WO2024139105A1 - Antibacterial peptide for non-specifically targeting and inhibiting pathogenic bacteria and use thereof - Google Patents

Abstract

Provided are an antibacterial peptide for non-specifically targeting and inhibiting pathogenic bacteria and the use thereof. The sequence of the antibacterial peptide is as shown in SEQ ID NO: 1. The antibacterial peptide is simple to prepare and low in cytotoxicity, can non-specifically target and inhibit pathogenic bacteria without affecting the normal growth of probiotics, and is suitable for preparing a bacteriostatic agent, such as an oral product for adjusting the balance of oral microecology, so as to use same for preventing and treating oral diseases, especially diseases such as caries, periodontitis, oral mucosa diseases or oral cancer. Moreover, the antibacterial peptide is relatively short, low in terms of chemical synthesis difficulty, can be directly synthesized into a high-purity product, and has clear effectiveness and wide application.

Description

An antimicrobial peptide for non-specific targeted inhibition of pathogenic bacteria and its application Technical Field

The present invention belongs to the technical field of antimicrobial peptides, and more specifically, relates to an antimicrobial peptide for non-specific targeted inhibition of pathogenic bacteria and its application.

Background technique

Cecropin A is an antimicrobial peptide composed of 37 amino acid residues with a molecular weight of approximately 4kDa. Unlike traditional antibiotics, Cecropin A can kill pathogens or inhibit their growth by targeting and inhibiting the synthesis of various biomolecules, destroying or penetrating the plasma membrane of target cells, and targeting metabolic enzymes. In addition, Cecropin A can also destroy the physical integrity of bacterial cell membranes, and bacterial resistance is closely related to its own biofilm, so Cecropin A is not prone to bacterial resistance.

At present, the main way to obtain cecropin A is to extract and purify it from organisms, which has problems such as limited sources and high cytotoxicity (Reddy K V R, Yedery R D, Aranha C. Antimicrobial peptides: premises and promises [J]. International journal of antimicrobial agents, 2004, 24 (6): 536-547.), which also leads to limited application of cecropin A.

Therefore, it is urgent to modify cecropin A to broaden its source and reduce its cytotoxicity.

Summary of the invention

The present invention aims to provide an antimicrobial peptide for non-specific targeted inhibition of pathogenic bacteria, which is modified from cecropin A and has significantly reduced cytotoxicity.

The first object of the present invention is to provide an antimicrobial peptide that non-specifically targets and inhibits pathogenic bacteria.

The second object of the present invention is to provide a polypeptide having more than 90% homology with the above antimicrobial peptide.

The third object of the present invention is to provide a polypeptide obtained by acetylation-modifying the N-terminus of the above antimicrobial peptide.

The fourth object of the present invention is to provide the use of the above antimicrobial peptide or polypeptide in the preparation of an antibacterial agent.

The fifth object of the present invention is to provide the use of the above antimicrobial peptide or polypeptide in the preparation of oral products.

A sixth object of the present invention is to provide an oral product.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention first discovered that the antimicrobial peptide with a sequence as shown in SEQ ID NO: 1 is not only simple to prepare and has low cytotoxicity, but also can non-specifically target and inhibit pathogenic bacteria without affecting the normal growth of probiotics. Therefore, the present invention provides an antimicrobial peptide with a sequence as shown in SEQ ID NO: 1 that can non-specifically target and inhibit pathogenic bacteria, A polypeptide having more than 90% homology with the above antimicrobial peptide, a polypeptide obtained by acetylation of the N-terminus of the above antimicrobial peptide, the use of the above antimicrobial peptide or polypeptide in the preparation of an antibacterial agent, and the use of the above antimicrobial peptide or polypeptide in the preparation of oral products.

The antimicrobial peptide includes 37 amino acids, has a molecular weight of 3911.69, a molecular formula of C ₁₇₈ H ₃₀₅ N ₅₁ O ₄₇ , and an overall average hydrophilicity of -0.349.

The antimicrobial peptide provided by the present invention has the characteristics of non-specific targeted inhibition of pathogenic bacteria, wherein non-specific refers to the characteristic that the antimicrobial peptide has the characteristics of simultaneously inhibiting multiple pathogenic bacteria, and targeted refers to the fact that the antimicrobial peptide can selectively inhibit pathogenic bacteria but not probiotics.

Preferably, the oral product has the function of treating and/or preventing oral diseases.

Further preferably, the oral disease is an oral disease caused by oral pathogenic bacteria, such as Porphyromonas gingivalis, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, Treponema denticola, Tannerella forsythia, Actinomyces viscosus, Staphylococcus aureus, Escherichia coli, Candida albicans, etc.

In the oral cavity, if the microecology is unbalanced, its microbial population is easily transformed into a pathogen-related population, which has a negative impact on oral health; at the same time, the increase of pathogenic bacteria is likely to cause bad breath in the oral cavity, which in turn leads to the occurrence of diseases such as dental caries, periodontitis, oral mucosal diseases or oral cancer. The above-mentioned antimicrobial peptides or polypeptides of the present invention can non-specifically target and inhibit pathogenic bacteria without affecting the normal growth of probiotics, thereby effectively regulating the balance of oral microecology. Therefore, oral products containing the above-mentioned antimicrobial peptides or polypeptides should be within the scope of protection of the present invention.

Preferably, the oral product is one or more of toothpaste, mouthwash, mouthwash effervescent tablets or oral care liquid.

The present invention has the following beneficial effects:

The antimicrobial peptide of the present invention is not only simple to prepare and has low cytotoxicity, but also can non-specifically inhibit pathogenic bacteria without affecting the normal growth of probiotics, and is suitable for preparing antibacterial agents, such as oral products, regulating the balance of oral microecology, and then used to prevent and treat oral diseases, especially dental caries, periodontitis, oral mucosal diseases or oral cancer. Moreover, the antimicrobial peptide of the present invention is short in length, has low difficulty in chemical synthesis, can be directly synthesized into a high-purity product, and has clear effects and wide applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the HPLC spectrum of acetylated antimicrobial peptides.

FIG2 is the LCMS spectrum of acetylated antimicrobial peptides.

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 Preparation of antimicrobial peptides for non-specific targeted inhibition of pathogenic bacteria

The amino acid No. 2 at the N-terminus of cecropin A (as shown in SEQ ID NO: 2: KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK) is replaced with lysine (K), the amino acids No. 34 to 36 at the C-terminus are replaced with 3 prolines (P), and the amino acid No. 37 is replaced with 1 asparagine (N), thereby obtaining the antimicrobial peptide sequence of the present invention: KKKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATPPPN (as shown in SEQ ID NO: 1).

Shanghai WuXi AppTec Pharmaceuticals Co., Ltd. was commissioned to synthesize the above antimicrobial peptide (as shown in SEQ ID NO: 1) by a protein chemical synthesis method (dehydration condensation). In order to improve the stability of the antimicrobial peptide of the present invention in the following test examples, the first lysine (K) at the N-terminus of the antimicrobial peptide was also acetylated. The basic physicochemical properties of the acetylated antimicrobial peptide are shown in Table 1 below.

Table 1 Basic physicochemical properties of acetylated antimicrobial peptides

The acetylated antimicrobial peptide was subjected to HPLC analysis and LCMS analysis. The analysis results are shown in Figures 1 and 2, showing that the purity of the acetylated antimicrobial peptide was 96.7% and the molecular weight was 3953.68.

Test Example 1 Hemolytic Evaluation

The hemolytic evaluation experiment of this test example was carried out in accordance with the Technical Guidelines for Drug Irritation, Allergy and Hemolytic Studies. The specific evaluation method is as follows: 20 mL of rabbit heart blood was taken and placed in a three-well plate containing glass beads. In a corner flask, shake for 10 minutes to remove fibrinogen to make defibrinated blood, add 10 times the volume of 0.9wt% sodium chloride solution, shake well, centrifuge at 1500r/min for 10 minutes, remove the supernatant, wash the precipitated red blood cells with 0.9wt% sodium chloride solution until the supernatant does not show red after centrifugation, and prepare a 2% red blood cell suspension with 0.9wt% sodium chloride solution by volume. Add 2.5mL of rabbit red blood cell suspension to 2.5mL of 0.9wt% sodium chloride solution as the negative group, and add 2.5mL of rabbit red blood cell suspension to 2.5mL of pure water as the positive group. Accurately weigh the original cecropin A and the acetylated antimicrobial peptide of the present invention, prepare 2.5mL of sodium chloride solution (sodium chloride concentration is 0.9wt%) with a sample concentration of 0.5wt% respectively, and then add 2.5mL of rabbit red blood cell suspension respectively as the sample group. Each group of solutions was shaken to mix evenly, placed in a (37±0.5)℃ water bath for 3h, taken out after the water bath, centrifuged at 3000r/min for 10min, and the supernatant was aspirated into a cuvette. The experiment was repeated three times, and the results were averaged. The negative group was used as a blank, and the absorbance of the positive group (Ayang), the negative group (Ayin) and the sample group (Asample) were measured at 542nm by colorimetry, and the hemolysis rate was calculated according to the hemolysis rate = (Asample-Ayin)/(Ayang-Ayin)×100%. The results are shown in Table 2.

Table 2 Hemolysis rate determination results

From the results in Table 2, it can be seen that the hemolytic activity of the acetylated antimicrobial peptide of the present invention is relatively low, only 1.03%, which is significantly lower than that of the original cecropin A, indicating that the antimicrobial peptide of the present invention has relatively low hemolytic activity.

Test Example 2 Cytotoxicity Evaluation

The toxicity of the acetylated antimicrobial peptide provided by the present invention to human oral epithelial cells HOEC (provided by Shenzhen Haodi Huatuo Biotechnology Co., Ltd.) was detected by MTT colorimetry. The specific cytotoxicity evaluation method is as follows: ① HOEC cell expansion culture: HOEC cells were digested with 1 mL of 0.25wt% trypsin for 2 min, centrifuged at 800 rpm for 5 min, and resuspended in complete culture medium (MEM culture medium), and then expanded and cultured for 24 h. The culture conditions were: 37°C, 5% volume fraction of CO ₂ and saturated humidity. ② Plating: Under the microscope, the cells in the culture bottle covered 90% of the bottom of the bottle, collected the cells, and then inoculated in a 96-well plate at 10 ⁴ /well for 24 h. ③Intervention: After removing the culture medium, add 30mL of complete culture medium (MEM culture medium) containing acetylated antimicrobial peptides at a final concentration of 0.5wt% and 30mL of complete culture medium (MEM culture medium) containing cecropin A at a final concentration of 0.5wt% as the sample group; at the same time, 30mL of complete culture medium (MEM culture medium) containing 10wt% fetal bovine serum without the test sample was used as the blank control group, and a negative control group (30mL 0.9wt% sodium chloride solution) and a positive control group (30mL 5% dimethyl sulfoxide solution) were set up. Each group was cultured for 24h under the same culture conditions as ①. ④Determination: Add 5mg/mL 20 μL of MTT solution was added, and the liquid in the wells was discarded after continued cultivation for 4 hours. 150 μL of DMSO was added, and the absorbance was measured at 570 nm and 630 nm using a microplate reader after shaking for 10 minutes. The relative proliferation rate (RGR) was calculated by the following formula: RGR = absorbance of the experimental group / absorbance of the blank control group × 100%. The results are shown in Table 3 below.

Table 3 Toxicity test results on human oral epithelial cells HOEC (n=6)

**: Compared with the blank control group, P<0.01.

From the data in Table 3, it can be seen that cecropin A has a certain inhibitory effect on the proliferation of human oral epithelial cells HOEC, while the acetylated antimicrobial peptide has no obvious inhibitory effect, indicating that the antimicrobial peptide of the present invention has no obvious toxic effect on human oral epithelial cells HOEC.

Test Example 3 Evaluation of the antibacterial effect on common pathogenic bacteria

With reference to 7.3 Antibacterial effect test method (suspension quantitative method) of antibacterial daily chemical products under "QBT2738-2012-Evaluation method for antibacterial and antibacterial effects of daily chemical products", the antibacterial activity of the acetylated antimicrobial peptide and the original cecropin A (both at an action concentration of 0.5wt%) of Example 1 of the present invention against 11 common pathogenic bacteria was evaluated, and a negative control group (sodium chloride solution, final concentration 0.09wt%) and a positive control group (cetylpyridinium chloride, final concentration 0.1wt%) were set up, and the action time was 2min.

The pathogenic bacteria used in this test example are shown in Table 4, and the antibacterial results are shown in Table 5.

Table 4 Types and sources of pathogenic bacteria

Table 5 Antibacterial activity test on pathogenic bacteria (n=3)

*: P<0.05 compared with cecropin A. **: P<0.01 compared with cecropin A.

From the data in Table 5 above, it can be seen that cecropin A has an inhibitory effect on most of the 11 bacteria, and has a stronger inhibitory effect on a few bacteria, while the acetylated antimicrobial peptide of the present invention has a stronger inhibitory effect on all of these 11 bacteria. The acetylated antimicrobial peptide of the present invention has an inhibitory effect on Treponema denticola and Tannerella forsythiae, and the inhibitory effect of the acetylated antimicrobial peptide on Treponema denticola and Tannerella forsythiae is stronger than that of cetylpyridinium chloride, indicating that the antimicrobial peptide of the present invention has excellent antimicrobial properties, can non-specifically target and inhibit pathogenic bacteria, and is suitable for preparing antibacterial agents.

Test Example 4 Evaluation of the antibacterial effect of common oral probiotics

In order to investigate whether the antimicrobial peptide of the present invention can be used to prepare oral products, this test example tests the inhibitory effect of acetylated antimicrobial peptide and cecropin A (both at an action concentration of 0.5wt%) on 9 common oral probiotics. The experimental method is carried out in accordance with the antibacterial effect test method (suspension quantitative method) of 7.3 antibacterial daily chemical products under "QBT2738-2012-Evaluation method for antibacterial and antibacterial effects of daily chemical products", and a negative control group (sodium chloride solution, final concentration 0.09wt%) and a positive control group (cetylpyridinium chloride, final concentration 0.1wt%) are set up, and the action time is 2min.

The oral probiotics used in this test example are shown in Table 6, and the antibacterial results are shown in Table 7.

Table 6 Types and sources of oral probiotics

Table 7 Antibacterial activity test on oral probiotics (n=3)

*: P<0.05 compared with cecropin A. **: P<0.01 compared with cecropin A.

It can be seen from the data in Table 7 that, compared with the original cecropin A, the acetylated antimicrobial peptides of the present invention have weaker or no inhibitory effect on common oral probiotics, indicating that the antimicrobial peptides of the present invention will not have a significant effect on the normal growth of oral probiotics.

Combining the results of Test Example 3 and Test Example 4, it can be seen that the antimicrobial peptide of the present invention can non-specifically target and inhibit pathogenic bacteria without affecting the normal growth of probiotics, and is suitable for preparing antibacterial agents, such as for preparing oral products, regulating the balance of oral microecology, and then for preventing and treating oral diseases, especially preventing and treating dental caries, periodontitis, oral mucosal diseases or oral cancer and the like.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. An antimicrobial peptide that non-specifically targets and inhibits pathogenic bacteria, characterized in that the sequence is as shown in SEQ ID NO:1.
2. A polypeptide having more than 90% homology with the antimicrobial peptide according to claim 1.
3. A polypeptide obtained by acetylation-modifying the N-terminus of the antimicrobial peptide according to claim 1.
4. Use of the antimicrobial peptide according to claim 1 or the polypeptide according to any one of claims 2 to 3 in the preparation of an antibacterial agent.
5. Use of the antimicrobial peptide according to claim 1 or the polypeptide according to any one of claims 2 to 3 in the preparation of oral products.
6. The use according to claim 5 is characterized in that the oral product has the function of treating and/or preventing oral diseases.
7. The use according to claim 6 is characterized in that the oral disease is an oral disease caused by oral pathogenic bacteria.
8. The use according to claim 7 is characterized in that the oral pathogenic bacteria are one or more of Porphyromonas gingivalis, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, Treponema denticola, Tannerella forsythia, Actinomyces viscosus, Staphylococcus aureus, Escherichia coli or Candida albicans.
9. An oral product, characterized in that it contains the antimicrobial peptide according to claim 1 or the polypeptide according to any one of claims 2 to 3.
10. The oral product according to claim 9 is characterized in that the oral product is one or more of toothpaste, mouthwash, mouthwash effervescent tablets or oral care liquid.