An antibacterial cyclic peptide containing unnatural amino acids and preparation method and application thereof

Abstract

The application belongs to the technical field of biology, and specifically discloses an antibacterial cyclic peptide containing non-natural amino acids and a preparation method and application thereof. The structure of the antibacterial cyclic peptide Pep-2307 is cyclo(Ile-Dab-Leu-Phe-Arg-Leu-Leu-Arg-Leu-Arg-Leu-Lys), and the amino acid sequence is shown as SEQ ID NO. 1. The antibacterial cyclic peptide has good antibacterial and antifungal activities, and has good enzymatic stability and biocompatibility. The antibacterial cyclic peptide can be prepared by using a solid-phase polypeptide synthesis and subsequent cyclization process, and the process route is clear, so that product purification, quality control and subsequent preparation development are facilitated. The antibacterial cyclic peptide has a good application prospect in preventing or treating fungal infection and bacterial infection related diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antimicrobial cyclic peptide containing non-natural amino acids, its preparation method, and its application. Background Technology

[0002] Bacterial and fungal infections pose significant challenges to clinical treatment and public health. With the long-term and widespread use of antibiotics and antifungal drugs, drug-resistant microorganisms are constantly emerging, significantly increasing the complexity of infection treatment. For example, opportunistic fungi such as Candida albicans can cause mucosal and skin infections, and lead to invasive infections in immunocompromised patients; drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) can cause wound and soft tissue infections. The continued development of drug resistance not only reduces the therapeutic efficacy of existing anti-infective drugs but also poses a serious challenge to clinical infection control. Therefore, developing antimicrobial molecules with novel chemical structures and unique mechanisms of action is an important direction for addressing drug-resistant infections and expanding anti-infective treatment strategies.

[0003] Antimicrobial peptides are a class of short peptide molecules that typically carry a positive charge and possess a certain degree of hydrophobicity. They exert their antimicrobial effects by interacting with bacterial cell membranes, disrupting membrane integrity, or interfering with intracellular bacterial processes. Compared to traditional small-molecule drugs, antimicrobial peptides generally have advantages such as faster onset of action, diverse target sites, and a relatively lower risk of inducing drug resistance. However, linear natural antimicrobial peptides still suffer from problems in practical applications, including insufficient stability, susceptibility to protease degradation, short in vivo half-life, and the need for improved selectivity. By introducing non-natural amino acids and constructing a cyclic backbone, the conformational constraint of the peptide can be enhanced, improving its enzymatic stability and antimicrobial activity.

[0004] Therefore, it is of great significance to develop a novel antimicrobial polypeptide containing non-natural amino acids with good biocompatibility and enzymatic stability. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention provides an antimicrobial cyclic peptide, Pep-2307, containing the non-natural amino acid Dab. The antimicrobial cyclic peptide Pep-2307 of this invention exhibits good antibacterial and antifungal activity, especially showing good inhibitory or bactericidal activity against Candida albicans, and also possesses good enzymatic stability and biocompatibility, showing promising application prospects in the prevention or treatment of fungal and bacterial infection-related diseases.

[0006] The present invention also provides a method for preparing the antimicrobial cyclic peptide Pep-2307.

[0007] This invention also provides the application of the antimicrobial cyclic peptide Pep-2307.

[0008] In a first aspect, the present invention provides an antimicrobial cyclic peptide Pep-2307, wherein the structure of the antimicrobial cyclic peptide is cyclo(Ile-Dab-Leu-Phe-Arg-Leu-Leu-Arg-Leu-Arg-Leu-Lys), and its amino acid sequence is shown in SEQ ID NO.1;

[0009] The structure of the antimicrobial cyclic peptide is abbreviated as: cyclo(I-Dab-LFRLLRLRLK);

[0010] The Dab is a 2,4-diaminobutyric acid residue, which is a non-natural amino acid residue.

[0011] According to some embodiments of the present invention, the antibacterial cyclic peptide Pep-2307 is a head-to-tail cyclic polypeptide, which forms an amide bond by condensation of the α-amino group of the N-terminal Ile group and the α-carboxyl group of the C-terminal Lys group of the linear precursor, thereby forming a cyclic structure, the structural formula of which is shown below:

[0012] .

[0013] A second aspect of the present invention provides a method for preparing the antimicrobial cyclic peptide Pep-2307 as described in the first aspect of the present invention, comprising the following steps:

[0014] A linear protective peptide with the amino acid sequence shown in SEQ ID NO.1 was prepared by solid-phase peptide synthesis. The peptide was then subjected to intramolecular cyclization, deprotection, and purification to obtain the target antibacterial cyclic peptide Pep-2307.

[0015] According to some embodiments of the present invention, the solid-phase polypeptide synthesis method is the Fmoc solid-phase polypeptide synthesis method.

[0016] According to some embodiments of the present invention, the purification method is high performance liquid chromatography.

[0017] A third aspect of the invention provides the use of the antimicrobial cyclic peptide Pep-2307 as described in the first aspect of the invention, or a pharmaceutically acceptable salt thereof, in the preparation of an antibacterial medicament.

[0018] According to some embodiments of the present invention, the bacteria include Gram-positive bacteria and Gram-negative bacteria;

[0019] The Gram-positive bacteria include Staphylococcus aureus and Streptococcus pneumoniae;

[0020] The Gram-negative bacteria include Escherichia coli and Klebsiella pneumoniae.

[0021] A fourth aspect of the invention provides the use of the antimicrobial cyclic peptide Pep-2307 as described in the first aspect of the invention, or a pharmaceutically acceptable salt thereof, in the preparation of an antifungal medicament.

[0022] According to some embodiments of the present invention, the fungus includes Candida albicans.

[0023] The antimicrobial cyclic peptide Pep-2307 of the present invention exerts its antimicrobial effect by disrupting the integrity of microbial cell membranes, altering cell membrane permeability, or interfering with normal physiological processes of microorganisms.

[0024] According to some embodiments of the present invention, the antimicrobial cyclic peptide Pep-2307 or a pharmaceutically acceptable salt thereof may be formulated into a suitable administration or treatment form according to the actual use scenario, such as a solution, gel, cream, spray, dressing loading form or coating form; the above forms may be prepared using conventional carriers or excipients in the art.

[0025] This invention has been verified through in vitro antimicrobial experiments, enzymatic stability experiments, biocompatibility experiments, and in vivo anti-infection related experiments. It has been found that the antimicrobial cyclic peptide Pep-2307 has good antibacterial and antifungal activities, especially showing good antifungal activity. At the same time, it has good stability and safety, and has the potential to be further developed into an antimicrobial drug.

[0026] A fifth aspect of the present invention provides the use of the antimicrobial cyclic peptide Pep-2307 as described in the first aspect of the present invention in the preparation of wound anti-infection agents, skin and mucous membrane anti-infection agents, or anti-infection agents for the treatment of vaginitis.

[0027] According to some embodiments of the present invention, the concentration of the antimicrobial cyclic peptide Pep-2307 in the wound anti-infection preparation is 1~100 μmol / L.

[0028] According to some embodiments of the present invention, the concentration of the antimicrobial cyclic peptide Pep-2307 in the anti-infective preparation for treating vaginitis is 10~1000 μg / mL.

[0029] The beneficial effects of this invention are:

[0030] 1) The antimicrobial cyclic peptide Pep-2307 of the present invention contains non-proteinogenic amino acid Dab residues. The introduction of Dab residues enriches the structural composition and side chain charge distribution of the peptide, which helps to regulate the interaction between the peptide and the microbial cell membrane, thereby enhancing its antimicrobial activity.

[0031] 2) The antimicrobial cyclic peptide Pep-2307 of the present invention has a head-to-tail cyclic backbone structure. Compared with the corresponding linear peptide, the cyclic structure has a stronger conformational constraint ability, which can reduce the flexibility of the peptide chain and is beneficial to improve the peptide's tolerance to protease or serum enzymatic hydrolysis and its in vitro stability.

[0032] 3) The antimicrobial cyclic peptide Pep-2307 of the present invention contains basic or cationic residues such as Arg, Lys and Dab, as well as hydrophobic residues such as Leu, Phe and Ile, and has amphiphilic structural characteristics, which can enhance its binding and interaction with microbial cell membranes, thereby endowing it with good antibacterial and antifungal activities.

[0033] 4) The antimicrobial cyclic peptide Pep-2307 of the present invention has particularly good inhibitory or killing activity against fungi, and can be used for the prevention and treatment of fungal infection-related diseases. At the same time, it also has inhibitory or killing effects on bacteria, and has the potential to be developed into a broad-spectrum antimicrobial drug.

[0034] 5) The antimicrobial cyclic peptide Pep-2307 of the present invention has good enzymatic stability. Enzymatic stability experiments have verified that the antimicrobial cyclic peptide has better stability than linear peptides or conventional antimicrobial peptides, which is beneficial to improving its application value in complex biological environments.

[0035] 6) The antimicrobial cyclic peptide Pep-2307 of the present invention has good biocompatibility. Within the effective antimicrobial concentration range, the antimicrobial cyclic peptide exhibits low hemolytic activity and low cytotoxicity, and has good safety.

[0036] 7) The antimicrobial effect of the antimicrobial cyclic peptide Pep-2307 of the present invention has been verified by in vitro and in vivo experiments, which can provide experimental basis for its further development as an anti-infective drug candidate.

[0037] 8) The antimicrobial cyclic peptide Pep-2307 of the present invention has a well-defined structure and can be prepared by solid-phase peptide synthesis and subsequent cyclization process. The process route is clear and facilitates product purification, quality control and subsequent formulation development.

[0038] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0040] Figure 1 This is the HPLC analysis report of the antimicrobial cyclic peptide Pep-2307 in Example 1 of the present invention;

[0041] Figure 2 This is the mass spectrum (MS) of the antibacterial cyclic peptide Pep-2307 from Example 1 of the present invention;

[0042] Figure 3This is a graph showing the in vitro antibacterial efficacy evaluation results of the antimicrobial cyclic peptide Pep-2307 in Example 2 of the present invention; where graphs a, b, c, d, e, and f show the antibacterial effects of Pep-2307 and the positive control drug against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, and multidrug-resistant Escherichia coli (MDR E. coli), respectively; the upper part of each graph shows the colony growth, and the lower part shows the corresponding bacterial survival rate statistics;

[0043] Figure 4 Figure 1 shows the in vitro anti-Candida albicans plate experiment and cell membrane permeability evaluation results of the antibacterial cyclic peptide Pep-2307 in Example 3 of this invention; Figure 2a shows the colony growth of Candida albicans after treatment with different concentrations of Pep-2307, clotrimazole, or fluconazole; Figures 3b, 4c, and 5d show the statistical results of the survival rate of Candida albicans in the Pep-2307 group, clotrimazole group, and fluconazole group, respectively; Figure 6e shows the Hoechst 33342 and YO-PRO-1 staining and combined images of Candida albicans in the control group, clotrimazole group, and Pep-2307 group.

[0044] Figure 5 Figure 4 shows the in vitro anti-enzymatic degradation effect and biocompatibility evaluation results of the antimicrobial cyclic peptide Pep-2307 in Example 4 of this invention; Figure a shows the bactericidal effect of Pep-2307 on MRSA and MDR E. coli after treatment with pepsin or trypsin; Figure b shows the colony count results of each treatment group; Figure c shows the chromatographic detection results of Pep-2307 after treatment with pepsin, trypsin or PBS; Figure d shows the hemolysis rate of different concentrations of Pep-2307; Figure e shows the cell viability after treatment with different concentrations of Pep-2307.

[0045] Figure 6 Figure 5 shows the in vivo experimental results of the antimicrobial cyclic peptide Pep-2307 promoting the healing of MRSA-infected wounds in Example 5 of this invention. Figure a shows the wound changes at different times in the control group, vancomycin group, and Pep-2307 group, with a schematic diagram of the wound contour changes on the right side. Figure b shows the statistical results of animal weight changes in each group. Figure c shows the statistical results of wound area changes in each group. Figure d shows the statistical results of wound closure rate in each group on day 14. Figure e shows the bacterial culture results of wounds in each group on days 2 and 14. Figures f and g show the bacterial colony count results of wounds on days 2 and 14, respectively.

[0046] Figure 7Figure 6 shows the in vivo therapeutic effect of the antimicrobial cyclic peptide Pep-2307 on the Candida albicans vaginal infection model in Example 6 of this invention. Figure a shows the animal model establishment and drug administration process; Figure b shows the changes in vaginal appearance at different time points in the control group, fluconazole group, clotrimazole group, and Pep-2307 group; Figure c shows the culture results of Candida albicans in vaginal samples collected from each group at different time points. Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0049] Example 1

[0050] This embodiment provides the chemical synthesis and purification of the antimicrobial cyclic peptide Pep-2307.

[0051] The antimicrobial cyclic peptide Pep-2307 in this embodiment was chemically synthesized by MCE (MedChemExpress). It achieved head-to-tail cyclization by forming an amide bond between the α-amino group of the N-terminal Ile and the α-carboxyl group of the C-terminal Lys. The molecular weight of the product was confirmed by mass spectrometry (MS), and the purity was determined to be 90.06% by HPLC. The HPLC analysis report is as follows. Figure 1 As shown;

[0052] The results of mass spectrometry analysis of the antimicrobial cyclic peptide Pep-2307 are as follows: Figure 2 As shown.

[0053] The structure of the antimicrobial cyclic peptide Pep-2307 in this embodiment is: cyclo(Ile-Dab-Leu-Phe-Arg-Leu-Leu-Arg-Leu-Arg-Leu-Lys), and its amino acid sequence is: IXLFRLLRLRLK, as shown in SEQ ID NO.1;

[0054] Among them, Dab is the non-natural amino acid residue 2,4-diaminobutyric acid.

[0055] The structural formula of the antimicrobial cyclic peptide Pep-2307 is shown below:

[0056] .

[0057] Example 2

[0058] This embodiment verifies the in vitro antibacterial activity of the antimicrobial cyclic peptide Pep-2307.

[0059] Gram-positive and Gram-negative bacteria were selected as experimental strains. Among them, Gram-positive bacteria were Staphylococcus aureus (ATCC29213), S. pneumoniae (ATCC49619), and MRSA (ATCC43300); Gram-negative bacteria were E. coli (ATCC25922), K. pneumoniae (ATCC700603), and MDR E. coli (Xiangya Hospital, Central South University).

[0060] Each bacterial strain was streaked into three zones and inoculated onto LB agar plates containing defibrinated sheep blood, and incubated overnight at 37 °C. Single colonies were then selected and transferred to LB liquid medium, and cultured at 37 °C and 180 rpm with shaking for 16 h. The bacterial suspension was then adjusted to the initial concentration (OD). 600 After diluting (=1.0), dilute 1000 times for later use.

[0061] The minimum inhibitory concentration (MIC) of the target peptide was determined using the micro-brothdilution method, with ceftazidime and polymyxin B used as positive controls. The experimental drug was dissolved in sterile PBS to prepare a 1.28 mM stock solution, which was then serially diluted to working concentrations (128–0.5 μM). PBS was used as a negative control. Susceptibility testing was performed in 96-well plates (final volume 200 μL) and incubated at 37 °C for 24 h. Results were considered valid only if bacteria grew well in the negative control group. The lowest drug concentration at which no obvious bacterial growth was observed was determined as the MIC. All experiments were independently repeated three times.

[0062] To further evaluate the bactericidal effect of the drug, 100 μL of liquid was transferred from each well of a 96-well plate at or above the MIC value and inoculated onto new LB agar plates using the plate spread method. After incubation at 37 °C for 24 h, colony counts (CFU) were performed. The lowest drug concentration at which no colonies grew on the plate was defined as the minimum bactericidal concentration (MBC). All experiments were independently repeated three times.

[0063] The results are as follows Figure 3 As shown, where Figure 3Figure a shows the inhibition of Staphylococcus aureus by different concentrations of Pep-2307 and the positive control ceftazidime, with 2 μM Pep-2307 showing strong antibacterial activity; Figures b and c show the inhibition of methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae by different concentrations of Pep-2307 and the positive control ceftazidime, respectively; Figure d shows the inhibition of Klebsiella pneumoniae by different concentrations of Pep-2307 and the positive control polymyxin B; Figures e and f show the inhibition of Escherichia coli and multidrug-resistant Escherichia coli by different concentrations of Pep-2307 and the positive control polymyxin B, respectively; the upper part of each figure shows the colony growth, and the lower part shows the corresponding bacterial survival rate statistics.

[0064] The test results above show that as the concentration of Pep-2307 increases, the number of colonies formed on the plates of each test strain gradually decreases, and the bacterial survival rate declines accordingly. Pep-2307 can inhibit the growth of Gram-positive and Gram-negative bacteria to varying degrees under different concentration conditions, including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, and multidrug-resistant Escherichia coli.

[0065] The above test results show that the Pep-2307 polypeptide provided by this invention has the potential for broad-spectrum antibacterial activity and can effectively inhibit the growth of different types of pathogens, providing experimental evidence for its application as an antimicrobial active molecule.

[0066] Example 3

[0067] This embodiment verifies the in vitro antifungal activity of the antimicrobial cyclic peptide Pep-2307.

[0068] The standard strain of *Candida albicans* (Xiangya Hospital, Central South University) was used as the experimental strain. The strain was first inoculated onto Sabouraud Dextrose Agar (SDA) agar plates and cultured under suitable conditions to obtain single colonies. Fresh single colonies were picked and inoculated into liquid culture at 37 ℃ and 180 rpm with shaking for 30 h. The bacterial suspension was then adjusted to the initial concentration (OD). 600 After diluting (=1.0), dilute 1000 times for later use.

[0069] The inhibitory activity of Pep-2307 against Candida albicans was evaluated using the broth microdilution method in 96-well plates. The test peptide was prepared as a stock solution and then serially diluted and added to 96-well plates. Fluconazole and clotrimazole were used as positive controls, and a blank control group without any added drugs and a culture medium control group were also included.

[0070] Then, an equal volume of standardized Candida albicans suspension was added to each well to achieve the same final volume. After incubation, fungal growth was evaluated by visual observation or absorbance measurement using a microplate reader. The lowest drug concentration at which no significant growth was observed was defined as the MIC. 100 μL of liquid was transferred from each well of the 96-well plate containing a concentration at or above the MIC and inoculated onto a new LB agar plate using the plate spread method. After incubation at 37 °C for 30 h, colony counts (CFU) were performed. The lowest drug concentration at which no colonies grew on the plate was defined as the MBC. All experiments were independently repeated three times.

[0071] After co-incubating Candida albicans culture with Pep-2307 for 1 h, the fungi were stained with Hoechst 33342 and YO-PRO-1, with Hoechst 33342 used to label total cells and YO-PRO-1 used to label dead cells. After staining, the distribution of fluorescence signals in the fungal cells was observed using a confocal laser scanning microscope.

[0072] The results are as follows Figure 4 As shown, where Figure 4 Figure a shows the colony growth of Candida albicans after treatment with 0, 2, 4, 8, 16, and 32 μM Pep-2307, clotrimazole, or fluconazole, respectively; Figures b, c, and d show the statistical results of the survival rate of Candida albicans in the Pep-2307 group, clotrimazole group, and fluconazole group, respectively; Figure e shows the Hoechst 33342 and YO-PRO-1 staining and combined images of Candida albicans in the blank control group, clotrimazole group, and Pep-2307 group, used to evaluate changes in cell membrane permeability.

[0073] The test results above show that as the concentration of Pep-2307 increases, the number of Candida albicans colonies on the plates gradually decreases, and the survival rate of the strains shows a significant concentration-dependent decreasing trend. Under the experimental conditions, Pep-2307 exhibits good in vitro inhibitory effects on Candida albicans and can effectively reduce the survival level of the fungus. Compared with the blank control group, after treatment with Pep-2307 and clotrimazole, the cell membrane integrity of Candida albicans was significantly disrupted, and membrane permeability was significantly increased, allowing YO-PRO-1 to enter the cell and bind to DNA, thereby producing a significantly enhanced fluorescence signal.

[0074] The above test results show that the Pep-2307 polypeptide provided by this invention has in vitro antifungal activity against Candida albicans, providing experimental basis for its application as an antimicrobial active molecule.

[0075] Example 4

[0076] In this embodiment, the enzymatic stability and biocompatibility of the antimicrobial cyclic peptide Pep-2307 were experimentally tested.

[0077] 1. In vitro enzymatic hydrolysis experiments were conducted using pepsin and trypsin, respectively;

[0078] Pep-2307 was dissolved in appropriate buffer systems, and pepsin and trypsin were added separately, and incubated at 37 °C. The pepsin group was prepared under acidic conditions (pH 2.0) to simulate the gastric fluid environment, and the trypsin group was prepared under weakly alkaline conditions (pH 8.0) to simulate the intestinal fluid environment. Samples were taken at preset time points, and collected after terminating the enzyme reaction. The residual amount and degradation status of Pep-2307 were analyzed by HPLC.

[0079] Meanwhile, the enzyme-treated Pep-2307 was used for subsequent antibacterial activity assays against MRSA and multidrug-resistant Escherichia coli to evaluate the retention of its biological activity after enzymatic hydrolysis.

[0080] The percentage of residual peptides was calculated based on the peak area of ​​the initial sample to characterize the tolerance of Pep-2307 to degradation by pepsin and trypsin.

[0081] 2. The hemocompatibility of Pep-2307 was evaluated using a erythrocyte hemolysis test;

[0082] Fresh red blood cells were washed with PBS and prepared into a suspension, which was then incubated with different concentrations of Pep-2307. The PBS-treated group served as a negative control, and the Triton X-100-treated group served as a positive control. After incubation, the supernatant was collected by centrifugation, and the absorbance was measured using a microplate reader. The hemolysis rate was calculated using the following formula:

[0083] ;

[0084] Wherein ODs, ODn, and ODp represent the absorbance values ​​of red blood cells after incubation with the sample, PBS, and Triton X-100, respectively.

[0085] 3. The MTT assay was used to evaluate the cytotoxicity of Pep-2307 on mammalian cells;

[0086] Cells were seeded in 96-well plates and cultured until adherent. Different concentrations of Pep-2307 were then added and incubated for 24 hours. MTT working solution was then added and cultured further. After discarding the culture medium, DMSO was added to fully dissolve the formazan crystals, and the absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated as the ratio of absorbance of the experimental group to the blank control group.

[0087] The results are as follows Figure 5As shown in Figure a, the plate culture results indicate that, compared with the control group without Pep-2307, Pep-2307 treated with either pepsin or trypsin reduced the colony formation of MRSA and MDR E. coli. However, compared with the untreated Pep-2307 group, the residual colonies in the enzyme-treated groups increased, indicating that enzyme treatment reduced its antibacterial activity, but did not completely eliminate it. Figure b shows the colony count results, consistent with the plate observations. The colony counts in both enzyme-treated groups were lower than those in the control group without Pep-2307, further supporting the fact that Pep-2307 retains its antibacterial activity against the two tested bacteria after enzyme treatment. Figure c shows the chromatographic results, indicating that, compared with the PBS-treated group, both the pepsin and trypsin-treated groups retained chromatographic peaks with retention times similar to the main peak of Pep-2307, while additional peaks with other retention times appeared. Furthermore, the positions and shapes of these additional peaks differed between the two enzyme-treated groups. This suggests that the chromatographic composition of the samples changed after enzyme treatment, but the main chromatographic signal corresponding to Pep-2307 was still preserved.

[0088] The test results above show that Pep-2307 still maintains a high residual ratio after treatment with pepsin and trypsin, and retains a certain antibacterial activity after enzyme treatment, indicating that it has good enzymatic stability and biological activity retention.

[0089] Figure 5 Figure d shows the hemolysis rate of different concentrations of Pep-2307; Figure e shows the cell survival rate after treatment with different concentrations of Pep-2307.

[0090] The above test results show that the hemolysis rate of erythrocytes remained at a low level after treatment with different concentrations of Pep-2307, and no obvious hemolysis phenomenon was observed. The MTT assay results show that Pep-2307 did not show obvious cytotoxicity to mammalian cells within the tested concentration range, and the cell survival rate remained at a high level.

[0091] The above test results show that the Pep-2307 polypeptide provided by this invention has good enzyme stability, biocompatibility and low mammalian cell toxicity, providing a safety basis for its application as an antimicrobial active molecule.

[0092] Example 5

[0093] This embodiment examines the effect of the antimicrobial cyclic peptide Pep-2307 on promoting the healing of infected wounds.

[0094] SPF-grade BALB / c mice (6-8 weeks old, weighing 18-22 g) were inoculated with MRSA in 5 mL of LB liquid medium and cultured at 30 °C with shaking at 200 rpm for 12-16 h until the logarithmic growth phase. The bacterial pellet was resuspended in sterile PBS. The concentration was determined using the McFarland turbidimetric method, and the bacterial concentration was finally adjusted to 1 × 10⁻⁶ with PBS. 8 For use, keep the working concentration of CFU / mL ready.

[0095] Mice were lightly anesthetized with isoflurane. Hair on the back or right flank was shaved off with clippers, and a suitable amount of depilatory cream was applied to ensure smooth, hairless skin. The following day, a full-thickness skin defect was prepared on the back using a 6 mm diameter biopsy punch. A mixed pathogen suspension (MRSA, 10 μL, OD) was then dripped into the wound. 600 =0.1), and covered with a sterile dressing for 24 hours. The appearance of pus on the wound the next day indicates that the mixed infection model has been successfully constructed.

[0096] Mice were randomly divided into four groups: a healthy control group, an infection model group (Vehicle), a positive control drug group (vancomycin), and a Pep-2307 intervention group. Treatment was administered topically to the wound every two days (10 µM, 50 µL / wound) for a total of three times. During treatment, wound photographs, wound area measurements, and mouse weight changes were recorded on days 0, 2, 4, 6, 8, 10, and 14. Wound tissue samples were collected on days 2 and 14. The wound tissue was placed in sterile PBS, minced with sterile scissors, and thoroughly homogenized. 100 μL of the homogenate was serially diluted and plated onto LB agar plates for quantitative analysis of bacterial load in the wound tissue.

[0097] Mice were sacrificed on day 14 of treatment, and wound tissue samples were collected, fixed in 4% paraformaldehyde, dehydrated with graded ethanol, and then embedded in paraffin and sectioned. The tissue sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome staining to observe the reepithelialization and collagen deposition in the wound tissue.

[0098] The results are as follows Figure 6 As shown, where Figure 6 Figure a shows the wound changes in the control group, vancomycin group, and Pep-2307 group on days 0, 2, 4, 6, 8, 10, and 14. The right side of the figure shows a schematic diagram of the wound contour changes. Figure b shows the statistical results of the changes in animal body weight in each group. Figure c shows the statistical results of the changes in wound area in each group. Figure d shows the wound closure rate of each group on day 14. Figure e shows the bacterial culture results of each group's wounds on days 2 and 14. Figures f and g show the bacterial colony count results of the wounds on days 2 and 14, respectively (***P < 0.001; ns indicates no statistical significance).

[0099] The test results above show that, in the wound infection model, compared with the untreated group, the wound area in the Pep-2307-treated group decreased significantly over time, and the wound closure rate increased on day 14, demonstrating a faster wound repair trend. Tissue colony counting results showed that the bacterial load in the wound tissue was significantly reduced after Pep-2307 treatment, indicating that it can effectively inhibit the proliferation of pathogens at the infection site. Further histological analysis showed that the wound tissue structure in the Pep-2307-treated group recovered more completely, inflammatory cell infiltration was reduced, and granulation tissue formation and tissue repair were promoted.

[0100] The above test results show that the Pep-2307 peptide provided by this invention has good anti-infective activity in an in vivo wound infection model and can promote the recovery of infected wounds, and has the potential to be used as a candidate molecule for antimicrobial therapy.

[0101] Example 6

[0102] This embodiment tests the efficacy of antimicrobial cyclic peptide Pep-2307 in treating vaginitis.

[0103] A Vulvovaginal Candidiasis model was established using female BALB / c mice (6-8 weeks old, weighing 20±1 g) to evaluate the in vivo therapeutic activity of Pep-2307 against vaginal infections.

[0104] After Candida albicans was cultured in Sabouraud dextrose agar to the logarithmic growth phase, it was centrifuged at 1000×g for 10 min, the supernatant was discarded, and the culture was washed twice with sterile physiological saline and resuspended in 30% glycerol. The final bacterial concentration was adjusted to 1×10⁻⁶. 7 CFU / mL.

[0105] Starting 6 days prior to infection, estradiol benzoate (0.2 mg / animal, dissolved in 0.1 mL of vegetable oil) was administered subcutaneously every two days. Subsequently, 20 μL of Candida albicans suspension (1×10⁻⁶) was inoculated vaginally via drip instillation. 7 A vaginal infection model was established using CFU / mL, and the successful establishment of the model was confirmed by counting colonies in the vaginal douche fluid.

[0106] Experimental animals were randomly divided into a normal control group, a model control group, a Pep-2307 group, a fluconazole control group, and a clotrimazole control group, with 6 mice in each group. After the model was established, 20 μL of the corresponding drug was administered vaginally daily for 9 consecutive days. The concentration of Pep-2307 was 100 μg / mL (dissolved in PBS and administered vaginally), the concentration of clotrimazole was 4 mg / mL, and the concentration of fluconazole was 10 mg / mL. On days 3, 5, and 9 after administration, 2 mice from each group were randomly sacrificed, and vaginal irrigation was performed using 20 μL of PBS. The irrigation fluid was collected, serially diluted, and plated onto Sabouraud agar containing chloramphenicol. After incubation at 37 °C for 30 h, colony-forming units (CFU) were counted to evaluate changes in vaginal fungal load.

[0107] Simultaneously, vaginal tissue was collected for H&E and PAS staining analysis to observe inflammatory infiltration and fungal colonization, and to comprehensively evaluate the in vivo therapeutic effect of Pep-2307 on Candida albicans vaginal infection.

[0108] The results are as follows Figure 7 As shown, where Figure 7 Figure a shows the animal model establishment and drug administration process: Candida albicans was inoculated on day -5, and Pep-2307, fluconazole, or clotrimazole were administered on days 0-9, respectively; Figure b shows the changes in vaginal appearance at different time points in the control group, fluconazole group, clotrimazole group, and Pep-2307 group; Figure c shows the culture results of Candida albicans in vaginal samples collected on days 0, 3, 5, and 9 in each group.

[0109] The above test results indicate that in the Candida albicans-induced mouse vaginal infection model, Pep-2307 administration reduced the Candida albicans burden at the infection site and improved vaginal tissue inflammation. Compared with the infection control group, the Pep-2307-treated group showed lower fungal colonization levels and better tissue condition.

[0110] The above test results demonstrate that the Pep-2307 peptide exhibits anti-Candida albicans infection activity in an in vivo model. In summary, the antimicrobial cyclic peptide Pep-2307 of this invention possesses broad-spectrum and excellent antimicrobial activity.

[0111] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An antibacterial cyclic peptide Pep-2307, characterized in that, The structure of the antimicrobial cyclic peptide is cyclo(Ile-Dab-Leu-Phe-Arg-Leu-Leu-Arg-Leu-Arg-Leu-Lys), and its amino acid sequence is shown in SEQ ID NO.

1.

2. The antibacterial cyclic peptide Pep-2307 according to claim 1, characterized in that, The Dab is a 2,4-diaminobutyric acid residue; the antibacterial cyclic peptide Pep-2307 is a head-to-tail cyclic polypeptide, which is formed by the condensation of the α-amino group of the N-terminal Ile group of the linear precursor with the α-carboxyl group of the C-terminal Lys group to form an amide bond, thereby forming a cyclic structure.

3. The antibacterial cyclic peptide Pep-2307 according to claim 1, characterized in that, The structural formula of the antimicrobial cyclic peptide Pep-2307 is shown below: 。 4. A method for preparing the antibacterial cyclic peptide Pep-2307 according to any one of claims 1 to 3, characterized in that, Includes the following steps: A linear protective peptide with the amino acid sequence shown in SEQ ID NO.1 was prepared by solid-phase peptide synthesis. The peptide was then subjected to intramolecular cyclization, deprotection, and purification to obtain the target antibacterial cyclic peptide Pep-2307.

5. The preparation method according to claim 4, characterized in that, The purification method is high-performance liquid chromatography (HPLC).

6. The use of the antimicrobial cyclic peptide Pep-2307 or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3 in the preparation of an antibacterial medicament.

7. Use according to claim 6, characterized in that, The bacteria include Gram-positive bacteria and Gram-negative bacteria; The Gram-positive bacteria include Staphylococcus aureus and Streptococcus pneumoniae. The Gram-negative bacteria include Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae).

8. The use of the antimicrobial cyclic peptide Pep-2307 or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3 in the preparation of an antifungal medicament.

9. Use according to claim 8, characterized in that, The fungi include Candida albicans.

10. The use of the antimicrobial cyclic peptide Pep-2307 as described in claims 1 to 3 in the preparation of wound anti-infection agents, skin and mucous membrane anti-infection agents, or anti-infection agents for treating vaginitis.

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