Beta-hairpin antibacterial peptide designed aiming at trypsin restriction enzyme cutting site as well as preparation method and application of beta-hairpin antibacterial peptide

Abstract

The invention discloses a beta-hairpin antibacterial peptide WIFrPG designed for a trypsin restriction enzyme cutting site as well as a preparation method and application of the beta-hairpin antibacterial peptide WIFrPG. A sequence of the antibacterial peptide is shown as SEQ ID No.1, the antibacterial peptide contains a PG corner unit, a C terminal adopts amino amidation, and arginine in the sequence is D-type amino acid. On the basis of arrangement of beta-hairpin amphiphilic peptides and trypsin restriction enzyme cutting sites, D-type amino acid is added into an antibacterial peptide sequence of a beta-hairpin structure, and the obtained antibacterial peptide is named WIFrPG; the invention further discloses application of the antibacterial peptide in preparation of drugs for treating infectious diseases caused by gram-negative bacteria or / and gram-positive bacteria. The antibacterial peptide has extremely high antibacterial activity and extremely low hemolysis on various bacteria, the therapeutic index reaches 216.95, in addition, the antibacterial peptide has high stability under the conditions of trypsin and simulated intestinal juice, and it is indicated that the antibacterial peptide has high potential for replacing antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a β-hair antimicrobial peptide designed targeting the trypsin cleavage site, its preparation method, and its application. Background Technology

[0002] Due to the overuse of antibiotics, bacterial antimicrobial resistance (AMR) has become one of the major public health threats of the 21st century, making the prevention and treatment of drug-resistant bacterial infections even more critical. While antibiotics have historically been effective in treating most bacterial infections, the emergence of AMR has diminished the effectiveness of existing antibiotics. Therefore, finding effective alternatives is crucial. Antimicrobial peptides (AMPs), with their broad-spectrum antibacterial properties, are currently being used in the clinical treatment of pathogen infections, wound healing, and cancer. Furthermore, because AMPs can electrostatically adsorb onto bacterial membranes and, due to their hydrophobic nature, easily penetrate and disrupt membrane structures, leading to bacterial cell death, this unique membrane-disrupting bactericidal mechanism makes them extremely difficult to induce resistance. Therefore, they represent one of the most promising antibiotic alternatives for the future.

[0003] However, AMPs have encountered many obstacles in clinical applications. For example, their poor stability against various inhibitory factors present in vivo (physiological salts, serum, and proteases) leads to a reduction or even loss of their antimicrobial activity. The most critical limitation is the impact of proteases on the stability of AMPs, as they significantly reduce their bioavailability in vivo. In recent years, various strategies have been explored to address the poor protease resistance of AMPs and overcome this key limitation. Introducing D-amino acids into the peptide sequence is a common strategy. AMPs containing D-amino acids generally exhibit better protease stability and demonstrate antibacterial and hemolytic activities comparable to their parent peptides. While replacing L-amino acids in peptides with D-amino acids can effectively enhance protease stability, excessive substitution can also lead to uncertain side effects, such as excessively high hemolytic activity and toxicity. How to rationally use D-amino acids for substitution, improving enzyme stability in vivo while maintaining the original biological activity and enhancing biosafety, is a problem that needs to be solved. Summary of the Invention

[0004] To address the above shortcomings, this invention provides a β-hairpin antimicrobial peptide, WIFrPG, designed targeting the trypsin cleavage site, in order to solve the problem of poor stability of β-hairpin antimicrobial peptides against trypsin.

[0005] The technical solution adopted in this invention is as follows: a β-hairpin antimicrobial peptide WIFrPG designed for trypsin cleavage sites, using PG as the turning unit, utilizing the interaction between Trp and Ile to form a stable β-hairpin structure, and employing the D-type positively charged amino acid Arg to provide resistance to trypsin digestion while ensuring the number of positive charges, and its amino acid sequence is shown in SEQ ID No. 1.

[0006] The purpose of this invention is to provide a method for preparing the β-hairpin antimicrobial peptide WIFrPG, designed targeting the trypsin cleavage site as described above, as follows: The peptide chain contains a PG turn unit, and a phenylalanine residue is placed at both the N-terminus and C-terminus of the peptide chain to increase the hydrophobicity of the peptide. Based on the arrangement of the β-hairpin amphiphilic peptide and the trypsin cleavage site, the cross-chain interaction between tryptophan and isoleucine is used to assist the PG turn unit in forming a stable β-hairpin structure. Then, a positively charged amino acid, D-Arg, is added to prevent enzyme cleavage, resulting in the amino acid sequence of the peptide as shown in SEQ ID No. 1. The peptide is then synthesized using a solid-phase chemical synthesis method. The peptide is subjected to antibacterial activity testing, cytotoxicity testing, and hemolytic activity testing. The effects of protease and simulated intestinal fluid on antibacterial activity are then evaluated. Finally, the peptide is named the antimicrobial peptide WIFrPG.

[0007] Another object of the present invention is to provide the use of the β-hair antimicrobial peptide WIFrPG, designed as described above for targeting the trypsin cleavage site, in the preparation of medicaments for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

[0008] Furthermore, the Gram-negative bacteria include: Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium.

[0009] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis.

[0010] The beneficial effects and advantages of this invention are as follows: The antimicrobial peptide WIFrPG of this invention has a short sequence length and stable structure. After testing the synthesized antimicrobial peptide WIFrPG for antibacterial, hemolytic, enzymatic, and salt stability, it was found that WIFrPG exhibited excellent inhibitory effects against various strains of bacteria, including *Escherichia coli*, *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Pseudomonas aeruginosa*, *Salmonella typhimurium*, and *Enterococcus faecalis*. The geometric mean of the minimum inhibitory concentration (MIC) against ten bacteria (five Gram-positive and five Gram-negative) reached 4.30 μM, and it also exhibited very low hemolytic activity. Furthermore, WIFrPG maintained excellent stability even under high concentrations of protease. In conclusion, WIFrPG is an antimicrobial peptide with high application value. Attached Figure Description

[0011] Figure 1 The high-performance liquid chromatogram of the antimicrobial peptide WIFrPG;

[0012] Figure 2 This is the mass spectrum of the antimicrobial peptide WIFrPG. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1

[0015] Design of antimicrobial peptide WIFrPG

[0016] The hydrophobicity of the antimicrobial peptide was increased by placing a phenylalanine residue at both the N-terminus and C-terminus of the sequence. Based on the arrangement of the β-hair amphiphilic peptide, the structural force of the β-hair antimicrobial peptide formed by the cross-chain interaction between tryptophan and isoleucine was used to assist the formation of the PG turn unit. The addition of the D-type positively charged amino acid Arg ensured the antimicrobial performance. The β-hair antimicrobial peptide FWrIrPGrWrIF-NH2 was designed and named antimicrobial peptide WIFrPG. Its amino acid sequence is shown in Table 1.

[0017] Table 1. Amino acid sequence of the antimicrobial peptide WIFrPG

[0018]

[0019]

[0020] The antimicrobial peptide WIFrPG has a 12-amino acid sequence with a PG unit as the turn unit. The C-terminus of WIFrPG is amidated to increase a positive charge of +5. Antimicrobial peptides designed in this way possess a stable β-hairpin structure and exhibit excellent trypsin stability while demonstrating high antibacterial activity, low hemolytic activity, and high antimicrobial activity.

[0021] Example 2

[0022] Solid-phase chemical synthesis of antimicrobial peptide WIFrPG

[0023] 1. The preparation of AMPs proceeded sequentially from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (where X is the first amino acid at the C-terminus of each antimicrobial peptide) was inoculated into Wang resin, and then the Fmoc group was removed to obtain X-Wang resin. Next, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxylic acid-trimethyl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide) was added. This procedure was followed sequentially from the C-terminus to the N-terminus until the synthesis was complete, yielding a resin with the side chains protected by the removed Fmoc group.

[0024] 2. Add the cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter. Wash the precipitate with TFA (trifluoroacetic acid), mix the washings with the above filtrate, concentrate by rotary evaporator, add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, and a white powder will precipitate. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain the peptide. The cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5.

[0025] 3. Equilibrate the column for 30 min using 0.2 mol / L sodium sulfate (adjusted to pH 7.4 with phosphoric acid). Dissolve the peptide in 90% acetonitrile aqueous solution, filter, and elute using a C18 reversed-phase atmospheric pressure column with gradient elution (eluting agent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), at a flow rate of 1 mL / min and a detection wavelength of 220 nm. Collect the main peak and lyophilize it. Further purify using a reversed-phase C18 column with eluent A being 0.1% TFA / aqueous solution and eluent B being 0.1% TFA / acetonitrile solution, with an elution concentration of 25% B to 40% B, an elution time of 12 min, and a flow rate of 1 mL / min. Collect the main peak again and lyophilize it.

[0026] 4. Identification of antimicrobial peptides: The AMPs obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weight shown in the mass spectrum (e.g., Figure 2 The molecular weight (as shown) is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide WIFrPG is greater than 95% (e.g., Figure 1 (As shown).

[0027] Example 3

[0028] Assay of antimicrobial activity of antimicrobial peptide WIFrPG

[0029] 1. Determination of antimicrobial activity: The method used to determine the minimum inhibitory concentration (MIC) of the antimicrobial peptide was the broth microdilution method specified by the Clinical Laboratory and Standards Institute (CLSI). Bacteria were cultured overnight in MHB broth at 37°C and 220 rpm on a shaker. The next day, the culture was transferred to fresh MHB broth and incubated for another 4 hours on a shaker until the logarithmic growth phase. The culture was then diluted to OD0.05. 600 =0.4, dilute the bacterial solution 1000 times before use. Volume (50 μL) of bacterial suspension and solutions containing different concentrations of peptides (0.25 × 10⁻⁶) -6 –128×10 -6Bovine serum albumin (BSA, 0.2%; acetic acid, 0.01%) solution was added to 96-well plates. MHB medium containing bacteria was used as a positive control, and uninoculated MHB medium was used as a negative control. The 96-well plates were incubated at 37°C for 16–18 hours. The minimum inhibitory concentration (MIC) was determined visually and using an OD500 reader. 492 The minimum peptide concentration for bacterial growth could not be observed under the specified optical density. The concentration of antimicrobial peptides that did not show microbial growth under either visual or spectrophotometric methods represents the minimum inhibitory concentration (MIC) of the antimicrobial peptide. The test results are shown in Table 2.

[0030] Table 2. Antibacterial activity (μM) of the antimicrobial peptide WIFrPG

[0031]

[0032] As shown in Table 2, the antimicrobial peptide WIFrPG exhibits high antimicrobial activity against both Gram-positive and Gram-negative bacteria.

[0033] 2. Determination of hemolytic activity: 1 mL of blood was drawn from a healthy individual, centrifuged at 3000–3500 rpm and 4°C for 10 min, the supernatant was discarded, and the lower layer of red blood cells was collected. The cells were washed three times with sterile PBS solution (pH = 7.4), and then... -3 Red blood cells were resuspended in ELISA (pH 7.4, medium). Serially diluted antimicrobial peptides (50 μL) were mixed with an equal volume of red blood cell suspension in a 96-well plate, and incubated at 37°C for 1 hour. Red blood cell suspension treated with 0.1% Triton X-100 served as a positive control, while untreated red blood cell suspension served as a negative control. After centrifugation (3000–3500 rpm, 4°C) for 10 minutes, the supernatant was transferred to a new sterile 96-well plate, and the OD values ​​were observed and measured using a microplate reader. 570 The absorbance was measured. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = [(Measured value - Negative control value) / (Positive control value - Negative control value)] × 100%. The minimum hemolysis concentration was the concentration of the antimicrobial peptide that caused a 10% hemolysis rate. The test results are shown in Table 3.

[0034] Table 3. Determination of hemolytic activity of the antimicrobial peptide WIFrPG

[0035]

[0036]

[0037] As shown in Table 3, the antimicrobial peptide WIFrPG did not exhibit hemolytic activity within the detection range. Its therapeutic index was calculated using the ratio of the geometric mean of the minimum hemolytic concentration and the minimum inhibitory concentration, and the therapeutic index reached 216.95.

[0038] 3. To evaluate the effects of protease and simulated intestinal fluid on antibacterial activity, the antimicrobial peptide was co-incubated with trypsin solution (8 mg / mL) and simulated intestinal fluid for 4 hours, and the antibacterial activity was then measured. The results are shown in Table 4.

[0039] Table 4. Antimicrobial activity (μM) of the antimicrobial peptide WIFrPG against Escherichia coli 25922 and Staphylococcus aureus 29213 under trypsin and simulated intestinal fluid conditions.

[0040]

[0041] As shown in Table 4, the antimicrobial peptide WIFrPG maintained good antibacterial activity under conditions of 8 mg / mL trypsin and simulated intestinal fluid.

[0042] Based on all the above results, the β-hair antimicrobial peptide WIFrPG, designed targeting the trypsin cleavage site, has a high therapeutic index and extremely strong stability, indicating its strong potential as an alternative to antibiotics.

Claims

1. A β-hairpin antimicrobial peptide, WIFrPG, designed targeting the trypsin cleavage site, characterized in that, The amino acid sequence is shown in SEQ ID No. 1, containing a PG turn unit, with aminoamidation at the C-terminus, and all arginine in the sequence is a D-type amino acid.

2. The method for preparing the β-hairpin antimicrobial peptide WIFrPG designed targeting the trypsin cleavage site according to claim 1, characterized in that, The method is as follows: The peptide chain contains a PG turn unit, and a phenylalanine residue is placed at both the N-terminus and C-terminus of the peptide chain to increase the hydrophobicity of the peptide. Based on the arrangement of the β-hairpin amphiphilic peptide and the trypsin cleavage site, the cross-chain interaction between tryptophan and isoleucine is used to assist the PG turn unit in forming a stable β-hairpin structure. Then, a positively charged amino acid, D-Arg, is added to prevent enzyme cleavage, and the amino acid sequence of the peptide is shown in SEQ ID No.

1. The peptide is then synthesized using a solid-phase chemical synthesis method. The peptide is subjected to antibacterial activity, cytotoxicity, and hemolytic activity tests. The effects of protease and simulated intestinal fluid on antibacterial activity are then evaluated. Finally, it is named the antimicrobial peptide WIFrPG.

3. The use of the β-hair antimicrobial peptide WIFrPG, designed targeting the trypsin cleavage site according to claim 1, in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

4. The application according to claim 3, wherein the Gram-negative bacteria comprise: Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium.

5. The application according to claim 3, wherein the Gram-positive bacteria comprise: Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis.

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