Construction and activity evaluation of antibacterial peptide inhibitor GLIP16 specifically recognized and combined with BamA

Abstract

The invention relates to preparation of an antibacterial peptide inhibitor GLIP16 with good biocompatibility, stability and excellent antibacterial activity and application of the antibacterial peptide inhibitor GLIP16 in treatment of bacterial infection, and belongs to the field of medicine. The amino acid sequence of the antibacterial peptide inhibitor GLIP16 is C16H28N2O-His-Arg-Trp-Lys-Lys-Leu-Lys-Leu-Trp-Trp-Gly-Trp-Lys-Tyr-Lys-Phe-C6H12NO5 (abbreviated as C12H20N2O-WKKLLKWWLKKFKKLD-C6H12NO5), and the antibacterial peptide inhibitor GLIP16 is designed on the basis of a plurality of important biological characteristics (net positive charge, amphipathy and alpha-helical structure) of the antibacterial peptide and a key conserved sequence of an outer membrane protein beta signal, so that the antibacterial peptide inhibitor GLIP16 can be used for preparing the antibacterial peptide inhibitor GLIP16. Imidazolyl ionic liquid C16H29N2O2 and monosaccharide C6H13NO5 are respectively coupled to the N end and the C end of the N-terminal and the C-terminal of the N-terminal and the C-terminal of the N-terminal and the C-terminal. The invention provides a technical scheme for solid-phase synthesis of an antibacterial peptide inhibitor skeleton. The GLIP16 has excellent antibacterial activity, good in-vitro stability and good biocompatibility. An in-vivo antibacterial experiment shows that the designed antibacterial peptide inhibitor can play an excellent antibacterial role in a mouse infection model and is an antibiotic substitute with a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the preparation of an amphiphilic cationic antimicrobial peptide inhibitor GLIP16 with excellent in vitro antibacterial activity, good in vitro stability and high biosafety, and its application in treating bacterial infections. Background Art

[0002] Overuse of antibiotics has led to the global spread of multidrug-resistant bacteria, necessitating the development of novel antimicrobial agents. Antimicrobial peptides (AMPs), with their unique broad-spectrum antimicrobial activity, low resistance induction, and targeted selectivity, are considered promising candidates for antibiotic replacement. However, their clinical application is still limited by bottlenecks such as insufficient chemical stability, host cytotoxicity risks, and delivery system limitations. Therefore, while ensuring the excellent antimicrobial activity of AMPs, their stability needs to be enhanced, their cytotoxicity reduced, and their bioavailability improved to expand their clinical application prospects.

[0003] Antimicrobial peptides (AMPs) are natural defense molecules present in nearly all life forms, acting directly against bacteria to exert antimicrobial effects. The cationicity and amphipathic structure of AMPs form the molecular basis for their membrane-targeting activity. Cationic amino acids (such as arginine and lysine) impart a positive charge (+2 to +9) to AMPs, enabling them to bind specifically to lipoteichoic acid (LTA) of Gram-positive bacteria or lipopolysaccharide (LPS) of Gram-negative bacteria through electrostatic interactions. AMPs, relying on the hydrophobic regions formed by hydrophobic amino acids (such as tryptophan and leucine), insert into the bacterial membrane lipid bilayer, triggering membrane lysis (pore formation or collapse) or interfering with intracellular targets (such as nucleic acids and enzyme activity), thereby playing an important role in maintaining host-microbe homeostasis. Unlike traditional antibiotics (which target a single specific protein or metabolic pathway), the multi-mechanistic activity of AMPs (membrane disruption, immune activation, and biofilm inhibition) enables them to significantly reduce the risk of drug resistance through synergistic effects. The positive charge (+4 to +9) of cationic AMPs (such as human defensins) can be partially neutralized by negatively charged components on the bacterial surface, but their amphipathic α-helical structure can still penetrate membrane barriers through hydrophobic interactions, leading to cytoplasmic leakage and cell death. Screening of AI peptides based on the leech transcriptome revealed that hirunipin 2 exerts broad-spectrum antimicrobial, antibiofilm, and anti-inflammatory activities through a dual mechanism of membrane disruption and intracellular aggregation, with low cytotoxicity, low resistance induction, and synergistic potential. The active core fragment of the human antimicrobial peptide LL-37 (LL-3717-29) self-assembles into thermostable helical fibrils. Its alternating hydrophobic-cationic surface bands target bacterial membrane structures and mimic the cross-α-amyloid assembly mechanism of the bacterial toxin PSMα3, exhibiting broad-spectrum antimicrobial activity (including membrane lysis and intracellular colocalization). Therefore, antimicrobial peptides, with their multi-mechanistic antimicrobial activity and low resistance propensity, exhibit excellent antimicrobial efficacy through membrane lysis and are considered a core strategy for addressing the threat of multidrug-resistant bacteria in the post-antibiotic era.

[0004] Despite their superior antimicrobial activity, antimicrobial peptides face dual stability challenges: degradation by serum proteases during systemic application (e.g., inactivation of LL-37 by binding to serum proteins) and destruction by environmental / wound factors (hydrolysis, oxidation, alkaline pH, and proteolysis) during topical application. Furthermore, some AMPs (e.g., melittin) induce hemolytic toxicity by non-selectively cleaving eukaryotic cell membranes. To overcome this bottleneck, multi-dimensional structural modification is required. Increasing the proportion of cationic amino acids, such as lysine (Lys), enhances binding to negatively charged bacterial membranes (LPS / LTA), and inserting tryptophan (Trp) and leucine (Leu) into the mid-peptide chain to form a hydrophobic core, promote membrane insertion, balance amphipathicity, and form an α-helical structure, thereby enhancing antimicrobial activity. N- / C-terminal amidation is a useful modification method for regulating the proteolytic stability and antimicrobial activity of AMPs. For example, fatty acid modification can increase the stability of AMPs by blocking regions susceptible to protease damage or forming supramolecular structures, further prolonging the efficacy of lipidated AMPs. Imidazolyl ionic liquid modification, which combines a long-chain alkyl hydrophobic backbone with an imidazolyl cationic group, can optimize the stability and biocompatibility of AMPs. Glycosylation modification can mask enzymatic cleavage sites and increase molecular rigidity, reducing AMP toxicity, increasing molecular stability, and improving physicochemical and pharmacological parameters. Combining nanocarriers with poorly soluble antibiotics (such as tetracycline) can reduce the risk of drug resistance through a dual mechanism of membrane disruption and drug penetration. The combined application of multiple strategies has synergistically improved the targeted antimicrobial activity, enzymatic stability, and biosafety of antimicrobial peptides, providing new perspectives for the development of highly effective and low-toxic antimicrobial peptide inhibitors.

[0005] In order to enable antimicrobial peptides to precisely target bacteria and reduce their damage to host cells, antimicrobial peptide sequences can be modified based on the unique bacterial structure. Outer membrane β-barrel proteins (β-OMPs) are important components of the outer membrane of Gram-negative bacteria, and the bacterial β-Barrel Assembly Machinery (BAM) is present in all Gram-negative bacteria and can serve as a potential new antibiotic target. The BAM transport assembly machinery assists in the folding, integration, and completion of the structural assembly of Gram-negative bacterial β-OMPs through dynamic conformational changes, thereby maintaining the integrity of the outer membrane and the normal operation of the barrier function and material exchange. Newly synthesized OMPs in the bacterial cytoplasm are first translocated across the inner membrane by the SecYEG transposon, and then escorted to the outer membrane by periplasmic chaperone proteins. In the outer membrane, they are assembled into β-barrels by BAM and transported to the outer membrane to form complete β-OMPs. Most β-OMPs contain a highly conserved β-signal motif at their C-termini: Hy-G-Hy-Po-Hy-Po-F (Po: polar amino acid; Hy: hydrophobic amino acid; Y is preferred at the third position from the C-terminus). Periplasmic OMPs are delivered to the BAM complex by chaperone proteins, where they specifically recognize and bind to the β1 strand of BamA via the C-terminal β-signal sequence, initiating folding and membrane insertion. OMPs fold in an antiparallel manner to form a β-sheet structure. Ultimately, as BamA's β16 strand interlocks with the β1 strand, the OMPs complete a head-to-tail cyclization, forming a functional β-barrel structure and integrating into the outer membrane. The natural cyclic peptide antibiotic darobactin mimics the OMP β-signal by forming a rigid, bicyclized β-strand conformation. It targets the lateral gate of the Gram-negative bacterial BamA protein, displacing lipid molecules at this site and utilizing the membrane environment as an expanded binding pocket, thereby blocking outer membrane protein folding and insertion. Therefore, targeted blocking of the binding process between BamA and the OMPsβ-signaling motif can effectively inhibit the folding and transport of bacterial OMPs, leading to defects in the outer membrane barrier function, thereby interfering with the basic physiological activities of bacteria and exerting antibacterial effects.

[0006] Therefore, based on the biological properties of antimicrobial peptides and the consensus motif of OMPsβ signaling, the present invention constructed a peptide inhibitor, GLIP16, with significant antimicrobial activity through solid-phase synthesis and C- and N-terminal amidation modification. In vitro experiments evaluated the stability and biocompatibility of GLIP16 and revealed its membrane disruption mechanism. Further studies revealed that GLIP16 can self-assemble into aggregates in aqueous solution. By loading the poorly soluble antibiotic TC to construct GLIP16@TC, the solubility of TC was enhanced, achieving a synergistic antimicrobial effect between the antimicrobial peptide inhibitor GLIP16 and the antibiotic TC. Summary of the Invention

[0007] One of the purposes of the present invention is to prepare an amphiphilic antimicrobial peptide inhibitor GLIP16 with good antimicrobial activity and stability based on polypeptide solid-phase synthesis technology. The second purpose of the present invention is to apply the prepared antimicrobial peptide inhibitor molecules to the treatment of bacterial infections.

[0008] To achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0009] The amino acid sequence of the amphiphilic cationic antimicrobial peptide inhibitor GLIP16 of the present invention is C 16 H 28 N2O-His-Arg-Trp-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Trp-Gly-Trp-Lys-Tyr-Lys-Phe-C6H 12 NO5, (abbreviated as C 16 H 28 N2O-HRWKKLLKKLWGWKYKF-C6H 12 NO5), where His (H) is histidine, Arg (R) is arginine, Trp (W) is tryptophan, Lys (K) is lysine, Leu (L) is leucine, Gly (G) is glycine, Tyr (Y) is tyrosine, Phe (F) is phenylalanine, and the N-terminal modification is C 16 H 29 N2O2, the C-terminal connected monosaccharide molecule is C6H 13 NO5. Its design method is as follows: Based on the OMPsβ-signaling motif Hy-G-Hy-Po-Hy-Po-F (Po: polar amino acid; Hy: hydrophobic amino acid; Y is preferred at the third position from the C-terminus) and several important biological characteristics of antimicrobial peptides (net positive charge, amphipathicity and α-helical structure), the positively charged hydrophilic amino acids Lys (K), Arg (R), His (H) and the hydrophobic amino acids Trp (W), Leu (L) and Tyr (Y) are introduced into the designed amino acid sequence, and the imidazolyl ionic liquid C is coupled to its N-terminus and C-terminus respectively. 16 H 29 N2O2 and monosaccharide molecule C6H 13 NO5 obtained antimicrobial peptide inhibitors. According to the designed peptide sequence, amino acids were coupled sequentially from C-terminus to N-terminus by peptide solid phase synthesis method and the Fmoc protecting group of the N-terminal amino acid was removed to obtain peptide resin. The synthetic imidazolyl ionic liquid C was coupled to the N-terminus of the peptide resin. 16 H 29 N2O2 and remove the resin, then couple the monosaccharide molecule C6H at the C-terminus of the antimicrobial lipopeptide 13 NO5, finally, all side chain protecting groups of the obtained antimicrobial peptide inhibitor are removed. The antimicrobial peptide inhibitor has the following structural formula:

[0010]

[0011] The secondary structure of the obtained antimicrobial peptide inhibitor GLIP16 was determined by circular dichroism spectroscopy; the in vitro antimicrobial activity of the prepared antimicrobial peptide inhibitor GLIP16 was tested by the microbroth dilution method; the serum stability, temperature stability, pancreatic enzyme stability and biocompatibility of the prepared antimicrobial peptide inhibitor GLIP16 were studied; the antimicrobial mechanism of the antimicrobial peptide inhibitor GLIP16 was studied; finally, the in vivo antimicrobial activity of the antimicrobial peptide inhibitor GLIP16 was evaluated. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : CD spectra of antimicrobial peptide inhibitor GLIP16 in pure water and SDS solution.

[0013] Figure 2 : The killing kinetics of Escherichia coli by the antimicrobial peptide inhibitor GLIP16.

[0014] Figure 3 : Determination of pancreatic enzyme stability of the antimicrobial peptide inhibitor GLIP16.

[0015] Figure 4 Biocompatibility of the antimicrobial peptide inhibitor GLIP16. (a) Relative cell viability of RAW264.7 cells treated with different concentrations of GLIP16 for 24 h; (b) Hemolytic activity of GLIP16 on mouse erythrocytes; (c) Morphology of mouse erythrocytes treated with NS (400×); (d) Morphology of mouse cells treated with GLIP16 (8 mg / kg) (400×).

[0016] Figure 5 : Membrane disruption mechanism studies. (a) Study on the outer membrane penetration mechanism of E. coli ATCC 25922 by GLIP16; (b) Study on the inner membrane penetration mechanism of E. coli ATCC 25922 by GLIP16; (c) Study on the inner membrane penetration mechanism of S. aureus ATCC 29213 by GLIP16.

[0017] Figure 6 :FCM detection of GLIP16 (4×MIC) uptake by E. coli ATCC 25922 at different time points: (a) 0h; (b) 0.5h; (c) 1h; (d) 2h; (e) 4h; (f) 6h.

[0018] Figure 7: CLSM fluorescence images (400×) of E. coli ATCC 25922 after treatment with LIP16-Fluorescein (4×MIC) at 0h, 0.5h, 1h, 2h, 4h and 6h.

[0019] Figure 8 TEM images of E. coli ATCC 25922 treated with different conditions for 2 h. (a) E. coli ATCC 25922 treated with PBS in the control group; (b) E. coli ATCC 25922 treated with 4×MIC glycolipopeptide GLIP16.

[0020] Figure 9 In vivo antimicrobial activity assays. (a) Survival rate in an acute peritonitis model in mice treated with NS and the glycolipid peptide GLIP (8 mg / kg); (b) Colony counts in peritoneal fluid of mice treated with NS and the glycolipid peptide GLIP16 (8 mg / kg) for different durations of acute peritonitis; (c) Eye recovery in mice infected with S. aureus ATCC 29213 and treated with NS and GLIP16 for different durations. DETAILED DESCRIPTION

[0021] The following are specific embodiments of the present invention to further illustrate the composition of the present invention, but the present invention is not limited to the following embodiments.

[0022] Example 1: Synthesis of the antimicrobial peptide inhibitor GLIP of the present invention

[0023] (1)Fmoc-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Le u-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-2-Chlorotrity Synthesis of Resin: with Fmoc-Phe-2-Chlorotrity Resin was used as the carrier and Fmoc-amino acid as the raw material. The peptide was synthesized using a solid phase synthesizer by the following method: First, Fmoc-Phe-2-Chlorotrity Resin (loading amount 0.566 mmol / g) was weighed and swelled for 30 min in DMF. After swelling, a DMF solution containing 20% ​​piperidine was added and stirred for 30 min. The mixture was then rinsed and filtered three times with DMF, DCM, and DMF to remove the Fmoc protecting group. Then, anhydrous DMF was added to swell the Phe-2-Chlorotrity Resin again. Resin, added 2 times the molar amount of Fmoc-Lys(Boc)-OH, and used 2.6 times the molar amount of DCC, HOBt, and DIEA as condensing agents, and stirred for 48 hours; after the reaction was completed, it was washed with DMF, DCM, and DMF three times each, and the above steps were repeated to couple the remaining Fmoc amino acids from the C-terminus to the N-terminus, and freeze-dried to obtain Fmoc-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-2-Chlorotrity Resin.

[0024] (2) Ionic Liquid C 16 H 29 Preparation of N2O2: Weigh 12-bromooctanoic acid and dissolve it in a mixture of 24 mL of anhydrous THF and 6 mL of anhydrous ethanol. Then, add a 10-fold molar amount of N-methylimidazole with stirring. Seal the mixture and react at 60°C for 32-48 hours. After the reaction is complete, remove the volatile components by rotary evaporation at 40°C. Once the solution volume remains constant (oil), add 6-fold the amount of glacial methyl tert-butyl ether to precipitate the crude product. Finally, wash the precipitate four times with glacial methyl tert-butyl ether by centrifugation and freeze-dry to obtain the desired product.

[0025] (3) Antimicrobial lipopeptide C 16 H 28Synthesis of N2O-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-COOH: First, remove the Fmoc protecting group of the polypeptide resin in (1), and weigh 1.5 times the molar amount of the ionic liquid C in (2) above. 16 H 29 N2O2 and 2.5 times the molar amount of NHS and EDC were placed in anhydrous DMF and stirred for 6 hours to activate the carboxyl group of the ionic liquid. Then, the freeze-dried resin was added to the above DMF solution and stirred for 48 hours. After the reaction was completed, it was rinsed and filtered with DMF, DCM, and DMF three times respectively, and placed in a dialysis bag (MW 8000-14000) and dialyzed with ethanol 20 times. After the dialysis was completed, lysis buffer (DCM: TFA = 99: 1, V / V) was added and stirred for 1.5 hours to remove the resin. The peptide solution was collected by filtration under reduced pressure, and the peptide solution was extracted with saturated NaHCO3 to neutralize TFA and collect the DCM phase. The DCM phase was then extracted with pure water multiple times, and finally the DCM solution was collected and concentrated by evaporation under reduced pressure, and freeze-dried to obtain C 16 H 28 N2O-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-C OOH.

[0026] (4) Antimicrobial glycolipid peptide GLIP16 (C 16 H 28 N2O-His-Arg-Trp-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Trp-Gly-Trp-Lys-Tyr-Lys-Phe-C6H 12 NO5) Synthesis: The antimicrobial lipopeptide in (3) above was dissolved in anhydrous DMF with 3 times the molar amount of NHS and EDC and stirred for 6 h to activate the carboxyl group of the peptide chain, and then 3 times the molar amount of C6H 13 NO5·HCl was added simultaneously with a 3-fold molar amount of triethylamine and stirred for 48 h. After the reaction was complete, the mixture was placed in a dialysis bag (MW 1500) and dialyzed several times with pure water, and freeze-dried to obtain C 12 H 21N2O-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-Trp(Boc)-Leu-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Asp(OtBu)-C6H 12 NO5; Finally, the reaction was stirred with a cutting solution (TFA:H2O:Tis=95:2.5:2.5, V / V) for 1.5h to remove the side chain protecting group, the filtrate was evaporated and concentrated under reduced pressure, precipitated by adding icy methyl tert-butyl ether, and then centrifuged and washed 3 times with icy methyl tert-butyl ether, and freeze-dried to obtain glycolipid peptide C 16 H 28 N2O-His-Arg-Trp-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Trp-Gly-Trp-Lys-Tyr-Lys-Phe-C6H 12 NO5.

[0027] Example 2: Characterization of the secondary structure of the antimicrobial peptide inhibitor GLIP16

[0028] The secondary structure of the glycolipid peptide GLIP16 was determined using circular dichroism (CD) spectroscopy. GLIP16, an antimicrobial peptide inhibitor, was dissolved in pure water (simulating a hydrophilic environment) and 30 mM SDS (simulating a negatively charged microbial membrane) to prepare a 150 μM glycolipid peptide solution. The sample was placed in a quartz cell (0.5 mm path length) and scanned over a wavelength range of 180–260 nm. The obtained CD spectra were converted to average residual ellipticity using the following formula:

[0029] θ M =(θ obs ×1000) / cln

[0030] Among them, θ M is the residual ellipticity (deg·cm 2 dmol -1 ),θ obs is the observed ellipticity (mdeg) corrected for buffer at a given wavelength, c is the peptide concentration (mM), l is the path length (mm), and n is the number of amino acids.

[0031] Example 3: Determination of in vitro antibacterial activity of the antimicrobial peptide inhibitor GLIP16

[0032] Minimum inhibitory concentration (MIC) determination: E. coli ATCC 25922 and S. aureus ATCC 29213 were inoculated into LB medium and cultured at 37°C overnight. The culture was then transferred to fresh LB and cultured until the logarithmic growth phase. The bacterial suspension was centrifuged, the pellet was washed twice with PBS, and the bacteria were resuspended in fresh LB to a concentration of approximately 1×10 5 Prepare a bacterial suspension with a concentration of 100 μM CFU / mL. A serial dilution series of peptide inhibitor GLIP16 stock solutions was performed to obtain concentrations ranging from 2 to 256 μM. 50 μL of the bacterial suspension and 50 μL of the peptide solution at various concentrations after serial dilution were added to a 96-well plate, with at least three replicates for each concentration. Wells containing only LB served as negative controls, while wells containing bacteria but no peptide solution served as positive controls. The plates were incubated at 37°C for 12 to 16 hours. The minimum inhibitory concentration (MIC) of GLIP16 was determined as the lowest concentration at which no turbidity was observed on the bottom of the 96-well plate.

[0033] Minimum Bactericidal Concentration (MBC) Assay: Bacterial suspensions from clear wells of the MIC 96-well plate were plated onto LB plates without further dilution and incubated at 37°C for 18 hours. The MBC in LB medium was defined as the lowest drug concentration required to eliminate 99.9% of viable bacteria.

[0034] Bactericidal kinetics: E. coli ATCC 25922 in the logarithmic growth phase was washed with PBS and centrifuged, then resuspended to a concentration of about 10 5 CFU / mL of bacterial solution. GLIP16 solutions of varying concentrations were mixed with an equal volume of bacterial solution (final GLIP16 concentrations were 4×MIC, 2×MIC, and 1×MIC) and incubated at 37°C. For the control group, PBS was mixed with an equal volume of bacterial solution. The mixture was aspirated at 0, 0.5, 1, 2, 4, 6, and 12 hours of incubation, diluted, and plated. The mixture was incubated at 37°C for 18 hours, and the number of colonies in each group at each time point was counted.

[0035] Example 4: In vitro stability determination of the antimicrobial peptide inhibitor GLIP16

[0036] Serum stability: GLIP16 solutions at various concentrations were mixed with an equal volume of FBS solution (FBS:PBS = 1:1) and incubated at 37°C for 30 min. The antimicrobial activity of the peptide inhibitors against E. coli ATCC 25922 and S. aureus ATCC 29213 in serum was determined using the MIC method described above.

[0037] Temperature stability: GLIP16 solutions of different concentrations were incubated at 4°C, room temperature, 37°C, and 60°C for 30 min. After returning to room temperature, the antibacterial activity of the peptide inhibitors against E. coli ATCC 25922 and S. aureus ATCC 29213 was determined according to the above-mentioned MIC method.

[0038] Stability of trypsin: 25% trypsin stock solution was diluted with PBS to different concentrations of trypsin solution (10 -3 ~10 3 μg / mL) and mixed with 4×MIC GLIP16 solution in equal volumes. After incubation at 37°C for 1 hour, the mixture was immediately transferred to 60°C for 20 minutes to inactivate trypsin. E. coli ATCC 25922 and S. aureus ATCC 29213 in the logarithmic growth phase were washed with PBS and centrifuged, and then resuspended to a concentration of about 10 5 ~10 6 CFU / mL of bacterial solution. 100 μL of peptide solution co-incubated with trypsin and 100 μL of bacterial solution were added to a 96-well plate. After incubation at 37°C for 18 h, the OD value of each well was measured at 600 nm to calculate the bacterial survival rate.

[0039] Example 5: Biocompatibility determination of antimicrobial peptide inhibitor GLIP16

[0040] Cytotoxicity assay: The cytotoxicity of antimicrobial peptide inhibitors against mouse macrophage RAW264.7 cells (mouse mononuclear leukemia cells) was determined using the MTT reduction assay. GLIP16 was prepared at various concentrations (256 μmol / L, 128 μmol / L, 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L, and 1 μmol / L). RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates and incubated in a cell culture incubator for 24 hours. The supernatant was discarded, and 100 μL of LIP16 or GLIP16 solution at various concentrations was added to each well. The cells were incubated in the incubator for 24 hours, with five replicates for each concentration. MTT solution (20 μL) was added to each well in the dark. After incubation for 4 hours, the supernatant was discarded, and 150 μL of DMSO medium was added to each well. The 96-well plate was shaken at 170 rpm in the dark for 7 min, and the OD value was measured at a wavelength of 485 nm. The relative survival rate of each well was calculated to evaluate the cytotoxicity of GLIP16.

[0041] Hemolytic assay: Blood was collected from the mouse eyeball and placed in an anticoagulant tube. The blood was centrifuged at 1000 rpm for 10 min at 4°C. The supernatant was discarded and the cell suspension was resuspended in saline and centrifuged three times. The red blood cells were prepared into a 2% cell suspension with saline and added to a 96-well plate at 100 μL per well. Different concentrations of GLIP16 solution (128 μmol / L, 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L, 1 μmol / L) were added to a 96-well plate at 100 μL per well. 100 μL of saline was added to each well of the negative control group, and 100 μL of sterile water was added to each well of the positive control group. Five replicates were set up and incubated at 37°C for 1 hour. Centrifuge the 96-well plate at 3000 rpm for 5 min, transfer 100 μL of the supernatant to another 96-well plate, measure the absorbance at 570 nm, and calculate the hemolysis rate at different concentrations according to the formula:

[0042] Hemolysis (%) = [(A-A0) / (A 100 -A0)]×100

[0043] Where A represents the absorbance measured in the glycolipid peptide GLIP solution, A0 and A 100 The absorbances at 0% and 100% hemolysis were measured in 0.9% NaCl and sterile water, respectively.

[0044] In vivo hemolysis assay: Two healthy mice were randomly selected and injected via the tail vein with 200 μL of GLIP16 solution (8 mg / kg) and 200 μL of sterile saline (negative control). Two hours later, blood was collected from the eyeballs of the mice. The blood was collected in anticoagulant tubes, diluted with saline, and then observed for red blood cell morphology under a fluorescence microscope.

[0045] Example 6: Study on the membrane disruption mechanism of the antimicrobial peptide inhibitor GLIP16

[0046] Outer membrane permeation assay: E. coli ATCC 25922 in the logarithmic growth phase was washed with PBS, centrifuged, and resuspended. NPN solution with a final concentration of 10 μM and inhibitor solutions with final concentrations of 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, and 0 μM were added to the bacterial suspension, and E was measured in a fluorescence spectrophotometer. x =350nm, E m = Fluorescence intensity at 420 nm. Polymyxin B (1 mg / mL) was used as a positive control. The assay was repeated every 30 seconds until the fluorescence intensity showed no significant change. The average value over 10 minutes was taken and the NPN uptake rate was calculated according to the following formula:

[0047] NPN uptake (%) = (F obs -F0) / (F 100 -F0)×100%

[0048] Among them, F obs is the fluorescence observed at a given peptide concentration, F0 is the initial fluorescence of the NPN in the absence of peptide, and F 100 It is the fluorescence of NPN after adding 1 mg / mL polymyxin B. "100%" represents the NPN uptake of polymyxin B as a positive control.

[0049] Inner membrane permeation assay: E. coli ATCC 25922 and S. aureus ATCC 29213 in logarithmic growth phase were washed with PBS and centrifuged, then resuspended in 1.5 mM ONPG in PBS. Fifty microliters of bacterial suspension and 50 microliters of peptide solutions of varying concentrations were added to a 96-well plate. The absorbance was measured at 420 nm using a microplate reader, every five minutes for 60 minutes.

[0050] FCM assay: E. coli ATCC 25922 cells in the logarithmic growth phase were washed with PBS, centrifuged, and resuspended. An 8×MIC GLIP16 solution was mixed with an equal volume of the bacterial suspension and incubated at 37°C for 0, 0.5, 1, 2, 4, and 6 hours. After centrifugation and washing, the supernatant was discarded and a 50 μg / mL PI solution was added. The cells were incubated at 37°C for 15 minutes, washed, centrifuged, and resuspended in 400 μL of PBS. Flow cytometry was performed at a wavelength of 488 nm.

[0051] CLSM experiment: E. coli ATCC 25922 in the logarithmic growth phase was washed with PBS, centrifuged, and resuspended. A 16×MIC LIP16-Fluorescein solution was mixed with an equal volume of the bacterial suspension and incubated at 37°C for 0, 0.5, 1, 2, 4, and 6 hours. After centrifugation and washing, the supernatant was discarded and a 50 μg / mL PI solution was added. The cells were incubated at 37°C for 15 minutes, washed, centrifuged, and resuspended in 400 μL of PBS. A 10 μL aliquot was placed on a glass slide, coverslipped, and observed under a confocal laser scanning microscope.

[0052] TEM experiment: Transmission electron microscopy (TEM) was used to observe changes in bacterial morphology and intracellular substances after GLP16 treatment. E. coli ATCC 25922 in the logarithmic growth phase was washed with PBS, centrifuged, and resuspended. A 4×MIC GLIP16 solution was mixed with an equal volume of bacterial suspension and incubated at 37°C for 4 hours. The negative control group was mixed with an equal volume of bacterial suspension and incubated with PBS. After incubation, the cells were washed and centrifuged twice with PBS, and then fixed overnight with 3% glutaraldehyde solution. They were washed four times with PBS and fixed with 1% osmium acid for 90 minutes. After washing four times with PBS, they were dehydrated with graded ethanol of 50%, 70%, 80%, 95%, and 100% for 15 minutes each, and replaced with acetone twice. The cells were treated with a mixture of acetone and permeabilizing agent for 3 hours and then incubated in pure permeabilizing agent overnight. The bacterial samples were embedded with embedding agent, sectioned, stained, and observed under TEM.

[0053] Example 7: In vivo antibacterial experiment

[0054] Mouse acute peritonitis model: E. coli ATCC 25922 was used to establish a mouse acute peritonitis model. Mice were randomly divided into two groups. E. coli ATCC 25922 in the logarithmic growth phase was washed with PBS and centrifuged three times, and then resuspended in NS to a concentration of approximately 10 8 CFU / mL of bacterial suspension. Each mouse was intraperitoneally injected with 200 μL of bacterial solution and the mice were observed. The model was successfully established when the mice showed symptoms such as curling up, less movement, listlessness, depressed abdomen, loss of appetite, sticky feces, and inverted fur. Each group of mice was injected with NS and GLIP16 (8 mg / kg), 200 μL per mouse. The survival status of each group of mice was observed and recorded, and the mouse survival rate curve was drawn. Peritoneal fluid was collected at 0h, 12h, 24h, and 48h after treatment, and the number of colonies was counted using the dilution plate count method.

[0055] Mouse bacterial keratitis model: A mouse bacterial keratitis model was established using S. aureus ATCC 29213. S. aureus ATCC 29213 in the logarithmic growth phase was washed with PBS and centrifuged three times, then resuspended in NS to a concentration of approximately 10 8CFU / mL of bacterial suspension. Before the experiment, the mouse eyeballs were observed with a slit lamp to ensure that their corneas were in normal physiological state. The mice were randomly divided into NS group and GLIP16 group. Each mouse was intraperitoneally injected with 4% chloral hydrate for general anesthesia, and a drop of 1% lidocaine was dripped into the mouse eyeball. After standing for 1 minute, it was absorbed with absorbent paper, and the operation was repeated 4 times, and then rinsed with NS 3 times. The cornea was scratched with a sterile blade, and the scratch was observed with a slit lamp. 20μL of bacterial solution was added to the corneal wound, and the eye infection was observed and recorded 12 hours after infection. 20μL of NS and GLIP16 (0.003g / mL) were added to each group of mice respectively, once a day for three consecutive days, and the recovery of the eyeball was observed and recorded.

[0056] The antimicrobial peptide inhibitors prepared by the method of the present invention have good biomedical properties:

[0057] (1) The antimicrobial peptide inhibitor GLIP16 has an α-helical structure in a prokaryotic biomembrane simulation environment

[0058] The secondary structure of GLIP16 in aqueous solution and SDS solution (simulating bacterial cell membrane environment in vitro) was detected by CD spectrum and it was found that its secondary structure would be significantly adjusted with the change of environment. Figure 1 As shown, in water, GLIP16 forms a negative peak at 200 nm, lacking the characteristic α-helical structure. However, in an SDS solution-simulated bacterial cell membrane environment, it forms a positive peak at 192 nm and two negative peaks at 208 nm and 222 nm, indicating typical α-helical structure peaks. The α-helical structure content of GLIP16 in aqueous and SDS solutions was also measured. The α-helical structure content in water was only 10%, but increased to 58.3% in SDS solution. This environmentally dependent structural transition suggests that GLIP16 spontaneously forms a regular α-helical conformation upon contact with bacterial membranes, enhancing its amphipathicity and enabling more efficient membrane disruption.

[0059] (2) This antimicrobial peptide inhibitor GLIP16 has excellent in vitro antimicrobial activity

[0060] The in vitro antimicrobial activity of the antimicrobial peptide inhibitor GLIP16 against E. coli ATCC 25922 and S. aureus ATCC 29213 was determined by the broth microdilution method. As shown in Table 1, the MIC values ​​of the antimicrobial peptide inhibitor GLIP16 against E. coli ATCC 25922 and S. aureus ATCC 29213 were 3 μM and 2 μM, respectively, demonstrating excellent in vitro antimicrobial activity.

[0061] To further evaluate the antibacterial activity of synthesized GLIP16, an MBC assay was performed (Table 1). The MBC values ​​of GLIP16 against E. coli ATCC 25922 and S. aureus ATCC 29213 were 4 μM and 2 μM, respectively, which were almost unchanged from their MICs, with an MBC / MIC ratio of <4, indicating that GLIP16 can exert excellent bactericidal activity as a bactericidal agent.

[0062] Table 1 MICs and MBCs of the antimicrobial peptide inhibitor GLIP16.

[0063]

[0064] Then, the antimicrobial peptide inhibitor GLIP16 was tested for its bactericidal kinetics against Gram-negative bacteria E. coli ATCC 25922 at concentrations of 4×MIC, 2×MIC, and MIC, respectively. Figure 2 As shown, during the initial exposure phase (0.5 h), all experimental groups (4×MIC, 2×MIC, and MIC) showed a significant decrease in colony counts compared to the PBS control group, but a complete bactericidal effect was not achieved. The 4×MIC group achieved complete sterilization within 1 h, while only a small number of bacteria remained in the 2×MIC and MIC groups at this time point. From 2 to 12 h, the 4×MIC and 2×MIC groups remained sterile, while the MIC group maintained an inhibitory effect (no significant increase in colony count) until 4 h. However, after 6 h, bacterial regrowth at subinhibitory concentrations was observed, which was still significantly lower than that of the PBS control group during the same period. The PBS control group, on the other hand, showed an increasing trend throughout the observation period. The bactericidal effect of GLIP16 showed a clear concentration- and time-dependency.

[0065] (3) This antimicrobial peptide inhibitor GLIP16 has good in vitro stability

[0066] The in vitro serum stability of GLIP16 was evaluated using a 25% FBS co-incubation assay. As shown in Table 2, GLIP16 exhibited good serum stability. Its MIC values ​​against E. coli ATCC 25922 and S. aureus ATCC 29213 remained unchanged after serum treatment, maintaining its antibacterial activity. This demonstrates that GLIP16 exhibits good resistance to proteolysis and excellent serum stability.

[0067] Table 2 MIC (μM) of GLIP16 in 25% FBS.

[0068]

[0069] At the same time, the MIC values ​​of GLIP16 after incubation at different temperatures for 30 min were determined. As shown in Table 3, the MIC value of GLIP16 did not change significantly, indicating that GLIP16 can maintain structural integrity in a wide temperature range (4-60°C) and has good thermal stability.

[0070] Table 3 MIC values ​​(μM) of GLIP16 after incubation at different temperatures for 30 min

[0071]

[0072] Furthermore, the in vitro trypsin stability of GLIP16 was evaluated by measuring the changes in the antibacterial activity of the inhibitor against bacteria after trypsin digestion. Figure 3 As shown, in the trypsin concentration gradient (10 -3 to 10 3 μg / mL) treatment conditions, E. coli ATCC 25922: high concentration enzyme treatment group (10 0 -10 3 μg / mL) bacterial survival rate was about 90%, while the low concentration group (10 -3 -10 -1 μg / mL) survival rate dropped sharply to about 3%, trypsin concentration ≥10 0 The antibacterial effect of GLIP16 may be significantly weakened by enzymatic hydrolysis at 10 μg / mL; S. aureus ATCC 29213: The survival rate increased stepwise with the increase of enzyme concentration. 2 -10 3 The survival rate of the 10 μg / mL group was 70%. 1 The 10 μg / mL group was 12%, 0 μg / mL group decreased to 6%, and the low concentration group (10 -3 -10 -1 The antibacterial activity of GLIP16 was significantly trypsin-dependent.

[0073] (4) This antimicrobial peptide inhibitor GLIP16 has good biocompatibility

[0074] The toxicity of GLIP16 to RAW264.7 macrophages was evaluated by MTT assay, and the results showed that its toxicity was significantly concentration-dependent. Figure 4As shown in a, when the inhibitor concentration is less than 32 μM, the relative survival rate of the cells is above 70% (when the inhibitor concentration is less than 16 μM, the relative survival rate of the cells is above 90%), and the toxicity to the cells is relatively low. Compared with the antibacterial MIC value, its therapeutic index (TI = cytotoxic concentration / antibacterial concentration) is as high as 10 times or more, showing a wide safety window. With the increase of concentration, the relative survival rate of the cells decreases. When the inhibitor concentration is greater than 128 μM, the relative survival rate of the cells is less than 30%, showing a clear concentration-dependent trend. Therefore, this result highlights the biosafety advantage of GLIP16 within the therapeutic window, and its selectivity can be further optimized through other strategies in the future.

[0075] To further test the biocompatibility of GLIP16, fresh mouse red blood cells were used to evaluate its hemolytic activity. The results showed that the hemolytic effect of GLIP16 was concentration-dependent. The in vitro hemolysis rate increased significantly with increasing concentration ( Figure 4 b), the hemolysis rate of the high concentration group (128 μM) of GLIP16 was as high as 94%, but it gradually decreased with the decrease of concentration, and the hemolysis rate of the low concentration group (1 μM) was only 4%. However, through morphological observation ( Figure 4 cd) showed that at a concentration of 8 mg / kg, the erythrocyte morphology of mice treated with GLIP16 was not significantly different from that of the saline control group, indicating that the risk of hemolysis is manageable at this therapeutic dose. In vivo injection doses far exceeding 128 μM did not exhibit significant hemolytic effects in mice, likely due to mechanisms such as plasma protein binding, rapid metabolism, or tissue distribution that mitigate the drug's direct effects on erythrocytes. Therefore, GLIP16 exhibits excellent biosafety.

[0076] (5) This antimicrobial peptide inhibitor GLIP16 has good membrane-breaking activity

[0077] The unique outer membrane structure of Gram-negative bacteria (composed of lipopolysaccharides, porins, and a phospholipid bilayer) is an important barrier to the penetration of antimicrobial drugs. This experiment used the hydrophobic fluorescent probe NPN (1-N-phenylnaphthylamine) to evaluate the outer membrane-disrupting efficacy of GLIP16: when the outer membrane is intact, NPN quenches fluorescence due to its hydrophilic extracellular environment; when drugs disrupt the integrity of the outer membrane, NPN can embed itself into the hydrophobic lipid region and emit fluorescence, the intensity of which is positively correlated with the degree of membrane damage ( Figure 5 a). Experimental data showed that as the GLIP16 concentration gradient increased, the NPN fluorescence intensity exhibited a dose-dependent increase, indicating that this glycolipid peptide can gradually destabilize the outer membrane of E. coli ATCC 25922. The permeability of GLIP16 may be related to its sugar chain-mediated targeting mode of action, achieving selective transmembrane access through specific recognition of BamA rather than relying on potent membrane lytic activity. This provides a new mechanistic perspective for balancing antimicrobial activity and host cytotoxicity.

[0078] To further explore the destructive properties of GLIP16 on the bacterial inner membrane, this study used an ONPG (o-nitrophenyl-β-D-galactoside) hydrolysis experiment for dynamic monitoring. As a polar molecule, ONPG cannot penetrate the intact membrane structure. When GLIP16 causes membrane damage, ONPG enters the cell through the damaged membrane structure and is hydrolyzed by β-galactosidase to produce o-nitrophenol (ONP). The increase in absorbance at 420nm can quantitatively reflect the degree of membrane permeability enhancement ( Figure 5 bc). Experimental results showed that GLIP16 exhibited significant concentration-dependent membrane-damaging effects on both E. coli ATCC 25922 and S. aureus ATCC 29213. However, the membrane damage to E. coli ATCC 25922 increased over time, while the extent of membrane damage to S. aureus ATCC 29213 remained largely unchanged. These results suggest that GLIP16 possesses excellent inner membrane permeability and can exert its antibacterial activity through membrane disruption.

[0079] The transmembrane action mechanism of GLIP16 on E.coli ATCC 25922 was revealed by FCM quantitative data and CLSM fluorescence imaging. Figure 6-7 In FCM, the PI positivity rate in the control group was 0% at 0 h, indicating that the intact cell membrane effectively blocked PI influx. After 0.5 h of inhibitor treatment, the PI permeability rate surged to 91.5% (fluorescence intensity 1807 au), indicating that GLIP16 rapidly disrupted the integrity of the cell membrane. Over time, the PI permeability rate gradually increased, reaching a peak at 4 h and then slightly decreasing at 6 h, although the fluorescence intensity continued to increase, indicating that the inhibitor could continue to accumulate within the bacteria. CLSM observations revealed no LIP16-Fluorescein (green) or PI (red) signals within the bacteria at 0 h, demonstrating an intact membrane structure and the infiltration of the inhibitor and PI, consistent with the FCM results. After 0.5 h, green and red fluorescence appeared within the bacteria, and they were almost overlapping. Similarly, the number of fluorescent bacteria increased over time, reaching a peak at 4 h and decreasing slightly at 6 h. This may be due to bacterial rupture, which allowed the outflow of the inhibitor and PI, resulting in a decrease in the number of fluorescent cells within the bacteria. Combined analysis of FCM and CLSM showed that GLIP16 can achieve an antibacterial mode of rapid membrane penetration and sustained penetration, providing a key basis for the development of highly efficient and low-toxic membrane-targeted antibacterial agents.

[0080] Transmission electron microscopy was used to observe the morphology and content changes of E.coli ATCC 25922 after GLIP16 treatment ( Figure 8After PBS treatment, E. coli ATCC 25922 maintained its typical rod-shaped morphology, with an intact and continuous cell membrane structure and a rich cytoplasm. However, after GLIP16 treatment, E. coli ATCC 25922 underwent significant morphological changes, with visible dissociation of the inner and outer membrane bilayers, incomplete membrane rupture, a transparent interior, the outflow of contents, and a shortening of the bacteria, losing their typical rod-shaped characteristics. This suggests that GLIP16 exerts its antibacterial effects by increasing membrane permeability and disrupting membrane integrity.

[0081] (5) This antimicrobial peptide inhibitor GLIP16 has excellent in vivo antimicrobial activity

[0082] The in vivo antibacterial effect of GLIP16 was determined by establishing an acute bacterial peritonitis model in mice through intraperitoneal injection of E. coli ATCC 25922. 1.5 hours after infection, mice in each group showed symptoms such as lethargy, huddling, refusal to eat, and slow reaction, as well as severe diarrhea, confirming the successful establishment of the model. Each group of mice was injected with 200 μL of NS and GLIP16 (8 mg / kg) for treatment. The survival of mice in each group at different time points was recorded, and the peritoneal fluid was collected to count the colonies. Figure 9 a, The survival rate of mice in the NS group was 0, while that in the GLIP16 group was 60%, which indicates that GLIP16 can effectively exert its antibacterial activity in vivo and improve the survival rate of mice. Figure 9 As shown in (b), the bacterial count in the peritoneal fluid of the NS group mice increased at 12 h, while the bacterial count in the peritoneal fluid of the GLIP16 group mice gradually decreased during the treatment period, indicating that GLIP16 can effectively exert its antibacterial effect in vivo in a short period of time.

[0083] The in vivo anti-Staphylococcus aureus activity of the inhibitors was evaluated by acute keratitis assay in mice ( Figure 9 c). Under normal physiological conditions, the mouse eyeballs and corneas are transparent, intact, smooth, and free of vascular proliferation. However, 12 hours after infection, significant increases in eye opacity, corneal congestion, and swelling were observed. Each group of mice was treated with the corresponding NS and GLIP16 (0.003g / mL). In the GLIP16 group, eye symptoms gradually subsided over time, with clear, transparent eyes and intact corneas after the third treatment. In contrast, in the NS group, eye opacity remained unchanged and progressively increased after treatment, indicating no therapeutic effect on inflammation. This suggests that GLIP16 is effective against eye infections caused by Staphylococcus aureus.

Claims

1. An antimicrobial peptide inhibitor GLIP16, wherein the sequence of the antimicrobial peptide inhibitor GLIP16 from N-terminus to C-terminus is C 16 H 28 N2O-His-Arg-Trp-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Trp-Gly-Trp-Lys-Tyr-Lys-Phe-C6H 12 NO5, (abbreviated as C 16 H 28 N2O-HRWKKLLKKLWGWKYKF-C6H 12 NO5), where His (H) is histidine, Arg (R) is arginine, Trp (W) is tryptophan, Lys (K) is lysine, Leu (L) is leucine, Gly (G) is glycine, Tyr (Y) is tyrosine, Phe (F) is phenylalanine, and the N-terminal modification is C 16 H 29 N2O2, the C-terminal connected monosaccharide molecule is C6H 13 NO5 (D-(+)-glucosamine), the structural formula is shown below:

2. An antimicrobial peptide inhibitor GLIP16 according to claim 1, wherein the preparation method is as follows: (1) Synthesis of Fmoc-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-2-Chlorotrity Resin: Fmoc-Phe-2-Chlorotrity Resin was used as a carrier and Fmoc-amino acid as a raw material. The peptide was synthesized using a solid phase synthesizer. First, Fmoc-Phe-2-Chlorotrity Resin (loading capacity 0.566mmol / g) was added with DMF and swelled for 30min. After swelling, a DMF solution containing 20% ​​piperidine was added and stirred for 30min. The mixture was rinsed and filtered three times with DMF, DCM, and DMF to remove the Fmoc protecting group. Then, anhydrous DMF was added to re-swell the Phe-2-Chlorotrity Resin, added 2 times the molar amount of Fmoc-Lys(Boc)-OH, and used 2.6 times the molar amount of DCC, HOBt, and DIEA as condensing agents, and stirred for 48 hours; after the reaction was completed, it was washed with DMF, DCM, and DMF three times each, and the above steps were repeated to couple the remaining Fmoc amino acids from the C-terminus to the N-terminus, and freeze-dried to obtain Fmoc-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-2-Chlorotrity Resin. (2) Ionic Liquid C 16 H 29 Synthesis of N2O2: Weigh 12-bromooctanoic acid and dissolve it in a mixed solution of 24 mL of anhydrous THF and 6 mL of anhydrous ethanol, then add 10 times the molar amount of N-methylimidazole under stirring, seal, and react at 60°C for 32-48 hours; after the reaction is completed, remove the volatile components by rotary evaporation at 40°C under reduced pressure, and wait until the solution volume remains unchanged (oil), add 6 times the amount of glacial methyl tert-butyl ether to precipitate the crude product; finally, centrifuge and wash the precipitate four times with glacial methyl tert-butyl ether, and freeze-dry to obtain the target product. (3) Antimicrobial lipopeptide C 16 H 28 Synthesis of N2O-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-COOH: First, remove the Fmoc protecting group of the polypeptide resin in (1), and weigh 1.5 times the molar amount of the ionic liquid C in (2) above. 16 H 29 N2O2 and 2.5 times the molar amount of NHS and EDC were placed in anhydrous DMF and stirred for 6 hours to activate the carboxyl group of the ionic liquid. Then, the freeze-dried resin was added to the above DMF solution and stirred for 48 hours. After the reaction was completed, it was rinsed and filtered with DMF, DCM, and DMF three times respectively, and placed in a dialysis bag (MW 8000-14000) and dialyzed with ethanol 20 times. After the dialysis was completed, lysis buffer (DCM: TFA = 99: 1, V / V) was added and stirred for 1.5 hours to remove the resin. The peptide solution was collected by filtration under reduced pressure, and the peptide solution was extracted with saturated NaHCO3 to neutralize TFA and collect the DCM phase. The DCM phase was then extracted with pure water multiple times, and finally the DCM solution was collected and concentrated by evaporation under reduced pressure, and freeze-dried to obtain C 16 H 28 N2O-His(Trt)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Trp(Boc)-Gly-Trp(Boc)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Phe-COOH. (4) Antimicrobial glycolipid peptide GLIP16 (C 16 H 28 N2O-His-Arg-Trp-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Trp-Gly-Trp-Lys-Tyr-Lys-Phe-C6H 12 NO5) Synthesis: The antimicrobial lipopeptide in (3) above was dissolved in anhydrous DMF with 3 times the molar amount of NHS and EDC and stirred for 6 h to activate the carboxyl group of the peptide chain, and then 3 times the molar amount of C6H 13 NO5·HCl was added simultaneously with a 3-fold molar amount of triethylamine and stirred for 48 h. After the reaction was complete, the mixture was placed in a dialysis bag (MW 1500) and dialyzed several times with pure water, and freeze-dried to obtain C 12 H 21 N2O-Trp(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-Trp(Boc)-Leu-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Asp(OtBu)-C6H 12 NO5; Finally, the reaction was stirred with a cutting solution (TFA:H2O:Tis=95:2.5:2.5, V / V) for 1.5h to remove the side chain protecting group, the filtrate was evaporated and concentrated under reduced pressure, precipitated by adding icy methyl tert-butyl ether, and then centrifuged and washed 3 times with icy methyl tert-butyl ether, and freeze-dried to obtain glycolipid peptide C 16 H 28 N2O-His-Arg-Trp-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Trp-Gly-Trp-Lys-Tyr-Lys-Phe-C6H 12 NO5.

3. According to claim 1, the secondary structure of GLIP16 is determined by circular dichroism spectroscopy, and the results show that the antimicrobial peptide inhibitor of the present invention exhibits an α-helical structure in a membrane simulation environment; the antimicrobial peptide inhibitor is subjected to in vitro antimicrobial activity determination by broth microdilution and plate coating methods, and the results show that the antimicrobial peptide inhibitor has excellent antimicrobial activity; serum stability testing and biocompatibility evaluation are also conducted, and the results show that the antimicrobial peptide inhibitor of the present invention has excellent proteolysis resistance and good biocompatibility; the antimicrobial mechanism is studied by membrane disruption experiments and bacterial uptake experiments, and the results show that the antimicrobial peptide inhibitor exerts antimicrobial activity by destroying bacterial membranes; and the prepared antimicrobial peptide inhibitor is confirmed to have excellent in vivo antimicrobial activity by an in vivo antimicrobial experiment in mice.

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