Antibacterial peptide mutant and application thereof

Abstract

The application discloses an antibacterial peptide mutant and application thereof, and the amino acid sequence of the antibacterial peptide mutant is shown as SEQ ID NO. 1. The application optimizes a honeycomb peptide sequence, obtains 10 potential candidate peptides, evaluates the antibacterial activity and hemolytic activity of the 10 potential candidate peptides, and obtains antibacterial peptides with high activity, low toxicity and long half-life. The antibacterial peptide molecules with high antibacterial activity obtained by the application not only significantly reduce the toxicity in a host, but also greatly improve the in-vivo stability, thereby becoming high-quality antibacterial peptide candidate molecules with high efficiency, safety and stability, and effectively increasing the clinical application potential.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, and in particular to an antimicrobial peptide mutant and its application. Background Technology

[0002] Antimicrobial peptides (AMPs) are considered important candidates for antibiotic alternatives due to their ability to rapidly disrupt microbial cell membranes and their low tendency to induce drug resistance. However, naturally occurring or artificially designed antimicrobial peptides are often limited by trade-offs between "high activity-high toxicity" or "low toxicity-low activity," and their short half-life due to easy degradation by proteases leads to insufficient drug-like properties. Antimicrobial peptides are a class of short peptides naturally found in animals, plants, and microorganisms, possessing important functions such as broad-spectrum antibacterial, antifungal, and antiviral activity. Because they primarily exert their effects by disrupting microbial membrane structures or regulating immune responses, they are less likely to induce bacterial resistance, thus holding significant strategic importance in the development of anti-infective drugs. However, the in vivo application of most naturally occurring or artificially designed antimicrobial peptides still faces significant limitations, mainly manifested in high toxicity, poor stability, and insufficient pharmacokinetic performance. For example, many peptides with high antimicrobial activity also exhibit strong hemolytic activity or toxic side effects on mammalian cells, leading to significant safety risks when used in vivo; while other peptides, although having lower toxicity, lack sufficient activity to achieve effective antibacterial levels. Meanwhile, antimicrobial peptides are readily degraded by proteases in vivo, have short half-lives, and are difficult to maintain at effective concentrations, further affecting their therapeutic efficacy. These factors significantly limit the drug development process of antimicrobial peptides.

[0003] Existing antimicrobial peptide optimization design methods often focus solely on enhancing antimicrobial activity, lacking systematic consideration of key indicators such as toxicity and half-life. This results in candidate peptides exhibiting poor overall performance. Some studies have attempted to use machine learning or deep learning models to predict peptide activity or toxicity, but their optimization processes still fail to achieve balance and synergy among multiple indicators, and remain insufficient in terms of screening efficiency and quality. Current antimicrobial peptide optimization methods based on rule-based design or single-point mutation strategies often fail to effectively model the nonlinear coupling relationships between the high-dimensional physicochemical features inherent in the sequence. The optimization process is prone to getting trapped in local optima and lacks global search capabilities, leading to performance bottlenecks in the generated peptide sequences in terms of activity, toxicity, or stability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an antimicrobial peptide mutant and its application. The antimicrobial peptide mutant, through a multi-objective computational optimization method, significantly reduces toxicity to the host and prolongs the in vivo half-life while enhancing antimicrobial activity, thus obtaining a candidate peptide sequence with greater drug development potential.

[0005] This invention provides an antimicrobial peptide mutant, the amino acid sequence of which is shown in SEQ ID NO.1 (PIGALMLKLHTGFPCMICAIKRKRVI), and the antimicrobial peptide mutant protein has the characteristics of high activity, low toxicity and long half-life.

[0006] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide mutant is 15 μM.

[0007] The present invention also provides an expression cassette or recombinant vector that can express the antimicrobial peptide mutant.

[0008] The present invention also provides a recombinant bacterium, a recombinant cell line, or a recombinant virus, wherein the recombinant bacterium, recombinant cell line, or recombinant virus comprises the recombinant expression vector described above.

[0009] The present invention also provides the use of the aforementioned antimicrobial peptide mutant in the preparation of any one of antibacterial products, animal feed additives, and cosmetic additives.

[0010] Furthermore, the antibacterial product can be any one of antibacterial daily necessities and antibacterial drugs. The antibacterial daily necessities include any one of hand sanitizer and disinfectant.

[0011] Furthermore, the antibacterial drug can be administered via any one of intravenous injection, oral administration, or topical application.

[0012] Furthermore, the antibacterial drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients are any one of water extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters, or aerosols.

[0014] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0015] This invention is based on the antimicrobial peptide mutant sequence of "high activity and high toxicity" or "low activity and low toxicity", and uses mutation, recombination and other technical means to modify and optimize it, so that the novel antimicrobial peptide mutant molecule can have the excellent characteristics of "high activity, low toxicity and long half life".

[0016] The antimicrobial peptide molecules obtained by this invention not only significantly reduce toxicity in the host, but also greatly improve stability in vivo, thus becoming high-quality antimicrobial peptide candidate molecules with high efficiency, safety and stability, effectively increasing their potential for clinical application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram showing the antibacterial effect of the cellular peptide and antimicrobial peptide mutants on Escherichia coli in Example 1 of the invention.

[0019] Figure 2 This is a diagram showing the hemolytic effect of the cellular peptide and antimicrobial peptide mutant on mouse erythrocytes in Example 2 of the invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0021] This invention uses melive venom peptides as the starting sequence. To achieve joint optimization of candidate peptides in multiple attributes such as antibacterial activity, host toxicity, and half-life, a comprehensive and accurate performance evaluation of the input peptide sequence is first required. Therefore, in the initial stage of the technical process, a multi-task learning model is introduced as a multi-attribute prediction module to simultaneously evaluate the antibacterial activity and hemolytic activity (as a toxicity indicator) of the peptide, providing quantitative feedback for the subsequent evolutionary optimization process.

[0022] This method first constructs a multi-attribute prediction module, including a multi-task prediction module and a multi-scale feature extraction structure, to support the evaluation of the pharmacological properties of peptide sequences and facilitate co-evolutionary optimization. Based on this, a multi-objective optimization method combining Monte Carlo Tree Search (MCTS) and co-evolutionary mechanisms is employed: MCTS constructs a state-action search tree, and the search proceeds through four steps: selecting potential branches according to the UCT strategy, expanding point mutations to generate new candidate peptide sub-nodes, simulation (the core stage, introducing a co-evolutionary mechanism to replicate candidate peptides to multiple subpopulations, optimizing locally for different attributes such as antibacterial activity, generating new sequences, and then combining with the prediction model for weighted summation and Pareto optimality screening and evaluation), and backpropagation (results are backpropagated to adjust the tree's evaluation and strategy). Finally, through the above optimization process, 10 potential peptides are obtained.

[0023] To verify the antibacterial efficacy and drug-likeness of the 10 potential candidate peptides, in vitro experiments were conducted to evaluate their antibacterial activity and hemolytic activity.

[0024] Example 1: Detection of antibacterial activity of potential candidate peptides

[0025] The minimum inhibitory concentration (MIC) of antimicrobial peptides against *Escherichia coli* ATCC 25922 was determined using the broth microdilution method. Ten potential antimicrobial peptides and cellular peptide samples were dissolved in sterile solution (e.g., ddH₂O) to prepare initial concentrations (e.g., 1000 μM), and then serially diluted in 96-well plates at a 1:2 ratio. *E. coli* bacterial culture in the logarithmic growth phase (OD) was then... 600 ≈0.5) diluted to approximately 5×10 4 After inoculating with CFU / mL, the solution was added to each well, with a final volume of 200 μL per well. Blank, negative, positive, and growth control samples were included. After incubation at 37°C for 18 hours, OD values ​​were read. 600 The minimum concentration required to completely inhibit visible bacterial growth was determined as the MIC value. The experiment was repeated three times, and the average result was used to analyze antibacterial activity.

[0026] Experimental conclusions: such as Figure 1 As shown, T1 is bee venom peptide (GIGAVLKVLTTGLPALISWIKRKRQQ), and T2 (GIQAVLHVLHTGFPAMICAIKRKRYI), T3 (GIGAVDFVLHHGFPNMICAIKRVRFQ), T4 (DIGALKLVLHTGFPCMICAIKRKRVI), T5 (GVGALNFVLHTGFPNMICAIKRKRVI), T6 (GEGALPLVLHTGFPCMICAIKRKRVI), T7 (PIGALMLKLHTGFPCMICAIKRKRVI), T8 (FIGAAYKVLLHRLNHFHYWIQRQMCW), T9 (FIGMAYKVLLVRLCHFHYWIQRQRCW), T10 (GCMGMLHDWHKGFHAWICAPKRKRWS), and T11 (GIPLMAYHLGRGFYSEICAIKRKLES) are 10 candidate antimicrobial peptides. KC served as the control group, DB as the known drug polymyxin B, and KONGBAI as the blank control group. Candidate peptides T2, T4, T5, T6, T7, T10, and T11 exhibited varying degrees of antibacterial activity against *Escherichia coli*. Among them, T7 (SEQ ID NO.1) showed a significant antibacterial effect, with a minimum inhibitory concentration (MIC) close to 15 μM.

[0027] Example 2: Assessment of the hemolytic activity of potential antimicrobial peptides

[0028] The hemolytic toxicity of antimicrobial peptides to mouse erythrocytes (MRBCs) was evaluated using a erythrocyte hemolysis assay. Blood was collected from healthy mice, anticoagulated, washed with PBS, and resuspended to a 5% MRBC suspension. The antimicrobial peptide sample was dissolved in PBS at a maximum concentration of 1000 μM, and then serially diluted 1:2 in 96-well plates. An equal volume of MRBC suspension and antimicrobial peptide solution was added to each well. PBS was used as a negative control, and ddH2O or Triton X-100 was used as a positive control. After incubation at 37°C for 60 minutes, the supernatant was collected by centrifugation, and the absorbance at 540 nm was measured. The hemolysis rate was calculated using the formula. A concentration-hemolysis rate curve was plotted, and the HC ratio was determined. 50 The experiment was repeated three times to ensure the reliability of the results.

[0029] Experimental conclusion: Figure 2 The results showed that T1 cellular peptides had the highest hemolytic activity among all substances and were more destructive to erythrocytes. The hemolytic activity of the 10 potential antimicrobial peptides obtained was lower than that of the T1 control group, with T2-T7 and T10-T11 showing the highest HC content. 50 The values ​​were all greater than 250 μM (the upper limit of detection), indicating that within the detection concentration range, they would not cause 50% hemolysis of red blood cells, exhibiting very low hemolytic activity and good safety. T8 HC 50 The concentration was 62.50 μM, indicating some hemolytic activity, but significantly lower than that of T1. T9 HC 50 The value was 250 μM, which is just the upper limit of detection, indicating that its hemolytic activity is also low and its safety is good.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0031]

Claims

1. An antimicrobial peptide mutant, characterized in that, The amino acid sequence of the antimicrobial peptide mutant is shown in SEQ ID NO.

1.

2. The antimicrobial peptide mutant according to claim 1, characterized in that, The minimum inhibitory concentration of the antimicrobial peptide mutant is 15 μM.

3. The application of the antimicrobial peptide mutant according to claim 1 in the preparation of anti-Escherichia coli products.

4. The application according to claim 3, characterized in that, The anti-E. coli product is an anti-E. coli drug.

5. The application according to claim 4, characterized in that, The anti-E. coli drug also contains pharmaceutically acceptable excipients.

6. The application according to claim 4, characterized in that, The drug is any one of the following: water extract, powder, lotion, tincture, oil, ointment, plaster, or aerosol.

Images