Efficient and safe antimicrobial peptide variant and application thereof

Abstract

This invention discloses a highly efficient and safe antimicrobial peptide variant and its applications. Based on the natural antimicrobial peptide Upperin 3.6 sequence (as shown in SEQ NO:1), it involves replacing the disordered N-terminal region with the basic amino acid arginine; repeating two amyloid C-terminal sequences and adding three arginine residues to each end; and inserting a four-amino acid sequence DPDG (which has the greatest tendency to form a β-turn structure) between the two repeated amyloid C-terminal sequences and adding three arginine residues to each end. The antimicrobial peptide of this invention enhances the activity and safety of antimicrobial peptides, exhibiting good antimicrobial properties against both Gram-negative and Gram-positive bacteria. This antimicrobial peptide has broad application prospects as a novel antimicrobial agent.

Description

[0001] This application is a divisional application. The original application was filed on February 14, 2023, with application number 2023101110084, and the invention title was "A Highly Effective and Safe Antimicrobial Peptide Variant and Its Application". Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a highly efficient and safe antimicrobial peptide variant and its application. Background Technology

[0003] Antimicrobial peptides (AMPs), also known as host defense peptides, are widely distributed in the biological world and are important components of the body's innate immune system, forming the first line of defense against invading pathogenic microorganisms. Most of these bioactive peptides possess strong acid and alkali resistance, thermal stability, and broad-spectrum antibacterial properties. In 1980, Swedish scientist G. Boman and others discovered the world's first antimicrobial peptide, named Cecropins. Since the discovery of antibiotics, they have played a vital role in human and animal health, significantly promoting animal production and growth. However, due to increasingly prominent issues such as drug resistance and residues, more and more countries and regions have gradually banned the addition of antibiotics to animal feed and are continuously regulating the clinical use of antibiotics. In recent years, research on new medical drugs and antibiotic alternatives in animal feed has become a hot topic of attention and research, and research on the application of antimicrobial peptides in medicine and animal production is also increasing.

[0004] Today, antimicrobial peptides have been successfully isolated and classified from most organic organisms, from prokaryotes to humans. Antimicrobial peptides typically act on bacteria and play a crucial role in the innate immunity of eukaryotes, considered to be effectively preserved immune molecules in mammals throughout ancient evolution. Therefore, as a class of polypeptides widely found in organisms in nature, antimicrobial peptides can serve as the first line of defense against pathogens. They possess various biological activities, including antibacterial, antifungal, and antiviral activity, as well as the ability to inhibit and kill cancer cells, and are less prone to inducing drug resistance.

[0005] Currently, antimicrobial peptides have achieved some satisfactory results in medical applications, and many new drugs are gradually entering the pharmaceutical market. The most thoroughly researched are breviculin, polymyxin, and lactic acid. Breviculin is limited to use in superficial wounds and upper respiratory tract infections; conversely, polymyxin can be used not only to treat eye infections but also to treat gastrointestinal and systemic infections caused by drug-resistant Gram-negative bacteria. Another effective cyclic AMP that plays a role in treating complicated skin and skin structure infections caused by Staphylococcus aureus is daptomycin, which is often used in combination therapy to improve treatment success rates. Nisin is used in dental care, gastric ulcer treatment, and treatment of colon infections. The amphibian antimicrobial peptide magainin-derived antimicrobial peptide drug MAI278 is nearing completion of Phase III clinical trials, showing good killing effects against viruses and tumor cells. Daptomycin is an anionic antimicrobial peptide developed by Cubit Pharmaceuticals and approved for marketing by the U.S. Food and Drug Administration in September 2003. It can be used to treat skin infections and sepsis caused by Gram-positive bacteria such as Staphylococcus aureus. In addition to their role in animal production, antibiotics, used as feed additives, have played a vital role in the development of animal husbandry. However, their residues in animals and animal products, as well as the antibiotic resistance developed by pathogens, have had negative impacts on human health and the environment. Therefore, antimicrobial peptides, as the most promising alternatives to traditional antibiotics, have excellent application prospects in the pharmaceutical industry and food additives. Summary of the Invention

[0006] The main objective of this invention is to provide a highly effective and safe antimicrobial peptide variant and its applications. Studies have shown that the natural antimicrobial peptide Uperin 3.6, derived from the skin gland secretions of the Australian toad (Toadlet Uperoleiamjobergii), exhibits good antimicrobial activity against some bacteria, but its antimicrobial effect against Escherichia coli is poor (MIC value > 100 μg / mL). Therefore, this invention, based on the antimicrobial peptide Uperin 3.6, has obtained a variant with higher antimicrobial performance than the natural antimicrobial peptide Uperin 3.6 through modification, serving as a strong candidate for next-generation antimicrobial drugs.

[0007] This invention provides a highly efficient and safe antimicrobial peptide variant. Based on the C-terminal sequence of the amyloid region (amino acids 8 to 17 of the natural antimicrobial peptide Uperin 3.6, as shown in SEQ NO:1), the original disordered N-segment (amino acids 1 to 7) of Uperin 3.6 is replaced with the basic amino acid arginine (which has a stronger inhibitory effect on aggregation compared to other amino acids) to obtain the antimicrobial peptide variant Uperin 3.6-N. 7R Its amino acid sequence is shown in SEQ NO:2.

[0008] The present invention provides a highly efficient and safe antimicrobial peptide variant, which is obtained by repeating the amyloid C-terminal sequence of Uperin3.6 (as shown in sequence SEQ NO:1) (the 8th to 17th amino acids of Uperin3.6) and adding 3 arginines to each end to obtain the antimicrobial peptide variant Uperin3.6C-C-6R, the amino acid sequence of which is shown in sequence SEQ NO:3.

[0009] The present invention provides a highly efficient and safe antimicrobial peptide variant, which is obtained by repeating the amyloid C-terminal sequence of Uperin 3.6 (as shown in sequence SEQ NO:1) (the 8th to 17th amino acids of Uperin 3.6) and adding 3 arginines to each end thereto, and inserting a four-amino acid sequence DPDG (which has a strong tendency to form β-turns and inhibits amyloid aggregation) between the two repeated Uperin 3.6 amyloid C-terminal sequences to obtain the antimicrobial peptide variant Uperin 3.6C-6R-DPDG, the amino acid sequence of which is shown in sequence SEQ NO:4.

[0010] Furthermore, the C-terminus of the antimicrobial peptide is modified with amidation, and the N-terminus is modified with acetylation.

[0011] Furthermore, the modified antimicrobial peptide Uperin 3.6-N 7R The molecular weight of is 2307.79 Da; the molecular weight of Upperin 3.6C-C-6R is 3307.04 Da; and the molecular weight of Upperin 3.6C-6R-DPDG is 3691.38 Da.

[0012] The polypeptide sequence encoding provided by this invention is also within the scope of protection of this invention.

[0013] The application of the antimicrobial peptide variants of the present invention is to prepare antimicrobial preparations and related preparations such as anticancer preparations using the said antimicrobial peptide variants.

[0014] The antibacterial agent has inhibitory and bactericidal effects on both Gram-negative and / or Gram-positive bacteria. Specifically, it includes *Escherichia coli*, *Bacillus subtilis*, *Micrococcus luteus*, *Escherichia coli* Fergusonian, *Staphylococcus epidermidis*, *Pseudomonas aeruginosa*, and *Listeria noviceae*.

[0015] The physicochemical properties of wild-type and modified antimicrobial peptides were evaluated and analyzed, including minimum inhibitory concentration (MIC), aggregation kinetics, secondary structure analysis, membrane interaction analysis, and hemolytic activity analysis. The MIC value is the lowest concentration value that inhibits Gram-positive bacteria such as Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, and Listeria monocytogenes, as well as Gram-negative bacteria such as Escherichia coli, Escherichia coli, and Pseudomonas aeruginosa. Aggregation kinetics involves incubating the antimicrobial peptide with thioflavin T (ThT). The β-sheets formed by the aggregation of the antimicrobial peptide specifically bind to thioflavin T (ThT), causing a change in fluorescence intensity. The change in fluorescence intensity indicates the change in the number of aggregates formed. Secondary structure analysis involves detecting the antimicrobial peptide in PBS buffer and 2,2,2-trifluoroethanol (TFEA) in a simulated membrane environment using far-ultraviolet circular dichroism spectroscopy. Membrane interaction analysis involves detecting changes in the bacterial cell membrane after incubation of the antimicrobial peptide with bacteria using scanning electron microscopy. Hemolytic activity analysis evaluates the cytotoxic effect of the antimicrobial peptide on normal mammalian cells by assessing its activity in disrupting the erythrocyte membrane and releasing hemoglobin.

[0016] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0017] This invention is based on existing natural antimicrobial peptide sequences. It modifies these peptides by inserting sequences with specific structures and effects (DPDG, which has a strong tendency to form β-turns and inhibits amyloid aggregation) and adding sequences with specific effects (three consecutive Args that can inhibit amyloid aggregation) to the ends. Short peptides are then obtained through artificial synthesis. The antimicrobial activity and other physicochemical properties of the resulting series of short peptides are measured to assess whether they are AMPs and their activity intensity. Based on existing AMP sequences and antimicrobial activity information, this invention rationally designs and modifies them, and verifies them experimentally. This allows for efficient and rapid screening of antimicrobial peptides, solving problems such as low activity and low success rate of modification of some natural AMPs.

[0018] The modified antimicrobial peptides provided by this invention have the following advantages: This series of antibacterial peptides exhibits inhibitory and bactericidal effects against both Gram-negative and Gram-positive bacteria, specifically including *Escherichia coli*, *Bacillus subtilis*, *Micrococcus luteus*, *Escherichia coli*, *Staphylococcus epidermidis*, *Pseudomonas aeruginosa*, and *Listeria monocytogenes*, demonstrating promising application prospects. The modified antimicrobial peptides of this invention can be used to prepare antibacterial drugs (antibacterial inhibitors, such as inhibitors against Gram-negative or Gram-positive bacteria). Because bacterial cells and cancer cells share many similarities, and many antimicrobial peptides also exhibit anticancer activity, the anticancer applications of the modified antimicrobial peptides of this invention are also within the scope of protection. Attached Figure Description

[0019] Figure 1 The aggregation kinetics of the natural antimicrobial peptide Uperin3.6 and its mutants.

[0020] Figure 2 Far-ultraviolet circular dichroism spectroscopy for the natural antimicrobial peptide Uperin 3.6 and its mutants.

[0021] Figure 3 This is a scanning electron microscope image of the interaction between the natural antimicrobial peptide Uperin 3.6 mutant and bacteria.

[0022] Figure 4 A comparison of the hemolytic activity of the natural antimicrobial peptide Uperin 3.6 and its mutants.

[0023] Figure 5 The hemolysis rate of the natural antimicrobial peptide Uperin 3.6 and its mutants. Detailed Implementation

[0024] This invention is not limited to the specific embodiments described below. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0025] The invention will now be described in further detail with reference to specific examples.

[0026] All experimental reagents used in this invention can be purchased through commercial channels. The models and specifications of some of the experimental instruments used in this invention are described below.

[0027] Antimicrobial peptides: The antimicrobial peptides used in this invention were prepared and synthesized by Shanghai Jier Biochemical Co., Ltd. using Fmoc solid-phase synthesis, with a purity of over 98%.

[0028] The bacterial strains and their sources were as follows: Escherichia coli and Bacillus subtilis were provided by the laboratory; Micrococcus luteus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Listeria innocua, Salmonella typhimurium, and Bacillus cereus were purchased from Beijing BioBio Biotechnology Co., Ltd.

[0029] Reagents used: PBS buffer, pure water, DMSO, ampicillin (100 mg / mL), thiamine.

[0030] Culture medium: LB liquid medium (sterilized at 121℃ for 20 min).

[0031] Experimental instruments: SpectraMax M5 fully automated microplate reader (Meigu Molecular Instruments (Shanghai) Co., Ltd.), shaking incubator (Shanghai Minquan Instruments Co., Ltd.), circular dichroism chromatograph.

[0032] The original amino acid sequence of Uperin 3.6 is detailed in SEQ NO:1; the antimicrobial peptide variant Uperin 3.6-N 7R The amino acid sequence of the antimicrobial peptide variant Upperin 3.6C-C-6R is detailed in SEQ NO:2; the amino acid sequence of the antimicrobial peptide variant Upperin 3.6C-6R-DPDG is detailed in SEQ NO:4.

[0033] Example 1: Design of antibacterial peptides

[0034] Table 1 below shows the sequences of the modified antimicrobial peptides.

[0035]

[0036] During synthesis, the natural antimicrobial peptide Uperin 3.6 and its variants undergo C-terminal amidation and N-terminal acetylation. N-terminal acetylation and C-terminal amidation reduce the peptide's total charge, potentially decreasing its overall solubility and making it more similar to the parent protein, thus enhancing its ability to enter cells. Furthermore, since terminal acetylation and amidation generate mimics that more closely resemble the natural protein, the peptide's stability is also improved, thereby increasing its resistance to exopeptidases such as proteases, telomerases, and synthases. Therefore, these modifications enhance the peptide's biological activity.

[0037] Example 2: Determination of the minimum inhibitory concentration (MIC) of antibacterial peptides

[0038] The minimum inhibitory concentration (MIC) was determined according to the standard method of CLSI. Single colonies of the strain were picked up using an inoculation stick and placed in LB liquid medium, incubated overnight at 37°C and 220 rpm in a constant temperature shaking incubator, and then a bacterial suspension of 10⁴ CFU / mL was prepared. 100 μL of the bacterial suspension was added to each well of a 96-well plate. The antimicrobial peptide was serially diluted 2-fold, with 100 μL added to each well to achieve final concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μg / mL. In the negative control group, 100 μL of the antimicrobial peptide solution was replaced with an equal volume of PBS buffer; in the positive control group, 100 μL of the antimicrobial peptide solution was replaced with an equal volume of ampicillin. Each treatment had three replicates. The 96-well plate was incubated in a constant temperature shaking incubator at 37°C and 220 rpm for 24 hours until visibly cloudy liquid appeared in the negative control wells. The concentration of the antimicrobial peptide that completely inhibits bacterial growth (with clear wells) is the MIC value of the antimicrobial peptide for that bacterium.

[0039] Table 2 shows the MIC values ​​(μg / mL) of the natural antimicrobial peptide Uperin 3.6 and its mutants against various bacteria.

[0040]

[0041] Antimicrobial activity assay results showed that, compared to the natural antimicrobial peptide Uperin 3.6, our designed and modified antimicrobial peptide mutant UP3.6-N... 7R The antibacterial activity of Uperin 3.6C-C-6R and Uperin 3.6C-6R-DPDG was significantly enhanced.

[0042] Example 3: Aggregation kinetics determination of antimicrobial peptides

[0043] β-sheet aggregates formed by proteins or peptides can specifically bind to thioflavin T (ThT), causing a change in fluorescence intensity. Changes in the fluorescence intensity due to thioflavin T binding can indicate changes in the number of aggregates formed. Detection of Uperin 3.6, Uperin 3.6-C, Uperin 3.6-N, and Uperin 3.6-N. 7RThe aggregation of Uperin 3.6C-6R-DPDG at different concentrations (20 μM, 50 μM, 100 μM) was investigated. ThT fluorescence kinetics samples were placed in 96-well black microplates, with three parallel experiments and three control experiments for each sample. ThT fluorescence intensity was measured at 25°C using a SpectraMax M5 multiplexer (Molecular Devices Limited) with an excitation wavelength of 440 nm, an emission wavelength of 480 nm, and a slit wavelength of 2 nm. The sample was shaken for 3 seconds to mix before each measurement, and fluorescence was read every two minutes.

[0044] Figure 1 The results show the aggregation kinetics of the natural antimicrobial peptide Uperin 3.6 and its mutants. The aggregation kinetics results indicate that the C-terminal peptide of the truncated natural antimicrobial peptide Uperin 3.6 aggregated; the disordered N-terminal peptide itself did not aggregate, but it inhibited C-terminal aggregation; while the designed and modified antimicrobial peptide mutants did not aggregate.

[0045] Example 4: Determination of the secondary structure of antimicrobial peptides

[0046] Far-ultraviolet circular dichroism was used to record the spectra of peptides in PBS aqueous solution and in 10% trifluoroethanol TFEA and 20% trifluoroethanol TFEA, respectively, with a wavelength range of 200~250 nm, a step size of 1 nm, and an optical path of 1 cm.

[0047] Figure 2 The images show far-ultraviolet circular dichroism chromatograms of the natural antimicrobial peptide Uperin 3.6 and its mutants. The results show that the natural antimicrobial peptide Uperin 3.6 formed a small number of helical structures in a simulated membrane environment, indicating that it can interact with the membrane, but the interaction is weak. In contrast, the designed and modified antimicrobial peptides were induced to form more helical structures in the simulated membrane environment, indicating a stronger interaction with the membrane. Therefore, it is inferred that the designed and modified antimicrobial peptides can interact more strongly with the bacterial cell membrane, folding into more regular secondary structures and inserting into the bacterial membrane, thus leading to more severe membrane damage and cell death.

[0048] Example 5: Analysis of the interaction between antimicrobial peptides and bacterial cell membranes - SEM characterization

[0049] Bacteria were cultured in LB at 37°C to the exponential phase, centrifuged at 1000 rpm for 10 min, washed twice with 10 mM PBS, and resuspended until OD600 was 0.2. The cell suspension was incubated at 37°C with different peptides at 1×MICs for 30 min. The control group was run without peptides. After incubation, cells were collected by centrifugation at 5000 rpm for 5 min at 4°C, and then washed three times with PBS. The bacterial cells were then fixed overnight at 4°C with 2.5% (v / v) glutaraldehyde. The fixed samples were washed twice with PBS and then dehydrated for 15 min in fractionated ethanol solutions (50, 70, 90, and 100%) (twice at 100% concentration, once at each of the other concentrations). They were then transferred to a mixture of anhydrous ethanol and tert-butanol (1:1, v / v) for 20 min, and then transferred to pure tert-butanol for 30 min. After lyophilization and gold plating, the cells were observed using a scanning electron microscope.

[0050] Figure 3 This is a scanning electron microscope (SEM) image of the interaction between the natural antimicrobial peptide Uperin 3.6 mutant and bacteria. SEM results show that upon contact with the modified antimicrobial peptide, the bacterial cell membranes ruptured, and contents leaked out. This further demonstrates that the modified antimicrobial peptide of this invention exerts its antimicrobial effect through interaction with the bacterial cell membrane. The mechanism may be that the antimicrobial peptide acts on the bacterial membrane, inserting its hydrophobic portion into the lipid bilayer core to significantly disrupt the bacterial membrane, leading to membrane damage and cell death.

[0051] Example 6: Determination of the hemolytic activity of antimicrobial peptides

[0052] The cytotoxic effect of antimicrobial agents on normal mammalian cells is generally evaluated by their hemolytic activity on erythrocytes. Fresh blood from mice was collected and an anticoagulant was added. The blood was diluted with physiological saline, transferred to centrifuge tubes, and centrifuged at 4°C and 3000 rpm for 5 min. The supernatant serum was discarded, leaving the erythrocyte pellet. The erythrocytes were washed three times with an equal volume of physiological saline to remove residual serum. The washed erythrocytes were diluted with physiological saline to 8% (v / v). 300 μL of the erythrocyte solution was transferred to a centrifuge tube, and then 300 μL of a series of two-fold dilutions of antimicrobial peptide solution were added. 100 μL of physiological saline was used as a negative control, and 100 μL of 0.1% Triton X-100 solution was used as a positive control. The centrifuge tubes were incubated in a shaker at 37°C and 220 rpm for 1 h. After the reaction, the erythrocytes were centrifuged at 4°C and 3000 rpm for 5 min to precipitate the pellet. Aspirate the supernatant from the centrifuge tube into a 96-well plate, and measure the absorbance at 540 nm using a microplate reader. The hemolysis rate is calculated using the following formula:

[0053] Hemolysis rate (%) = (Abs peptide - Abs negative) / (Abs positive - Abs negative) × 100%.

[0054] Figure 4 A comparison of the hemolytic activity of the natural antimicrobial peptide Uperin 3.6 and its mutants.

[0055] Figure 5 The hemolysis rate of the natural antimicrobial peptide Uperin 3.6 and its mutants.

[0056] The results are as follows Figure 4 , Figure 5 As shown, the antimicrobial peptide UP3.6C-C-6R exhibits a hemolytic activity of 5.54% at a concentration of 3.125 μg / mL (hemolysis is considered to be greater than 5%). UP3.6C-C-6R only shows antibacterial activity at concentrations above 3.125 μg / mL, and hemolysis has already occurred at this concentration, indicating that this antimicrobial peptide does not have good safety. Meanwhile, the antimicrobial peptide UP3.6-N... 7R Both UP3.6C-6R-DPDG and UP3.6C-6R-DPDG showed hemolytic activity below 5% at a high concentration of 100 μg / mL. These results indicate that the modified antimicrobial peptide UP3.6-N exhibits significantly improved hemolytic activity. 7R UP3.6C-6R-DPDG exhibits high antibacterial activity while maintaining high safety. The insertion of the four-amino acid sequence DPDG can significantly reduce the hemolytic activity of UP3.6C-C-6R and improve the safety of the antimicrobial peptide.

[0057] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide variant, characterized in that: The antimicrobial peptide variant is based on the C-terminal sequence of the amyloid region of the natural antimicrobial peptide Uperin3.

6. Three arginine residues are added to both ends of its tandem repeat sequence to obtain the antimicrobial peptide variant Uperin3.6C-C-6R, whose amino acid sequence is shown in SEQ NO:

3. Alternatively, the antimicrobial peptide variant is based on the C-terminal sequence of the amyloid region of the natural antimicrobial peptide Uperin3.6, with three arginine residues added to both ends of its tandem repeat sequence, and a four-amino acid sequence DPDG inserted between the two repeating Uperin3.6 amyloid C-terminal sequences to obtain the antimicrobial peptide variant Uperin3.6C-6R-DPDG, whose amino acid sequence is shown in sequence SEQ NO:4; The C-terminus of the antimicrobial peptide variant is modified with amidation, and the N-terminus is modified with acetylation.

2. The use of the antimicrobial peptide variant of claim 1 in the preparation of antimicrobial agents, characterized in that: The amino acid sequence of the antimicrobial peptide variant is shown in SEQ NO:3; The antibacterial agent has inhibitory and bactericidal effects on both Gram-negative and / or Gram-positive bacteria; The Gram-negative and / or Gram-positive bacteria are one or more of Escherichia coli, Bacillus subtilis, Escherichia fenestration, Micrococcus luteus, Glucocortisone epidermidis, Pseudomonas aeruginosa, and Listeria monocytogenes.

3. The use of the antimicrobial peptide variant of claim 1 in the preparation of antimicrobial agents, characterized in that: The amino acid sequence of the antimicrobial peptide variant is shown in SEQ NO:4; The antibacterial agent has inhibitory and bactericidal effects on both Gram-negative and / or Gram-positive bacteria; The Gram-negative and / or Gram-positive bacteria are one or more of Escherichia coli, Bacillus subtilis, Micrococcus luteus, Glucocortis epidermidis, and Listeria monocytogenes.

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