotrypsin at 37°C for 1 h, and then the enzymes were inactivated at 60°C (15 min). Then the change in the antibacterial activity of the peptides to bacteria was detected, and the results are shown in Figure 2 .

[0028] Figure 2The results show that the inhibitory ability of N2W3 on bacteria is lost by more than 50% after incubation with 0.1 μg / mL chymotrypsin or trypsin for 1 h, and is completely lost when incubated with 1 μg / mL and above, indicating that N2W3 is very easy to be degraded by enzymes and has poor stability. The enzyme degradation stability results of LL-N2W3, IL-N2W3, II-N2W3, and LI-N2W3 obtained by terminal amino acid substitution are similar to those of N2W3, and all show poor stability to trypsin and chymotrypsin. D-IL-N2W3 obtained by D-type amino acid substitution of IL-N2W3 with the highest antibacterial activity can still completely inhibit the growth of bacteria after incubation with the highest test concentration of 1000 μg / mL enzyme, indicating that D-IL-N2W3 has excellent enzyme degradation stability.

[0029] The present application designs a peptide N2W2 (WKWKWWKWKW-NH2) with "WW" as the symmetry center and "WK" repeat sequence symmetrically distributed, and reduces its toxicity by terminal amino acid substitution. The four antibacterial peptides obtained by replacing the tryptophan at the C-terminal and N-terminal of N2W2 with leucine and / or isoleucine can basically maintain the excellent antibacterial activity of the parent peptide, and their hemolytic activity is greatly reduced, improving the safety. Among them, IL-N2W2 has the highest antibacterial activity, but its stability needs to be improved, so D-type amino acid substitution is performed on IL-N2W2 to obtain D-IL-N2W2. D-IL-N2W2 maintains the high antibacterial activity and low toxicity of IL-N2W2, and can still completely inhibit bacterial growth after incubation with high concentration of trypsin or chymotrypsin, and has great potential in alleviating antibiotic resistance and good application prospect in preparing clinical antibacterial drugs. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The results of the hemolytic activity of the antibacterial peptides of the present application on red blood cells after incubation with red blood cells for 1 h;

[0031] Figure 2 The antibacterial activity of the antibacterial peptides of the present application on E. coli after incubation with chymotrypsin or trypsin for 1 h;

[0032] Figure 3 The mass spectrum of the antibacterial peptide N2W2 of the present application;

[0033] Figure 4 The mass spectrum of the parent peptide LL-N2W2 of the present application;

[0034] Figure 5 The mass spectrum of the antibacterial peptide IL-N2W2 of the present application;

[0035] Figure 6 The mass spectrum of the antibacterial peptide II-N2W2 of the present application;

[0036] Figure 7 Mass spectrum of the antibacterial peptide LI-N2W2 of the present application;

[0037] Figure 8 Mass spectrum of the antibacterial peptide D-IL-N2W2 of the present application. DETAILED DESCRIPTION

[0038] The synthesis of the low-toxicity broad-spectrum antibacterial peptide of the present application with WW as the symmetric center is further described below by specific examples.

[0039] Example 1: Synthesis of the antibacterial peptide N2W2

[0040] (1) Activation and pretreatment of the resin

[0041] The MBHA resin was weighed and added to the polypeptide solid-phase synthesizer, swelled in DCM for 30 min, washed with DMF, and then identified by the ninhydrin coloration method. If it was colorless, the resin was qualified.

[0042] (2) Synthesis of Fmoc-N2W2-MBHA

[0043] The Fmoc protecting group was removed using a DMF solution containing 20% piperidine, and the resin was blue-violet, indicating successful deprotection to expose the amino group. After washing with DMF four times, 3 times the amount of Trp, 3 times the amount of HOBt, HBTU, and 6 times the amount of DIEA were dissolved in DMF and added to the synthesizer for stirring and condensation for 1 h. After the reaction time, the resin was washed with DMF three times, and the resin was colorless and transparent, indicating successful condensation, and Fmoc-Trp-MBHA was obtained.

[0044] According to the above method, Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, and Trp were sequentially condensed to obtain Fmoc-Trp-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-MBHA.

[0045] (3) Cleavage of the polypeptide

[0046] After the obtained Fmoc-Trp-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-MBHA was treated with a DMF solution containing 20% piperidine to remove the Fmoc protecting group, it was washed with DCM and methanol in sequence, and the resin was completely dried. 10 mL of cleavage reagent (TFA:Tris:water = 95:2.5:2.5 (v:v:v)) was added, and the reaction was carried out for 3 h. After extraction with ether, it was freeze-dried.

[0047] (4) Purification of the polypeptide

[0048] RP-HPLC purification conditions are mobile phase A: 0.1% TFA / acetonitrile, mobile phase B: 0.1% TFA / water, using linear gradient elution, collecting the target peak effluent, and lyophilizing to obtain the antibacterial peptide N2W2, the mass spectrum of which is shown in Figure 3

[0049] Example 2: Synthesis of antibacterial peptide LL-N2W2

[0050] (1) Activation and pretreatment of resin

[0051] The same as in Example 1.

[0052] (2) Synthesis of Fmoc-LL-N2W2-MBHA

[0053] The Fmoc protecting group was removed using a solution of DMF containing 20% piperidine, and the indenyl resin turned blue-violet, indicating successful deprotection to expose the amino group. After washing four times with DMF, 3 times the amount of Leu, 3 times the amount of HOBt, HBTU, and 6 times the amount of DIEA were dissolved in DMF and added to the synthesizer for stirring and condensation for 1 h. After the reaction time, the resin was washed three times with DMF, and the indenyl resin turned colorless and transparent, indicating successful condensation, to obtain Fmoc-Leu-MBHA.

[0054] Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, Leu were sequentially condensed according to the above method to obtain Fmoc-Leu-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Leu-MBHA.

[0055] (3) Peptide cleavage

[0056] The same as in Example 1.

[0057] (4) Peptide purification

[0058] The same as in Example 1, to obtain the antibacterial peptide LL-N2W2, the mass spectrum of which is shown in Figure 4

[0059] Example 3: Synthesis of antibacterial peptide IL-N2W2

[0060] (1) Activation and pretreatment of resin

[0061] The same as in Example 1.

[0062] (2) Synthesis of Fmoc-IL-N2W2-MBHA

[0063] The synthesis of Fmoc-Leu-MBHA was the same as in Example 2.

[0064] ​​Fmoc-Ile-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Leu-MBHA was obtained by sequentially condensing Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, Ile according to the above method.

[0065] (3) Peptide cleavage

[0066] The same as Example 1.

[0067] (4) Peptide purification

[0068] The same as Example 1, to obtain the antibacterial peptide II-N2W2, the mass spectrum thereof is shown in Figure 5 .

[0069] Example 4: Synthesis of antibacterial peptide II-N2W2

[0070] (1) Activation and pretreatment of resin

[0071] The same as Example 1.

[0072] (2) Synthesis of Fmoc-II-N2W2-MBHA

[0073] The Fmoc protecting group was removed with a DMF solution containing 20% piperidine, and the indenyl resin turned blue-violet, indicating successful deprotection and exposing the amino group. After washing with DMF four times, 3 times the amount of Ile, 3 times the amount of HOBt, HBTU, and 6 times the amount of DIEA were dissolved in DMF and added to the synthesizer for stirring and condensation for 1 h. After the reaction time, the resin was washed with DMF three times, and the indenyl resin turned colorless and transparent, indicating successful condensation, to obtain Fmoc-Ile-MBHA.

[0074] Fmoc-Ile-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Ile-MBHA was obtained by sequentially condensing Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, Ile according to the above method.

[0075] (3) Peptide cleavage

[0076] The same as Example 1.

[0077] (4) Peptide purification

[0078] The same as Example 1, to obtain the antibacterial peptide II-N2W2, the mass spectrum thereof is shown in Figure 6 .

[0079] Example 5: Synthesis of antibacterial peptide II-N2W2

[0080] (1) Activation and pretreatment of resin

[0081] The same as Example 1.

[0082] (2) Synthesis of Fmoc-LI-N2W2-MBHA

[0083] The same as Example 4.

[0084] The same as Example 1.

[0085] (3) Peptide cleavage

[0086] The same as Example 1.

[0087] (4) Peptide purification

[0088] The same as Example 1, to obtain the antibacterial peptide LI-N2W2, the mass spectrum thereof is shown in Figure 7 .

[0089] Example 6: Synthesis of antibacterial peptide D-IL-N2W2

[0090] (1) Activation and pretreatment of resin

[0091] The same as Example 1.

[0092] (2) Synthesis of Fmoc-D-IL-N2W2-MBHA

[0093] The Fmoc protecting group on the resin was removed with a DMF solution containing 20% piperidine, and the indenyl resin turned blue-violet, indicating that the deprotection was successful, and the amino group was exposed. The piperidine solution was removed by washing with DMF four times, and then 3 times the amount of D-Leu, 3 times the amount of HOBt, HBTU, and 6 times the amount of DIEA were dissolved in DMF and added to the synthesizer for stirring and condensation for 1 h. After the reaction was completed, the resin was washed with DMF three times, and the indenyl resin turned colorless and transparent, indicating that the condensation was successful, and Fmoc-D-Leu-MBHA was obtained.

[0094] According to the above method, D-Lys, D-Trp, D-Lys, D-Trp, D-Trp, D-Lys, D-Trp, D-Lys, D-Ile were sequentially condensed to obtain Fmoc-D-Ile-D-Lys-D-Trp-D-Lys-D-Trp-D-Trp-D-Lys-D-Trp-D-Lys-D-Leu-MBHA.

[0095] (3) Peptide cleavage

[0096] The same as Example 1.

[0097] (4) Polypeptide purification

[0098] As in Example 1, the antibacterial peptide D-IL-N2W2 was obtained, and its mass spectrum is shown in Figure 8 Figure 2.

Claims

1. A class of low-toxicity, broad-spectrum antimicrobial peptides with WW as the symmetry center, characterized in that, The antimicrobial peptide is obtained by repeating WK ​​twice and symmetrically distributing it on both sides, with WW as the center of symmetry. WKWKWWKWKW-NH2 (N2W2), whose amino acid sequence is shown in SEQ ID No.1; then, the C-terminus and N-terminus of N2W2 are replaced with leucine and / or isoleucine to obtain the following: That is, the antimicrobial peptide is LKWKWWKWKL-NH2, labeled as LL-N2W2, and its amino acid sequence is shown in SEQ ID No.2; Alternatively: IKWKWWKWKL-NH2, labeled as IL-N2W2, with its amino acid sequence shown in SEQ ID No. 3; Alternatively: IKWKWWKWKI-NH2, labeled II-N2W2, with its amino acid sequence shown in SEQ ID No. 4; Alternatively: LKWKWWKWKI-NH2, labeled LI-N2W2, with its amino acid sequence shown in SEQ ID No.

5.

2. The low-toxicity, broad-spectrum antimicrobial peptide with WW as the symmetry center as described in claim 1, characterized in that, The antimicrobial peptide is IKWKWWKWKL-NH2, labeled IL-N2W2, and its amino acid sequence is shown in SEQ ID No.

3.

3. The low-toxicity, broad-spectrum antimicrobial peptide with WW as the symmetry center as described in claim 2, characterized in that, The antimicrobial peptide is obtained by replacing all L-type amino acids in the IL-N2W2 sequence with D-type amino acids, and its structural formula is as follows: D I D K D W D K D W D W D K D W D K D L-NH2, labeled as D-IL-N2W2.

4. The application of a class of low-toxicity, broad-spectrum antimicrobial peptides with WW as the symmetry center as described in any one of claims 1-3 in the preparation of clinical antimicrobial drugs, characterized in that, The bacteria inhibited by the low-toxicity broad-spectrum antimicrobial peptide are either Gram-positive or Gram-negative bacteria. The Gram-positive bacteria are Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermidis, and the Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

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