Low-toxicity broad-spectrum antibacterial peptide with WW as symmetric center and application thereof

Abstract

The invention discloses application of a low-toxicity broad-spectrum antibacterial peptide taking WW as a symmetric center, WK is repeated twice and symmetrically distributed on two sides, WW is taken as the symmetric center to obtain an antibacterial peptide WKWKWWKWKW-NH2 (N2W2), and tryptophan at the C terminal and the N terminal of the N2W2 is replaced with leucine and / or isoleucine to obtain the antibacterial peptide. The antibacterial peptide can basically maintain the excellent antibacterial activity of the parent peptide, and the hemolytic activity of the antibacterial peptide is greatly reduced, so that the safety is improved. Wherein the antibacterial activity of IL-N2W2 is the highest, but the stability of IL-N2W2 needs to be improved, so that D-IL-N2W2 is obtained by carrying out D-type amino acid substitution on IL-N2W2. The D-IL-N2W2 keeps the high antibacterial activity and low toxicity of IL-N2W2, can still completely inhibit the growth of bacteria after being incubated with high-concentration trypsin or chymotrypsin, has great potential in the aspect of relieving the drug resistance of antibiotics, and has good application prospects in preparation of clinical antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and relates to a class of low-toxic broad-spectrum antimicrobial peptides with KWWK as the symmetry center. The invention also relates to the application of the antimicrobial peptides in the preparation of clinical antimicrobial drugs. Background Art

[0002] Antibiotic resistance poses a serious threat to global public health, but few new antibiotics have been developed and approved by the U.S. Food and Drug Administration. It is estimated that the cost of developing a new antibiotic is about $1.5 billion, and it takes more than 10 years from basic research discovery to FDA approval. Antimicrobial peptides (AMPs) are a potential antibiotic because they are not prone to drug resistance due to their unique mechanism of action on bacterial membranes. However, AMPs have some inherent defects, such as the difficulty of combining high antimicrobial activity, low toxicity and high stability, as well as high cost. Therefore, there is an urgent need to develop new antimicrobial peptides with simple sequences and easy synthesis.

[0003] Hydrophilicity and hydrophobicity are key factors affecting the antibacterial activity and toxicity of cationic AMPs. The cation helps AMPs bind to the negatively charged regions of the bacterial membrane, and the hydrophobic part promotes the insertion of AMPs into the hydrophobic phospholipid layer of the bacterial membrane. The alternating arrangement of hydrophobic amino acids and cationic amino acids can promote AMPs to form an amphiphilic surface with one side hydrophilic and the other side hydrophobic, and "K" and "W" are used as a golden combination in the design of many AMPs. Some studies have shown that AMPs with repetitive sequences and symmetry have high antibacterial activity and low toxicity. Therefore, the applicant initially designed a peptide in which "WK" was repeated twice and symmetrically distributed on both sides. In order to improve the antibacterial activity of the peptide, tryptophan, which can interact strongly with the bacterial membrane, was repeated twice as the symmetry center of the peptide, thereby obtaining the antibacterial peptide WKWKWWKWKW-NH2 (N2W2). N2W2 has high-efficiency and broad-spectrum antibacterial activity, but its hemolytic activity is relatively high. Studies have shown that excessive hydrophobicity can lead to increased hemolytic activity, and the hydrophobicity of N2W2 is as high as 0.954, so appropriately reducing its hydrophobicity may improve its toxicity. Summary of the invention

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

[0005] The second object of the present invention is to provide the use of the above antimicrobial peptide in the preparation of clinical antimicrobial drugs.

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

[0007] 1. Structural design of a class of low-toxic broad-spectrum antimicrobial peptides with WW as the symmetry center

[0008] The present invention provides a class of low-toxic broad-spectrum antimicrobial peptides with WW as the symmetry center, which is obtained by repeating WK ​​twice and symmetrically distributing it on both sides, and taking WW as the symmetry center.

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

[0010] That is, the antimicrobial peptide is LKWKWWKWKL-NH2, labeled as LL-N2W2, and its amino acid sequence is shown in SEQ ID No. 2;

[0011] or: IKWKWWKWKL-NH2, labeled as IL-N2W2, whose amino acid sequence is shown in SEQ ID No.3;

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

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

[0014] As a further preferred embodiment of the technical solution of the present invention, the antimicrobial peptide is: IKWKWWKWKL-NH2, marked as IL-N2W2, and its amino acid sequence is shown in SEQ ID No.3.

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

[0016] The above newly designed antimicrobial peptides were all prepared by classical solid-phase synthesis.

[0017] II. Application of a class of low-toxic broad-spectrum antimicrobial peptides with WW as the symmetry center in the preparation of clinical antimicrobial drugs

[0018] 1. In vitro antibacterial experiment

[0019] In order to evaluate the in vitro antibacterial activity of the newly designed antimicrobial peptides, the minimum inhibitory concentration of the peptides was tested by the microbroth dilution method. The peptides were prepared into a gradient concentration of 256 μM to 2 μM using the two-fold dilution method. Bacteria grown to the logarithmic phase were diluted to 1×10 5 CFU / mL, equal volumes of peptide solution and bacterial solution were added to a 96-well plate and incubated at 37°C for 18 hours. The lowest peptide concentration at which no bacteria grew was observed by naked eye was the minimum inhibitory concentration of the peptide against the bacteria. This experiment used polymyxin B, ciprofloxacin and streptomycin as positive controls, and the test was repeated three times independently, with three parallels each time. The test results are shown in Table 1.

[0020] Table 1 Minimum inhibitory concentration (μM) of the antimicrobial peptides of the present invention against common standard strains

[0021]

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

[0023] 2. Hemolytic activity test

[0024] In order to evaluate the toxicity of the newly designed antimicrobial peptides of the present invention to mammalian erythrocytes, the antimicrobial peptides were diluted twice with PBS and then incubated with equal volumes of mouse erythrocytes (8% v / v) at 37°C for 1 h. The absorbance of the supernatant was detected, with 1% TritonX-100 as the positive control and PBS as the negative control. The hemolysis rate results are shown in Figure 1 .

[0025] Figure 1 The results showed that the parent peptide N2W2 caused more than 10% hemolysis at a concentration of 128 μM, while the hemolytic activity of the analogue after terminal amino acid replacement was significantly reduced, still below 10% at 1024 μM, indicating that the antimicrobial peptide provided by the present invention has good biocompatibility.

[0026] 3. Determination of enzymatic stability

[0027] In order to evaluate the stability of the antimicrobial peptides of the present invention, the antimicrobial peptides with a final concentration of 2×MIC were incubated with trypsin or chymotrypsin at different concentrations at 37°C for 1 h, and then the enzymes were inactivated at 60°C (15 min). Then, the changes in the antibacterial activity of the peptides against bacteria were detected. The results are shown in Figure 2 .

[0028] Figure 2The results showed that N2W3 lost more than 50% of its inhibitory ability against bacteria after incubation with 0.1μg / mL chymotrypsin or trypsin for 1h, and completely lost its antibacterial ability when incubated with enzymes at concentrations of 1μg / mL and above, indicating that N2W3 is very easily degraded by enzymes and has poor stability. The enzymatic stability results of LL-N2W3, IL-N2W3, II-N2W3, and LI-N2W3 obtained by terminal amino acid replacement were similar to those of N2W3, all of which showed poor stability to trypsin and chymotrypsin. However, D-IL-N2W3, obtained by replacing the D-amino acid of IL-N2W3 with the highest antibacterial activity, was still able to completely inhibit the growth of bacteria after incubation with the highest tested enzyme concentration of 1000μg / mL, indicating that D-IL-N2W3 has excellent enzymatic stability.

[0029] The present invention designs a peptide N2W2 (WKWKWWKWKW-NH2) with "WW" as the symmetry center and "WK" repeating sequence symmetrically distributed, and reduces its toxicity by replacing the terminal amino acids. The tryptophan at the C-terminus and N-terminus of N2W2 is replaced with leucine and / or isoleucine. The four antimicrobial peptides obtained can basically maintain the excellent antimicrobial activity of the parent peptide, and their hemolytic activity is greatly reduced, which improves safety. Among them, IL-N2W2 has the highest antimicrobial activity, but the stability needs to be improved. Therefore, IL-N2W2 is replaced with D-type amino acids to obtain D-IL-N2W2. D-IL-N2W2 maintains the high antimicrobial activity and low toxicity of IL-N2W2, and can still completely inhibit bacterial growth after incubation with high concentrations of trypsin or chymotrypsin. It has great potential in alleviating antibiotic resistance and has good application prospects in the preparation of clinical antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of the hemolytic activity of the antimicrobial peptide of the present invention on mouse erythrocytes after incubation with erythrocytes for 1 hour;

[0031] Figure 2 The antibacterial activity of the antimicrobial peptide of the present invention against Escherichia coli after incubation with chymotrypsin or trypsin for 1 hour;

[0032] Figure 3 is the mass spectrum of the antimicrobial peptide N2W2 of the present invention;

[0033] Figure 4 is the mass spectrum of the mother peptide LL-N2W2 of the present invention;

[0034] Figure 5 is the mass spectrum of the antimicrobial peptide IL-N2W2 of the present invention;

[0035] Figure 6 is the mass spectrum of the antimicrobial peptide II-N2W2 of the present invention;

[0036] Figure 7 is the mass spectrum of the antimicrobial peptide LI-N2W2 of the present invention;

[0037] Figure 8 It is the mass spectrum of the antimicrobial peptide D-IL-N2W2 of the present invention. DETAILED DESCRIPTION

[0038] The synthesis process of the low-toxic broad-spectrum antimicrobial peptide with WW as the symmetry center of the present invention is further described below through specific examples.

[0039] Example 1: Synthesis of antimicrobial peptide N2W2

[0040] (1) Activation and pretreatment of resin

[0041] Weigh MBHA resin and add it to the peptide solid phase synthesizer. Swell it with DCM for 30 minutes. After washing with DMF, identify the resin with ninhydrin colorimetric method. If it is colorless, it indicates that the resin is qualified.

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

[0043] The Fmoc protecting group was removed with a DMF solution containing 20% ​​piperidine. The indene resin was blue-purple, indicating successful deprotection and exposure of 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 hour. After the reaction time was reached, it was washed with DMF three times. The indene resin was colorless and transparent, indicating that the condensation was successful, and Fmoc-Trp-MBHA was obtained.

[0044] According to the above method, Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, Trp, Lys, Trp are condensed in sequence to obtain Fmoc-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-MBHA.

[0045] (3) Peptide cleavage

[0046] The obtained Fmoc-Trp-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-MBHA was removed from the Fmoc protecting group with a DMF solution containing 20% ​​piperidine, washed with DCM and methanol in sequence, and the resin was completely drained. 10 mL of a cleavage reagent (TFA:Tris:water=95:2.5:2.5 (v:v:v)) was added and reacted for 3 h, and then extracted with ether and freeze-dried.

[0047] (4) Peptide purification

[0048] The RP-HPLC purification conditions were as follows: mobile phase A: 0.1% TFA / acetonitrile, mobile phase B: 0.1% TFA / water, linear gradient elution was used, the target peak effluent was collected, and freeze-dried to obtain the antimicrobial peptide N2W2, whose mass spectrum is shown in FIG. Figure 3 shown.

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

[0050] (1) Activation and pretreatment of resin

[0051] Same as Example 1.

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

[0053] The Fmoc protecting group was removed with a DMF solution containing 20% ​​piperidine. The indene resin was blue-purple, indicating successful deprotection and exposure of the amino group. After washing with DMF four times, 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 hour. After the reaction time was reached, it was washed with DMF three times. The indene resin was colorless and transparent, indicating that the condensation was successful, and Fmoc-Leu-MBHA was obtained.

[0054] According to the above method, Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, and Leu are condensed in sequence to obtain Fmoc-Leu-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-Lys-Leu-MBHA.

[0055] (3) Peptide cleavage

[0056] Same as Example 1.

[0057] (4) Peptide purification

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

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

[0060] (1) Activation and pretreatment of resin

[0061] Same as Example 1.

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

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

[0064] According to the above method, Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, and Ile were condensed in sequence to obtain Fmoc-Ile-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-Lys-Leu-MBHA.

[0065] (3) Peptide cleavage

[0066] Same as Example 1.

[0067] (4) Peptide purification

[0068] The same method as in Example 1 was used to obtain the antimicrobial peptide IL-N2W2, the mass spectrum of which is shown in Figure 5 shown.

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

[0070] (1) Activation and pretreatment of resin

[0071] 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. The indene resin was blue-purple, indicating successful deprotection, 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 hour. After the reaction time was reached, it was washed with DMF three times. The indene resin was colorless and transparent, indicating that the condensation was successful, and Fmoc-Ile-MBHA was obtained.

[0074] According to the above method, Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, and Ile were condensed in sequence to obtain Fmoc-Ile-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-Lys-Ile-MBHA.

[0075] (3) Peptide cleavage

[0076] Same as Example 1.

[0077] (4) Peptide purification

[0078] The same method as in Example 1 was used to obtain antimicrobial peptide II-N2W2, the mass spectrum of which is shown in Figure 6 shown.

[0079] Example 5: Synthesis of antimicrobial peptide LI-N2W2

[0080] (1) Activation and pretreatment of resin

[0081] Same as Example 1.

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

[0083] The synthesis of Fmoc-Ile-MBHA is the same as in Example 4.

[0084] According to the above method, Lys, Trp, Lys, Trp, Trp, Lys, Trp, Lys, and Leu are condensed in sequence to obtain Fmoc-Leu-Lys-Trp-Lys-Trp-Trp-Lys-Trp-Lys-Trp-Lys-Ile-MBHA.

[0085] (3) Peptide cleavage

[0086] Same as Example 1.

[0087] (4) Peptide purification

[0088] The same method as in Example 1 was used to obtain the antimicrobial peptide LI-N2W2, the mass spectrum of which is shown in Figure 7 shown.

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

[0090] (1) Activation and pretreatment of resin

[0091] 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. The indene resin was blue-purple, 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 hour. After the reaction was completed, it was washed with DMF three times. The indene resin was 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, and D-Ile are condensed in sequence to obtain Fmoc-D-Ile-D-Lys-D-Trp-D-Lys-D-Trp-D-Trp-D-Trp-D-Lys-D-Trp-D-Lys-D-Leu-MBHA.

[0095] (3) Peptide cleavage

[0096] Same as Example 1.

[0097] (4) Peptide purification

[0098] The same method as in Example 1 was used to obtain the antimicrobial peptide D-IL-N2W2, the mass spectrum of which is shown in Figure 8 shown.

Claims

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

5.

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

3.

3. A low-toxic broad-spectrum antimicrobial peptide with WW as the symmetry center as claimed 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 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. Use of a low-toxic broad-spectrum antimicrobial peptide with WW as the symmetry center as described in any one of claims 1 to 3 in the preparation of clinical antimicrobial drugs.

5. The use of a low-toxic broad-spectrum antimicrobial peptide with WW as the symmetry center in the preparation of clinical antimicrobial drugs as claimed in claim 4, characterized in that: The bacteria inhibited by the low-toxic broad-spectrum antimicrobial peptide are Gram-positive bacteria or Gram-negative bacteria.

6. The use of a low-toxic broad-spectrum antimicrobial peptide with WW as the symmetry center in the preparation of clinical antimicrobial drugs as claimed in claim 5, characterized in that: 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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