An antibacterial peptide and its use in the prevention and treatment of periodontal problems

Abstract

The present application relates to the field of polypeptide and biological medicine technology, and in particular to an antibacterial peptide and its application in preventing and treating periodontal problems, wherein, specifically, through analyzing and transforming the sequence and structure of the antibacterial peptide KR-12-a5, an antibacterial peptide KR-20 containing 20 amino acids is designed and synthesized, the antibacterial peptide has good bacteriostatic effect on common oral pathogenic bacteria such as Streptococcus mutans, Porphyromonas gingivalis and Fusobacterium nucleatum, can inhibit the formation of bacterial biofilm, has low hemolyticity, good biological safety, and has application potential in preventing and / or treating gingivitis and periodontitis, and therefore has good practical application value.

Description

Technical Field

[0001] This invention relates to the fields of polypeptide and biomedical technology, specifically to an antimicrobial peptide and its application in the prevention and treatment of periodontal problems. Background Technology

[0002] Most oral diseases are caused by an imbalance in the oral flora. The virulence factors and metabolites produced by the imbalanced flora can induce tooth decay, periodontitis, oral mucosal diseases, and even oral cancer, posing a significant threat to global public health and imposing a heavy economic burden. Traditional oral disease treatment mainly relies on antibiotics, but the overuse of antibiotics has led to increasingly prominent problems of bacterial resistance, resulting in a continuous decline in treatment effectiveness. Therefore, the development of safe, effective, and less resistance-prone novel antibacterial drugs is urgently needed.

[0003] Antimicrobial peptides are small molecule polypeptides widely found in nature, mostly cationic and amphiphilic. Based on their secondary structure, they can be classified into various types, including α-helical peptides. They exhibit broad-spectrum antimicrobial activity against oral pathogens such as cariogenic bacteria and periodontal pathogens, and can also regulate the oral microecology, reduce inflammation, and promote tissue repair. Unlike the targeted antibacterial mechanism of antibiotics, antimicrobial peptides disrupt bacterial cell membranes through electrostatic attraction, making them less likely to induce drug resistance and thus an ideal alternative to antibiotics.

[0004] KR-12-a5 is an amphiphilic α-helical antimicrobial peptide derived from LL-37. Current research confirms that KR-12-a5 and some of its analogues possess antimicrobial activity against a variety of drug-resistant bacteria, including clinically isolated methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococci. However, there are currently no studies on its antibacterial activity, and its biosafety in oral applications lacks clear research data. Summary of the Invention

[0005] In view of the shortcomings of the prior art, this invention provides an antimicrobial peptide and its application in the prevention and treatment of periodontal problems. Specifically, this invention analyzes and modifies the sequence and structure of the antimicrobial peptide KR-12-a5, and designs and synthesizes an antimicrobial peptide KR-20 containing 20 amino acids. This antimicrobial peptide has good antibacterial effects against common oral pathogens such as Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum, while also inhibiting bacterial biofilm formation, exhibiting low hemolytic activity, good biocompatibility, and potential application in the prevention and / or treatment of gingivitis and periodontitis. Based on the above research results, this invention is thus completed.

[0006] Specifically, the present invention adopts the following technical solution: In a first aspect, the present invention provides an antimicrobial peptide having an amino acid sequence as shown in SEQ ID NO.1.

[0007] Specifically, this invention has found that the bioactivity of antimicrobial peptides is closely related to their physicochemical properties, among which the regulation of parameters such as charge properties, hydrophobicity, and the stability of the polypeptide helical structure has a key impact on their function. From a molecular interaction perspective, an increased positive charge density carried by antimicrobial peptides can enhance their binding ability to the bacterial cell membrane through electrostatic attraction, stemming from the structural characteristics of bacterial cell membranes rich in negatively charged phospholipid components. Simultaneously, enhanced hydrophobicity and improved helical conformation stability can promote the membrane insertion behavior of antimicrobial peptides, thereby driving the physical segmentation and disintegration of the bacterial cell membrane.

[0008] Based on the above theoretical foundation, the C-terminus of the KR-12-a5 sequence (SEQ ID NO.2: KRIVKLILKWLR) was lengthened, taking into account factors such as positive charge, hydrophobicity, and α-helical conformation, thus obtaining the antimicrobial peptide of this invention. In this invention, the antimicrobial peptide is named antimicrobial peptide KR-20, which is derived from antimicrobial peptide KR-12-a5, comprises 20 amino acids, and has the molecular formula C 111 H 193 N 35 O 24 It has a molecular weight of 2401.94 Da, an isoelectric point of 12.81, a net charge of 6.21 at pH 7.0, and a hydrophobic residue ratio of 45%.

[0009] A second aspect of the present invention provides a method for synthesizing the above-mentioned antimicrobial peptide, the method comprising synthesizing the above-mentioned antimicrobial peptide using a solid-phase synthesis method.

[0010] Furthermore, the synthesis method includes: synthesizing the above-mentioned antimicrobial peptide using the Fmoc solid-phase synthesis method, and then performing desalting purification.

[0011] A third aspect of the present invention provides the use of the above-mentioned antimicrobial peptide in the preparation of products for the prevention and treatment of periodontal problems.

[0012] The prevention and treatment of periodontal problems specifically manifests as follows: (a) Anti-oral pathogens; (b) Inhibits the formation of oral pathogenic bacteria biofilm; (c) Antagonizes the inflammatory stimulation of host cells by oral pathogens; (d) Prevention and / or treatment of gingivitis and periodontitis.

[0013] The oral pathogens include at least Fusobacterium nucleatum (… Fusobacterium nucleatum ), Porphyromonas gingivalis ( Porphyromonas gingivalis ) and Streptococcus mutans ( Streptococcus mutans Any one or more of the following.

[0014] The host cell may be gingival fibroblasts (HGF), and the host may be a human or a non-human animal, wherein the non-human animal may be a non-human mammal.

[0015] The antagonistic effect of oral pathogens on host cells' inflammation is specifically manifested in inhibiting the expression of inflammatory factors in host cells induced by oral pathogens. These inflammatory factors include IL-6 and IL-8.

[0016] In this invention, the products for preventing and treating periodontal problems include, but are not limited to, pharmaceuticals and oral care products; the oral care products include toothpaste, mouthwash, whitening strips, denture cleaning solutions / tablets, lozenges, effervescent tablets, breath freshening sprays, etc., and are not specifically limited here.

[0017] Therefore, the dosage form of the product for preventing and treating periodontal problems can be ointment, film, gel, liquid, effervescent, spray, aerosol, etc., without specific limitations.

[0018] In a fourth aspect, the present invention provides a product for preventing and treating periodontal problems, said product comprising at least the above-mentioned antimicrobial peptide.

[0019] The products mentioned for preventing and treating periodontal problems include, but are not limited to, pharmaceuticals and oral care products.

[0020] The beneficial effects of one or more of the above technical solutions: The antimicrobial peptide KR-20 provided by the above technical solution is formed by sequence modification and optimization based on KR-12-a5. Compared with the original antimicrobial peptide, it has better antibacterial effect and lower hemolytic activity. At the same time, the antimicrobial peptide KR-20 has a short sequence, is easy to prepare, and has high drug-likeness. It can be used to prepare a variety of oral care products, thereby preventing and treating various oral diseases such as dental caries, periodontitis, and oral cancer caused by oral pathogens. Therefore, it has good practical application value. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 The results show the synthesis, purification, and identification of the antimicrobial peptide KR-20: A is the high-performance liquid chromatogram of KR-20; B is the mass spectrometry identification chromatogram of KR-20.

[0023] Figure 2 The image shows the predicted results for the antimicrobial peptide KR-20: A is the α-helix diagram of KR-20 analyzed by HeliQuest software; B is the three-dimensional structure of KR-20 predicted by Alpha fold 3.

[0024] Figure 3 The graph shows the antibacterial effects of antimicrobial peptides KR-20 and KR-12-a5 against common oral pathogens.

[0025] Figure 4 The image shows the bactericidal results of antimicrobial peptides KR-20 and KR-12-a5 against common oral pathogens.

[0026] Figure 5 This is a graph showing the inhibitory effect of the antimicrobial peptide KR-20 on the formation of biofilms by common oral pathogens.

[0027] Figure 6 The graph shows the hemolysis test results of antimicrobial peptides KR-20 and KR-12-a5. In the graph, A represents the hemolysis results of the antimicrobial peptides; B represents the statistical graph of the hemolysis rate of the antimicrobial peptides.

[0028] Figure 7 The effect of antimicrobial peptide KR-20 on the expression level of inflammatory factors mRNA in Pg-induced HGF cells. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0032] The raw materials used in the following examples are all commercially available products that can be purchased.

[0033] Among them, Fusobacterium nucleatum (ATCC 25586), Porphyromonas gingivalis (ATCC 33277) and Streptococcus mutans (ATCC 25175) were purchased from Beijing BioBio Biotechnology Co., Ltd.

[0034] Example 1: Design and Synthesis of Antimicrobial Peptide KR-20 The bioactivity of antimicrobial peptides is closely related to their physicochemical properties, among which the regulation of parameters such as charge properties, hydrophobicity, and the stability of the polypeptide helical structure has a key impact on their function. From the perspective of molecular interactions, the increased positive charge density carried by antimicrobial peptides can enhance their binding ability to bacterial cell membranes through electrostatic attraction, which stems from the structural characteristics of bacterial cell membranes rich in negatively charged phospholipid components. At the same time, the enhanced hydrophobicity and improved helical conformation stability can promote the membrane insertion behavior of antimicrobial peptides, thereby driving the physical segmentation and disintegration of bacterial cell membranes.

[0035] Based on the above theoretical foundation, the C-terminus of the KR-12-a5 (SEQ ID NO.2: KRIVKLILKWLR) sequence was lengthened, taking into account factors such as positive charge, hydrophobicity and α-helical conformation, to obtain the sequence of the antimicrobial peptide KR-20 of the present invention: KRIVKLILKWLRHAGDLVSR (SEQ ID NO:1).

[0036] The above-mentioned antimicrobial peptide was synthesized by Nanjing Taopu Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method, and then desalted and purified. The antimicrobial peptide was then analyzed by HPLC and LCMS. The high-performance liquid chromatogram and mass spectrum of the antimicrobial peptide KR-20 prepared in this embodiment are shown below. Figure 1 As shown.

[0037] Example 2: Determination of the antibacterial and bactericidal activities of antimicrobial peptides KR-20 and KR-12-a5 The periodontal pathogenic strains involved in this embodiment include Fusobacterium nucleatum (… Fusobacterium nucleatum Fn; ATCC 25586), Porphyromonas gingivalis ( Porphyromonas gingivalis Pg; ATCC 33277) and Streptococcus mutans ( Streptococcus mutans The methods for determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides (Sm; ATCC 25175) are as follows: (1) Determination of MIC MIC refers to the lowest concentration at which a drug can inhibit the growth of microorganisms. It is determined using the micro-broth dilution method, and the steps are as follows: Glycerol-containing bacteria stored at -80℃ were streaked onto BHI blood agar solid medium and incubated statically in a 37℃ anaerobic incubator. Once colonies reached a suitable size, single colonies were randomly selected and inoculated onto BHI liquid medium, and cultured in a 37℃ anaerobic incubator until the logarithmic growth phase (Streptococcus mutans should be cultured in a standard incubator at 37℃ and 200 rpm). The logarithmic growth phase bacterial culture was centrifuged at 5000 rpm for 5 min, and the bacterial cells were adjusted to a final concentration of 1×10⁻⁶ using BHI liquid medium. 7CFU / mL. In 96-well plates, the antimicrobial peptide was serially diluted half-fold with BHI liquid medium; 100 μL of the bacterial suspension was added to each well. Wells containing 200 μL of culture medium served as the blank experimental group, and wells containing 200 μL of bacterial suspension served as the negative control group. The 96-well plates were incubated at 37℃ in an anaerobic incubator or a regular constant temperature incubator for 24–48 h. The turbidity of the culture medium in each well was observed visually; the drug concentration corresponding to the well that became completely clear was the MIC value of the sample. (2) Measurement of MBC MBC refers to the lowest concentration at which a drug can kill microorganisms. The determination procedure is as follows: Take 10 μL of bacterial culture from each well corresponding to the concentration at or above the MIC value, streak it on the surface of a BHI blood agar plate, invert the plate, and incubate it in a 37°C anaerobic incubator or a regular constant temperature incubator for 3 to 4 days. The minimum drug concentration corresponding to the absence of bacterial growth on the plate surface is the MBC value.

[0038]

[0039] Table 1. MIC and MBC of antimicrobial peptides KR-20 and KR-12-a5 against three oral pathogens Through Table 1, Figure 3 and Figure 4 The results showed that the MIC and MBC ranges of the template antimicrobial peptide KR-12-a5 against the three tested bacteria were 250–4000 μg / mL and 250–4000 μg / mL, respectively, while the modified antimicrobial peptide KR-20 had MIC and MBC ranges of 31.25–125 μg / mL and 31.25–500 μg / mL, respectively. Compared to the template antimicrobial peptide KR-12-a5, the antimicrobial peptide KR-20 showed an 8–64 fold increase in antimicrobial activity against the tested oral pathogens. Specifically, the geometric mean (GM, the root of the product of the MIC values ​​of all tested bacteria) of KR-12-a5 was 1000 μg / mL, while the GM of KR-20 was 62.5 μg / mL. Therefore, after modification, the antimicrobial peptide KR-20 has a better antibacterial effect against three oral pathogens than KR-12-a5.

[0040] Example 3: Inhibitory effect of antimicrobial peptide KR-20 on bacterial biofilm formation The inhibitory effect of the antimicrobial peptide KR-20 on the formation of oral pathogenic bacteria biofilm was evaluated using crystal violet staining. The specific steps of the method are as follows: (1) Prepare bacterial suspensions of oral pathogens according to the method in Example 2; (2) Take a 96-well plate and add 50 μL of bacterial suspension containing periodontal pathogens and 50 μL of antimicrobial peptide solution of different concentrations to each well, so that the final concentration of antimicrobial peptide PA9 in the well plate is 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL. At the same time, set up a positive control (oral pathogens and culture medium) and a negative control (culture medium only), and set up three replicates for each group; (3) Place the 96-well plate in an anaerobic incubator at 37°C or a regular constant temperature incubator and incubate for 24 h; (4) After culturing, discard the supernatant, rinse slowly three times with 200 μL of PBS buffer to remove airborne oral pathogens, and air dry at room temperature; (5) Add 100 μL of methanol to each well, fix for 15 min, remove the methanol, and air dry; (6) Add 100 μL of 0.1% crystal violet staining agent to each well to stain the bacterial biofilm at room temperature for 20 min; (7) After staining, remove the crystal violet staining agent, rinse off the excess staining agent with PBS, and air dry; (8) Then add 100 μL of 30% acetic acid to each well, gently shake to decolorize for about 10 min at room temperature, and record the OD value at 595 nm using a microplate reader. The results are as follows. Figure 5 As shown. Using the formula: Inhibition rate (%) = [1 - (OD)] 测试 / OD 对照 )]×100%, calculate the biofilm formation inhibition rate.

[0041] Depend on Figure 5 It was found that, compared with the control group, the antimicrobial peptide KR-20 significantly inhibited the formation of biofilms of Fn, Pg, Sm, and the mixed biofilms of the three bacteria. At a concentration of 15.625 μg / mL, the antimicrobial peptide KR-20 significantly inhibited the formation of Fn and Sm biofilms; at 31.25 μg / mL, it significantly inhibited the formation of Pg biofilms; and at 62.5 μg / mL, it significantly inhibited the formation of the mixed biofilm. All differences were statistically significant (P < 0.05). At concentrations of 15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, and 250 μg / mL, the inhibition rates of the antimicrobial peptide KR-20 against Fn, Pg, Sm, and the mixed biofilm were greater than 50%, all lower than their MIC concentrations. These data fully demonstrate that the antimicrobial peptide KR-20 provided by this invention can effectively inhibit the formation of biofilms by oral pathogenic bacteria.

[0042] Example 4: Detection of hemolytic activity of antimicrobial peptides KR-20 and KR-12-a5 Take 1 mL of sterile anticoagulated sheep blood, centrifuge at 3000 rpm for 5 min, discard the supernatant and retain the red blood cell pellet. Resuspend the pellet in sterile PBS buffer, centrifuge at 3000 rpm for 5 min, discard the supernatant, and repeat the operation 3 times. The final concentration of antimicrobial peptide was set to 0, 31.25, 62.5, and 125 μg / ml. Red blood cells were placed in PBS buffer containing different concentrations of antimicrobial peptide and incubated at 37℃ for 1 h. After centrifugation at 3000 rpm for 5 min, the supernatant was collected and the absorbance was measured at 570 nm using a microplate reader. Positive and negative control groups were set up simultaneously. The positive control was PBS buffer containing 1% Triton X-100; the negative control was sterile PBS buffer, and the operation was the same. Each group had 3 replicates.

[0043] The formula for calculating the hemolysis rate is: Hemolysis rate (%) = (AT-AO) / (AC-AO) × 100%.

[0044] Wherein, AT represents the absorbance at 570 nm for different concentrations of antimicrobial peptide treatment groups, and AC and AO refer to the absorbance at 570 nm for the positive control and negative control groups, respectively.

[0045] Table 2. Hemolytic activity of antimicrobial peptides at different concentrations

[0046] Within the testing range of 0–62.5 μg / ml, the hemolysis rate of antimicrobial peptide KR-20 was less than 2%. Compared with the template antimicrobial peptide KR-12-a5, the hemolysis rate of antimicrobial peptide KR-20 was slightly increased, but within the minimum inhibitory concentration range for inhibiting oral pathogens, the hemolysis rate of antimicrobial peptide KR-20 was less than or equal to 5%, indicating good safety and significant application potential. Further in-depth research and development are warranted.

[0047] Example 5: Inhibitory effect of antimicrobial peptides on the inflammatory response of *Porphyromonas gingivalis*-induced human gingival fibroblasts. (1) Cell culture: Primary human gingival fibroblasts (HGF) were obtained from clinically collected isolated gingival tissue. HGF was passaged to 5-8 generations for experiments. After trypsin digestion, 250,000 cells / well were seeded into 6-well plates and allowed to adhere overnight. (2) Bacterial treatment: Porphyromonas gingivalis Pg in the logarithmic growth phase was centrifuged at 5000 rpm for 5 min, and the bacterial pellet was resuspended in DMEM until OD=1 (10⁻¹⁰). 9(CFU / ml), and dispensed into 1.5 ml sterile EP tubes according to the ratio of 1 ml DMEM + 6.25 μL bacterial suspension (multiple of infection (MOI) = 25). Different concentrations of antimicrobial peptide were added to make the final concentration of antimicrobial peptide Chrysophsin3-2 in the EP tubes 3.9~125 μg / mL. The tubes were then incubated in an anaerobic incubator at 37℃ for 3 h. (3) Stimulate cells: Discard the culture medium in each well, wash twice with PBS, add all the bacterial suspension containing antimicrobial peptides in the EP tube into the cells, and incubate at 37°C for 3 h. (4) RT-qPCR detection: Total RNA was extracted from cells using a kit and reverse transcribed into cDNA. GAPDH was used as an internal reference gene to detect the expression of inflammatory genes in cells after co-culturing with HGF and antimicrobial peptide-treated Pg. (5) Calculate relative gene expression levels: 2 -( Ct) The value represents the expression level of the target gene. The primer sequences are as follows: Table 5. Reaction primer sequences

[0048] Inflammatory cytokines are major factors contributing to the periodontal inflammatory microenvironment. Among them, IL-6 can induce periodontal tissue lesions by promoting osteoclast formation and bone destruction. IL-8, an important chemokine that accumulates extensively in neutrophils, has been shown to play a crucial role in acute inflammatory responses. Figure 7 As shown, Pg stimulation significantly increased IL-6 and IL-8 in human gingival fibroblasts. Compared to the Pg-stimulated group alone, the expression levels of inflammatory genes induced by Pg treatment with antimicrobial peptide KR20 (15.6 μg / ml) showed a significant decreasing trend. This indicates that the ability of bacteria pretreated with antimicrobial peptide KR20 to induce HGF cells to produce inflammatory factors was significantly inhibited. KR20 can effectively antagonize the inflammatory stimulation of host cells by *Porphyromonas gingivalis*, blocking the key link in the occurrence of periodontal inflammation at its source by directly inhibiting the pathogen and its pathogenicity. Therefore, this antimicrobial peptide has clear application value and broad development prospects in the preparation of drugs, oral care products, or medical devices for the prevention, relief, and / or treatment of gingivitis, periodontitis, and related inflammatory responses.

[0049] In summary, by optimizing the amino acid sequence of the existing antimicrobial peptide KR-12-a5, a new antimicrobial peptide sequence, KR-20, was obtained. Antimicrobial activity testing revealed that KR-20 exhibited significantly reduced MIC and MBC, indicating a marked improvement in antibacterial and bactericidal activity. Simultaneously, hemolytic activity testing showed that KR-20 possessed low hemolytic activity. Since the antimicrobial target of antimicrobial peptide KR-20 is the main pathogens causing periodontal disease and dental caries, it can be further developed into oral disease prevention and treatment drugs, as well as daily care products such as toothpaste and mouthwash.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection 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. A method for synthesizing the antimicrobial peptide according to claim 1, characterized in that, The synthesis method includes synthesizing the antimicrobial peptide of claim 1 using a solid-phase synthesis method.

3. The application of the antimicrobial peptide according to claim 1 in the preparation of products for preventing and treating periodontal problems, wherein the prevention and treatment of periodontal problems specifically manifests as follows: (a) Anti-oral pathogens; (b) Inhibits the formation of oral pathogenic bacteria biofilm; (c) Antagonizes the inflammatory stimulation of host cells by oral pathogens; (d) Prevention and / or treatment of gingivitis and periodontitis.

4. The application as described in claim 3, characterized in that, The oral pathogens include Fusobacterium nucleatum (… Fusobacterium nucleatum ), Porphyromonas gingivalis ( Porphyromonas gingivalis ) and Streptococcus mutans ( Streptococcus mutans Any one or more of the following.

5. The application as described in claim 3, characterized in that, The host cells are gingival fibroblasts.

6. The application as described in claim 3, characterized in that, The antagonistic effect of oral pathogens on host cells is specifically manifested in inhibiting the expression of inflammatory factors in host cells induced by oral pathogens.

7. The application as described in claim 6, characterized in that, The inflammatory factors include IL-6 and IL-8.

8. The application as described in claim 3, characterized in that, The products for preventing and treating periodontal problems include pharmaceuticals and oral care products; the oral care products include toothpaste, mouthwash, whitening strips, denture cleaning solution / tablets, lozenges, effervescent tablets, and breath freshening sprays.

9. A product for preventing and treating periodontal problems, characterized in that, The product contains at least the antimicrobial peptide as described in claim 1.

Images