An antibacterial peptide and its use in the treatment of ulcerative colitis
By modifying the amino acid sequence of antimicrobial peptides, highly efficient, stable, and non-toxic antimicrobial peptides were designed, solving the treatment problem of ulcerative colitis and achieving highly efficient bactericidal effect against intestinal pathogens and therapeutic effect on ulcerative colitis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2026-04-07
AI Technical Summary
There is a lack of effective, stable and non-toxic antibacterial drugs in the current technology for the treatment of ulcerative colitis, and long-term use of antibiotics may lead to drug resistance problems.
By screening natural antimicrobial peptides targeting intestinal pathogens from an antimicrobial peptide database, and modifying them using bioinformatics and computer-aided design techniques, a new antimicrobial peptide was designed with the amino acid sequence LWRDLICLCRNRRCNRGQLFPGWCPGWWLRCCRR. The positive charge number was reduced, and the hydrophobicity and amphiphilicity were increased. The peptide was then chemically synthesized and its bactericidal activity and cytotoxicity were verified.
It achieved highly efficient bactericidal activity against both Gram-positive and Gram-negative bacteria, with a bactericidal rate of over 99% at a concentration of 7.5 μM and 100% at a concentration of 25 μg/mL. In an ulcerative colitis model, it significantly improved weight loss and spleen index in mice and reduced cytotoxicity.
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Figure CN120209086B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to an antimicrobial peptide and its application in the treatment of ulcerative colitis. Background Technology
[0002] Ulcerative colitis is a lifelong inflammatory disease affecting the rectum and colon. Its global incidence is gradually increasing. This disease can significantly impact patients' lives, leading to long-term complications. Currently, the exact mechanisms of the disease remain unclear, but its causes are related to lifestyle, diet, genetic factors, immune deficiencies, and gut microbiota dysbiosis. There is currently no specific drug to treat this disease, although treatment techniques have advanced, with medications including 5-aminosalicylic acid, antibiotics, immunosuppressants, and biologics now used clinically. However, these treatments are not universally applicable, and long-term use of these drugs can lead to a range of side effects. In particular, antibiotic resistance can develop when treating ulcerative colitis.
[0003] Antimicrobial peptides are a class of polypeptides widely found in nature with broad-spectrum antimicrobial activity. Due to their unique antimicrobial mechanisms, they are considered novel alternatives to traditional antibiotics. However, the research and application of natural antimicrobial peptides are limited by drawbacks such as low activity and the potential cytotoxicity of highly charged peptides, including limitations related to their source, instability, toxicity, and bioavailability. Therefore, the design and optimization of natural antimicrobial peptides has become a recent research hotspot.
[0004] Due to their unique mechanisms of action, antimicrobial peptides are completely different from antibiotics. Among the reported mechanisms of action of antimicrobial peptides, some antimicrobial peptides kill bacteria by disrupting the bacterial cell membrane structure, causing the bacterial contents to leak out; others can inhibit bacterial specific enzymes or DNA transcription and protein translation, affecting intracellular protein interactions. These mechanisms of action are less likely to cause bacterial resistance. Therefore, antimicrobial peptides are expected to replace antibiotics in solving the problem of bacterial resistance and a series of problems caused by antibiotic abuse. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an antimicrobial peptide that has the advantages of high efficiency, stability and non-toxicity, and can be used in the treatment of ulcerative colitis.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0007] In a first aspect, the present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.1.
[0008] This invention screens natural antimicrobial peptides targeting intestinal pathogens from an antimicrobial peptide database. Using bioinformatics and computer-aided drug design techniques, it designs and modifies existing natural antimicrobial peptides to improve their activity and reduce their cytotoxicity. The invention modifies the natural antimicrobial peptide as follows: deleting the positively charged amino acid arginine; replacing the uncharged serine residue in the hydrophilic region with tryptophan; and replacing tyrosine and threonine with proline and tryptophan, ultimately designing a new antimicrobial peptide with the amino acid sequence: LWRDLICLCRNRRCNRGQLFPGWCPGWWLRCCRR. The antimicrobial peptide obtained by this invention through N-terminal amidation modification and amino acid substitution reduces the positive charge of the original antimicrobial peptide, thus reducing its cytotoxicity. Simultaneously, it enhances the antimicrobial peptide's efficacy by increasing its hydrophobicity and amphiphilicity. The antimicrobial peptide obtained by this invention has the advantages of high efficiency, stability, and non-toxicity.
[0009] Secondly, the present invention provides the application of the aforementioned antimicrobial peptide in the preparation of antimicrobial agents.
[0010] The antimicrobial peptide obtained in this invention was tested for bactericidal activity, and the results showed that the antimicrobial peptide has good bactericidal activity. At a concentration of 7.5 μM, the bactericidal rate can reach over 99%; at a concentration of 25 μg / mL, the bactericidal rate can reach 100%.
[0011] Preferably, the bacteria inhibited by the antibacterial agent include Gram-positive bacteria and Gram-negative bacteria.
[0012] Preferably, the Gram-positive bacteria include Staphylococcus aureus; the Gram-negative bacteria include Escherichia coli and drug-resistant Acinetobacter baumannii.
[0013] Thirdly, the present invention provides an antibacterial agent comprising the aforementioned antibacterial peptide.
[0014] Preferably, the effective concentration of the antimicrobial peptide is not less than 7.5 μM.
[0015] Fourthly, the present invention provides the use of the aforementioned antimicrobial peptide in the preparation of a medicament or formulation for treating ulcerative colitis.
[0016] This invention experimentally verified the effects of the antimicrobial peptide on the intestinal flora of animals and the therapeutic effect of the antimicrobial peptide on ulcerative colitis.
[0017] Preferably, the antimicrobial peptide treats ulcerative colitis by acting on the intestinal flora.
[0018] Fifthly, the present invention provides a medicament or preparation for treating ulcerative colitis, comprising the aforementioned antimicrobial peptide.
[0019] The beneficial effects of this invention are as follows:
[0020] This invention screens natural antimicrobial peptides targeting intestinal pathogens from an antimicrobial peptide database. Using bioinformatics and computer-aided drug design techniques, a novel antimicrobial peptide is designed and modified from existing natural antimicrobial peptides, with its amino acid sequence shown in SEQ ID NO.1. This invention chemically synthesizes the described antimicrobial peptide and experimentally verifies its antimicrobial activity, cytotoxicity, and hemolytic activity. Simultaneously, it verifies the therapeutic effect of this antimicrobial peptide on ulcerative colitis, demonstrating its potential application in the treatment of ulcerative colitis. Attached Figure Description
[0021] Figure 1 The mass spectrometry results are for the antimicrobial peptides chemically synthesized in this invention.
[0022] Figure 2 This is a predicted secondary structure diagram of the antimicrobial peptide described in this invention.
[0023] Figure 3 Figure A shows the sterilization plates after treatment with different concentrations of antimicrobial peptides; Figure B shows the results for Staphylococcus aureus; Figure C shows the results for drug-resistant Acinetobacter baumannii; Figure C shows the results for Escherichia coli.
[0024] Figure 4 Histograms showing bacterial survival rates after treatment with different concentrations of antimicrobial peptides; Figure A shows the results for Staphylococcus aureus; Figure B shows the results for drug-resistant Acinetobacter baumannii; Figure C shows the results for Escherichia coli.
[0025] Figure 5 The results show the cytotoxicity of the antimicrobial peptides described in this invention.
[0026] Figure 6 The results show the cell hemolysis of the antimicrobial peptide described in this invention.
[0027] Figure 7 The change in body weight of mice after modeling.
[0028] Figure 8 The change in body weight of mice after treatment.
[0029] Figure 9 Results of colon length comparison.
[0030] Figure 10 The results show the comparison of spleen indices. Detailed Implementation
[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0032] Example 1:
[0033] This invention searches for natural antimicrobial peptides that can act on intestinal pathogens in the Antimicrobial Peptide Database. One natural antimicrobial peptide was screened and its amino acid sequence is: LSRDLICLCRNRRCNRGELFYGTCAGPFLRCCRRRR. According to the database information, this antimicrobial peptide was isolated from the small intestine of C57BL / 6 mice, with a hydrophobicity of 31% and a net charge of +10. This natural antimicrobial peptide is active against intestinal Salmonella, Typhoid Fever (phoP strain), Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. However, high-charged antimicrobial peptides exhibit strong cytotoxicity. Therefore, this invention modifies the natural antimicrobial peptide as follows: the positively charged amino acid arginine is deleted; the amphiphilicity of the antimicrobial peptide significantly affects its antimicrobial activity and selectivity, so the uncharged serine residues in the hydrophilic region are replaced with tryptophan to enhance amphiphilicity; to increase hydrophobicity, tyrosine and threonine are replaced with proline and tryptophan, ultimately resulting in a new antimicrobial peptide with the amino acid sequence: LWRDLICLCRNRRCNRGQLFPGWCPGWWLRCCRR. After modification, an algorithm was used for prediction, and the prediction results showed that the correctness of identifying it as an antimicrobial peptide was 99.9%, its net charge was +7, and its hydrophobicity remained at 50%. The modified and screened antimicrobial peptide sequence was then chemically synthesized and subsequently analyzed by mass spectrometry. The mass spectrometry results showed that (…). Figure 1 The antimicrobial peptides designed in this invention have almost the same molecular weight as chemically synthesized antimicrobial peptides. ， The concentrations were 4293.13 g / mol and 4294.11 g / mol, respectively, with purities exceeding 95%. Alphafold3 was used to predict its secondary structure, which was shown to be an α-helix structure. Figure 2 This invention uses the chemically synthesized antimicrobial peptide to conduct subsequent experiments.
[0034] Example 2:
[0035] This embodiment tests the bactericidal activity of the antimicrobial peptide chemically synthesized in Example 1. The bacteria tested included: typical Gram-positive bacteria—Staphylococcus aureus, typical Gram-negative bacteria—Escherichia coli, and multidrug-resistant Acinetobacter baumannii. The minimum bactericidal concentration (MBC) is the lowest drug concentration that kills bacteria, determined using a colony counting method.
[0036] Sterilization experiment process:
[0037] The bacterial suspensions of Staphylococcus aureus, Escherichia coli, and multidrug-resistant Acinetobacter baumannii were diluted with PBS to a viable count of 10-1. 5 cfu / mL. The antimicrobial peptide was diluted with PBS to different concentrations: 400 μg / mL, 200 μg / mL, 100 μg / mL and 50 μg / mL.
[0038] The diluted bacterial suspension and antimicrobial peptide were mixed at a volume ratio of 1:1 and incubated together at 37°C for 2 hours. The mixture was then inoculated onto solid LB medium and incubated at 37°C for 12 hours. PBS was used as a control. The microbial cytotoxicity (MCC) was determined by the number of colonies formed on the solid medium. The bactericidal rate of the antimicrobial peptide was calculated using formula (1) based on the number of colonies on each plate by colony counting.
[0039] Sterilization rate (%) = (number of colonies on experimental plate / number of colonies on control plate) × 100% — Equation (1)
[0040] Sterilization results as follows Figure 3-4 As shown in the figure. The results from the sterilization plates indicate that the antimicrobial peptides described in this invention exhibit significant bactericidal activity against three representative bacteria, achieving a sterilization rate of 100% at a concentration of 25 μg / mL. Figure 3 ).
[0041] A survival rate histogram was constructed using GraphPad Prism 8. The results showed that the antimicrobial peptide achieved a bactericidal rate of over 99% at a concentration of 7.5 μM. Figure 4 ).
[0042] Example 3:
[0043] This embodiment presents a cytotoxicity test on the antimicrobial peptides (AMPs) chemically synthesized in Example 1.
[0044] Mouse macrophages RAW264.7 were cultured to the logarithmic growth phase, with 10 cells per well. 5 Cells were seeded at a density of 1000 μg / mL in 96-well plates and incubated in a cell culture incubator (5% CO2, 37°C) for 24 hours. The supernatant was discarded, and AMPs were added to DMEM medium to achieve final concentrations of 12.5, 25, 50, 100, and 200 μg / mL. Samples without AMP served as blank controls. After 12 hours of incubation, 10 μL of CCK-8 was added to each well, followed by another 2 hours of incubation. Finally, absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated according to equation (1), and graphs were plotted using GraphPad Prism 8.
[0045] Cell viability (%) = (OD experimental wells - OD blank wells) / (OD control wells - OD blank wells) × 100% — Equation (2)
[0046] The toxicity results of RAW264.7 macrophages showed that the antimicrobial peptide had no toxic effect on macrophages at concentrations of 0-200 μg / mL. Figure 5 ).
[0047] Example 4:
[0048] This embodiment presents a cell hemolysis experiment on the antimicrobial peptides (AMPs) chemically synthesized in Example 1.
[0049] Fresh blood was collected, centrifuged, and the supernatant was discarded. After washing with PBS 4-6 times, red blood cells were collected until the supernatant was reached. A 2% V / V red blood cell suspension dispersed in PBS was mixed with AMPs to achieve final AMP concentrations of 12.5, 25, 50, 100, and 200 μg / mL. PBS samples were used as negative controls, and 2% Triton X-100 samples were used as positive controls. After gentle incubation at 37°C for 2 hours, the supernatant was collected by centrifugation, and the absorbance at 570 nm was measured using a microplate reader. The hemolysis rate was calculated according to equation (3), and a histogram of the hemolysis rate was plotted using GraphPadPrism 8.
[0050] Hemolysis rate (%) = [OD(experimental group) - OD(PBS group)] / [OD(positive control group) - OD(PBS group)] — Equation (3)
[0051] Cell hemolysis results as follows Figure 6 As shown, when the antimicrobial peptide (LR) of the present invention is compared with other antimicrobial peptides (KC, amino acid sequence: KWWIKKVFKWIKGIGKEVVIRTGIEIAACKIKGEC), it can be seen that at a concentration of 100 μg / mL, LR hardly produces hemolysis, while the hemolysis rate of KC is much higher than that of LR.
[0052] Example 5:
[0053] This embodiment verifies the application of the chemically synthesized antimicrobial peptides (AMPs) in Example 1 in ulcerative colitis.
[0054] Experimental procedure:
[0055] Healthy female C57 mice (18-20g) aged 6-8 weeks were randomly divided into two groups. The experimental group was given 2.5% DSS water with free access to water for 7 days, while the control group was given normal water. The establishment of the ulcerative colitis (UC) model was determined by daily weight measurement and observation of fecal occult blood. Spleen index and colon length are important phenotype indicators of UC. Mice in the experimental group with successful UC were treated with AMPs at a dose of 5 mg / kg by gavage for 7 days, while the control group was treated with the same dose of PBS by gavage for 7 days. As the duration of AMP treatment increased in the experimental group, mouse weight gradually increased. At the end of the treatment period, the colon length of the mice in the experimental group significantly increased compared to the PBS treatment group, and the spleen index also significantly decreased after the AMP treatment period.
[0056] Spleen index determination: First, measure the weight (grams) of the mouse: weigh the mouse and record its weight; then measure the weight (grams) of the mouse's spleen: weigh the spleen using a balance and record its weight. Calculate the spleen index according to formula (4).
[0057] Spleen index = Spleen weight (g) / Mouse body weight (g) — Equation (4)
[0058] The results are as follows Figure 7-10 As shown, DSS water treatment led to a significant decrease in body weight in mice. Changes in body weight, colon length, and spleen index indicated that ulcerative colitis symptoms improved after AMP treatment.
[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. An antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.
1.
2. The application of the antimicrobial peptide as described in claim 1 in the preparation of antimicrobial agents, characterized in that, The antibacterial agent inhibits Staphylococcus aureus, Escherichia coli, and drug-resistant Acinetobacter baumannii.
3. An antibacterial agent, characterized in that, Includes the antimicrobial peptide as described in claim 1.
4. The antibacterial agent as described in claim 3, characterized in that, The effective concentration of the antimicrobial peptide is not less than 7.5 μM.
5. The use of the antimicrobial peptide as described in claim 1 in the preparation of a medicament for treating ulcerative colitis.
6. The use of the antimicrobial peptide as described in claim 1 in the preparation of a formulation for treating ulcerative colitis.
7. A drug for treating ulcerative colitis, characterized in that, Includes the antimicrobial peptide as described in claim 1.
8. A preparation for treating ulcerative colitis, characterized in that, Includes the antimicrobial peptide as described in claim 1.