Antibacterial nonapeptide and use thereof

Abstract

The application discloses an antibacterial nonapeptide and application thereof, and belongs to the field of antibacterial peptide synthesis. The application designs an antibacterial peptide with an amino acid sequence as shown in SEQ ID NO. 1; and further provides application of the antibacterial peptide in inhibiting growth of bacteria and the like. Experimental results show that the antibacterial peptide can efficiently inhibit staphylococcus aureus and escherichia coli, and the MIC is as low as 4-8 mu g / mL; meanwhile, the antibacterial peptide still maintains low cytotoxicity and hemolyticity under high concentration. Moreover, the antibacterial peptide provided by the application has a length of only 9 amino acids, and is easy to synthesize and prepare. The application provides a new antibacterial peptide for developing bacteriostatic agents and drugs for preventing or treating bacterial infections, and the antibacterial peptide has advantages of strong bacteriostatic capacity, broad-spectrum bactericidal activity, safety performance, low-cost synthesis and application and the like. The antibacterial peptide provided by the application has outstanding practical value and wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide synthesis, and in particular to an antimicrobial nonapeptide and its applications. Background Technology

[0002] Bacterial infections are a major threat to public health, especially among the immunocompromised, hospitalized, and elderly, where they can easily lead to serious complications. While the widespread use of traditional antibiotics has been effective in controlling many infections, it has also led to the rapid development of drug resistance in pathogens, resulting in multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and drug-resistant Escherichia coli (ESBLs), severely limiting the clinical effectiveness of anti-infective treatments.

[0003] To address this challenge, researchers have increasingly turned to exploring antimicrobial agents with novel mechanisms. Among these, antimicrobial peptides are considered highly promising alternatives due to their natural presence in various organisms and their combination of rapid bactericidal action, broad-spectrum antimicrobial activity, and low risk of drug resistance. Antimicrobial peptides exert their bactericidal effects primarily through disruption of bacterial cell membranes, charge adsorption, and pore formation.

[0004] However, most known antimicrobial peptides suffer from problems such as long sequences (often 20-50 amino acid residues), high synthesis costs, poor stability, short in vivo half-life, or toxicity to mammalian cells, which limit their industrial application. For example:

[0005] The patent with publication number CN117106034A discloses a class of artificially designed antimicrobial peptides that have certain antimicrobial effects. However, all of its peptide segments have more than 13 amino acids, and some modification sites still cause the problem of high hemolysis rate, which limits its direct use in the human body.

[0006] The Caerin1.1 modified peptide developed in the patent with publication number CN114395026A effectively improves the antibacterial ability against Gram-negative bacteria, but its peptide segment has 32 amino acids, making it difficult to reduce the synthesis cost and posing a potential risk of immunogenicity.

[0007] In summary, the design of antimicrobial peptides in existing publicly available technologies still faces the challenge of balancing synthetic economy, safety, and antimicrobial efficacy. Therefore, developing short-chain, structurally optimized antimicrobial peptides with high bactericidal activity and low toxicity is a key research focus. Thus, there is an urgent need to develop novel antimicrobial peptides that are short-chain, highly stable, low in toxicity, and possess broad-spectrum antimicrobial effects. Summary of the Invention

[0008] The purpose of this invention is to provide an antimicrobial nonapeptide and its application to solve the problems existing in the prior art. This invention screens and designs an antimicrobial peptide composed of 9 amino acid residues, which has both antibacterial efficacy and safety, and is inexpensive to prepare. It provides a new antimicrobial peptide for the development of antibacterial agents and drugs for the prevention or treatment of bacterial infections, and has outstanding practical value and broad application prospects.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] The present invention provides an antimicrobial nonapeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0011] The present invention also provides the application of the above-mentioned antimicrobial nonapeptide in the preparation of products that inhibit bacterial growth.

[0012] Furthermore, the bacteria are Staphylococcus aureus or Escherichia coli.

[0013] The present invention also provides an antibacterial agent, wherein the antibacterial agent has the above-mentioned antimicrobial nonapeptide as the main active ingredient.

[0014] Furthermore, it also includes excipients acceptable in the field of antibacterial agents.

[0015] The present invention also provides the use of the above-mentioned antimicrobial nonapeptide in the preparation of a medicament for the prevention and / or treatment of bacterial infections.

[0016] Furthermore, the bacteria are Staphylococcus aureus or Escherichia coli.

[0017] The present invention also provides a medicament for the prevention and / or treatment of bacterial infections, wherein the medicament has the above-mentioned antimicrobial nonapeptide as the main active ingredient.

[0018] Furthermore, it also includes pharmaceutically acceptable excipients.

[0019] Furthermore, the dosage form of the drug is a spray, gel, capsule, tablet, suppository, or lotion.

[0020] The present invention discloses the following technical effects:

[0021] This invention designs an antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.1; further, it provides the application of this antimicrobial peptide in inhibiting bacterial growth. Experimental results show that the antimicrobial peptide provided by this invention can effectively inhibit Staphylococcus aureus and Escherichia coli, with a MIC as low as 4-8 μg / mL; simultaneously, the antimicrobial peptide maintains low cytotoxicity and hemolytic activity even at high concentrations. Moreover, the antimicrobial peptide provided by this invention is only 9 amino acids long, making it easy to synthesize. This invention provides a novel antimicrobial peptide for the development of antibacterial agents and drugs for the prevention or treatment of bacterial infections. This antimicrobial peptide has advantages such as strong antibacterial ability, broad-spectrum bactericidal activity, safety, and low-cost synthesis and application. The antimicrobial peptide provided by this invention has outstanding practical value and broad application prospects. Attached Figure Description

[0022] 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.

[0023] Figure 1 The HPLC results of the antibacterial nonapeptide of the present invention are shown in the figure.

[0024] Figure 2 This is the mass spectrometry result of the antibacterial nonapeptide of the present invention;

[0025] Figure 3 The graph shows the hemolytic activity of the antibacterial nonapeptide of the present invention at a concentration of 25 μg / mL. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] The antimicrobial peptide of the present invention contains 9 amino acid residues and is also named antimicrobial nonapeptide, the sequence of which is shown in SEQ ID NO.1.

[0032] SEQ ID NO.1: IFLKIWRLW.

[0033] Example 1

[0034] 1. Solid-phase synthesis of antimicrobial peptides

[0035] 1.1 Resin Pretreatment

[0036] Take 0.6 g of 2-chlorotriphenylmethyl chloride resin (2-Chlorotrityl Chloride Resin, degree of substitution 0.4 mmol / g), place it in a reaction tube, add dichloromethane (DCM) at a volume of 15 mL / g resin, and shake thoroughly on a shaker for 30 min to allow the resin to swell.

[0037] 1.2 Grafting the first amino acid

[0038] After removing DCM by filtration, 3 molar equivalents of Fmoc-L-Leu-OH, 10 molar equivalents of diisopropylethylamine (DIEA), and an appropriate amount of dimethylformamide (DMF) were added to the resin sequentially to dissolve them. The mixture was then stirred and reacted for 1 hour. After the reaction was completed, the resin was washed 6 times alternately with DMF and DCM.

[0039] 1.3 Removal of Fmoc group

[0040] The deprotection treatment was performed twice using 20% ​​piperidine DMF solution (15 mL / g), with the first reaction lasting 5 min and the second reaction lasting 15 min.

[0041] 1.4 Grafting effect test

[0042] After the piperidine solution is completely removed, take a portion of the resin and wash it three times with anhydrous ethanol. Add one drop each of ninhydrin, KCN, and phenol, and heat at 105-110℃ for 5 minutes. If a deep blue color appears, it indicates that amino acids have been successfully loaded onto the resin.

[0043] 1.5 Resin Cleaning

[0044] Two rounds of washing were performed alternately with DMF and methanol (10 mL / g each) to remove residual reagents.

[0045] 1.6 Subsequent amino acid condensation

[0046] Dissolve 3 molar equivalents of Fmoc-L-Gly-OH and 3 molar equivalents of HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate) in a small amount of DMF, add them to the reaction tube, and immediately add 10 molar equivalents of N-methylmorpholine (NMM). The reaction time is 30 min.

[0047] 1.7 Repeat the cleaning steps

[0048] After condensation, the reaction tube was cleaned sequentially with DMF and methanol to remove residual activator.

[0049] 1.8 Sequence Stepwise Assembly

[0050] Repeat steps 1.2-1.6, sequentially linking the required amino acids from the C-terminus to the N-terminus onto the resin support until peptide chain synthesis is complete.

[0051] 1.9 End-of-line protection removal and final resin cleaning

[0052] After linking the last amino acid, terminal Fmoc removal was performed, followed by washing twice each with DMF, methanol, and DCM (10 mL / g each), and then the mixture was dried for 10 min before use.

[0053] 1.10 Peptide cleavage

[0054] Prepare the cutting solution (TFA 94.5%, water 2.5%, EDT 2.5%, TIS 1%), using 10 mL / g resin. Place the resin in a flask or centrifuge tube, add the cutting solution, and shake the mixture at a constant temperature for 2 hours to release the peptide.

[0055] 1.11 Pyrolysis solution treatment

[0056] The lysis buffer was dried by blowing with nitrogen gas, and the crude peptide fragments were precipitated by adding diethyl ether. The buffer was washed with diethyl ether six times and then evaporated to dryness at room temperature.

[0057] 1.12 HPLC Purification Procedure

[0058] 1) Dissolve 200 mg of crude peptide in 2-5 mL of 50% acetonitrile-water solution, and sonicate briefly (about 2 min) to aid dissolution;

[0059] 2) The solution obtained by filtration using a 0.45 μm filter membrane;

[0060] 3) Analytical purity: Inject 3 μL of the sample into the analytical HPLC system, using water / acetonitrile as the mobile phase, and perform gradient elution for 30 min. The initial ratio is 95% water / 5% acetonitrile, and the final ratio is 5% water / 95% acetonitrile. Equilibrate for 5 min before injection.

[0061] 4) Preparation and purification: The sample was subjected to preparative HPLC with an equilibration time of 10 min, an initial gradient of 95% water / 5% acetonitrile, an ending gradient of 25% water / 75% acetonitrile, and a gradient time of 40 min. The target peak was then collected.

[0062] 5) Purity and molecular weight identification: The collected samples were subjected to HPLC purity detection and mass spectrometry (MS) analysis.

[0063] 1.13 Freeze-drying treatment

[0064] The purified peptide solution is freeze-dried to obtain the finished antimicrobial peptide product.

[0065] 1.14 Saving Method

[0066] The obtained polypeptide is a white powder, which is sealed in packaging and stored at -20°C for a long time.

[0067] The HPLC analysis results of the synthesized antimicrobial nonapeptide in this embodiment are as follows: Figure 1 As shown, the mass spectrometry results are as follows: Figure 2 As shown.

[0068] Example 2

[0069] Detection of the antimicrobial activity of antimicrobial peptides.

[0070] The antimicrobial performance of the antimicrobial nonapeptide prepared in Example 1 was evaluated using a 96-well plate assay. The bacterial strain used was purchased from the Guangdong Provincial Microbial Culture Collection Center.

[0071] The experimental steps are as follows:

[0072] 1. Preparation of inoculum

[0073] Staphylococcus aureus and Escherichia coli were inoculated onto sterile MHA solid medium and incubated overnight at 37°C. Single colonies were picked and inoculated into MHB liquid medium and cultured at 37°C and 120 rpm for 18 h with shaking.

[0074] 2. Preparation and inoculation of antimicrobial peptide gradients

[0075] The antimicrobial peptide was prepared as a stock solution of 1024 μg / mL using PBS. Working solutions with concentrations of 1024, 512, 256, 128, 64, 32, 16, 8, and 4 μg / mL were prepared by 2-fold dilution. 100 μL of each concentration was added to columns 2 through 10 (BG rows) of a 96-well plate, with 6 replicates.

[0076] Bacterial culture medium diluted to 5×10 5 CFU / mL, 100 μL was added to wells B2 through D10 as the experimental group. The working concentration (μg / mL) of the antimicrobial peptide in each well during this stage is detailed in Table 1.

[0077] Table 1. Antimicrobial peptide concentrations in each column of a 96-well plate.

[0078] Number of columns 2 3 4 5 6 7 8 9 10 Concentration (μg / mL) 512 256 128 64 32 16 8 4 2

[0079] 3. Compare settings

[0080] 100 μL of MHB was added to wells E2-G10 as a blank control; PBS and MHB solutions were added to wells B11-D11 as negative controls; PBS and diluted bacterial solution were added to wells E11-G11 as positive controls.

[0081] After sealing, the plate was sealed with sealing film, placed in a sealed bag, and incubated at 37°C and 120 rpm for 18 hours.

[0082] 4. OD value detection and MIC determination

[0083] After cultivation, the OD600 values ​​of each well (B2-D10) were measured using a microplate reader. The concentration of the antimicrobial peptide corresponding to the lowest OD600 value is the minimum inhibitory concentration (MIC) for that strain. Table 2 shows the minimum inhibitory concentration (MIC, unit: μg / mL) of the antimicrobial nonapeptide described in this invention against the target strain.

[0084] Table 2 Calculation results of antimicrobial peptide MIC

[0085] target strain S.aureus E. coli MIC (μg / mL) 4 8

[0086] The smaller the MIC value, the stronger the inhibitory effect of the antimicrobial peptide on the strain. As shown in Table 2, the antimicrobial peptide described in this invention has significant antibacterial effects against both Gram-positive and Gram-negative bacteria, exhibiting good broad-spectrum antibacterial properties.

[0087] Example 3

[0088] Evaluation of the hemolytic effect of antimicrobial peptides at specific concentrations.

[0089] To evaluate the potential membrane toxicity of the antimicrobial peptide of this invention to mammalian cells at effective concentrations, a mouse erythrocyte model was used to detect its hemolytic activity. The experimental concentration of the antimicrobial peptide was set at 25 μg / mL.

[0090] 1. Collection and processing of red blood cells

[0091] Healthy BALB / c mice were selected, and blood was collected from their necks in test tubes containing EDTA anticoagulant. Red blood cells were separated by centrifugation at 1500 rpm for 5 min. After discarding the supernatant and leukocyte layer, the red blood cells were washed three times with sterile PBS (pH 7.4) until the supernatant was clear. A 4% (v / v) red blood cell suspension was prepared for use.

[0092] 2. Sample preparation and experimental design

[0093] The antimicrobial peptide prepared in Example 1 was dissolved in PBS buffer at a concentration of 25 μg / mL. 100 μL of the antimicrobial peptide solution was added to each well, followed by 100 μL of 4% erythrocyte suspension. After mixing, this was used as the experimental group, and the mixture was repeated 5 times. An additional Gramicidin experimental group was included, consisting of 100 μL of 25 μg / mL Gramicidin mixed with 100 μL of 4% erythrocyte suspension. This was used to evaluate the hemolytic effects of commonly published antimicrobial peptides at the same concentration and to compare their hemolytic activity with that of the antimicrobial nonapeptide of this invention.

[0094] The following control groups were set up in this experiment: the negative control group was a mixture of 100 μL PBS and 100 μL red blood cell suspension, which was used to represent the non-hemolytic state; the positive control group was a mixture of 100 μL 0.5% Triton X-100 and 100 μL 4% red blood cell suspension, which was used to represent the complete hemolytic state.

[0095] 3. Incubation and Detection

[0096] The 96-well plate was incubated at 37°C for 1 hour, followed by centrifugation at 1500 rpm for 10 minutes. The supernatant from each well was collected, and its absorbance (OD) was measured at 542 nm using a microplate reader. 542 ).

[0097] 4. Data Calculation and Results

[0098] The hemolysis rate is calculated using the following formula:

[0099]

[0100] A1: OD of the experimental group 542 value;

[0101] A2: OD in the negative control group 542 value;

[0102] A3: OD in the positive control group 542 value.

[0103] Figure 3 The results of the hemolytic activity of the antimicrobial nonapeptide of the present invention at a concentration of 25 μg / mL were demonstrated. Figure 3 The results showed that at a concentration of 25 μg / mL, the hemolysis rate of the antimicrobial peptide described in this invention on BALB / c mouse erythrocytes was consistently below 5%, significantly lower than that of the Gramicidin experimental group at the same concentration, and far lower than the complete hemolysis level induced by 0.5% Triton X-100. These results indicate that the antimicrobial peptide of this invention has relatively good biocompatibility.

[0104] In summary, the antimicrobial peptide of this invention exhibits significant in vitro antibacterial activity based on its unique amino acid composition and sequence. Particularly noteworthy is that even at high concentrations, its cytotoxicity and hemolytic activity remain at low levels. Therefore, this antimicrobial nonapeptide is suitable for preparing drugs for treating infectious diseases such as bacterial pneumonia and sepsis.

[0105] The broad-spectrum antibacterial activity of the antimicrobial nonapeptide of this invention stems from its mechanism of action in disrupting bacterial cell membranes. The antimicrobial nonapeptide molecule carries a positive charge and can adsorb onto the negatively charged bacterial cell membrane through electrostatic interactions. When the concentration of the antimicrobial nonapeptide adsorbed on the membrane surface accumulates to a critical threshold, its inherent amphiphilic structure causes the polypeptide molecule to insert into the phospholipid bilayer of the cell membrane and self-assemble therein to form transmembrane channels, ultimately leading to membrane disintegration and bacterial death. This mode of action enables it to effectively kill a variety of pathogens.

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antibacterial nonapeptide, characterized in that, The amino acid sequence of the antibacterial nonapeptide is shown as SEQ ID NO.

1.

2. Use of an antibacterial nonapeptide according to claim 1 for the preparation of a product for inhibiting the growth of bacteria, characterized in that, The bacteria are Staphylococcus aureus or Escherichia coli.

3. A bacteriostatic agent, characterized in that, The bacteriostatic agent takes the antibacterial nonapeptide of claim 1 as the main effective component.

4. The bacteriostatic agent of claim 3, wherein, It also includes adjuvants acceptable in the field of bacteriostatic agents.

5. Use of the antibacterial nonapeptide according to claim 1 for the preparation of a medicament for the prevention and / or treatment of bacterial infections, characterized in that, The bacteria are Staphylococcus aureus or Escherichia coli.

6. A medicament for preventing and / or treating a bacterial infection, characterized by, The drug takes the antibacterial nonapeptide of claim 1 as the main effective component.

7. The medicament according to claim 6, wherein It also includes pharmaceutically acceptable adjuvants.

8. The medicament according to claim 7, wherein The dosage form of the drug is spray, gel, capsule, tablet, suppository or lotion.

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