Cationic antibacterial peptide with amidated terminal as well as preparation method and application of cationic antibacterial peptide

Abstract

The invention provides a cationic antibacterial peptide with an amidated terminal as well as a preparation method and application of the cationic antibacterial peptide, and particularly belongs to the technical field of antibacterial peptide products. The invention provides a cationic antibacterial peptide with an amidated terminal, the amino acid sequence of the cationic antibacterial peptide is as shown in SEQ ID NO.1 or SEQ ID NO.2, and the C terminal of the cationic antibacterial peptide is amidated by adopting-NH2. The cationic antibacterial peptide directly acts on cell membranes of bacteria, has no specific target spot, can effectively reduce the risk of drug resistance in the use process, is high in antibacterial activity, low in hemolytic activity and easier in safety control, can be used in anti-infection clinical treatment, and has a wide development prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of antimicrobial peptide products, specifically relating to a terminally amidated cationic antimicrobial peptide, its preparation method, and its application. Background Technology

[0002] Antimicrobial drugs play a crucial role in the clinical treatment of diseases in humans and animals. The discovery and application of antibiotics have extended human lifespan, significantly improved quality of life, and brought revolutionary progress to the medical field. However, with the widespread and even irrational use of antimicrobial drugs, pathogens are becoming increasingly resistant to them. Currently, bacterial resistance worldwide is trending towards multidrug resistance, and controlling the harm caused by drug-resistant bacterial infections has become a major focus of attention in the medical field. Finding and developing new strategies and drugs to effectively control drug-resistant bacterial infections has become a common goal for scientists worldwide. In stark contrast, the development of new antibiotics has been extremely slow, with very few structurally novel antibiotics appearing in clinical treatment. Furthermore, new antibiotics such as streptozotocin, oxazolidinone, and lipopeptide antibiotics primarily target Gram-positive bacteria. Therefore, developing new antibacterial strategies and finding new antibiotic alternatives has become a matter of urgency. Antimicrobial peptides are small, bioactive polypeptides produced in vivo through induction. They are encoded by specific genes in various biological cells and generated under external conditions. They have relatively small molecular weights, with most ranging from 10 to 50 amino acids in length. Antimicrobial peptides possess antimicrobial activity, and their unique antimicrobial mechanisms have attracted widespread interest. However, broad-spectrum antimicrobial peptides are currently lacking. Summary of the Invention

[0003] The purpose of this invention is to provide a terminally amidated cationic antimicrobial peptide, its preparation method, and its applications. This cationic antimicrobial peptide acts directly on the bacterial cell membrane without a specific target. During use, it effectively reduces the risk of drug resistance, exhibits high antimicrobial activity, low hemolytic activity, high safety, and broad-spectrum activity, effective against both Gram-negative and Gram-positive bacteria, and also effective against clinically isolated drug-resistant strains. It can be used in clinical anti-infective treatment and has broad development prospects.

[0004] The present invention provides a terminally amidated cationic antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2, and the C-terminus of which is amidated with -NH2.

[0005] The present invention also provides a method for preparing the cationic antimicrobial peptide described in the above technical solution, comprising the following steps: synthesizing a crude polypeptide using microwave-promoted solid-phase synthesis according to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and purifying it using preparative liquid chromatography to obtain the cationic antimicrobial peptide.

[0006] The present invention also provides the application of the cationic antimicrobial peptide described in the above technical solution or the cationic antimicrobial peptide prepared by the preparation method described in the above technical solution in the preparation of antibacterial or anti-infective products.

[0007] The present invention also provides the application of the cationic antimicrobial peptide described in the above technical solution or the cationic antimicrobial peptide prepared by the preparation method described in the above technical solution in the preparation of antibacterial or antibacterial infection products.

[0008] Preferably, the bacteria include Gram-positive bacteria and / or Gram-negative bacteria, wherein the Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Escherichia coli and / or Pseudomonas aeruginosa.

[0009] Preferably, the product includes a drug.

[0010] Preferably, when the product is a drug, the product also includes pharmaceutically acceptable excipients.

[0011] Preferably, the drug includes an oral drug or a topical drug; the oral drug includes an oral preparation or an injection; the topical drug includes a patch or a topical preparation.

[0012] The present invention also provides an antibacterial product, which includes the cationic antimicrobial peptide described in the above technical solution or the cationic antimicrobial peptide or its salt and excipients prepared by the preparation method described in the above technical solution.

[0013] Preferably, the antibacterial product also includes other substances with antibacterial properties.

[0014] This invention provides a terminally amidated cationic antimicrobial peptide. This invention relates to a novel cationic antimicrobial peptide that interacts with the negatively charged lipopolysaccharides and phospholipids in the bacterial cell wall and outer membrane through the positive charge of the basic amino acids in its structure. This disrupts the integrity of the bacterial cell membrane, causing perforation and resulting in the leakage of cell contents, thus killing the bacteria. Because this cationic antimicrobial peptide acts directly on the bacterial cell membrane without a specific target, it effectively reduces the risk of drug resistance during use. This cationic antimicrobial peptide has broad-spectrum activity, effective against both Gram-negative and Gram-positive bacteria, including clinically isolated drug-resistant strains. It exhibits low hemolytic activity and high safety. This cationic antimicrobial peptide is a short peptide with fewer than 15 amino acids, characterized by its small molecular weight, simple synthesis, and low hemolytic activity. Compared to long peptides or protein-based antimicrobial peptides, short peptides have lower immunogenicity, a lower risk of inducing allergic reactions or immune responses, and easier safety control. Therefore, this cationic antimicrobial peptide and its pharmaceutically acceptable salts have broad development prospects for clinical anti-infective treatment. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The concentration-hemolysis rate curves of the compounds in Examples 1 and 2 provided by this invention are shown in the figure. Detailed Implementation

[0017] This invention provides a terminally amidated cationic antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2, and the C-terminus of the cationic antimicrobial peptide is amidated with -NH2. The cationic antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.1 is: Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Arg-Leu-NH2 (RRLFRRILRRL-NH2); the cationic antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.2 is Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Arg-Arg-Leu-NH2 (KKLFKKILKYLRRL-NH2). The cationic antimicrobial peptide of this invention is chemically stable and has the characteristics of small molecular weight, simple synthesis, and low hemolytic activity. Specifically, the cationic antimicrobial peptide of the present invention has a novel structure and stable chemical properties. It can interact with the positive charge of the basic amino acids in its structure and the negative charge of lipopolysaccharides and phospholipids in the bacterial cell wall and outer membrane, thereby destroying the integrity of the bacterial cell membrane and causing perforation, resulting in the leakage of cell contents and killing bacteria.

[0018] This invention also provides a method for preparing the cationic antimicrobial peptide described in the above-mentioned technical solution, comprising the following steps: synthesizing a crude polypeptide using microwave-promoted solid-phase synthesis according to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and purifying it using preparative liquid chromatography to obtain the cationic antimicrobial peptide. This invention does not specifically limit the specific methods and conditions for microwave-promoted solid-phase synthesis; conventional microwave-promoted solid-phase synthesis methods are acceptable. In a specific embodiment, the cationic antimicrobial peptide is synthesized efficiently and rapidly using microwave-promoted Fmoc / tBu orthogonal protected solid-phase synthesis. Compared with traditional liquid-phase preparation, the preparation method of this invention has the advantages of high coupling efficiency and short synthesis cycle, which is beneficial for industrial-scale synthesis. This invention does not specifically limit the methods and conditions for preparative liquid chromatography; conventional preparative liquid chromatography methods are acceptable.

[0019] This invention also provides the application of the cationic antimicrobial peptide described in the above-described technical solutions or the cationic antimicrobial peptide prepared by the preparation method described in the above-described technical solutions in the preparation of antibacterial or anti-infective products. The anti-infective products of this invention can be used for clinical anti-infective treatment, and the cationic antimicrobial peptides of this invention are suitable as active ingredients in clinical anti-infective therapeutic drugs. In specific embodiments, the product includes a drug. In specific embodiments, when the product is a drug, the product also includes pharmaceutically acceptable excipients. In specific embodiments, the drug includes an oral drug or a topical drug; the oral drug includes an oral preparation or an injection; the topical drug includes a patch or a topical application.

[0020] This invention also provides the application of the cationic antimicrobial peptide described in the above-described technical solutions or the cationic antimicrobial peptide prepared by the preparation method described in the above-described technical solutions in the preparation of antibacterial or antibacterial infection products. In a specific embodiment, the bacteria include Gram-positive bacteria and / or Gram-negative bacteria, the Gram-positive bacteria including Staphylococcus aureus, and the Gram-negative bacteria including Escherichia coli and / or Pseudomonas aeruginosa. In a specific embodiment, the product includes a drug. In a specific embodiment, when the product is a drug, the product also includes pharmaceutically acceptable excipients. In a specific embodiment, the drug includes an oral drug or a topical drug; the oral drug includes an oral preparation or an injection; the topical drug includes a patch or a topical application.

[0021] This invention also provides an antibacterial product comprising the cationic antimicrobial peptide described in the above-described technical solution or the cationic antimicrobial peptide prepared by the preparation method described in the above-described technical solution, or its salt and excipients. In a specific embodiment, the salt may be a pharmaceutically acceptable salt of the cationic antimicrobial peptide described in the above-described technical solution or the cationic antimicrobial peptide prepared by the preparation method described in the above-described technical solution. In a specific embodiment, the excipient may be a diluent or a carrier. In a specific embodiment, the antimicrobial product further includes other substances with antimicrobial activity.

[0022] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a terminally amidated cationic antimicrobial peptide, its preparation method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0023] The following abbreviations are used throughout this specification: DCM: Dichloromethane; NMP: N-methylpyrrolidone; DIPEA: N,N-diisopropylethylamine; TFA: Trifluoroacetic acid; EDT: Ethylene dithiol; Fmoc: N-9-fluorenylmethoxycarbonyl; HBTU: Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; HOBT: 1-hydroxy-benzotriazole; ESI-MS: Electrospray ionizer; Leu: Leucine; Lys: Lysine; Phe: Phenylalanine; Ile: Isoleucine; Tyr: Tyrosine; Arg: Arginine.

[0024] Unless otherwise specified, the test reagents and materials used in the embodiments of this invention are all commercially available products.

[0025] Example 1 Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Arg-Leu-NH2 (SEQ ID NO.1) (1) Swelling of resin Weigh 50 mg of Fmoc-Rink amide-MBHA Resin (substitution amount 0.4 mmol / g), swell with 7 mL of DCM for 30 min, filter to remove DCM, swell with 10 mL of NMP for 30 min, and finally rinse with 7 mL of NMP, DCM and NMP respectively.

[0026] (2) Microwave-promoted removal of Fmoc protecting groups The swollen resin was placed in a reactor, and 7 mL of a 25% piperidine / NMP (v / v) solution containing 0.1 M HOBT was added. The reaction was carried out in a microwave reactor for 1 min at a microwave power of 15 W, with the reaction temperature controlled below 50 °C. Compressed air was used for cooling. After the reaction, the solution was filtered off. Another 7 mL of the 25% piperidine / NMP (v / v) solution containing 0.1 M HOBT was added, and the reaction was carried out in the microwave reactor for another 4 min at a microwave power of 25 W, with the reaction temperature controlled at 50 °C. Compressed air was used for cooling. After the reaction, the solution was filtered off, and the resin was washed thoroughly with NMP. The resin with the initially attached Fmoc protecting group removed was obtained.

[0027] (3) Microwave-promoted synthesis of Fmoc-Leu-Rink amide-MBHA Resin Fmoc-Leu-OH (0.04 mmol), HBTU (0.04 mmol), HOBT (0.04 mmol), and DIPEA (0.08 mmol) were dissolved in 10 mL of NMP. This solution was then added to the resin obtained in step (2) after the initial Fmoc protecting group was removed. The reaction was carried out in a microwave reactor for 7 min at a microwave power of 25 W and a reaction temperature of 50 °C, using compressed air from an air compressor for cooling. After the reaction was completed, the reaction solution was filtered off, and the resin was washed three times with 7 mL each of DCM and NMP.

[0028] (4) Detection of coupling efficiency Take a small amount of the resin particles obtained in step (3), wash them with ethanol, put them in a transparent vial, and add 5% ninhydrin ethanol and 2×10 5 Add 2 drops each of mol / L KCN pyridine solution and 80% phenol ethanol solution, and heat at 80℃ for 5 minutes. If the resin turns blue, it is positive, indicating the presence of free amino acids, and the coupling time needs to be extended. If the resin turns white or light yellow, it is negative, the coupling is complete, and the next amino acid coupling cycle can begin.

[0029] (5) Elongation of peptide chains Following the order of the peptide chains, the above deprotection and coupling steps were repeated from the C-terminus to the N-terminus to connect the corresponding amino acids. The coupling reaction was promoted by microwave for 5 to 25 minutes to obtain the peptide-resin complex.

[0030] (6) Cleavage of peptides on resin The obtained peptide-resin complex was placed in a reaction flask, and 10 mL of cleavage agent Reagent K (TFA / anisole / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added to each. The mixture was first shaken at 0 °C for 30 min, and then reacted at room temperature for 3 h. After the reaction, the mixture was filtered, washed three times with a small amount of TFA and DCM, and the filtrates were combined. The filtrate was added to a large amount of ice-cold diethyl ether to precipitate a white flocculent precipitate, which was then centrifuged at freeze temperature to obtain the crude target peptide. Finally, 43.2 mg of the crude target compound was obtained, with a yield of 78.5%.

[0031] (7) Purification of polypeptides The crude polypeptide was dissolved in 50% acetonitrile / water and purified by preparative liquid chromatography (HPLC). The chromatographic conditions were: C18 reversed-phase column (320 mm × 28 mm, 5 µm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40%–90%, 60 min; flow rate 5 mL / min; detection wavelength 214 nm. The collected solution was lyophilized to obtain 26.8 mg of pure product. The theoretical relative molecular mass was 1554.98. ESI-MS m / z: calculate [M+2H] 2+ 777.99, [M+3H] 3+ 518.99; found [M+2H] 2+ 778.79, [M+3H] 3+ 519.15.

[0032] Example 2 Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Arg-Arg-Leu-NH2 (SEQ ID NO. 2) The cationic antimicrobial peptides of Example 2 were synthesized according to the general method described in Example 1, based on the corresponding sequences, and their molecular weights were confirmed by ESI-MS. The theoretical relative molecular mass is 1846.44. ESI-MS m / z: calculate [M+2H] 2+ 924.22, [M+3H] 3+ 616.48; found [M+2H] 2+ 924.23, [M+3H] 3+ 616.51.

[0033] Example 3 This invention uses a dilution method for drug susceptibility testing to determine the antibacterial activity of the compounds in Examples 1 and 2: Choose Escherichia coli ( E. coli ATCC 25922), Pseudomonas aeruginosa ( P. aeruginosa ATCC27853), Staphylococcus aureus ( S. aureus ATCC 25923), clinical strain of Escherichia coli ( E. coli IS, sourced from the Department of Laboratory Medicine, Zhongda Hospital Affiliated to Southeast University), clinically resistant strains of Pseudomonas aeruginosa ( P. aeruginosa IS, DR, sourced from the Department of Laboratory Medicine, Zhongda Hospital Affiliated to Southeast University), clinically resistant strains of Staphylococcus aureus ( S. aureusIS, DR (from the Department of Laboratory Medicine, Zhongda Hospital Affiliated to Southeast University), after resuscitation, was inoculated onto slant culture and incubated overnight at 37°C. The bacteria were then inoculated onto ordinary LB medium and incubated overnight at 37°C. The bacterial suspension was diluted to a concentration of 1×10⁻⁶. 4 ~10 5 CFU / ml, 100 μL of bacterial culture was inoculated into 96-well plates, divided into experimental group, positive control group, and blank control group. 10 μL of different concentrations of the compounds from Examples 1 and 2, ampicillin, and pexiganan were added to the experimental group and positive control group, respectively, at concentration gradients of 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL. The plates were then incubated at 37°C for 12 h, and the OD value at 620 nm was measured using a microplate reader. The drug concentration at which the ratio of the OD value of the experimental group / positive control group to the OD value of the blank control group was greater than 90% was defined as the minimum inhibitory concentration (MIC).

[0034] Table 1. MIC values ​​(μg / mL) of the compounds in the examples.

[0035] As shown in Table 1, the smaller the minimum inhibitory concentration (MIC) value, the stronger the antibacterial ability. The antibacterial activity of the compounds in Examples 1 and 2 was significantly higher than that of the positive controls ampicillin and pecidganam.

[0036] Example 4 This invention uses a hemolytic assay to determine that the compounds in Examples 1 and 2 have low hemolytic toxicity to erythrocytes: Take 20 ml of fresh rabbit blood into a 50 ml centrifuge tube containing anticoagulant, add 40 ml of PBS buffer, centrifuge at 1500 rpm for 15 min at 4°C, and discard the supernatant. Wash three times with ice-cold PBS buffer, then add 50 ml of PBS buffer and gently pipette to prepare a red blood cell suspension with a concentration of approximately 4%. Then add 200 μl of red blood cell suspension and 100 μl of different concentrations of the test compound to each well of a 48-well plate. PBS buffer is used as a negative control, and 1% Triton X-100 is used as a positive control. After incubation at 37°C for 1 h, remove the 48-well plate, centrifuge at 1500 rpm for 15 min at 4°C, and transfer 200 μl of supernatant to another 48-well plate. Detect the OD value at 540 nm using a microplate reader. Hemolysis rate (%) = (OD dosing well - OD negative well) / (OD positive well - OD negative well) × 100%. Based on the formula, the hemolysis rate of compounds of different concentrations in Examples 1 and 2 was calculated. Finally, the concentration-hemolysis rate curve was plotted using GraphPad Prism 7.0.

[0037] Figure 1 Concentration-hemolysis rate curves for different concentrations of the compounds in Examples 1 and 2 are shown. Since most cationic polypeptides can alter the permeability of erythrocyte cell membranes, leading to K... + Na + Ca 2+ Imbalances in intracellular ion concentrations can lead to cell lysis, exhibiting strong hemolytic toxicity and limiting its clinical use. However, if... Figure 1 As shown, when the concentration of the compounds in Examples 1 and 2 was 512 μmol / L, the hemolysis rate of erythrocytes was less than 10%, indicating that the compounds in Examples 1 and 2 had low hemolytic toxicity to erythrocytes.

[0038] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A terminally amidated cationic antimicrobial peptide, characterized in that, The amino acid sequence of the cationic antimicrobial peptide is shown in SEQ ID NO.1 or SEQ ID NO.2, and the C-terminus of the cationic antimicrobial peptide is amidated with -NH2.

2. The method for preparing the cationic antimicrobial peptide according to claim 1, characterized in that, Includes the following steps: The crude polypeptide was synthesized using microwave-promoted solid-phase synthesis based on the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and purified using preparative liquid chromatography to obtain the cationic antimicrobial peptide.

3. The use of the cationic antimicrobial peptide of claim 1 or the cationic antimicrobial peptide prepared by the preparation method of claim 2 in the preparation of antibacterial or anti-infective products.

4. The use of the cationic antimicrobial peptide of claim 1 or the cationic antimicrobial peptide prepared by the preparation method of claim 2 in the preparation of antibacterial or antibacterial infection products.

5. The application according to claim 4, characterized in that, The bacteria include Gram-positive and / or Gram-negative bacteria, the Gram-positive bacteria including Staphylococcus aureus, and the Gram-negative bacteria including Escherichia coli and / or Pseudomonas aeruginosa.

6. The application according to claim 3 or 4, characterized in that, The products include pharmaceuticals.

7. The application according to claim 6, characterized in that, When the product is a drug, the product also includes pharmaceutically acceptable excipients.

8. The application according to claim 6, characterized in that, The drug includes oral or topical medications; the oral medication includes oral preparations or injections; the topical medication includes patches or ointments.

9. An antibacterial product, characterized in that, The antimicrobial product includes the cationic antimicrobial peptide of claim 1 or the cationic antimicrobial peptide prepared by the preparation method of claim 2, or its salt and excipients.

10. The product according to claim 9, characterized in that, The antibacterial products also include other substances with antibacterial effects.

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