CN116063391B - Tryptophan and arginine enriched antibacterial peptide WRT6 with high cell selectivity and preparation method and application thereof - Google Patents
Tryptophan and arginine enriched antibacterial peptide WRT6 with high cell selectivity and preparation method and application thereof Download PDFInfo
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- 239000004475 Arginine Substances 0.000 title claims abstract description 30
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- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 4
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- KQIADDMXRMTWHZ-UHFFFAOYSA-N chloro-tri(propan-2-yl)silane Chemical compound CC(C)[Si](Cl)(C(C)C)C(C)C KQIADDMXRMTWHZ-UHFFFAOYSA-N 0.000 description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a tryptophan and arginine enriched antibacterial peptide WRT6 with high cell selectivity and a preparation method and application thereof, and belongs to the technical field of biology. The amino acid sequence of the antibacterial peptide WRT6 is shown as SEQ ID No. 1. The preparation method comprises the following steps: the antibacterial peptide WRT6 with high cell selectivity is designed and obtained according to the structure-activity relationship of the antibacterial peptide by introducing hydrophilic uncharged threonine and combining hydrophobic tryptophan and hydrophilic charged arginine, and then is synthesized by a solid phase synthesis method. The application of the antibacterial peptide WRT6 in preparing medicines for treating infectious diseases caused by gram-negative bacteria or gram-positive bacteria. The antibacterial peptide WRT6 has strong antibacterial activity on various bacteria and low cytotoxicity, so that the cell selectivity index is as high as 61.05, and the application potential is extremely high.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a tryptophan and arginine enriched antibacterial peptide WRT6 with high cell selectivity, and a preparation method and application thereof.
Background
Antibiotics play an important role in the fields of medical treatment, food, livestock and the like, but the problems of pathogenic microorganism drug resistance, environmental pollution and the like caused by long-term large-scale and high-dose abuse of antibiotics severely limit the development prospect of the antibiotics, even seriously crisis the health of human beings and animals, and become a global public problem. Therefore, the development of green, safe and efficient antibiotic substitution products is urgent. The antibacterial peptide is taken as an important component of a host immune system, has various biological functions such as antibacterial, antifungal, immunoregulation and the like, and is different from the traditional antibiotics which mainly act on intracellular targets, most antibacterial peptides kill bacteria through a multiple action mechanism mainly comprising membrane cleavage, and the multiple action mechanism is not easy to cause the bacteria to generate drug resistance, so that the antibacterial peptide has great potential for replacing antibiotics.
In recent years, extremely simplified and completely new design strategies for antimicrobial peptides have been proposed and have attracted considerable attention. In this design strategy, the combination of the hydrophobic amino acid tryptophan and the hydrophilic charged amino acid arginine is known as the most classical and effective "golden partner". Because the unique indole side chain of tryptophan compared to other hydrophobic amino acids is believed to promote interfacial association between the antimicrobial peptide and the lipid bilayer and mediate cohesive hydrophobic interactions with the lipid acyl chains during membrane rupture. While the guanidino side chain of arginine has a stronger electrostatic attraction to cell membranes bearing negatively charged components than other hydrophilic charged amino acids. While this combination may confer potent antimicrobial activity on the antimicrobial peptide, it also results in severe hemolysis and cytotoxicity, which results in loss of cell selectivity between bacterial cells and mammalian cells, which greatly limits its potential for use. It is therefore a challenge to overcome the problem of reducing cytotoxicity without reducing antibacterial activity.
Disclosure of Invention
Based on the defects, the invention aims to provide the antibacterial peptide WRT6 with high cell selectivity and enriched tryptophan and arginine, and solve the problem of low cell selectivity of the antibacterial peptide enriched tryptophan and arginine.
The technical scheme adopted by the invention is as follows: a tryptophan and arginine enriched antibacterial peptide WRT6 with high cell selectivity has an amino acid sequence shown in SEQ NO.1, and an amino amidation at the C-terminal, and has a molecular formula shown in formula (I):
another object of the present invention is to provide a method for preparing tryptophan and arginine enriched antibacterial peptide WRT6 with high cell selectivity as described above, comprising the steps of:
(1) Tryptophan with unique indole side chains is selected as a hydrophobic amino acid to provide the necessary hydrophobicity while enhancing the interaction between the polypeptide and the lipid bilayer;
(2) Arginine having a guanidino side chain is selected as a cationic amino acid to provide the requisite net positive charge while enhancing electrostatic interaction between the polypeptide and the negative membrane surface charge component;
(3) Threonine is placed at the C-terminal side of tryptophan and arginine, and the cytotoxicity of the tryptophan and arginine enriched antibacterial peptide is reduced and the cell selectivity of the tryptophan and arginine enriched antibacterial peptide is improved by regulating the balance between hydrophilicity and hydrophobicity;
(4) The template for designing the polypeptide sequence is as follows: (WRX) n -NH 2 When X=T and n=6, the amino acid sequence is shown as SEQ NO. 1;
(5) The preparation of the antibacterial peptide adopts a solid-phase chemical synthesis method, a polypeptide synthesizer is used for synthesizing polypeptide resin, then trifluoroacetic acid is used for cutting, reverse-phase high performance liquid chromatography is used for purifying, then the obtained polypeptide is subjected to mass spectrum identification, the polypeptide is obtained, and finally the polypeptide is named as antibacterial peptide WRT6 through antibacterial activity measurement and cytotoxicity measurement.
It is another object of the present invention to provide the use of a highly cell-selective tryptophan and arginine-enriched antibacterial peptide WRT6 as described above for the preparation of a medicament for the treatment of infectious diseases caused by gram-negative or gram-positive bacteria.
Further, the gram negative bacteria are escherichia coli, pseudomonas aeruginosa, salmonella typhimurium or salmonella choleraesuis.
Further, the gram positive bacteria is staphylococcus aureus or staphylococcus epidermidis.
The invention has the following advantages and beneficial effects: the invention can improve the cell selectivity of the tryptophan and arginine enriched antibacterial peptide by introducing threonine without complex chemical modification or unnatural amino acid substitution; the prepared antibacterial peptide WRT6 is subjected to antibacterial activity and cytotoxicity measurement, and the WRT6 is found to have strong inhibition effect on various bacteria such as escherichia coli, pseudomonas aeruginosa, salmonella typhimurium, salmonella choleraesuis, staphylococcus aureus, staphylococcus epidermidis and the like, almost has no toxicity on erythrocytes, macrophages, renal epithelial cells and intestinal epithelial cells, has higher application value, has a selectivity index as high as 61.05 and has great application potential.
Drawings
FIG. 1 is a reverse phase high performance liquid chromatogram of antibacterial peptide WRT6.
FIG. 2 is a mass spectrum of the antibacterial peptide WRT6.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
Design of antibacterial peptides
Tryptophan with unique indole side chains is selected as a hydrophobic amino acid to provide the necessary hydrophobicity while enhancing the interaction between the antimicrobial peptide and the lipid bilayer; arginine with a guanidino side chain is selected as a cationic amino acid to provide the requisite net positive charge while enhancing the electrostatic interaction between the antimicrobial peptide and the negative membrane surface charge component; threonine is placed on the C-terminal side of tryptophan and arginine, and the cytotoxicity of the tryptophan and arginine-enriched antimicrobial peptide is reduced by adjusting the balance between hydrophilicity and hydrophobicity, so that the cell selectivity of the tryptophan and arginine-enriched antimicrobial peptide is improved. The antibacterial peptide sequence template with high cell selectivity designed based on the technical scheme is as follows: (WRX) n -NH 2 When x=t, n=6, the antibacterial peptide is named WRT6.As a control antimicrobial peptide, i.e. (WR) when threonine is removed from the sequence, the sequence contains only tryptophan and arginine n -NH 2 When n=6, the control antibacterial peptide is named WR6.
The amino acid sequence of the antibacterial peptide WRT6 is as follows:
the amino acid sequence of the antibacterial peptide WR6 is as follows:
TABLE 1 amino acid sequence of peptides
Example 2
Synthesis of antibacterial peptide by solid phase chemical synthesis method
1. The synthesis of the antibacterial peptide is carried out one by one from the C end to the N end by a polypeptide synthesizer. Fmoc-X (X is the first amino acid at the C-terminal of each antibacterial peptide) is firstly accessed into Wang resin, and then Fmoc groups are removed to obtain X-Wang resin; fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y being the second amino acid at the C-terminus of each antimicrobial peptide); sequentially synthesizing from the C end to the N end according to the procedure until the synthesis is completed, and obtaining the side chain protected resin from which Fmoc groups are removed;
2. adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; washing the precipitated trifluoroacetic acid, mixing the washing liquid with the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous diethyl ether with volume about 10 times, precipitating for 3 hours at-20 ℃, separating out white powder, centrifuging for 10min at 2500g, collecting the precipitate, washing the precipitate by using anhydrous diethyl ether, and vacuum drying to obtain polypeptide, wherein the cutting reagent is formed by mixing trifluoroacetic acid, water and triisopropylchlorosilane according to a mass ratio of 95:2.5:2.5;
3. performing column balancing with 0.2mol/L sodium sulfate (pH=7.4), dissolving polypeptide with 90% acetonitrile water solution, filtering, performing C18 reverse phase normal pressure column, gradient eluting (eluting agent is methanol and sodium sulfate water solution mixed according to volume ratio of 30:70-70:30) at flow rate of 1mL/min, detecting wave of 220nm, collecting main peak, and lyophilizing; further purifying with reversed phase C18 column, wherein eluent A is 0.1% trifluoroacetic acid/water solution; the eluent B is 0.1 percent trifluoroacetic acid/acetonitrile solution, the elution concentration is 25 percent B-40 percent B, the elution time is 12min, the flow rate is 1mL/min, and then the main peak is collected and freeze-dried;
4. identification of antibacterial peptides: the antibacterial peptide obtained above was analyzed by electrospray mass spectrometry, and the molecular weight shown in the mass spectrum (shown in fig. 1) was substantially identical to the theoretical molecular weight shown in table 1, and the purity of the antibacterial peptide was greater than 95% (shown in fig. 2).
Example 3
Determination of antibacterial Activity of antibacterial peptides
1. Determination of bacteriostatic Activity: the Minimum Inhibitory Concentration (MIC) of the peptides was determined using standard micro broth dilution. Diluting log phase bacteria to-2×10 5 CFU/mL. Mu.l of peptides of different concentrations (final concentration of peptide 1-128. Mu.M) and equal volumes of bacterial suspension were added to each well of a 96-well plate, while negative controls (medium only) and positive controls (bacteria and medium) were set, and then the 96-well plate was placed in a constant temperature incubator at 37℃for 18-20 hours. With a microplate reader at 492nm (OD 492 ) And (5) measuring absorbance value, and determining the minimum inhibitory concentration. Three independent replicates were performed, two replicates each. The results are shown in Table 2.
TABLE 2 minimum inhibitory concentration (μM) of antibacterial peptides
As can be seen from Table 2, WRT6 and WR6 show strong antibacterial activity against a variety of bacteria such as E.coli, pseudomonas aeruginosa, salmonella typhimurium, salmonella choleraesuis, staphylococcus aureus, staphylococcus epidermidis, etc.
Example 4
Determination of cytotoxicity of antibacterial peptides
1. Red blood cells: fresh human red blood cell suspensions were collected and diluted 10-fold with PBS (ph=7.4). Mu.l of peptides of different concentrations (final concentration of peptide 1-128. Mu.M) and equal volumes of red blood cell suspension were placed in each well of a 96-well plate, human red blood cell suspension treated with 0.1% Triton X-100 was used as positive control, untreated human red blood cell suspension was used as negative control, and the 96-well plate was placed in a 37℃incubator for 1 hour. After centrifugation (1000 g,5min,4 ℃) 50. Mu.l of supernatant were collected from the mixture and transferred to a new 96-well plate and dried with an enzyme-labeled instrument at 570nm (OD 570 ) The absorbance value was measured. The haemolysis 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％
1. Macrophages, kidney epithelial cells, intestinal epithelial cells: diluting cells to-2X 10 4 Cells/well, 50. Mu.L of diluted cells were added to 1-11 columns of 96-well plates and mixed with 5% CO 2 The culture was continued in a 37℃cell incubator until the cells were grown fully adherent. In the new 96-well plate, the polypeptide concentrations in the 1-10 columns of wells were diluted to 512-1. Mu.M, respectively, in a volume of 50. Mu.L. 50. Mu.L of the corresponding cell culture medium was added to column 11, and 100. Mu.L of the corresponding cell culture medium was added to column 12. After the cells are grown completely, transferring the solution in each well of the new 96-well plate to the corresponding well of the 96-well plate containing the cells, and adding 5% CO 2 Is cultured in a 37℃cell incubator for 4 hours. Next, 25. Mu.L of MTT (0.5 mg/mL) was added to each well and the culture was continued for 3 hours. Finally, the supernatant was discarded and 150 μl DMSO was added to each well to dissolve formazan crystals. Columns 11 and 12 are positive and negative controls, respectively. Absorbance was measured at 570nm using a microplate reader. Two replicates per trial were run and three independent replicates were run. The cell viability was calculated as follows:
cell viability (%) = [ (sample OD) 570 Negative-negativeControl OD 570 ) /(positive control OD) 570 Negative control OD 570 )]×100％
The cytotoxicity test results of the antibacterial peptide on erythrocytes, macrophages, renal epithelial cells and intestinal epithelial cells are shown in Table 3.
TABLE 3 cytotoxicity and cell selectivity index of antibacterial peptides
Note that: a GM MIC is the geometric mean of the minimum inhibitory concentration. b HC 10 Is the minimum concentration of antimicrobial peptide that results in greater than or equal to 10% of red blood cell disruption. c IC 10 Is the minimum concentration of antimicrobial peptides that results in ≡10% cell death. d The formula for calculating the cell selectivity index is: HC (HC) 10 Or IC (integrated circuit) 10 With GM (GM) MIC When HC is the ratio of 10 Or IC (integrated circuit) 10 Above 256. Mu.M, the value of 512. Mu.M is taken for calculation. e GM SI GM is the geometric mean of the cell selectivity index SI The larger the cell, the higher its cell selectivity.
Taken together, the cell selectivity index of the antibacterial peptide WRT6 was as high as 61.05, whereas the cell selectivity index of the antibacterial peptide WR6 was only 1.27. The antibacterial peptide WRT6 has an approximately 48-fold improvement in cell selectivity index over the antibacterial peptide WR6. This suggests that the introduction of threonine significantly increases the cell selectivity of tryptophan and arginine-enriched antimicrobial peptides. The antibacterial peptide WRT6 has great application potential because of extremely high cell selectivity index.
Claims (4)
1. The high cell selectivity tryptophan and arginine enriched antibacterial peptide WRT6 is characterized in that the amino acid sequence is shown as SEQ NO.1, the C-terminal of the peptide is amidated by amino, and the molecular formula is shown as formula (I):
2. the method for preparing the high cell selectivity tryptophan and arginine enriched antibacterial peptide WRT6 according to claim 1, wherein the method comprises the following steps:
(1) Tryptophan with unique indole side chains is selected as a hydrophobic amino acid to provide the necessary hydrophobicity while enhancing the interaction between the polypeptide and the lipid bilayer;
(2) Arginine having a guanidino side chain is selected as a cationic amino acid to provide the requisite net positive charge while enhancing electrostatic interaction between the polypeptide and the negative membrane surface charge component;
(3) Threonine is placed at the C-terminal side of tryptophan and arginine, and the cytotoxicity of the tryptophan and arginine enriched antibacterial peptide is reduced and the cell selectivity of the tryptophan and arginine enriched antibacterial peptide is improved by regulating the balance between hydrophilicity and hydrophobicity;
(4) The template for designing the polypeptide sequence is as follows: (WRX) n -NH 2 When X=T and n=6, the amino acid sequence is shown as SEQ NO. 1;
(5) The preparation of the antibacterial peptide adopts a solid-phase chemical synthesis method, a polypeptide synthesizer is used for synthesizing polypeptide resin, then trifluoroacetic acid is used for cutting, reverse-phase high performance liquid chromatography is used for purification, then mass spectrum identification is carried out on the obtained polypeptide, the polypeptide is obtained, antibacterial activity and cytotoxicity are measured, and finally the polypeptide is named as antibacterial peptide WRT6.
3. Use of a highly cell-selective tryptophan and arginine-enriched antimicrobial peptide WRT6 according to claim 1, for the preparation of a medicament for the treatment of infectious diseases caused by gram-negative or gram-positive bacteria, which are staphylococcus aureus or staphylococcus epidermidis.
4. The use according to claim 3, wherein the gram-negative bacteria are escherichia coli, pseudomonas aeruginosa, salmonella typhimurium or salmonella choleraesuis.
Priority Applications (1)
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| JP2005247721A (en) * | 2004-03-02 | 2005-09-15 | Pacgen Biopharmaceuticals Inc | Antimicrobial peptide having reduced hemolysis, and method for using the same |
| WO2013100721A1 (en) * | 2011-12-30 | 2013-07-04 | 조선대학교산학협력단 | Novel antibacterial and fungicidal peptide in which lysine and tryptophan residues are repeated four times, and use thereof |
| CN111533786A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Beta-hairpin antibacterial peptide with tryptophan and arginine cross-chain interaction and preparation method thereof |
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| JP2005247721A (en) * | 2004-03-02 | 2005-09-15 | Pacgen Biopharmaceuticals Inc | Antimicrobial peptide having reduced hemolysis, and method for using the same |
| WO2013100721A1 (en) * | 2011-12-30 | 2013-07-04 | 조선대학교산학협력단 | Novel antibacterial and fungicidal peptide in which lysine and tryptophan residues are repeated four times, and use thereof |
| CN111533786A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Beta-hairpin antibacterial peptide with tryptophan and arginine cross-chain interaction and preparation method thereof |
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