An antibacterial peptide 16253 or derivative thereof, pharmaceutical composition and application

Abstract

The application discloses a kind of antibacterial peptide 16253 or its derivative, pharmaceutical composition and application in the biotechnology field, the amino acid sequence of the antibacterial peptide 16253 is as shown in SEQ ID NO:1 (AAWRKWLGKVLRKAKKYRRIKK), with broad-spectrum, efficient bacteriostatic activity, Escherichia, pseudomonas, salmonella, streptococcus, bacillus and Campylobacter etc. Multiple gram-negative bacteria and gram-positive bacteria show significant bacteriostatic and bactericidal effect, and it is not easy to induce bacterial drug resistance, hemolyticity is very low, cytotoxicity is low, and selectivity index is good.The application also provides the derivative, salt of the antibacterial peptide, the pharmaceutical composition comprising the same, and the application thereof in the preparation of bacteriostatic product or antibacterial drug, especially in the preparation of drug for preventing or treating Campylobacter infection, with wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antimicrobial peptide 16253 or its derivatives, pharmaceutical compositions, and applications. Background Technology

[0002] With the widespread use of antibiotics in aquaculture and clinical treatment, the problem of bacterial resistance is becoming increasingly serious, with multidrug-resistant strains constantly emerging. Due to the complexity of resistance mechanisms, the effectiveness of traditional antibiotic treatments is limited, making the development of novel antimicrobial strategies an urgent need in the field of public health.

[0003] Antimicrobial peptides (AMPs), as key components of the body's innate immune defense system, possess broad-spectrum antimicrobial activity. Because they primarily exert their effects through physical killing mechanisms (such as disrupting bacterial cell membranes), they are less likely to induce bacterial resistance, thus gaining increasing attention as "secondary antibiotics." Antimicrobial peptides are widely available, found in animals, plants, and bacteria, forming a natural immune barrier against pathogen invasion. Their high antimicrobial activity, broad antimicrobial spectrum, diverse types, and low tendency to induce resistance suggest broad application prospects in combating drug-resistant pathogens. Furthermore, antimicrobial peptides can produce synergistic effects when used in combination with existing drugs or act as immunomodulators to mobilize the body's immune response, demonstrating significant clinical application potential.

[0004] However, despite the advantages of antimicrobial peptides over traditional antibiotics, their clinical application still faces many challenges: for example, large-scale production is costly; peptide drugs are easily hydrolyzed by proteases, resulting in poor in vivo stability; and some antimicrobial peptides have significant cytotoxicity, which can lead to side effects such as hemolysis of red blood cells.

[0005] Therefore, it is necessary to develop stable, low-toxicity, and highly effective broad-spectrum antimicrobial drugs by artificially designing and synthesizing new antimicrobial peptides in order to effectively solve the problem of drug resistance in pathogenic bacteria. Summary of the Invention

[0006] The purpose of this invention is to provide an antimicrobial peptide 16253 or its derivatives, a pharmaceutical composition and its application, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides an antimicrobial peptide 16253, the amino acid sequence of which is as follows:

[0008] AAWRKWLGKVLRKAKKYRRIKK (SEQ ID NO:1); or

[0009] The amino acid sequence of SEQ ID NO: 1 that has undergone substitution and / or deletion and / or addition of one or more amino acid sequences and has the same function as SEQ ID NO: 1; or

[0010] The amino acid sequence of SEQ ID NO:1 that has undergone substitution and / or deletion and / or addition of one or more amino acid sequences and has the same function as SEQ ID NO:1.

[0011] Preferably, the above-mentioned antimicrobial peptide 16253 is configured as an L-type polypeptide, a D-type polypeptide, a Retroinverso polypeptide, or a cyclic peptide.

[0012] This invention provides a biological derivative of antimicrobial peptide 16253, which is a chemical modification of the amino acid sequence of the above-mentioned antimicrobial peptide 16253. The chemical modification includes acetylation, amination, methylation, phosphorylation, glycosylation, liposylation, ubiquitination, biotin labeling, or fluorescent protein labeling.

[0013] The present invention provides a salt of antimicrobial peptide 16253, including the metal salt of the above-mentioned antimicrobial peptide 16253 and the salt formed by the above-mentioned antimicrobial peptide 16253 and an inorganic acid or an organic acid.

[0014] Preferably, the metals mentioned above include lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum; the inorganic acids mentioned above include hydrochloric acid, sulfuric acid, boric acid, or carbonic acid; and the organic acids mentioned above include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, or oxalic acid.

[0015] This invention provides the application of the above-mentioned antimicrobial peptide 16253, its derivatives, and its salts in the preparation of antimicrobial products or antimicrobial drugs.

[0016] Preferably, the bacteria mentioned above are one or more of Escherichia coli, Pseudomonas aeruginosa, Salmonella, Streptococcus, Bacillus, and Campylobacter.

[0017] The present invention provides a pharmaceutical composition comprising at least one of the above-mentioned antimicrobial peptide 16253, a derivative of antimicrobial peptide 16253, and a salt of antimicrobial peptide 16253.

[0018] Preferably, the dosage form of the pharmaceutical composition is any one of aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion or liniment.

[0019] This invention provides the use of the above-mentioned antimicrobial peptide 16253, its derivatives, salts, or pharmaceutical compositions in the preparation of a medicament for the prevention or treatment of Campylobacter infections.

[0020] Preferably, the above-mentioned Campylobacter is Campylobacter jejuni.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The antimicrobial peptide 16253 of this invention has a broad-spectrum antibacterial effect, exhibiting strong antibacterial activity against a variety of bacteria, including Escherichia coli, Pseudomonas aeruginosa, Salmonella, Streptococcus, Bacillus, and Campylobacter. It has strong antibacterial activity, a broad antibacterial spectrum, and low toxicity, making it less likely to induce drug resistance. It can be used to develop and prepare various antimicrobial drugs and to treat bacterial infectious diseases, especially showing significant advantages in the treatment of Campylobacter infections, with broad application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of 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.

[0024] Figure 1 This is the mass spectrum of the present invention;

[0025] Figure 2 This is the high-performance liquid chromatogram of the present invention;

[0026] Figure 3 This is a graph showing the in vitro bactericidal kinetics of the present invention;

[0027] Figure 4 This is a diagram illustrating the development of drug resistance in this invention;

[0028] Figure 5 This is a graph showing the survival rate of HCT116 cells in this invention.

[0029] Figure 6 This is a graph showing the survival rate of HIEC-6 cells in this invention;

[0030] Figure 7 This is a graph showing the survival rate of Caco-2 cells in this invention.

[0031] Figure 8 This is a diagram showing the results of the hemolytic experiment of this invention. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Antimicrobial peptide 16253

[0034] This embodiment provides an antimicrobial peptide 16253, whose amino acid sequence is AAWRKWLGKVLRKAKKYRRIKK (SEQ ID NO:1). This antimicrobial peptide 16253 is prepared using amino acid derivatives as synthetic raw materials, and the amino acid residues in the sequence have no side chain modifications or skeletal modifications. This antimicrobial peptide 16253 was synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. (product name: TG-LG-16253 SAMP15, batch number: TP-20081937, molecular weight: 2783.46), with a purity greater than 95%.

[0035] The mass spectrum of the antimicrobial peptide 16253 is as follows: Figure 1 As shown, the mass spectrometry detection conditions are as follows:

[0036] The flow rate was 0.2 mL / min, the running time was 1 min, the mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile solution.

[0037] The high-performance liquid chromatogram of the antimicrobial peptide 16253 is shown below. Figure 2 As shown, the high-performance liquid chromatography (HPLC) detection conditions are as follows:

[0038] Chromatographic column: 4.6×150mm, Kromasil C18-5; mobile phase A was 0.1% trifluoroacetic acid in acetonitrile solution, and mobile phase B was 0.1% trifluoroacetic acid in aqueous solution.

[0039] Elution conditions: 0–0.01 min: mobile phase A 5%, mobile phase B 95%; 0.01–25 min: mobile phase A increased from 5% to 50%, mobile phase B decreased from 95% to 50%; 25–30 min: mobile phase A increased from 50% to 90%, mobile phase B decreased from 50% to 10%; flow rate: 1 mL / min; wavelength: 214 nm; injection volume: 20 μL.

[0040] Example 2: Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0041] In this embodiment, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptide 16253 in Example 1 were determined. The specific process is as follows:

[0042] Each test strain cultured to the logarithmic growth phase was scraped off the plate, and the bacterial suspension with an OD600 of 0.1 was diluted with MH medium at a ratio of 1:1000 for later use; the concentration of antimicrobial peptide 16253 was adjusted to 256 μg / mL.

[0043] Add 100 μL of culture medium to each of columns 2 to 10 of a 96-well plate. Add 200 μL of antimicrobial peptide 16253 solution to the first column. Take 100 μL of bacterial solution from the first well and dilute it twofold to the next well up to the 10th well. Add 200 μL of sterile MHB to the 11th well as a negative control and add 200 μL of bacterial solution to the 12th well as a positive control. Repeat each test compound in 3 wells. Incubate statically at 42°C under microaerophilic conditions for 48 h.

[0044] The minimum inhibitory concentration (MIC) was determined by taking the concentration of the antimicrobial peptide at which no bacteria grew. After interpreting the MIC results, 0.1 mL of the mixture from all wells where no bacterial growth was observed was added to drug-free nutrient agar medium and incubated upside down at 42°C for 48 h.

[0045] If the number of colonies of the test bacteria on the plate is ≤0.1% of the inoculum, the concentration of the antimicrobial peptide can be considered as the minimum bactericidal concentration (MBC).

[0046] When MBC / MIC≤2, the antimicrobial peptide is considered to have bactericidal effect;

[0047] When MBC / MIC≥4, the antimicrobial peptide is considered to have antibacterial effect.

[0048] The test results are shown in the table below:

[0049]

[0050] Conclusion: Antimicrobial peptide 16253 exhibits good antibacterial and bactericidal activities against both Gram-positive and Gram-negative bacteria, demonstrating a broad-spectrum antibacterial effect. In particular, it shows excellent bactericidal activity against Campylobacter strains (MBC / MIC≤2), with MICs as low as 2-4 μg / mL against several Campylobacter strains.

[0051] Example 3: In vitro bactericidal kinetics experiment

[0052] This embodiment conducts an in vitro bactericidal kinetics experiment on the antimicrobial peptide 16253 from Example 1. The specific process is as follows:

[0053] After streaking the frozen Campylobacter jejuni 11168 onto an MHA plate, the plate was incubated at 42°C. Colonies with uniform morphology were picked and inoculated into MHA broth for overnight enrichment. The OD600 value was adjusted to 0.1 with MH broth the next day for later use.

[0054] Based on the MIC value (4 μg / mL) of the test bacteria, the final concentrations of the antimicrobial peptide 16253 to be tested were set to 1×MIC, 2×MIC, and 4×MIC, and the bacteria were cultured in a shaker at 42℃. The bacterial culture without the addition of antimicrobial peptide 16253 served as the control group, and colony counting was performed.

[0055] At five time points (1h, 2h, 3h, and 5h), 0.1mL samples were taken from all tubes, diluted 10-10000 times accordingly, and added to the surface of drug-free nutrient agar medium. The agar was then spread evenly using a disposable spreader. All plates were incubated overnight at 42℃ for 48h. Colony counts were performed the following day to plot a time-kill curve. The results are as follows: Figure 3 As shown.

[0056] Conclusion: Antimicrobial peptide 16253 has good bactericidal effect. It shows antibacterial effect against Campylobacter jejuni 11168 in a short period of time and bactericidal effect in a 48-hour test period (see MBC experimental results).

[0057] Example 4: Drug Resistance Evaluation Experiment

[0058] This embodiment conducts a drug resistance evaluation experiment on the antimicrobial peptide 16253 in Example 1. The specific process is as follows:

[0059] After performing a minimum inhibitory concentration (MIC) experiment on Campylobacter jejuni, bacterial suspensions from the sub-inhibitory concentration (0.5×MIC) treatment group were diluted to approximately 1×10⁻⁶. 5 CFU / mL was used as the inoculum for the next round of MIC determination.

[0060] The process of "low-dose exposure-MIC determination" was repeated, and the cells were passaged for 30 generations. Ciprofloxacin and enrofloxacin were used as control drugs, and drug resistance induction experiments were carried out simultaneously.

[0061] Conclusion: Figure 4 As shown, after 30 generations of continuous treatment with the antimicrobial peptide 16253 on Campylobacter jejuni strains 11168 and LZ, the MIC values ​​remained stable at around 4 μg / mL, and no drug resistance was observed. In contrast, the control group showed rapid induction of drug resistance to fluoroquinolones (enrofloxacin and ciprofloxacin) in a short period of time: after 30 generations, the MIC values ​​of strain 11168 against both drugs increased to more than 4000 times the original values.

[0062] This result indicates that 16253 is less likely to induce drug resistance in Campylobacter, has significant advantages in clinical treatment, and can be considered a potential candidate drug for treating Campylobacter jejuni infection.

[0063] Example 5: Cytotoxicity Experiment

[0064] This embodiment conducts a cytotoxicity experiment on the antimicrobial peptide 16253 from Example 1. The specific procedure is as follows:

[0065] After being thawed from cryopreservation in liquid nitrogen, HCT116, HIEC-6, and Caco-2 cells were seeded into complete culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. HCT116 and HIEC-6 cells were cultured in RPMI 1640, and Caco-2 cells were cultured in DMEM. The cells were passaged at 37°C and 5% CO2.

[0066] Select T25 culture flasks that have reached 90% confluence. Discard the culture medium and wash with PBS 1-2 times. Add trypsin digestion solution to digest the adherent cells to make them round. Add complete culture medium to stop digestion. Use a pipette to wash off the adherent cells and transfer them to 1.5 mL centrifuge tubes. Centrifuge at 700g for 5 min and discard the supernatant. Add fresh complete culture medium to prepare a cell suspension. After counting the cells with a cell counting chamber, aspirate 100 μL of the cell suspension and seed 3000 cells per well into 96-well plates. Incubate overnight at 37°C until the cell confluence reaches 60%.

[0067] Dilute the antimicrobial peptide with complete culture medium and add different final concentrations of antimicrobial peptide to intervene in cells for 48 hours. Discard the culture medium in the wells, add 90 μL of serum- and antibiotic-free culture medium, add 10 μL of CCK8 solution, and incubate the 96-well plate in a 37°C incubator for 40-60 minutes.

[0068] The absorbance at 450 nm was measured using an ELISA reader. Wells without antimicrobial peptide 16253 were used as the control group, and wells without antimicrobial peptide 16253 and cells were used as the blank group.

[0069] Cell viability was calculated based on OD values:

[0070] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0071] Where As is the OD value of the experimental group, Ac is the OD value of the control group, and Ab is the OD value of the blank well.

[0072] The half-maximal inhibitory concentrations (IC50) of cells are shown in the table below:

[0073]

[0074] The cell selectivity index is shown in the table below:

[0075]

[0076] The survival rates of HCT116 cells, HIEC-6 cells, and Caco-2 cells are as follows: Figure 5-7 As shown.

[0077] Conclusion: The 16253 antimicrobial peptide exhibits good cellular safety in Caco-2 and HCT116 colorectal cancer cells, with an IC50 above 50 μg / mL. However, it shows high cytotoxicity in normal human intestinal epithelial cells HIEC-6, with an IC50 of 15.14 μg / mL. The selectivity index SI (IC50 / MIC) was calculated to better assess cytotoxicity. The selectivity index of 16253 against HIEC-6 cells was <10, while the selectivity indices for the other two cell types were >10, indicating that the 16253 antimicrobial peptide has good selectivity and potential for further development.

[0078] Example 6: Hemolytic Experiment

[0079] This embodiment conducts a hemolytic experiment on the antimicrobial peptide 16253 from Example 1. The specific process is as follows:

[0080] Six-week-old healthy male C57BL / 6 mice were selected, and whole blood was collected and 3.2% sodium citrate anticoagulant was added (blood to anticoagulant volume ratio of 9:1). The mixture was gently mixed to prevent clotting, and the mice were euthanized immediately after blood collection.

[0081] Centrifuge the anticoagulated blood at 1000×g for 10 min, discard the supernatant, wash the red blood cells 2-3 times with 0.9% saline until the supernatant is clear, then resuspend the red blood cells with saline and dilute to a final volume fraction of 2%, add different concentrations of antimicrobial peptides (prepared with 0.9% saline) to the red blood cell suspension, and incubate at 37℃ for 1 h.

[0082] After incubation, centrifuge at 1000×g for 10 min, and add 100 μL of the supernatant to a 96-well plate, with 3 replicates per group; measure the absorbance (OD) at 450 nm using a BioTek Synergy H1 microplate reader. 450 ).

[0083] In the experiment, the 2% Triton X-100 treatment group was used as a positive control (complete hemolysis), and the 0.9% saline treatment group was used as a negative control (no hemolysis).

[0084] The formula for calculating the hemolysis rate is as follows:

[0085] Hemolysis rate (%) = (sample OD) 450 - saline OD 450 ) / (Triton X-100 OD 450 - saline OD 450 )×100%

[0086] Conclusion: Figure 8As shown, antimicrobial peptide 16253 has extremely low hemolytic activity and good safety profile.

[0087] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0088] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An antimicrobial peptide 16253, characterized in that, The amino acid sequence of the antimicrobial peptide 16253 is shown in SEQ ID NO:

1.

2. The antimicrobial peptide 16253 according to claim 1, characterized in that, The antimicrobial peptide 16253 is configured as an L-type polypeptide, a D-type polypeptide, a Retroinverso polypeptide, or a cyclic peptide.

3. A derivative of antimicrobial peptide 16253, characterized in that, The derivative is a chemically modified amino acid sequence of the antimicrobial peptide 16253 according to claim 1 or 2, wherein the chemical modification includes acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, ubiquitination, biotin labeling, or fluorescent protein labeling.

4. A salt of antimicrobial peptide 16253, characterized in that, The salt includes the metal salt of the antimicrobial peptide 16253 according to claim 1 or 2, or the salt formed by the antimicrobial peptide 16253 according to claim 1 or 2 with an inorganic acid or an organic acid. The metal includes lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum; The inorganic acids include hydrochloric acid, sulfuric acid, boric acid, or carbonic acid; The organic acids include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, or oxalic acid.

5. A pharmaceutical composition, characterized in that, It contains at least one of the antimicrobial peptide 16253 of claim 1 or 2, the derivative of claim 3, or the salt of claim 4.

6. The pharmaceutical composition according to claim 1, characterized in that, The dosage form of the pharmaceutical composition is any one of aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion, or liniment.

7. The use of an antimicrobial peptide 16253 as described in claim 1 or 2, a derivative as described in claim 3, or a salt as described in claim 4 in the preparation of an antimicrobial product or an antimicrobial drug.

8. The application according to claim 7, characterized in that, The bacteria are one or more of Escherichia coli, Pseudomonas aeruginosa, Salmonella, Streptococcus, Bacillus, and Campylobacter.

9. Use of an antimicrobial peptide 16253 as claimed in claim 1 or 2, a derivative as claimed in claim 3, a salt as claimed in claim 4, or a pharmaceutical composition as claimed in claim 5 or 6 in the preparation of a medicament for the prevention or treatment of Campylobacter infection.

10. The application according to claim 9, characterized in that, The Campylobacter is Campylobacter jejuni.

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