Preparation of cationic antibacterial glycolipopeptide GLP6 and application thereof in treatment of bacterial infection

Abstract

This invention relates to the preparation of an amphiphilic cationic antimicrobial glycolipid GLP6 with good biocompatibility, stability, and antibacterial activity, and its application in the treatment of bacterial infections, belonging to the pharmaceutical field. The sequence of the antimicrobial glycolipid GLP6 is C8H. 15 O‑Ala‑Lys‑Ala‑Phe‑Leu‑Pro‑Ile‑Ile‑Arg‑Trp‑Lys‑Lys‑Arg‑Trp‑C6H 12 NO5 (abbreviated as C8H) 15 O-AKAFLPIIRWKKRW-C6H 12 NO5) is designed based on the biological characteristics of natural antimicrobial peptides (positive charge, hydrophobicity, and α-helical structure), the AXA motif recognized by SPase I, and the FLPII motif that reduces hemolysis, while being coupled with C8H. 16 O2 and C6H 12 NO5 was obtained. This invention provides a technical solution for the synthesis of GLP6. The antibacterial glycolipid GLP6 exhibits excellent in vitro antibacterial activity (MIC values ​​of 9 μmol / L for Gram-negative E. coli ATCC 25922 and 5 μmol / L for Gram-positive S. aureus ATCC 29213), good serum and pancreatic enzyme stability, and good biocompatibility. In vivo antibacterial experiments show that the designed antibacterial glycolipid GLP6 can effectively inhibit or kill bacteria in mice and has a certain therapeutic effect on acute peritonitis induced by E. coli ATCC 25922 in mice. It is a promising antibiotic alternative with potential for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the preparation of an amphiphilic cationic antimicrobial glycolipid GLP6 with good biocompatibility, stability and antibacterial activity and its application in the treatment of bacterial infections. Background Technology

[0002] The discovery and application of antibiotics was a great revolution in human medicine. However, the overuse of antibiotics has led to increasing drug resistance in many strains, giving rise to a series of superbugs. The emergence of multidrug-resistant superbugs, in particular, poses a serious threat to human health. Therefore, there is an urgent need to develop new therapeutic agents that can circumvent these resistance mechanisms and be used against drug-resistant strains. Among these, antimicrobial peptides (AMPs), as a potential candidate extracted from natural sources and used to combat drug-resistant bacteria, have attracted widespread attention. Antimicrobial peptides possess broad-spectrum antimicrobial activity, especially effective against certain drug-resistant pathogens. In addition, antimicrobial peptides also exhibit activities that promote tissue healing and regulate the body's immune system.

[0003] Studies have shown that antimicrobial peptides can exert their bactericidal effects through membrane-targeting mechanisms. Specifically, the cationic residues of the antimicrobial peptide initially contact the negatively charged bacterial surface through electrostatic interactions, while the hydrophobic domains and phospholipid tails interact hydrophobically, creating larger pores in the bacterial membrane and leading to bacterial death. In addition, antimicrobial peptides can also exert their effects through non-membrane-targeting mechanisms. Most bacterial proteins are synthesized as preproteins with an amino-terminal signal peptide (SP). These preproteins are typically transported across the membrane via secretion and diarginine translocation. Once transported to the membrane, a membrane-bound serine protease—Type I signal peptidases (SPase I)—cleaves the SP, separating it from the translocated preprotein. This releases the secreted protein onto the membrane, allowing it to localize to the periplasm, outer membrane, or the bacterial external environment. Inhibition of SPase I activity hinders protein secretion, interferes with bacterial function, and ultimately leads to bacterial death. Since the AXA motif is the SPase I recognition site, it is proposed to design the AXA motif into the membrane-targeting antimicrobial peptide backbone to enhance the specific binding ability of the antimicrobial peptide to pathogens (i.e., SPase I targeting ability), thereby improving its antimicrobial activity and reducing toxic side effects. The AXA motif is located at the left amino terminus of the peptide chain. When the amide bond to the right of AXA is cleaved, the entire peptide chain can be divided into two parts, and the remaining peptide chain can continue to exert its effect through membrane targeting.

[0004] The pentapeptide motif (FLPII) is a conserved sequence at the N-terminus of peptides with excellent antimicrobial and immunomodulatory activities. To design synthetic peptides with superior bioactivity, the FLPII motif can be incorporated into the N-terminus of some peptides to reduce hemolysis and exhibit immunomodulatory properties by increasing leukocyte migration to the infection site and inhibiting pro-inflammatory factors crucial for infection clearance. Proline can induce the formation of a tortuous structure, reducing the hemolytic activity of α-helical peptides. Finally, fatty acid modification can increase the hydrophobic interaction between amphiphilic peptides and bacterial membranes, thereby enhancing the bactericidal activity of antimicrobial peptides. Glycosylation can increase the targeting of antimicrobial peptides, improving their bioavailability and stability. Glucosamine (GlcN) can accelerate protein glycosylation at moderate temperatures (25°C and 37°C) and is a potential candidate for peptide glycosylation. Therefore, the designed and synthesized cationic linear antimicrobial peptide backbone with an amphiphilic α-helical structure was modified by lipolysis and glycosylation.

[0005] It is known that the antibacterial activity of antimicrobial peptides is closely related to their cationic and hydrophobic properties. The positive charge of antimicrobial peptides is fundamental to their binding to the bacterial outer membrane. For Gram-negative bacteria, cationic antimicrobial peptides readily interact with the negatively charged lipopolysaccharides on their outer membrane, thereby disrupting the membrane structure. Therefore, increasing the amount of positive charge on antimicrobial peptides can enhance their binding ability to the membrane, thus improving antimicrobial activity. The positive charge of antimicrobial peptides also helps them accumulate on the membrane surface, achieving an effective bactericidal concentration. Due to their amphiphilic nature, antimicrobial peptide molecules form channels on the plasma membrane, disrupting the original ordered structure of the lipid bilayer. Bacteria lose membrane potential, cannot maintain normal osmotic pressure, and die. Under certain conditions, the greater the hydrophobicity, the stronger the antimicrobial activity of the antimicrobial peptide. However, excessive hydrophobicity will lead to the aggregation of antimicrobial peptides, resulting in precipitation and reduced antimicrobial activity, while also increasing hemolytic activity. Therefore, there is an optimal range for the hydrophobicity ratio, within which the antimicrobial activity is highest. Furthermore, circular dichroism spectroscopy analysis of the secondary structure of antimicrobial peptides revealed that they form α-helical structures under certain conditions. The α-helix is ​​an excellent amphiphilic structure, with one side of its cylindrical molecule's longitudinal axis being a positively charged hydrophilic region and the symmetry plane being a hydrophobic region. This amphiphilic structure is key to the bactericidal activity of antimicrobial peptides, and altering the α-helical structure will affect their activity.

[0006] Therefore, based on the characteristics of the above-mentioned antimicrobial peptides, this study designed and synthesized a novel cationic amphiphilic antimicrobial glycolipid GLP6 with excellent antimicrobial activity. The positively charged amino acids lysine (Lys) and arginine (Arg) can provide positive charges to electrostatically attract the negatively charged bacterial membrane. The hydrophobic tryptophan (Trp), due to the strong quadrupole moment of the π-electron system of its aromatic residues, generates a negatively charged electron cloud, which interacts with cationic molecules (e.g., choline groups) in the bacterial lipid bilayer, leading to a prolonged association time with the bacterial lipid membrane. The aromatic amino acid phenylalanine (Phe) interacts hydrophobically with phospholipids, generating biological activity. The inventors searched and compared the complete amino acid sequence of the antimicrobial glycolipid of this invention using the NCBI protein database and found no identical polypeptides. This antimicrobial glycolipid of the present invention, while exerting targeted antimicrobial therapy, inhibits SPase I activity (non-membrane targeted), exerting a synergistic antimicrobial effect, effectively enhancing the sensitivity of drug-resistant bacteria to the targeted antimicrobial glycolipid, thereby further solving the problem of drug resistance in drug-resistant bacteria. Summary of the Invention

[0007] One objective of this invention is to prepare a novel cationic amphiphilic antimicrobial glycolipid GLP6 with excellent antibacterial activity using solid-phase synthesis technology, based on membrane-targeted and non-membrane-targeted (targeting SPase I) mechanisms of action. A second objective of this invention is to apply this antimicrobial glycolipid to the treatment of bacterial infections.

[0008] To achieve the objectives of this invention, the following technical solution is provided:

[0009] The sequence of the cationic amphiphilic antibacterial glycolipid peptide GLP6 of this invention is C8H. 15 O-Ala-Lys-Ala-Phe-Leu-Pro-Ile-Ile-Arg-Trp-Lys-Lys-Arg-Trp-C6H 12 NO5 (abbreviated as C8H) 15 O-AKAFLPIIRWKKRW-C6H 12 NO5), where Ala(A) is alanine, Lys(K) is lysine, Phe(F) is phenylalanine, Leu(L) is leucine, Pro(P) is proline, Ile(I) is isoleucine, Arg(R) is arginine, Trp(W) is tryptophan, and the attached fatty acid is octanoic acid (C8H). 16 O2, the attached sugar molecule is C6H 13 NO5. Its design method is as follows: First, based on the biological characteristics of natural antimicrobial peptides (positive charge, hydrophobicity, and α-helix structure), positively charged Lys, Arg, and hydrophobic amino acids Ala, Phe, Ile, Pro, Leu, and Trp are designed and introduced, while simultaneously coupling C8H... 16O2 and C6H 13 NO5 yielded the antibacterial glycolipid GLP6. Based on the designed antibacterial glycolipid sequence, the protected amino acids on the side chains were coupled sequentially from the C-terminus to the N-terminus on 2-Chlorotrityl Resin using solid-phase synthesis, followed by the removal of the Fmoc protecting group to obtain a peptide resin. This peptide resin was then reacted with octanoic acid (C8H2O). 16 O2) coupling yields a lipopeptide resin, which is then cleaved and coupled with D-(+)-glucosamine (C6H) 13 NO5), and finally, the side chain protecting groups are removed. The molecular structure of the antibacterial glycolipid GLP6 is as follows:

[0010]

[0011] The in vitro antibacterial activity of the prepared antimicrobial glycolipid GLP6 was detected by micro-broth dilution method; the secondary structure of the antimicrobial glycolipid GLP6 was determined by circular dichroism spectroscopy; serum and pancreatic enzyme stability, biocompatibility and antimicrobial mechanism of GLP6 were studied; finally, the in vivo antimicrobial activity of the antimicrobial glycolipid GLP6 was evaluated. Attached Figure Description

[0012] Figure 1 CD spectra of antibacterial glycolipid GLP6 in H2O and SDS.

[0013] Figure 2 : The minimum inhibitory concentration (MIC) of antimicrobial glycolipid peptide GLP6.

[0014] Figure 3 : Bactericidal kinetics of antibacterial glycolipid GLP6.

[0015] Figure 4 MIC (μM) of antimicrobial glycolipid GLP6 in 50% FBS.

[0016] Figure 5 Stability of antimicrobial glycolipid GLP6 in pancreatic enzymes.

[0017] Figure 6 Biocompatibility of antimicrobial glycolipid GLP6. (a) Hemolytic activity of NS on mouse erythrocytes; (b) Hemolytic activity of antimicrobial glycolipid GLP6 (8 mg / kg) on ​​mouse erythrocytes; (c) Relative cell viability of RAW264.7 cells treated with different concentrations of antimicrobial glycolipid GLP6.

[0018] Figure 7Bacterial biofilm clearance and bacterial membrane permeability assays. (a) Effects of different concentrations of antimicrobial glycolipid 6 on the biofilms of *S. aureus* ATCC 29213 and *E. coli* ATCC 25922; (b) Evaluation of the outer membrane permeability of *E. coli* ATCC 25922 using an NPN uptake assay; (c) Evaluation of the inner membrane permeability of *E. coli* ATCC 25922 using an ONPG solution; (d) Evaluation of the inner membrane permeability of *S. aureus* ATCC 29213 using an ONPG solution.

[0019] Figure 8 TEM images of bacteria before and after GLP6 treatment. (a) TEM image of E. coli ATCC 25922 control group; (b) TEM image of E. coli ATCC 25922 treated with 2×MIC antimicrobial glycolipid GLP6 for 24 h; (c) TEM image of E. coli ATCC 259224 h treated with 8×MIC antimicrobial glycolipid GLP6; (d) TEM image of S. aureus ATCC 29213 control group; (e) TEM image of S. aureus ATCC 292134 h treated with 2×MIC antimicrobial glycolipid GLP6; (f) TEM image of S. aureus ATCC 292134 h treated with 8×MIC antimicrobial glycolipid GLP6.

[0020] Figure 9 Membrane damage of E. coli ATCC 259220h, 2h, 4h, 6h, 8h, and 10h after treatment with 2×MIC antimicrobial glycolipid peptide GLP6.

[0021] Figure 10 Fluorescence images (400×) of E. coli ATCC 259220h, 4h, 8h, and 12h after treatment with antimicrobial lipopeptide LP6-fluorescein (4×MIC).

[0022] Figure 11 In vivo antibacterial activity in mice. (a) Survival rate of mice with acute peritonitis (E. coli ATCC 25922 infection) treated with NS and antimicrobial glycolipopeptide GLP6 (8 mg / kg); (b) Colony counts in peritoneal fluid of mice after different treatment times with NS and GLP6, respectively. Detailed Implementation

[0023] The following provides specific embodiments of the present invention to further illustrate the structure of the present invention, but it is not intended that the present invention be limited to the embodiments described below.

[0024] Example 1: Synthesis method of GLP6 molecule of the present invention

[0025] (1) Synthesis of Fmoc-Ala-Lys(Boc)-Ala-Phe-Leu-Pro-Ile-Ile-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Trp(Boc)-2-Chlorotrityl Resin. Fmoc-amino acids were used as the starting material, and Trp(Boc)-2-Chlorotrityl Resin with a loading of 0.59 mmol / g protected by Fmoc was used as the synthetic support. DCC / HOBt / DIEA were used as the condensation reagents. First, Fmoc-Trp(Boc)-2-Chlorotrityl Resin was weighed into a reaction vessel, and DMF was added at room temperature and under anhydrous conditions to swell the resin. After the resin was fully swollen, excess DMF was removed by filtration. The ninhydrin test (ninhydrin:pyridine:phenol = 1:2:1) is performed. If colorless, proceed to the next step. Add a mixed solution of V(piperidine):V(DMF) = 1:4 and stir to remove the Fmoc protecting group. After stirring, filter and wash sequentially with DMF, DCM, and DMF. Transfer the resin to a reaction vessel and perform the ninhydrin test. If a blue-purple color is observed, add DMF again to swell the resin. Take 2 molar amounts of Fmoc-Arg(Pbf)-OH, using 2.6 molar amounts of DCC, HOBt, and DIEA as condensing agents, and anhydrous DMF as the reaction solvent. Add the amino acid and condensing agent separately and stir for 48 hours. Perform the ninhydrin test. If colorless, proceed to the coupling of the next amino acid. The reacted resin was washed with DMF, DCM, and DMF to remove unreacted amino acids and catalyst. The sample was then transferred to a dialysis bag (MW: 8000-14000) and dialyzed with ethanol. The above steps were repeated, coupling amino acids one by one from the C-terminus to the N-terminus until Fmoc-Ala-Lys(Boc)-Ala-Phe-Leu-Pro-Ile-Ile-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Trp(Boc)-2-Chlorotrityl Resin was synthesized.

[0026] (2) Lipopeptide resin LP6-2-Chlorotrityl Resin (C8H 15Synthesis of O-Ala-Lys-Ala-Phe-Leu-Pro-Ile-Ile-Arg-Trp-Lys-Lys-Arg-Trp-2-Chlorotrityl Resin: Remove the Fmoc protecting group from the polypeptide resin obtained in (1). Take 2 molar amounts of n-octanoic acid, and use 2.5 molar amounts of NHS and EDC as linkers. Add fatty acids and linkers to anhydrous DMF as the reaction solvent and stir to activate. After activation, add the freeze-dried polypeptide resin and stir for 48 h. Wash the resin after reaction with DMF, DCM and DMF respectively, filter to remove unreacted fatty acids and linkers, and dialyze with ethanol. After dialysis, remove the resin from the dialysis bag, wash with anhydrous ethanol, freeze-dry to obtain LP6-2-Chlorotrityl Resin (C8H 15 O-Ala-Lys(Boc)-Ala-Phe-Leu-Pro-Ile-Ile-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Trp(Boc)-2-Chlorotrityl Resin).

[0027] (3) Antimicrobial glycolipid GLP6 (C8H 15 O-Ala-Lys-Ala-Phe-Leu-Pro-Ile-Ile-Arg-Trp-Lys-Lys-Arg-Trp-C6H 12 NO5): The lipopeptide resin LP6-2-Chlorotrityl Resin obtained in (2) was added to a DCM solution containing 1% TFA and stirred for 1.5 h to remove 2-Chlorotrityl Resin. After filtration, the filtrate was added to a saturated NaHCO3 solution to remove excess TFA, and the DCM phase was collected. Then, the DCM phase was mixed with pure water several times and extracted to remove all TFA and sodium salt. Finally, the DCM phase was collected and lyophilized to obtain C8H 15 O-Ala-Lys(Boc)-Ala-Phe-Leu-Pro-Ile-Ile-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys-(Boc)-Arg(Pbf)-Trp(Boc) sample. Weigh out 1 molar amount of C8H 15O-Ala-Lys(Boc)-Ala-Phe-Leu-Pro-Ile-Ile-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Trp(Boc), 3 molar amounts of NHS and EDC were added to anhydrous DMF and stirred for 6 h to activate the carboxyl groups. After activation, 3 molar amounts of D-(+)-glucosamine hydrochloride (C6H) were added. 13 NO5·HCl and triethylamine were added to the above mixed solution and stirred for 48 hours. After the reaction was complete, the solution was dialyzed with pure water (MW: 2000) to remove excess C6H. 13 NO5·HCl, NHS, EDC, and triethylamine were centrifuged to obtain C8H. 15 O-Ala-Lys(Boc)-Ala-Phe-Leu-Pro-Ile-Ile-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Trp(Boc)-C6H 12 NO5 sample. Add appropriate amount of cutting fluid (V TFA :V Tis :V H2O =95:2.5:2.5) was stirred for 1.5 h to remove all side chain protecting groups. After stirring, the mixture was concentrated under reduced pressure and precipitated in ice-cold diethyl ether. The precipitate was washed several times with diethyl ether and finally freeze-dried to obtain the antibacterial glycolipid GLP6 (C8H 15 O-AKAFLPIIRWKKRW-C6H 12 NO5).

[0028] Example 2: Secondary structure characterization of antibacterial glycolipid GLP6

[0029] The secondary structure of GLP6 was characterized using a Chirascan circular dichroism spectroscopy (CD spectroscopy). Specific experimental parameters were: scanning wavelength range 180-260 nm, band width 1 nm, scanning speed 0.5 s / point, and optical path 0.5 mm. First, a certain amount of GLP6 was weighed and dissolved in ultrapure water and sodium dodecyl sulfate (SDS, 30 mM) solution to prepare solutions with a final concentration of 150 μM, respectively. These solutions simulated the secondary conformation of the peptide in aqueous and lipid environments, respectively. The spectral absorption of GLP6 in the 180-260 nm range was detected. Then, the obtained spectra were converted to average residue ellipticity using the following equation:

[0030] θ M =(θobs×1000) / cln

[0031] Where θ M It is the average residue ellipticity (deg·cm) 2·dmol -1 ); θobs is the ellipticity (mdeg) observed after buffer correction at a given wavelength; c is the concentration of the antimicrobial glycolipid (mM); l is the path length (mm); n is the number of amino acids in the antimicrobial glycolipid GLP6.

[0032] Example 3: In vitro antibacterial activity assay of antimicrobial glycolipid GLP6

[0033] Minimum inhibitory concentration (MIC): For the antibacterial activity assay, Gram-positive bacteria (S. aureus ATCC 29213) and Gram-negative bacteria (E. coli ATCC 25922) were used. The minimum inhibitory concentration (MIC) of the glycolipid peptide GLP6 was determined using the standard two-fold dilution method. The specific method was as follows: The bacterial culture in the logarithmic growth phase was centrifuged, washed with PBS, and resuspended to obtain a concentration of approximately 10. 5 -10 6 CFU / mL bacterial suspension. GLP-6 was dissolved in sterile water and serially diluted to prepare solutions with concentrations of 1-256 μM. The solutions were then transferred to sterile 96-well plates (50 μL / well), and an equal volume of bacterial suspension (50 μL / well) was added to each well. The 96-well plates were incubated at 37°C for 12-16 hours, and the results were observed. The minimum inhibitory concentration (MIC) was defined as the lowest concentration observed to be clear to the naked eye. Throughout the experiment, the positive control group (containing only sterile culture medium) wells should remain clear and transparent, while the negative control group wells (containing no antimicrobial peptide) should show obvious bacterial growth characteristics (turbidity in the solution, precipitation at the bottom of the well, etc.).

[0034] Bactericidal kinetics: E. coli ATCC 25922 in the logarithmic growth phase was centrifuged, washed, and resuspended in LB broth to obtain a concentration of approximately 10. 5 -10 6 Prepare bacterial suspensions at CFU / mL for later use. Prepare initial concentrations of GLP6 at 8×MIC, 4×MIC, 2×MIC, and 1×MIC. Mix 700 μL of peptide solution with an equal volume of bacterial suspension (final peptide concentrations of 4×MIC, 2×MIC, 1×MIC, and 0.5×MIC). Incubate at 37℃ for 0 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h. At each of these time points, take 100 μL of the mixture, dilute it several times with physiological saline, and take 50 μL from the diluted liquid to plate. Incubate at 37℃ for 18 h. Calculate the bacterial content at each time point based on the dilution factor, colony count on the petri dish, and the aspirated volume, expressing the result as CFU / mL.

[0035] Example 4: Stability test of antimicrobial lipopeptide GLP6

[0036] Serum stability: To assess the serum stability of the antimicrobial glycolipid, the antimicrobial glycolipid was pre-incubated with fetal bovine serum (FBS), and the changes in antimicrobial activity of the FBS-treated antimicrobial glycolipid were then detected to evaluate its serum stability. First, the antimicrobial glycolipid GLP6 was prepared at different concentrations and mixed with an equal volume of 50% FBS solution (FBS:PBS = 1:1), and incubated at 37°C for 0.5 h and 1.5 h, respectively. The bacterial culture in the logarithmic growth phase was then diluted to 10⁻¹⁰ with LB medium. 6 CFU / mL was added to a 96-well plate, and the MIC value was determined.

[0037] Trypsin stability: 0.25% (0.25 g / 100 mL) trypsin solution was diluted with PBS to 0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, and 1000 μg / mL. The trypsin was mixed with an equal volume ratio of 4×MIC glycolipid GLP6 solution (i.e., the final GLP6 concentration was 2×MIC), incubated at 37°C for 1 h, and then incubated at 60°C for 20 min to inactivate the trypsin. The bacterial culture in the logarithmic growth phase was diluted with LB broth to 10... 6 CFU / mL. Add 100 μL of the co-incubation solution of trypsin and peptide to a 96-well plate, then add 100 μL of bacterial culture and mix well. Incubate at 37°C for 18 h. Measure the OD value of each well at 600 nm using a microplate reader and calculate the bacterial survival rate.

[0038] Example 5: Biocompatibility of antimicrobial glycolipid peptide GLP6

[0039] Hemolytic and cytotoxic properties of peptides are important indicators for evaluating their in vivo safety. The biocompatibility of antimicrobial glycolipid GLP6 was studied by measuring its hemolytic and cytotoxic properties on mammalian cells.

[0040] Hemolytic activity: Two male healthy mice (23-25g) were randomly selected. GLP6 (8mg / kg, 200μL) was injected into one mouse via the tail vein, while the other mouse was injected with 200μL of sterile saline as a negative control. Blood was collected by enucleation 2 hours later, diluted with saline, and the morphology of red blood cells was observed under a fluorescence microscope.

[0041] Cytotoxicity: GLP6 glycolipid was serially diluted to different concentrations (1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM, and 256 μM) using DMEM medium. RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The old medium was aspirated, and 100 μL of medium containing different concentrations of GLP6 was added to each well. To ensure the accuracy and reliability of the experimental results, five replicates were generally set up per column. After culturing for 24 h and 48 h, 20 μL (5 mg / mL) of medium was added to each well. -1 MTT solution was incubated in a cell culture incubator for 4 hours. After discarding the supernatant, 150 μL LDMSO was added to each well to dissolve the MTT crystals. The cells were then shaken in the dark to ensure complete dissolution. The cells were transferred to an enzyme-linked immunosorbent assay (ELISA) reader for OD value detection at a wavelength of 485 nm. The relative cell viability was calculated to determine the cytotoxicity of GLP-6.

[0042] Example 6: Membrane disruption effect of antibacterial glycolipid GLP6

[0043] Biofilm eradication assay: The inhibitory effect of the antimicrobial glycolipid GLP6 on bacterial biofilms was determined by crystal violet staining. Second-passaged Staphylococcus aureus (S. aureus ATCC 29213) and Escherichia coli (E. coli ATCC 25922) were centrifuged, washed, and resuspended in 10% LB medium. The bacterial suspension was added to 96-well plates (100 μl / well) and incubated for 72 h. The supernatant was aspirated and washed with PBS. A concentration gradient of 256–2 μM was established (100 μl / well). The negative control group received only sterile 10% LB medium, while the positive control group contained bacterial suspension but not the antimicrobial glycolipid GLP6. After 24 h of incubation, the supernatant was removed, washed with PBS, and aspirated. The plates were fixed with methanol (100 μl / well) for 15 min, the methanol was aspirated, and the plates were air-dried. Add crystal violet solution (100 μl / well) and stain for 15-20 min. Aspirate and wash 2-3 times with PBS. Aspirate and air dry. After drying, add 95% ethanol (100 μl / well) and react for 20 min. Measure the absorbance at 580 nm and calculate the bacterial biofilm eradication rate using the following formula.

[0044] Biofilm eradication rate (%) = [(A 100 ―A) / (A 100 —A 0) ]×100%

[0045] A represents the absorbance of the antimicrobial glycolipid GLP6 at a given concentration; A0 represents the absorbance of the negative control; A 100 This indicates the absorbance of the positive control group.

[0046] Outer membrane (OM) permeation assay: The outer membrane permeability of the antibacterial glycolipid GLP6 was determined using the fluorescent dye 1-N-phenylnaphthylamine (NPN). E. coli was centrifuged in ATCC 25922, resuspended in PBS, and OD was adjusted. 600 =0.4-0.6 for later use. Add NPN (final concentration 10 μM) to the bacterial suspension in a quartz cuvette and record the background fluorescence using a fluorescence spectrophotometer. Prepare the drug at different concentrations and add it to the cuvette. After mixing, use fluorescence detection to detect the fluorescence change under the conditions of excitation wavelength of 350 nm and emission wavelength of 420 nm. Measure once every 30 seconds, and continue for 10 minutes.

[0047] Inner membrane (IM) permeation assay: The permeability of the antimicrobial glycolipid GLP6 to the inner membrane was evaluated using an ONPG (o-nitrophenol-β-galactoside) solution assay. ONPG was dissolved in PBS to prepare a 1.5 mM ONPG solution. E. coli ATCC25922 and S. aureus ATCC 29213 were centrifuged and washed, then resuspended in the prepared ONPG solution, and added at 50 μL / well to a 96-well plate. The drug was diluted to different concentrations, and 50 μL / well was added to each well of the 96-well plate to final concentrations of 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM. In the control group, 50 μL of PBS was added to each well. The absorbance at 420 nm was measured using a microplate reader every 5 min for 100 min.

[0048] Transmission electron microscopy (TEM) experiments: The morphology and intracellular changes of bacteria treated with the antimicrobial glycolipid GLP6 were observed using TEM. E. coli ATCC 25922 grown overnight was centrifuged, washed, and resuspended in PBS. GLP6 was prepared with sterile water at concentrations of 4×MIC and 16×MIC for later use. Equal volumes of bacterial suspension were mixed with GLP6 solution to achieve final concentrations of 2×MIC and 8×MIC of the antimicrobial glycolipid, and incubated at 37°C for 4 h. The group without antimicrobial glycolipid served as a negative control. After incubation, the precipitate was collected by centrifugation, washed with PBS, and fixed overnight at 4°C with 3% glutaraldehyde solution. The precipitate was washed three times with 0.1M PBS, fixed with 1% osmium tetroxide at 4°C for 90 min, and washed three times with 0.1M PBS. Elution was performed sequentially with fractionated ethanol at concentrations of 50%, 70%, 80%, 90%, 100%, 100%, and 100%, followed by two acetone replacements. The samples were then treated with a mixture of acetone and penetrant for 3 hours (acetone:penetrant = 2:1 for 1 hour, acetone:penetrant = 1:2 for 2 hours), and incubated overnight at 35°C with the lid off. Bacterial samples were then embedded and incubated at 37°C for 12 hours, 45°C for 12 hours, and 60°C for 24 hours. Sections were prepared using an ultramicrotome. After staining with uranium acetate and lead citrate, the samples were observed and photographed under a JEM-1400 transmission electron microscope.

[0049] Flow cytometry (FCM) assay: The uptake of the antimicrobial glycolipid GLP6 by E. coli ATCC 25922 was quantitatively assessed by flow cytometry (FCM). E. coli ATCC 25922 bacterial culture was mixed with GLP6 (final concentration 4×MIC) and incubated at 37℃ for 2 h, 4 h, 6 h, 8 h, and 10 h. The negative control was bacterial culture without added peptide. After centrifugation and washing, PI solution at a concentration of 50 μg / mL was added, and the mixture was incubated at 37℃ in the dark for 30 min. After centrifugation and washing, the cells were resuspended in 400 μL of PBS. The cells were then appropriately diluted and analyzed using a BDCanto II flow cytometer (FCM) at 488 nm, and the results were recorded.

[0050] Laser confocal microscopy (CLSM) experiment: The uptake of the nuclear dye PI was observed using a laser confocal microscope to understand the integrity of the bacterial membrane and the distribution of LP6 in the bacteria after LP6 treatment. The antimicrobial lipopeptide LP6-fluorescein (8×MIC) was mixed with an equal volume of E. coli ATCC 25922 bacterial suspension (final concentration 4×MIC) and incubated at 37℃ for 4 h, 8 h, and 12 h. The negative control was bacterial suspension without peptide. After centrifugation and washing, 50 μg / mL PI solution was added, and the mixture was incubated at 37℃ in the dark for 30 min. After centrifugation and washing, the suspension was resuspended in 400 μL of PBS. 10 μL of the mixture was transferred to a glass slide, spread, and observed under a microscope.

[0051] Example 7: In vivo antibacterial experiment

[0052] Peritonitis Model: E. coli ATCC 25922 was used to infect mice with peritoneal infection. Twelve female Kunming mice were randomly divided into two groups: a control group (saline) and an experimental group (antimicrobial glycolipid lipopeptide GLP6, 8 mg / kg, 200 μL / mouse). Log-phase E. coli was centrifuged, washed, and resuspended in saline. The resulting solution was injected intraperitoneally (200 μL / mouse) to establish a mouse peritonitis model. Successful modeling was indicated by lethargy, loss of appetite, abdominal retraction, viscous excretions, and forked fur. After successful model establishment, mice were injected intraperitoneally with either saline or GLP6. Survival was monitored in both groups, and survival curves were plotted. Mice were sacrificed at 12, 24, and 48 hours after drug and saline injections, respectively. 1 mL of sterile saline was injected intraperitoneally, and the abdomen was gently massaged for 2 minutes. The peritoneal fluid was then aspirated, diluted, and analyzed using a plate count method to determine bacterial count.

[0053] The method of this invention prepares antibacterial glycolipid GLP6, which has good biomedical properties.

[0054] (1) This antibacterial glycolipid GLP6 has an α-helical structure in a membrane-simulated environment.

[0055] The α-helix structure is crucial for maintaining the bioactivity of AMPs. The secondary structure of the antibacterial glycolipid was detected using CD spectroscopy in aqueous solution (aqueous environment) and SDS solution (membrane simulation environment). In the UV region (180-260 nm), the CD spectrum of the α-helix showed a positive peak near 192 nm and two negative characteristic peaks at 208 nm and 222 nm. SDS contains anionic groups and was used to simulate the bacterial membrane environment; the results showed… Figure 1 The CD spectrum of GLP6 in 30mM SDS solution showed a strong positive peak at about 190nm and two negative peaks at about 208nm and 222nm, indicating that GLP6 presents an α-helical structure in the membrane environment, suggesting that the α-helical structure of GLP6 may play an important role in its antibacterial activity.

[0056] (2) This antibacterial glycolipid GLP6 has excellent in vitro antibacterial activity.

[0057] The antibacterial activity of GLP6 against different bacterial colonies was investigated by determining the MICs of GLP6 against E. coli ATCC 25922 and S. aureus ATCC 29213 in vitro. The results are as follows: Figure 2As shown, the antibacterial glycolipid GLP6 of the present invention exhibits strong activity against both Gram-negative E. coli and Gram-positive S. aureus standard strains, with MICs of 9 μM and 5 μM, respectively, indicating its excellent in vitro antibacterial activity.

[0058] This project further investigated the bactericidal kinetics of GLP6. For example... Figure 3 As shown, at 0 min, the colony counts at 4×MIC, 2×MIC, 1×MIC, and 0.5×MIC were essentially the same as those in PBS. From 10 min to 12 h, the colony counts at all drug concentrations were lower than those in PBS. Specifically, at 1 h, 2 h, 3 h, and 6 h, the colony count at the 4×MIC concentration was 0. This means that at a concentration of 4×MIC, GLP6 kills all *E. coli* within 1-6 h. The bactericidal kinetics results indicate that GLP6 can kill *E. coli* in a relatively short time, and its antibacterial activity against *E. coli* exhibits a certain time dependence.

[0059] (3) This antibacterial glycolipid GLP6 has good serum and pancreatic enzyme stability.

[0060] Antimicrobial peptides have some drawbacks, such as short in vivo half-life, susceptibility to enzymatic hydrolysis or clearance, reduced bioavailability, and hindered clinical application. Therefore, their serum stability was studied by co-incubating GLP-6 with 50% serum. Figure 4 As shown, after incubation with 50% serum for 0.5 h and 1.5 h, the MIC values ​​of the antimicrobial glycolipid against E. coli ATCC 25922 were both 11 μM, and the MIC value against S. aureus ATCC 29213 was both 5 μM. These values ​​were almost unchanged compared to the antimicrobial glycolipid without 50% serum (E. coli ATCC 25922: MIC = 9 μM, S. aureus ATCC 29213: MIC = 5 μM). This indicates that the antimicrobial glycolipid maintains good antimicrobial activity against both Gram-positive and Gram-negative bacteria in the presence of serum, thus demonstrating its high serum stability.

[0061] The stability of pancreatic enzymes was investigated by co-incubating GLP6 with different concentrations of pancreatic enzymes. Figure 5 The results show that, under the same conditions and time, after co-incubation of antibacterial glycolipids and trypsin, when the concentration of trypsin increased from 10... -3 Gradually increase μg / mL to 10 3At μg / mL, the relative survival rate of bacteria gradually increased, indicating that with the increase of trypsin concentration, the amount of peptides degraded gradually increased, leading to a decrease or even loss of antibacterial activity. However, for S. aureus ATCC 29213, the antibacterial activity of the antimicrobial glycolipid was basically unaffected under low concentration conditions, proving that it has good trypsin stability.

[0062] (3) This antibacterial glycolipid peptide GLP6 has good biocompatibility.

[0063] Hemolytic activity and cytotoxicity are important indicators for verifying drug safety and cell selectivity. Hemolytic activity was analyzed by examining morphological changes in erythrocytes after interaction with physiological saline and glycolipid peptides. Figure 6 a, b) Figure 6 a represents the morphology of red blood cells after injection of physiological saline. Figure 6 b shows the morphology of erythrocytes after interaction with GLP6. It can be seen that after injection of physiological saline and GLP6, the erythrocytes exhibited normal biconcave disc-like morphology, with no leakage of contents. This demonstrates that the GLP6 studied in this experiment has almost no hemolytic activity.

[0064] In addition to the hemolysis assay, the MTT assay was used to detect the survival rate of mouse macrophage-like cell line RAW264.7 cells after treatment with antimicrobial glycolipids, in order to evaluate the toxicity of antimicrobial glycolipids to eukaryotic cells. Figure 6 As can be seen from c, regardless of whether the incubation time was 24h or 48h, when the GLP6 concentration was below 64μM, the relative cell viability of RAW264.7 was greater than 87%, which is far greater than the MIC value (E. coli ATCC 25922: MIC = 9μM, S. aureus ATCC 29213: MIC = 5μM). This means that while exerting normal bactericidal activity, it basically does not cause damage to normal cells. Moreover, as the concentration decreased, the cell viability of RAW264.7 cells gradually increased. When the concentration reached 128μM, the cell viability dropped sharply. At 24h, the relative cell viability of RAW264.7 was 51.07%; at 48h, the relative cell viability was 50.29%. This indicates that within a certain concentration range, GLP6 antimicrobial glycolipids have virtually no killing effect on normal mammalian cells, meaning they have high selectivity for bacteria, almost no cytotoxicity, and the killing effect does not increase over time.

[0065] (4) This antibacterial glycolipid GLP6 has good membrane-breaking activity.

[0066] Biofilms are aggregates of microorganisms that protect bacteria from being killed and cleared by antibiotics and the body's immune defense system. This study investigated the disruptive effect of GLP-6 on biofilms formed by *S. aureus* ATCC 29213 and *E. coli* ATCC 25922 using crystal violet staining. Figure 7 The study showed that after 24 hours of treatment with different concentrations of GLP-6 on established *S. aureus* and *E. coli* biofilms, the biofilm-disrupting ability of both was significantly enhanced with increasing peptide concentration, exhibiting a concentration-dependent effect. For *S. aureus*, a biofilm clearance rate of 59% was achieved at a concentration of 4 μM, exceeding 60% and 70% at 16 μM and 128 μM, respectively. For *E. coli*, a biofilm clearance rate of 40% was achieved at a concentration of 4 μM, reaching 50% at 8 μM. This demonstrates that GLP-6 can disrupt biofilms and shows promise for use in biofilm infections. The strong biofilm-clearing ability of GLP-6 against *S. aureus* biofilms may be due to the protective outer membrane of *E. coli*, preventing its removal by the drug.

[0067] Gram-negative bacteria are more difficult to penetrate than Gram-positive bacteria, mainly because Gram-negative bacteria possess a special outer membrane, which is closely related to bacterial resistance, adhesion, invasion, and biofilm formation. The fluorescent dye 1-naphthylaminobenzene (NPN), as a hydrophobic fluorescent dye, shows almost no fluorescence in an aqueous environment but fluoresces in a hydrophobic environment. When the bacterial outer membrane is intact, NPN shows almost no fluorescence in an aqueous environment; when the drug disrupts the bacterial outer membrane, it enters the hydrophobic environment of the membrane and fluoresces, with the fluorescence intensity directly proportional to the degree of membrane damage. Figure 7 As shown in b, GLP6 can disrupt the outer membrane of negative bacteria, and the trend is generally that the fluorescence intensity gradually increases with increasing drug concentration, indicating a concentration-dependent effect. Furthermore, the fluorescence intensity reaches its peak within approximately 4 minutes, and there are no significant fluctuations in fluorescence intensity thereafter, suggesting that GLP6 disrupts the membrane relatively rapidly.

[0068] according to Figure 7 c and Figure 7As can be seen from Figure d, the absorbance increased with increasing concentration of the antimicrobial glycolipid peptide in both the E. coli ATCC 25922 and S. aureus ATCC 29213 groups. In the E. coli ATCC 25922 group, the absorbance continued to increase after 100 min, indicating that the membrane-disrupting effect of the antimicrobial peptide was ongoing. However, in the S. aureus ATCC 29213 group, the absorbance did not change significantly over time. This may be because Staphylococcus aureus does not have an outer membrane barrier, allowing the glycolipid peptide to rapidly disrupt its inner membrane, causing the absorbance to peak quickly. In contrast, the presence of an outer membrane in E. coli ATCC 25922 resulted in a slower effect. These results indicate that this antimicrobial glycolipid peptide possesses good membrane lysis or permeation capabilities and exerts its antimicrobial effect through a membrane disruption mechanism.

[0069] To understand the antibacterial mechanism of glycolipids, this study incubated 4×MIC and 16×MIC GLP6 (final concentrations of 2×MIC and 8×MIC) with *E. coli* ATCC 25922 and *S. aureus* ATCC 29213 for 4 h, respectively. TEM was used to observe changes in bacterial morphology before and after drug administration and after treatment with different drug concentrations. The results showed that the blank control group of *E. coli* (… Figure 8 a) The E. coli membrane was intact and smooth, consisting of short, rounded-end bacilli with distinct membrane boundaries, exhibiting a normal morphology. After treatment with 2×MIC GLP-6 ( Figure 8 b) The bacteria exhibit diverse morphologies, blurred membrane boundaries, wrinkles, and uneven internal material dispersion. After treatment with 8×MIC GLP-6 ( Figure 8 c) In E. coli, the bacterial membrane rupture was more severe, with surrounding vesicles appearing and a large amount of internal material leaking out. In the S. aureus group, the blank control group ( Figure 8 d) The S. aureus membrane was intact and smooth, with tight plasma membrane binding, exhibiting a normal spherical shape. After treatment with 2×MIC GLP-6 ( Figure 8 e) The bacterial morphology remained largely unchanged, but some leakage of contents was observed. After treatment with 8×MIC of GLP6 ( Figure 8 f) shows that a large amount of S. aureus contents leaked out, and some bacteria contained almost no contents. Overall, the antimicrobial glycolipid peptide caused severe damage to the bacterial membrane, demonstrating that GLP6 has excellent membrane permeability and lytic activity. Moreover, both 2×MIC and 8×MIC GLP6 showed stronger membrane-disrupting effects on E. coli, proving that GLP6's killing ability against E. coli is greater than that against S. aureus.

[0070] Flow cytometry was used to detect the uptake of antimicrobial glycolipids by E. coli ATCC 25922 at different time points, thereby investigating the disruptive effect of antimicrobial glycolipids on bacterial membranes. To maintain consistent concentrations, the final concentration was set to 36 μM (i.e., 4 × MIC). Figure 9 It can be seen that after E. coli ATCC 25922 was treated with glycolipid for 2h, 4h, 6h, 8h, and 10h, the percentages of fluorescent bacteria with disrupted membranes were 90.8%, 88.1%, 85.6%, 86.8%, and 84.5%, respectively. The fluorescence intensity reached its maximum within 2h, proving that PI had entered the bacterial interior in a relatively short time, further demonstrating that the antibacterial glycolipid has a strong ability to disrupt membranes.

[0071] The membrane-disrupting ability of the antimicrobial lipopeptide LP6 on E. coli ATCC 25922 and the distribution of LP6 in E. coli ATCC 25922 bacteria were analyzed by laser confocal microscopy. Figure 10 Fluorescence imaging showed that at 0 h, no bacteria exhibited red fluorescence of PI, indicating that no PI entered the bacterial interior; simultaneously, no green fluorescence of luciferin-labeled LP6 (LP6-luciferin) was observed within the bacteria. However, after treatment with LP6-luciferin for 4 h, 8 h, and 12 h, significant red fluorescence of PI and green fluorescence of LP6-luciferin appeared inside the bacteria. The fluorescence intensity gradually increased with treatment time, peaking at 12 h, indicating that the amount of antimicrobial lipopeptide LP6 entering the bacterial interior gradually increased with treatment time; that is, the stronger the membrane disruption, the more bacterial death occurred, exhibiting a time-dependent effect. Since GLP6 is a monosaccharide-modified LP6 compound, GLP6 should also possess almost the same properties and functions as those shown in CLSM.

[0072] (3) This antibacterial glycolipid GLP6 has a significant antibacterial effect in vivo.

[0073] A mouse peritonitis model was established by intraperitoneal injection of E. coli ATCC 25922, and its in vivo antibacterial effect was evaluated by injection of the antimicrobial glycolipid GLP6. Following intraperitoneal infection with E. coli ATCC 25922, mice exhibited lethargy, loss of appetite, abdominal retraction, viscous excretions, and forked fur. Figure 11 The results showed that after 24 hours, all mice in the saline group died, while the survival rate in the GLP-6 group was 66.7%; after 48 hours, the survival rate in the GLP-6 group was 50%. Furthermore, compared to the antibacterial GLP-6 group, the surviving mice in the control group were lethargic, had slower recovery of appetite, and exhibited severe abdominal retraction. Peritoneal fluid was collected at different time points for bacterial counting. Figure 11Results showed that the bacterial count in the peritoneal fluid of mice in the NS group was approximately the same as the initial bacterial count at 12h and 24h, while the bacterial count in the GLP6 group gradually decreased in a time-dependent manner. This indicates that the designed antimicrobial glycolipopeptide GLP6 can effectively inhibit or kill bacteria in mice and has a certain therapeutic effect on acute peritonitis induced by E. coli ATCC 25922 in mice.

Claims

1. An antibacterial glycolipid peptide GLP6, characterized in that, The sequence of the antimicrobial glycolipid GLP6 from the N-terminus to the C-terminus is C8H. 15 O-Ala-Lys-Ala-Phe-Leu-Pro-Ile-Ile-Arg-Trp-Lys-Lys-Arg-Trp-C6H 12 NO5 (abbreviated as C8H) 15 O-AKAFLPIIRWKKRW-C6H 12 NO5), where Ala (A) is alanine, Lys (K) is lysine, Phe (F) is phenylalanine, Leu (L) is leucine, Pro (P) is proline, Ile (I) is isoleucine, Arg (R) is arginine, Trp (W) is tryptophan, and the attached fatty acid is octanoic acid (C8H). 16 O2, the attached sugar molecule is D-(+)-glucosamine C6H 13 The structural formula of NO5, an antibacterial glycolipid peptide, is shown in the figure below: 。

Images