A highly effective and safe Aβ42 variant antimicrobial peptide and its application

Abstract

This invention discloses a highly efficient and safe Aβ42 variant antimicrobial peptide and its applications. Based on existing natural amyloid peptide sequences, this invention modifies the peptide by inserting sequences with specific structures and effects (YNGK, which has a strong tendency to form β-turns and inhibits amyloid aggregation) and adding sequences with specific effects at both ends (3 / 5 consecutive Args that can inhibit amyloid aggregation). Short peptides are obtained through artificial synthesis, and the antimicrobial activity and other related physicochemical properties of the resulting series of short peptides are measured to assess whether they are AMPs and their activity intensity. Based on existing amyloid peptide sequences and antimicrobial activity information, this invention rationally designs and modifies them, and verifies them experimentally. This method can efficiently and rapidly screen antimicrobial peptides, solving the problems of difficulty in obtaining antimicrobial peptides and poor antimicrobial effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly efficient and safe Aβ42 variant antimicrobial peptide and its applications. Background Technology

[0002] Since their discovery, antibiotics 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 are gradually banning the addition of antibiotics to animal feed and 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. Antimicrobial peptides (AMPs) are components of the innate immune system and are a class of drugs with high clinical application potential, having been proven to have effective antimicrobial activity against multidrug-resistant (MDR) strains. AMPs are key components in nature for organisms to combat pathogenic invasion and are found in all species. The diverse biological functions of antimicrobial peptides, coupled with their wide distribution and diversity, make them a rich source for discovering potential novel lead drugs.

[0003] Due to the presence of numerous hydrophobic amino acid residues, membrane interaction is a characteristic of amyloid peptides. There is a correlation between peptide amyloidosis and antibacterial activity. Aβ-amyloid peptides associated with Alzheimer's disease (AD) are produced in the brain and surrounding tissues, and are fragments of 39-43 amino acids in length, cleaved from β precursor protein (APP) via proteolytic pathways. Among them, Aβ42 exhibits greater neurotoxicity and stronger aggregation, forming the core of Aβ deposits. Aβ42 aggregates display different structural features during aggregation, including changes in β-sheet content and surface hydrophobicity, which are closely related to their cytotoxicity. Recent studies have shown that Aβ42 possesses antimicrobial and antimicrobial activity and may function in vivo as an AMP, acting as an innate immune effector molecule. Because of its similar structure and mechanism of action to typical antimicrobial peptides, we hope to modify Aβ42 into an antimicrobial peptide with antimicrobial activity. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a highly efficient and safe Aβ42 variant antimicrobial peptide and its applications.

[0005] Studies have shown that the natural amyloid peptide Aβ42 derived from the human brain has certain antibacterial activity against some bacteria, but its antibacterial effect is poor against others (MIC>50μM). Therefore, this invention uses Aβ42 amyloid peptide as a base and modifies it to obtain variants with higher antibacterial properties, as a strong candidate for next-generation antibacterial drugs.

[0006] The present invention relates to a variant antimicrobial peptide Aβ42, which is based on the amyloid aggregation segment sequence (amino acids 16 to 22 of Aβ42) of the natural amyloid peptide Aβ42 (as shown in SEQ ID NO:1). This aggregation segment is repeated, and three arginines (which have a stronger inhibitory effect on aggregation than other amino acids) are added to each end of it. A four-amino acid sequence YNGK (which has a strong tendency to form β-turns and inhibits amyloid aggregation) is inserted between the two repeated aggregation segments to obtain the variant antimicrobial peptide Aβ-6R-YNGK, whose amino acid sequence is shown in SEQ ID NO:2.

[0007] The present invention relates to a variant antimicrobial peptide Aβ42, which repeats the amyloid aggregation region sequence of Aβ42 (as shown in sequence SEQ ID NO:1) (amino acids 16 to 22 of Aβ42), adds 5 arginines to each end thereof, and inserts a four-amino acid sequence YNGK between the two repeated aggregation regions to obtain the variant antimicrobial peptide Aβ-10R-YNGK, the amino acid sequence of which is shown in sequence SEQ ID NO:3.

[0008] The present invention relates to a variant antimicrobial peptide of Aβ42, which is obtained by inverting the amyloid aggregation region sequence of Aβ42 (as shown in sequence SEQ ID NO:1) (amino acids 16 to 22 of Aβ42), adding 3 arginines to each end thereof, and inserting the four amino acid sequence YNGK between the two inverted aggregation regions to obtain the variant antimicrobial peptide Aβ-6R-YNGK-F, the amino acid sequence of which is shown in sequence SEQ ID NO:4.

[0009] The present invention relates to a variant antimicrobial peptide Aβ42, which is obtained by inverting the amyloid aggregation region sequence of Aβ42 (as shown in sequence SEQ ID NO:1) (amino acids 16 to 22 of Aβ42), adding 5 arginines to each end thereof, and inserting the four amino acid sequence YNGK between the two inverted aggregation regions to obtain the variant antimicrobial peptide Aβ-10R-YNGK-F, the amino acid sequence of which is shown in sequence SEQ ID NO:5.

[0010] Furthermore, the C-terminus of the Aβ42 variant antimicrobial peptide is modified with amidation, and the N-terminus is modified with acetylation.

[0011] Furthermore, the molecular weight of the modified antimicrobial peptides Aβ-6R-YNGK and Aβ-6R-YNGK-F is 3087.67 Da; the molecular weight of Aβ-10R-YNGK and Aβ-10R-YNGK-F is 3712.42 Da.

[0012] The present invention relates to the application of the Aβ42 variant antimicrobial peptide in the preparation of antimicrobial agents and related preparations such as anticancer agents.

[0013] The antibacterial agent has inhibitory and bactericidal effects on both Gram-negative and / or Gram-positive bacteria. Specifically, it includes Staphylococcus epidermidis, Escherichia coli, Escherichia coli Fergusonian, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Listeria innocense, and Pseudomonas aeruginosa.

[0014] Physicochemical properties of wild-type and modified Aβ42 antimicrobial peptides were evaluated, including minimum inhibitory concentration (MIC), aggregation kinetics, secondary structure analysis, membrane interaction analysis, hemolytic activity analysis, and anticancer activity analysis. The MIC value is the lowest concentration value that inhibits Gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, 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 the fluorescence intensity of thioflavin T binding indicates the change in the number of aggregates formed. Secondary structure analysis involves detecting the antimicrobial peptide in PBS buffer and liposomes 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. Anticancer activity analysis evaluates the cytotoxic effect of the antimicrobial peptide on cancer cells by assessing its activity in inhibiting cancer cell growth.

[0015] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0016] This invention is based on existing natural amyloid peptide sequences. It modifies these sequences by inserting sequences with specific structures and effects (YNGK, which has a strong tendency to form β-turns and inhibits amyloid aggregation) and adding specific-effect sequences (3 / 5 consecutive Args that can inhibit amyloid aggregation) to both ends. Short peptides are then artificially synthesized. The antibacterial 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 amyloid peptide sequences and antibacterial activity information, this invention rationally designs and modifies them, and verifies them experimentally. This allows for efficient and rapid screening of antimicrobial peptides, solving the problems of difficulty in obtaining antimicrobial peptides and poor antibacterial effects.

[0017] 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 *Staphylococcus epidermidis*, *Escherichia coli*, *Escherichia coli*, *Staphylococcus aureus*, *Bacillus subtilis*, *Bacillus cereus*, *Listeria ingrainedii*, and *Pseudomonas aeruginosa*, 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

[0018] Figure 1 Aggregation kinetics of natural amyloid peptide Aβ42 and its modified peptides.

[0019] Figure 2 Far-ultraviolet circular dichroism spectroscopy for natural amyloid peptide Aβ42 and its modified peptide.

[0020] Figure 3 Scanning electron microscope image of the interaction between the modified natural amyloid peptide Aβ42 and bacteria.

[0021] Figure 4 A comparison of the hemolytic activity of natural amyloid peptide Aβ42 and its modified peptide.

[0022] Figure 5 The hemolysis rate of natural amyloid peptide Aβ42 and its modified peptide.

[0023] Figure 6 Cell survival rate for the anticancer activity of natural amyloid peptide Aβ42 and its modified peptides. 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; Staphylococcus epidermidis, Pseudomonas aeruginosa, Listeria innocense, Salmonella typhimurium, and Bacillus cereus were purchased from Beijing Bio-BioBiotechnology Co., Ltd.

[0029] Reagents used: PBS buffer, pure water, acetonitrile, 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 Aβ42 is detailed in SEQ ID NO:1; the amino acid sequence of the variant antimicrobial peptide Aβ-6R-YNGK is detailed in SEQ ID NO:2; the amino acid sequence of the variant antimicrobial peptide Aβ-10R-YNGK is detailed in SEQ ID NO:3; the amino acid sequence of the variant antimicrobial peptide Aβ-6R-YNGK-F is detailed in SEQ ID NO:4; and the amino acid sequence of the variant antimicrobial peptide Aβ-10R-YNGK-F is detailed in SEQ ID NO:5.

[0033] Example 1: Design of antibacterial peptides

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

[0035]

[0036] During synthesis, the natural amyloid peptide Aβ42 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 closely resemble 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, then incubated overnight in a constant temperature shaking incubator at 37°C and 220 rpm. A bacterial suspension of 10⁴ CFU / mL was then 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 50, 20, 10, 5, and 1 μM. For the negative control group, 100 μL of the antimicrobial peptide solution was replaced with an equal volume of PBS buffer. For 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 plates were incubated in a constant temperature shaking incubator at 37°C and 220 rpm for 12–18 h, until visibly turbid liquid appeared in the negative control wells. The concentration of an antimicrobial peptide that can completely inhibit bacterial growth (with the well clear) is the MIC value of the antimicrobial peptide against that bacterium.

[0039] Table 2 shows the MIC values ​​(μM) of natural amyloid peptide Aβ42 and its modified peptide against various bacteria.

[0040]

[0041] The antibacterial activity test results showed that, compared with the natural amyloid peptide Aβ42, the antibacterial activity of our designed and modified variants Aβ-6R-YNGK, Aβ-6R-YNGK-F, Aβ-10R-YNGK, and Aβ-10R-YNGK-F was significantly enhanced.

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

[0043] β-sheet aggregates formed by proteins or peptides can specifically bind to thioflavin T (ThT), causing changes in fluorescence intensity. Changes in thioflavin T binding fluorescence intensity indicate changes in the number of aggregates formed. To detect the aggregation of Aβ42, Aβ-6R-YNGK, Aβ-6R-YNGK-F, Aβ-10R-YNGK, and Aβ-10R-YNGK-F at different concentrations (25 μM, 50 μM, 100 μM), ThT fluorescence kinetic samples were placed in 96-well black ELISA plates, with three parallel experiments for each sample. ThT fluorescence intensity was measured at 25°C using a Spectra Max M5 multiplex reader (Molecular Devices Limited) with an excitation wavelength of 440 nm, an emission wavelength of 485 nm, and a slit wavelength of 2 nm. The sample was shaken for 3 seconds before each measurement, and fluorescence was read every two minutes.

[0044] Figure 1 This is the result of aggregation kinetic monitoring of the natural amyloid peptide Aβ42 and its mutants. The aggregation kinetic results show that Aβ42 aggregated, while the designed 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 solution and liposomes in simulated membranes, with a wavelength range of 200–250 nm, a step size of 1 nm, and an optical path length of 1 cm.

[0047] Figure 2 The images show far-ultraviolet circular dichroism chromatograms of the natural amyloid peptide Aβ42 and its variants. The results show that Aβ42 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 abundant secondary structures in the simulated membrane environment, indicating that their interaction with the membrane is stronger. 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, thereby 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] The bacteria were cultured in LB at 37°C until the exponential phase, centrifuged at 1000 rpm for 10 min, washed twice with 10 mM PBS, and then resuspended at OD. 600The concentration was 0.2. Cell suspensions were incubated at 37°C with different peptides at 1×MICs for 1 h. The control group was run without peptides. After incubation, cells were collected by centrifugation at 4°C and 5000 rpm for 5 min, and then washed three times with PBS. Bacterial cells were then fixed with 2.5% (v / v) glutaraldehyde at 4°C for 2 h. The fixed samples were washed twice with PBS and then dehydrated for 10 min in fractionated ethanol solutions (50%, 70%, 90%, and 100%). 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, they were observed using a scanning electron microscope.

[0050] Figure 3 This is a scanning electron microscope (SEM) image showing the interaction between the natural amyloid peptide Aβ42 mutant and bacteria. SEM results show that upon contact with the modified antimicrobial peptide, the bacterial cell membranes ruptured, and the contents leaked out. This further demonstrates that the antimicrobial peptide designed and modified in this invention exerts its antimicrobial effect through interaction with the bacterial cell membrane.

[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. 10 mL of 4% mouse erythrocyte blood was transferred to a centrifuge tube and centrifuged at 3000 rpm for 5 min at 4°C. 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 then diluted to 5% (v / v) with physiological saline. 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 3000 rpm for 5 min at 4°C 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 natural amyloid peptide Aβ and its variants.

[0055] Figure 5 The hemolysis rate of natural amyloid peptide Aβ and its variants.

[0056] like Figure 4 , Figure 5 As shown, the hemolysis rates of antimicrobial peptides Aβ-10R-YNGK and Aβ-10R-YNGK-F at a concentration of 5 μM were 8.71% and 8.25%, respectively (hemolysis is considered to be greater than 5%). Aβ-10R-YNGK and Aβ-10R-YNGK-F only exhibited antibacterial activity at concentrations above 5 μM, and hemolysis occurred at this concentration, indicating that these antimicrobial peptides do not possess good safety. The hemolysis rate of antimicrobial peptide Aβ-6R-YNGK-F at a concentration of 10 μM was 3.86%, while the hemolytic activity of antimicrobial peptide Aβ-6R-YNGK remained below 5% even at a high concentration of 50 μM. These results demonstrate that the modified antimicrobial peptide Aβ-6R-YNGK achieved high antimicrobial activity while maintaining high safety.

[0057] Example 7: Determination of the anticancer activity of antimicrobial peptides

[0058] The anticancer activity of antimicrobial peptides was evaluated using the CCK-8 assay. U937 (human lymphoma cell line) cells were cultured in 1640 medium (supplemented with 9% fetal bovine serum and 1% penicillin-streptomycin solution) at 37°C in a CO2 incubator. When the cells reached 80%-90% confluence, they were collected by centrifugation. The single-cell suspension was mixed, and the cells were counted using a hemocytometer to determine the cell concentration. The cells were seeded at a density of 10,000 cells / well in 96-well plates. 90 μL of culture medium and 10 μL of antimicrobial peptide solutions of different concentrations were added (the final concentrations of the antimicrobial peptides in the 96-well plates were 50 μM, 20 μM, 10 μM, 5 μM, and 1 μM). Each concentration was used in 6 replicates. 100 μL of PBS was added to the outermost wells to prevent water evaporation from the plate and affecting the drug concentration. After co-culturing the antimicrobial peptide with cells for 24 hours, 20 μL of CCK-8 solution was added, and the cells were incubated in a CO2 incubator at 37℃ for 1-4 hours. The plates were then removed, shaken for 10 minutes, and the absorbance was measured at 450 nm using a microplate reader from Meigu Molecular Instruments (Shanghai) Co., Ltd. The viability was calculated using the following formula:

[0059] Survival rate (%) = (OD of dosing well) 450 - Zeroing hole OD 450 ) / (Blank Hole OD) 450 - Zeroing hole OD 450 ) × 100%

[0060] The dosing wells contain added antimicrobial peptides, the blank wells contain only pure cells without added antimicrobial peptides, and the zeroing wells contain pure culture medium without added antimicrobial peptides or cells.

[0061] Figure 6Cell survival rate as a measure of the anticancer activity of natural amyloid peptide Aβ and its mutants.

[0062] like Figure 6 As shown, the modified antimicrobial peptides all reduced cancer cell survival in a dose-dependent manner. Specifically, at a concentration of 50 μM, the cancer cell survival rate of all antimicrobial peptides was below 20%. Notably, compared to other peptides, the antimicrobial peptides Aβ-10R-YNGK and Aβ-10R-YNGK-F, which exhibit higher hemolytic activity, showed significantly higher anticancer activity, especially at low concentrations. This may be due to the similarities between blood cells and cancer cells in certain aspects of their membranes.

[0063] 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 Aβ42 variant antimicrobial peptide, characterized in that: The Aβ42 variant antimicrobial peptide, abbreviated as Aβ-6R-YNGK-F, has the amino acid sequence shown in SEQ ID NO:

4.

2. The application of the Aβ42 variant antimicrobial peptide according to claim 1 in the preparation of antimicrobial agents, characterized in that: The antibacterial agent has inhibitory and bactericidal effects on Gram-negative and / or Gram-positive bacteria; The Gram-negative and / or Gram-positive bacteria are selected from one or more of Staphylococcus epidermidis, Escherichia coli, Escherichia fenrenz, and Bacillus subtilis.

3. The use of the Aβ42 variant antimicrobial peptide of claim 1 in the preparation of anti-lymphoma agents.

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