An antibacterial peptide modified from signal peptide and application thereof

Abstract

The application discloses an antibacterial peptide reformed from a signal peptide and application thereof, and belongs to the field of biological medicines. The application renews and reforms the signal peptide by using an artificial intelligence model, and provides an antibacterial peptide Perceptide17 reformed from the signal peptide, wherein the sequence of the antibacterial peptide is VKKRLLFRKPLLKLFLLFGRRLKA. The antibacterial peptide has good bacteriostatic activity to gram-positive bacterial strains and gram-negative bacterial strains, and the minimum bacteriostatic concentration can reach 1 muM; the hemolytic activity is low, the half hemolysis concentration HC 50 is greater than 128 muM, the interaction with the inner membrane of bacteria and the human cell membrane shows selectivity, and the antibacterial peptide can be used for the development of antibacterial drugs and antibacterial products.

Description

TECHNICAL FIELD

[0001] The present application relates to an antibacterial peptide and its application, in particular to an antibacterial peptide modified from a signal peptide and its application in the preparation of a drug, and belongs to the field of biological medicine. BACKGROUND

[0002] Drug resistance caused by antibacterial drugs is threatening the drug treatment effect relied on by human beings. According to the statistics of the World Health Organization, about 1.3 million people die from this every year, and this number may reach 10 million in 2050. Commonly used small molecule antibiotics have the problem of causing pathogen resistance during use, and antibacterial peptides mainly play an antibacterial role by destroying the bacterial cell membrane mechanism, have a broad-spectrum inhibitory effect on pathogens such as bacteria, and are not easy to cause drug resistance. According to its characteristic of destroying the cell membrane, one strategy for designing antibacterial peptides is to modify the existing signal peptide sequence. The signal peptide itself has the characteristics of being located on the biological membrane and plays an important role in correctly guiding the processing and secretion of endogenous proteins in different organelles. For example, Porto et al. changed six residues of a signal peptide EcDBS1 in Escherichia coli by mode substitution, obtained an antibacterial peptide EcDBS1R6 that destroys the bacterial cell membrane, and under electron microscopy, it can be observed that it causes morphological changes of Pseudomonas aeruginosa and is accompanied by content leakage (BBA - General Subjects, 1864, 129633, 2020).

[0003] At the same time, since the mechanism of antibacterial peptides destroying the membrane may be broad-spectrum, the current antibacterial peptides face another problem of having high hemolytic activity. An ideal antibacterial peptide drug candidate should show species selectivity when it works, that is, only destroy the cell membranes of pathogens such as bacteria, and have no effect on human cells. The signal peptide itself has characteristics related to species, and according to the differences in the secretion pathway guided, some signal peptides only play a role in gram-positive or gram-negative bacteria, and do not affect eukaryotes. Therefore, the antibacterial peptide obtained by modifying the signal peptide has a great possibility of showing the characteristics of selectivity. SUMMARY

[0004] In order to obtain an antibacterial peptide with selectivity, the present application uses an artificial intelligence model independently developed by the present application to mutate and modify the signal peptide, and provides an antibacterial peptide Perceptide17 modified from the signal peptide, and the sequence thereof is VKKRLLFRKPLLKLFLLFGRRLKA (SEQ ID No: 1).

[0005] Perceptide 17 is derived from the known bacterial signal peptide MNKKRLLFRTPLDALFLLFGTALSA (SEQ ID No: 2). After the first M is truncated from the signal peptide, 7 mutations (N1V, T20R, T9K, D12L, S23K, A13K, A21R) are introduced by using artificial intelligence model, and Perceptide 17 is obtained, which has a length of 23 amino acids, a molecular weight of 2954.78, and 9 positive charges.

[0006] The antibacterial peptide Perceptide 17 can be synthesized by polypeptide solid-phase synthesis and other chemical methods, and applied in antibacterial drugs and other antibacterial products (such as disinfectants, detergents).

[0007] Through testing, the antibacterial peptide Perceptide 17 has good antibacterial activity on both gram-positive and gram-negative bacterial strains, including but not limited to Escherichia coli 、 Pseudomonas aeruginosa 、 Staphylococcus aureus 、 Bacillus subtilis, and the minimum inhibitory concentration can reach 1 μM; and the hemolytic activity is low, and the half hemolysis concentration HC 50 is greater than 128 μM, and the interaction with the bacterial inner membrane and the human cell membrane shows selectivity, which can be used for the development of antibacterial drugs and antibacterial products. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 The three-dimensional structure of the antibacterial peptide Perceptide 17 predicted by AlphaFold2 for Example 1.

[0009] Figure 2 The heavy atom interaction ratio of the antibacterial peptide Perceptide 17 simulated by molecular dynamics in Example 2 with the bacterial inner membrane and the human cell membrane.

[0010] Figure 3 The hemolytic activity determination results of the antibacterial peptide Perceptide 17 in Example 4. DETAILED DESCRIPTION

[0011] The present application will be further described below by examples in conjunction with the drawings. However, it should be understood that these descriptions are only to further illustrate the features and advantages of the present application, and are not a limitation on the claims of the present application.

[0012] Example 1 Source and properties of antibacterial peptide

[0013] The antibacterial peptide Perceptide 17 is evolved from the known bacterial signal peptide MNKKRLLFRTPLDALFLLFGTALSA based on an independently developed artificial intelligence model. First, the first M of the signal peptide is translated as the start codon and has no direct connection with function, so M is removed. Then the sequence is input into the artificial intelligence model, and Perceptide 17 is obtained by sequentially performing 7 mutations: VKKRLLFRKPLLKLFLLFGRRLKA, which has a length of 23 amino acids, a molecular weight of 2954.78, 9 positive charges, an isoelectric point of 12.61, an aromatic amino acid proportion of 0.13, and a hydrophobic amino acid proportion of 0.54. The three-dimensional structure of Perceptide 17 predicted using AlphaFold2 is shown in Figure 1 , in which the N-terminal part is a random coil, and the remaining part exhibits an α-helix secondary structure.

[0014] Example 2: Molecular dynamics simulation verifies the selectivity of antibacterial peptides

[0015] The complex interaction process between antibacterial peptides and cell membranes can be studied using molecular dynamics simulation. The antibacterial peptide structure used in the simulation is derived from AlphaFold2 prediction, and the simulation system is built using CHARMM-GUI. In the initial system, the polypeptide is placed about 2 nm above the parallel phospholipid bilayer membrane. Two systems, bacterial inner membrane and human cell membrane, are built for antibacterial peptide Perceptide 17, and the phospholipid composition is referred to Pandi et al. (Nature Communications, 14:7197, 2023). 150 mM NaCl is added to maintain the system as electrically neutral. The simulation process is run using GROMACS 2018, with a force field of CHARMM36m and a step size of 2 fs. The Nose-Hoover temperature coupling algorithm is used to maintain the temperature at 310 K, and the Parinello-Rahman pressure coupling algorithm is used to maintain the system pressure stable at 1.0 bar. The cutoff value of the Lennard-Jones potential is 1.2 nm, and the Ewald method is used for long-range electrostatic interactions. The NVT and NPT equilibrium processes use the default steps of CHARMM-GUI. Each system is run for 500 ns of simulation. The proportion of antibacterial peptide heavy atoms interacting with phospholipid heavy atoms (i.e., within 3.5 angstroms) is calculated for the last 100 ns of each trajectory, and the results are shown in Figure 2 , with three repeated simulations performed for each system. The interaction proportion of antibacterial peptide Perceptide 17 with the bacterial inner membrane is significantly higher than that with the human cell membrane, indicating that antibacterial peptide Perceptide 17 may exert antibacterial function by interacting with the bacterial inner membrane without affecting the human cell membrane, and has selectivity.

[0016] Example 3 Minimum inhibitory concentration determination of antibacterial peptide

[0017] The bacteria used in the minimum inhibitory concentration determination experiment include two gram-negative bacterial strains and two gram-positive bacterial strains, which are Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538, and Bacillus subtilis ATCC 6633, respectively. Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Bacillus subtilis The minimum inhibitory concentration of the antibacterial peptide was determined using the Hancock micro broth dilution method. Four standard strains were activated by overnight culture, diluted after about 3 hours of culture at 37°C in a shaking incubator at 220 rpm, so that the OD value was in the interval of 0.08-0.13. 625

[0018] The dry powder of antibacterial peptide Perceptide 17 was synthesized by Wuhan Dangang Biotechnology Co., Ltd. by solid-phase chemical synthesis method, and the relative molecular mass was correct by mass spectrometry detection, and the purity was more than 95%. The antibacterial peptide solution stock was prepared using sterile PBS solution (pH 7.4), and the concentration was 512 μM. In the 96-well plate, 100 μL of LB culture medium and 100 μL of antibacterial peptide solution were added, and the antibacterial peptide solution was diluted to a final concentration of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μM in a two-fold concentration gradient. Each 100 μL of diluted 100-fold bacterial solution was added, mixed well, and then cultured in a 37°C incubator for 16 hours. The lowest antibacterial peptide solution concentration value observed by naked eye without obvious bacterial growth was the minimum inhibitory concentration value of the antibacterial peptide under the condition. The test results are shown in Table 1, and the minimum inhibitory concentrations of Perceptide 17 for the four strains are 8, 32, 2, and 1 μM, respectively, showing strong antibacterial activity.

[0019] Table 1. Minimum inhibitory concentration of antibacterial peptide for four strains (unit: μM)

[0020]

[0021] Example 4 Hemolytic activity determination of antibacterial peptide

[0022] ​​​​Fresh rat blood was collected, mixed in PBS buffer (pH 7.4) and centrifuged at 500 g for 5 min to make 10% erythrocyte suspension. The antimicrobial peptides were prepared in a two-fold dilution series to a final concentration of 1-128 μM and added to 100 μL of the erythrocyte suspension. After incubation at 37 °C for 1 min, the samples were centrifuged at 2500 g for 6 min and 75 μL of the supernatant was added to each well of a 96-well plate. Triton X-100 at 1%, 0.1%, 0.001% was used as positive control and PBS as blank control. The absorbance of the supernatant at 450 nm was then measured with a microplate reader. All tests were performed in triplicate. The hemolysis rate of each well was calculated as (OD 450,多肽 - OD 450, PBS ) / (OD 450,阳性对照 - OD 450, PBS ), where OD 450,多肽 represents the absorbance at 450 nm of the well treated with the current concentration of antimicrobial peptide, OD 450, PBS represents the absorbance at 450 nm of the well treated with PBS and OD 450,阳性对照 represents the absorbance at 450 nm of the well treated with Triton X-100. The results of the hemolytic activity test of Perceptide 17 are shown in Table 1. Figure 3 The hemolytic concentration HC 50 50 of Perceptide 17 was greater than 128 μM, confirming that Perceptide 17 has good biological safety.

Claims

1. An antibacterial peptide, characterized in that, The amino acid sequence of the antibacterial peptide is as follows: VKKRLLFRKPLLKLFLLFGRRLKA.

2. Use of the antibacterial peptide of claim 1 in the manufacture of an antibacterial medicament against one or more of the following bacteria: Escherichia coli 、 Pseudomonas aeruginosa 、 Staphylococcus aureus 、 Bacillus subtilis.

3. Use according to claim 2, wherein the compound is ###0002### The effective concentration of the antibacterial peptide in the antibacterial drug is greater than or equal to 1 μM.

4. An antibacterial agent, characterized by, The antibacterial product comprises the antibacterial peptide of claim 1.

5. Use of the antibacterial peptide of claim 1 in the manufacture of an antibacterial product directed against one or more of the following bacteria: Escherichia coli 、 Pseudomonas aeruginosa 、 Staphylococcus aureus 、 Bacillus subtilis.

6. The use according to claim 5, wherein the compound is ###0002### The antibacterial product is a disinfectant or a detergent.

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