High stability broad-spectrum antibacterial peptide and application thereof in prevention and treatment of drug-resistant bacteria

Abstract

The application belongs to the field of polypeptide drugs, and particularly relates to a high-stability broad-spectrum antibacterial peptide and application thereof in drug-resistant bacteria prevention and treatment. The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 2-10. The antibacterial effect of the antibacterial peptide is better than that of Ursoricin, and the stability is further enhanced.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drugs, specifically relating to a highly stable broad-spectrum antimicrobial peptide and its application in the prevention and control of drug-resistant bacteria. Background Technology

[0002] Antimicrobial peptides, as a class of small positively charged peptides that naturally exist in organisms, play an important role in defending against bacterial, fungal, and viral infections. Due to their diverse mechanisms of action and low drug resistance, they have become important candidates for antibiotic alternatives.

[0003] Ursoricin is a naturally derived, leaderless antimicrobial peptide with good antimicrobial activity against G. + Bacterial spectrum and certain G - It has strong inhibition capabilities, with compact sequences and high spatial folding efficiency, but the original sequence still has the following shortcomings:

[0004] 1. Contains multiple Trypsin / Chymotrypsin-sensitive sites, resulting in low enzyme stability;

[0005] 2. The lack of a circular structure design is detrimental to the stability of expression and the integration of the delivery system.

[0006] Therefore, we optimized Ursoricin at the molecular level by modifying / reducing enzyme recognition sites while retaining its original structure and function, making it more resistant to enzymatic digestion, easier to engineer, and more structurally stable. Summary of the Invention

[0007] This invention provides a highly stable broad-spectrum antimicrobial peptide or its pharmaceutically acceptable salt. The antimicrobial peptide is structurally optimized from a natural antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO: 1. The natural antimicrobial peptide has the following structural functional regions: an N-terminal hydrophobic anchoring region MWGRILAFVA, a mid-segment polarity regulation region KYGTKAVQWA, an attack effect region WKNKWFLLSL, and a C-terminal stabilizing transition region GEAVFDYIRSIWGG.

[0008] (Forms folded surfaces / loops and modulates variability), (Densely charged bactericidal regions), (Provides overall peptide stability and folding and structural closure functions)

[0009] Preferably, the N-terminal hydrophobic anchoring region retains W, I, L and V, enabling it to rapidly bind to the bacterial membrane and exert a penetrating or membrane-breaking effect.

[0010] Preferably, the N-end hydrophobic anchoring region further retains F.

[0011] Preferably, the mid-section polarity adjustment region balances flexibility and charge density and improves structural stability through one or more reconfigurable polarity control regions of K, W, Y, T and Q.

[0012] Preferably, the attack effect region is enriched with K and / or W to form a positively charged attack surface, which has a killing effect on both Gram-positive and Gram-negative bacteria.

[0013] Preferably, the C-terminal stable transition region constructs a stable spatial folding structure, enhancing the overall conformational closure and resistance to enzymatic degradation.

[0014] Preferably, the spatial folding structure is GEVKIWG.

[0015] Preferably, the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 2-10.

[0016] In a preferred embodiment of the present invention, the present invention also provides a polynucleotide encoding the highly stable broad-spectrum antimicrobial peptide described herein.

[0017] In a preferred embodiment of the present invention, an expression vector containing the polynucleotides described herein is also provided.

[0018] In another preferred embodiment of the present invention, a host cell is provided containing the polynucleotide or expression vector described in the present invention.

[0019] Preferably, the host cell is a lactic acid bacterium or an Escherichia coli.

[0020] In another preferred embodiment of the present invention, a pharmaceutical composition is provided comprising the highly stable broad-spectrum antimicrobial peptide, polynucleotide, expression vector or host cell described herein, and a pharmaceutically acceptable vector.

[0021] In another preferred embodiment of the present invention, the present invention provides the use of the highly stable broad-spectrum antimicrobial peptide and / or the pharmaceutical composition of the present invention in the preparation of antimicrobial agents.

[0022] Preferably, the antibacterial agent is widely applicable to medical anti-infection, skin repair, daily chemical preservation, and food antibacterial applications.

[0023] This application optimizes the structure of wild-type antimicrobial peptides. The mid-section polarity regulation region is optimized to achieve folding / loop variability regulation. The attack effect region is optimized to construct a positively charged, densely packed bactericidal region. The C-terminal stabilizing transition region is optimized to improve the overall stability of the peptide and provide folding and structural closure functions. The structure-optimized antimicrobial peptide C0-C8 has better antibacterial effect than Ursoricin, and its stability is further enhanced.

[0024] The following will further explain the concept, specific structure, and technical effects of the present invention in order to fully understand the purpose, features, and effects of the present invention. Detailed Implementation

[0025] The present invention will be described in detail below with reference to specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0026] Example 1: Determination of antibacterial spectrum and study of minimum inhibitory concentration (MIC)

[0027] The antimicrobial spectrum was determined using the soft agar overlay method. Antimicrobial peptides were spotted onto tryptone soybean agar (TSA) plates and incubated at 37°C and 5% CO2 for 20 hours. Subsequently, plates containing each indicator strain (10...) were... 7 3.5 mL of preheated half-strength TSB soft agar (0.75%) was poured onto TSA plates and incubated at 37°C for 20 hours. The diameter of the growth inhibition zone was measured in three directions. The minimum inhibitory concentration (MIC) was determined using purified bacteriocin according to the modified method of the CLSI 2021 guideline. MIC was determined in 96-well microplates using serial 2-fold dilutions of the antimicrobial peptide preparation (100 μL TSB medium per well). Each bacterial culture (10 cells / mL) was then used as a test. 5 A total of 100 bacteria were inoculated into 100 μL TSB medium containing different concentrations of purified antimicrobial peptides. After 24 hours of incubation, the minimum inhibitory concentration (MIC) was determined. The MIC was defined as the lowest concentration that inhibits bacterial growth. This experiment was repeated three times.

[0028] The antibacterial spectrum results are shown in Table 1, indicating that Urosoricn has effective bactericidal activity against most Gram-positive bacteria, especially drug-resistant Staphylococcus aureus and vancomycin-resistant Enterococci at a concentration of μM.

[0029] Table 1. Results of antibacterial spectrum detection

[0030] Note: The experiment was repeated 12 times, and the data are expressed as mean and standard error (SEM). - indicates no obvious antimicrobial zone.

[0031] The results of the minimum inhibitory concentration (MIC) study are shown in Table 2. The results show that the antimicrobial peptide C0-C8 has a better inhibitory effect on Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa than Ursoricin.

[0032] Table 2. Results of bactericidal activity tests of highly stable broad-spectrum antimicrobial peptides

[0033]

[0034] Example 2: Stability Study of Antimicrobial Peptides

[0035] The effect of temperature on activity was investigated by incubating the antimicrobial peptide solution (100 μg / mL) at 40°C, 60°C, and 80°C for 20 min, and at 100°C for 20, 40, and 60 min. The effect of pH was assessed by adjusting the pH of the antimicrobial peptide solution (100 μg / mL) to 3–9 using 6 M hydrochloric acid or 6 M sodium hydroxide solution and incubating at 25°C for 2 h. The effect of protease on activity was investigated by mixing the antimicrobial peptide solution (100 μg / mL) with proteinase K solution to a final concentration of 1 mg / mL and incubating at 37°C for 1 h. Samples not treated with temperature or protease served as untreated controls. The antimicrobial activity of the treated and untreated samples was tested on *E. faecium* EF151 strain using the dot assay described above.

[0036] The experimental results are shown in Table 3: the antimicrobial peptides retained 100% activity after incubation at 40℃, 60℃, and 80℃ for 20 minutes, and even maintained activity after incubation at 100℃ for 60 minutes. They performed well under both acidic (pH 3 to 5) and alkaline (pH 9) conditions, with residual activity exceeding 93.17%. After treatment with 1 mg / mL proteinase K for 2 hours, approximately 63.7% activity was still retained.

[0037] Table 3. Retention rate of bactericidal efficacy of Ursoricin after treatment under different physical / biological conditions.

[0038]

[0039] Note: Residual bactericidal efficacy = Diameter of inhibition zone in the treated group / Diameter of inhibition zone in the untreated control group x 100

[0040] Furthermore, the retention rate of antimicrobial peptide efficacy was further determined under pepsin, proteinase K, high temperature (100℃ for 60 min), acid-base stability (pH 2-12), and blood microenvironment (bovine) conditions.

[0041] The results are shown in Table 4. The activity retention of antimicrobial peptide C0-C8 under pepsin, proteinase K, high temperature (100℃ for 60 min), acid-base stability (pH 2-12) and blood microenvironment (bovine) conditions was significantly higher than that of Ursoricin, indicating that the stability of the modified antimicrobial peptide C0-C8 was enhanced.

[0042] Table 4. Stability test results of highly stable broad-spectrum antimicrobial peptides

[0043]

[0044] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A highly stable broad-spectrum antimicrobial peptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 2-10.

2. A polynucleotide encoding the highly stable broad-spectrum antimicrobial peptide of claim 1.

3. An expression vector containing the polynucleotide of claim 2.

4. A host cell containing the polynucleotide of claim 2 or the expression vector of claim 3.

5. A pharmaceutical composition comprising the highly stable broad-spectrum antimicrobial peptide of claim 1 or a pharmaceutically acceptable salt thereof, the polynucleotide of claim 2, the expression vector of claim 3 or the host cell of claim 4, and a pharmaceutically acceptable carrier.

6. The use of the highly stable broad-spectrum antimicrobial peptide of claim 1 and / or the pharmaceutical composition of claim 5 in the preparation of antibacterial agents, characterized in that: The bacteria are Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, or Pseudomonas aeruginosa.

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