Bacterial phospholipid-induced random β-sheet structures can be transformed into antimicrobial peptides and their preparation methods.

Abstract

The application belongs to the technical field of antibacterial polypeptides, and provides a bacterial phospholipid-induced random-β-folding structure convertible antibacterial peptide and a preparation method thereof. The bacterial phospholipid-induced random-β-folding structure convertible antibacterial peptide provided by the application has the following structural general formula: wherein n represents the number of amino acid residues, and the value range is 5-15; R1 is selected from the group consisting of an amino acid residue, a short peptide, an alkyl group, an aromatic group or a polyether chain substituted carbonyl group. The antibacterial peptide provided by the application can selectively activate antibacterial activity on the surface of bacterial cell membranes. The application constructs a series of random-β-folding structure convertible antibacterial peptides (Cβ-AMPs) R1-AG n -CA targeting bacterial phospholipid PG through a solid-phase synthesis method and a thiol-ene click reaction.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to antimicrobial peptides with bacterial phospholipid-induced random-β-sheet structures that can be converted and their preparation methods. Background Technology

[0002] Antimicrobial peptides are a class of naturally occurring antimicrobial active substances widely found in the biological world. They form the body's first line of defense against bacterial infection and possess broad-spectrum antimicrobial activity. Antimicrobial peptide molecules typically consist of 10–50 positively charged and hydrophobic amino acid residues. Antimicrobial peptides primarily adsorb onto bacterial cell membranes through electrostatic interactions, subsequently inserting into the membrane through hydrophobic interactions and causing membrane structure disruption. Because the basic components of bacterial cell membranes are relatively conserved evolutionarily, bacteria are less likely to develop resistance to antimicrobial peptides. Compared to other antimicrobial polymers, the prominent structural feature of antimicrobial peptides lies in their specific secondary structures, such as α-helices and β-sheets. Furthermore, antimicrobial peptides also exhibit good killing effects against antibiotic-resistant strains and biofilm-associated low-metabolic bacteria, thus being considered an important direction for the development of novel antimicrobial drugs.

[0003] Antimicrobial peptides (AMPs) are widely considered promising therapeutic options against multidrug-resistant bacterial infections due to their broad-spectrum antimicrobial activity and low tendency to develop resistance. These peptides typically consist of cationic and hydrophobic amino acid residues that bind to the phospholipid bilayer via electrostatic interactions, subsequently inserting into the membrane through hydrophobic interactions, thus disrupting membrane integrity. Bacterial cell membranes are rich in anionic phospholipids, such as phosphatidylglycerol (PG, 20%–90%) and cardiolipin (CL, 5%–50%). However, mammalian cell membranes also contain negatively charged phospholipids—phosphatidylserine (PS, <15%), primarily located in the inner lobes of the cell membrane, and CL (~20%) in the mitochondrial membrane. These characteristics make AMPs nonspecifically cytotoxic to mammalian cells, as similar membrane lysis mechanisms can cause local or systemic toxicity. Despite significant differences in the types, abundances, and spatial distribution of anionic phospholipids between bacterial and mammalian cell membranes, achieving selective targeting of bacterial cell membranes with AMPs remains a significant challenge.

[0004] The secondary structure of AMPs is a key determinant of their membrane interactions. Well-defined structural motifs, particularly α-helices and β-sheets, promote the formation of amphiphilic structures, which are crucial for efficient membrane insertion and disruption. Radially amphiphilic helical AMPs can distinguish phospholipid components, exhibiting a significantly higher affinity for prostaglandins (PGs) than for phospholipids (PSs) (Yangbin Liang et al., Helicity-directed recognition of bacterial phospholipid via radiallyamphiphilic antimicrobial peptides). Sci. Adv. 10, eadn9435(2024).DOI:10.1126 / sciadv.adn9435). In contrast, random AMPs exhibit lower lipid-binding activity and negligible membrane-disrupting activity. However, helical AMPs readily enter mammalian cells and disrupt CL-rich mitochondrial membranes, causing off-target toxicity. A key challenge in this field is to rationally modulate the secondary structure of AMPs to maximize bactericidal selectivity while minimizing nonspecific damage to normal tissue cells and organelles. Summary of the Invention

[0005] The purpose of this invention is to overcome the lack of antimicrobial selectivity in existing antimicrobial peptides and to provide a bacterial phospholipid-induced atlanto-β-sheet structure that can be converted into an antimicrobial peptide, enabling the peptide to selectively activate antimicrobial activity on the bacterial cell membrane surface. This invention constructs a series of atlanto-β-sheet structure-convertible antimicrobial peptides (Cβ-AMPs) R1-AG that target bacterial phospholipid PG through solid-phase synthesis and thiol-olefin click reaction. n -CA.

[0006] This invention provides a bacterial phospholipid-induced random-β-sheet structure that can be converted into an antimicrobial peptide, the general structural formula of which is shown below:

[0007] (I) Wherein, n represents the number of amino acid residues, and its value ranges from 5 to 15; R1 is selected from: amino acid residues, short peptides, alkyl groups, and aromatic groups substituted with carbonyl groups.

[0008] Preferably, R1 is selected from: amino acid residues with alkyl or aromatic side chains, short peptides consisting of 2-4 amino acids, C1-C 15 Alkyl-substituted carbonyl groups, aromatic-substituted C1-C 15 Carbonyl group.

[0009] More preferably, R1 is selected from: phenylalanine residues, tryptophan residues, tyrosine residues, glycine residues, alanine residues, C6-C... 12 Alkyl and aromatic substituted C6-C 12 alkyl.

[0010] Preferably, R1 is selected from: , , , .

[0011] Preferably, R1 is selected from: , , , , , , , One of them.

[0012] This invention also provides a method for preparing the above-mentioned antimicrobial peptide with bacterial phospholipid-induced random-β-sheet structural transition, comprising the following steps: (1) Dissolve Fmoc-allyl glycine, terminal molecules, DIC, and Oxyma in DMF respectively, install them in the corresponding positions of the CEM peptide synthesizer, and add MBHA resin to the reaction cell. (2) After setting the microwave heating time, number of coupling cycles and number of amino acid residues of the target polypeptide, start the reaction; (3) After the reaction is complete, the resin carrying the peptide is mixed with mercaptoethylamine molecules and added to DMF. After purging with nitrogen, 2,2-dimethoxy-2-phenylacetophenone is added, and a click reaction is performed under ultraviolet light to modify the mercaptoethylamine part to the side chain of the peptide. (4) Wash the resin with DMF and methanol, and then soak it in methanol and dry it under vacuum. (5) The peptide was cleaved from the resin using a lysis buffer obtained by mixing trifluoroacetic acid, triisopropylsilane and water, precipitated in a mixed solution of diethyl ether and n-hexane, and then dried under vacuum. (6) The obtained product was mixed with mercaptoethylamine and added to DMSO. 2,2-Dimethoxy-2-phenylacetophenone was added, and a click reaction was performed under ultraviolet light to completely modify the side chain of the polypeptide with mercaptoethylamine, so as to obtain the antimicrobial peptide R1-AGn-CA with bacterial phospholipid-induced random-β-sheet structure transformation.

[0013] Preferably, the allyl glycine polypeptide in step (1) has 5-15 residues and the terminal groups are n-hexyl, dodecyl, phenylpropyl, tryptophan, and phenylalanine.

[0014] More preferably, the allyl glycine polypeptide in step (1) has 12 residues.

[0015] More preferably, the end group in step (1) is phenylpropyl.

[0016] Preferably, the microwave heating time in step (2) is 1 to 4 minutes, and the coupling is performed 1 to 3 times.

[0017] More preferably, step (2) involves microwave heating for 2 minutes and coupling once.

[0018] Preferably, the molar ratio of the polypeptide to mercaptoethylamine in step (3) is 0.01-0.1:1, and the ultraviolet irradiation time is 1-4 h.

[0019] More preferably, the molar ratio of the polypeptide to mercaptoethylamine in step (3) is 0.02:1.

[0020] Further preferred, the ultraviolet irradiation time in step (3) is 2 h.

[0021] Preferably, the methanol soaking time in step (4) is 5~20 min.

[0022] More preferably, the methanol soaking time in step (4) is 15 min.

[0023] Preferably, in step (5), the volume ratio of trifluoroacetic acid, triisopropylsilane, and water is 95:2.5:2.5, and the volume ratio of diethyl ether to n-hexane is 1:1.

[0024] More preferably, in step (5), the volume ratio of diethyl ether to n-hexane is 1:1.

[0025] This invention also provides the application of the above-mentioned bacterial phospholipid-induced random-β-sheet structure convertible antimicrobial peptide material in antimicrobial activity.

[0026] The preparation method provided by this invention uses solid-phase synthesis combined with click chemistry to obtain antimicrobial peptides with bacterial phospholipid-induced random-β-sheet structures that can be converted. The process is simple, convenient to operate, low in cost, and effectively achieves selective antibacterial activity.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The method of solid-phase synthesis and click chemistry produces peptides with accurate structures and is simple; (2) The present invention can significantly reduce the toxicity of antimicrobial peptides to normal cells while maintaining high antimicrobial activity. Attached Figure Description

[0028] Figure 1 H-AG, a polypeptide that transitions from a random to a β-sheet structure. n -The synthetic route of CA.

[0029] Figure 2 For AG n -CA 1 H-NMR spectrum.

[0030] Figure 3 For AG n -Mass spectrum of CA.

[0031] Figure 4 R1-AG 12 -CA 1 H-NMR spectrum.

[0032] Figure 5 R1-AG 12 -Mass spectrum of CA.

[0033] Figure 6 For Bn-AG 12 -Circular dichroism chromatograms before and after CA is combined with DPPG and DPPS.

[0034] Figure 7 For Bn-AG 12 -Dose-dependent antibacterial activity of CA against Staphylococcus aureus.

[0035] Figure 8 For Bn-AG 12 -Graph showing the results of CA killing clinically isolated drug-resistant bacteria.

[0036] Figure 9 For Bn-AG 12 -Dose-dependent cytotoxicity of CA on sheep blood cells and NIH / 3T3 cells.

[0037] Figure 10 For Bn-AG 12 -Isothermal titration calorimetry results of the interaction between CA and DPPG and DPPS.

[0038] Figure 11 For Bn-AG 12 - Image showing the results of dye leakage from liposomes after CA treatment of liposomes with different phospholipid components.

[0039] Figure 12 For Bn-AG 12 - Results of CA inhibiting Staphylococcus aureus biofilm formation (a) and clearing mature biofilm (b).

[0040] Figure 13 Staphylococcus aureus biofilm via Bn-AG 12 - Three-dimensional fluorescence imaging results after CA processing.

[0041] Figure 14 For Bn-AG12 -CA has bactericidal activity against bacteria in biofilms.

[0042] Figure 15 Bn-AG was administered intravenously. 12 - Blood biochemistry results of mice after CA treatment.

[0043] Figure 16 For Bn-AG 12 -Graph showing the tissue bacterial load results after CA treatment in mice with acute bacterial cystitis.

[0044] Figure 17 For Bn-AG 12 -Graph showing the tissue bacterial load results after CA treatment for bacterial biofilm-induced cystitis in mice.

[0045] Figure 18 For Bn-AG 12 -Graph showing the tissue bacterial load after CA treatment for infection of subcutaneous implant biofilm in mice. Detailed Implementation

[0046] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0047] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0048] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0049] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0050] In the compounds of this invention, when any variable (e.g., R2, R3, etc.) appears more than once in any component, the definition of each occurrence is independent of the definition of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. A line drawn from a substituent into the ring system indicates that the bond referred to can be attached to any substituted ring atom. If the ring system is polycyclic, it means that such a bond is attached only to any suitable carbon atom of a neighboring ring. It is to be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is substituted by more than one group, it should be understood that these groups can be on the same carbon atom or different carbon atoms, as long as the structure is stable. The phrase "optionally substituted by one or more substituents" is considered equivalent to the phrase "optionally substituted by at least one substituent," and in this case, the preferred embodiment will have 0-3 substituents.

[0051] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0052] The drug of the present invention for treating bacterial infections can be used in non-human mammals or humans.

[0053] The pharmaceutically acceptable excipients used in the medicaments for treating bacterial infections of the present invention refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0054] "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate with the active ingredient of the present invention (the compound shown in Formula I or its host-guest complex or its pharmaceutically acceptable salt) without significantly reducing the efficacy of the active ingredient.

[0055] The pharmaceutically acceptable excipients used in the medicament for treating bacterial infections of the present invention include, but are not limited to, one or more of the following materials: solvents, excipients, fillers, compatibilizers, binders, humectants, disintegrants, slow solvents, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents, and fragrances.

[0056] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0057] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0058] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0059] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silica; (b) Adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) Moisturizers, such as glycerin; (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginate, certain complex silicates, and sodium carbonate; (e) Slow solvents, such as paraffin; (f) Absorption accelerators, for example, quaternary ammonium compounds; (g) Wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) Adsorbents, such as kaolin; (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0060] The solid dosage form can also be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient from this composition can be delayed in a portion of the digestive tract. Examples of suitable encapsulating components are polymers and waxes.

[0061] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0062] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0063] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0064] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0065] The following are specific examples.

[0066] In the following examples, polypeptides with double bonds in their side chains were obtained by solid-phase synthesis. Subsequently, a series of cationic antibacterial polypeptide materials of formula (I) were synthesized by reacting the double bonds with thiol groups. The reaction formulas are as follows:

[0067] The solid-phase synthesis was carried out using a CEM peptide synthesizer. The required Fmoc-allylglycine, MBHA resin, amidation catalyst, and mercaptoethylamine were all commercially available.

[0068] In the following examples, "1 eq" means 0.2 mmol.

[0069] Example 1: n=5, 8, 10, 12, 15, R1 is H Cβ-AMPs (AG n Preparation and characterization of -CA See the synthetic route. Figure 1A CEM peptide synthesizer was used, with MBHA resin as the solid-phase support and Fmoc-L-allylglycine as the amino acid monomer. The coupling reaction used N,N′-diisopropylcarbodiimide (DIC) and ethyl oxime cyanoacetate (Oxyma) as catalysts, and dimethylformamide (DMF) as the solvent. During the synthesis, the Fmoc protecting group was removed using a 10% (v / v) piperidine / DMF solution. The target peptides had 5, 8, 10, 12, and 15 amino acid residues. The MBHA resin was swollen with DMF before the reaction. The amide coupling reaction was carried out at 90 °C under microwave assistance for 1 min, with three coupling cycles (or one coupling cycle for 4 min). After the reaction, the resin was washed three times with DMF. Peptides R1-AG modified with different alkyl chains or aromatic groups were synthesized through the above steps. 12 .

[0070] For peptides with n = 5, 8, 10, 12, and 15, 1.14 g, 1.82 g, 2.27 g, 2.73 g, and 3.41 g of mercaptoethylamine hydrochloride were weighed out and dissolved in 10 mL of DMF, respectively, and placed in a 25 mL round-bottom flask. Then, 400 mg of AG-loaded peptides were added. n MBHA resin (loading 0.5 mmol / g) was stirred for 15 min under a nitrogen atmosphere. 2,2-Dimethoxy-2-phenylethyl ketone (DMPA, 5 mg) was added, and stirring continued for 15 min under light-protected and nitrogen atmosphere. Then, a click reaction was carried out under a 365 nm UV point light source for 2 h. After the reaction, the resin was washed three times with DMF and methanol, soaked in methanol for 5 min, and the solvent was removed under vacuum. The resin was collected and dried for later use.

[0071] Subsequently, a peptide lysis buffer (volume ratio: 95% trifluoroacetic acid, 2.5% triisopropylsilane, 2.5% deionized water) was prepared, and the dried resin was soaked for 2 h to cleave the peptides on the resin. The resulting lysis buffer was transferred to a 50 mL centrifuge tube, and a mixture of diethyl ether and n-hexane (volume ratio 1:1) was added. The mixture was centrifuged (4 ℃, 3000 rpm), and the supernatant was discarded. This operation was repeated 3 times, and the precipitate was collected. The solvent was removed under vacuum.

[0072] The precipitate was collected and dissolved in DMSO (15 mL) with mercaptoethylamine hydrochloride (10 mmol), and the click reaction was repeated as described above. After the reaction was completed, the product was purified by dialyzing in deionized water using a dialysis bag with a molecular weight cutoff of 1000 Da, and then lyophilized to obtain the final product AG. n -CA (n=5, 8, 10, 12, 15). The prepared AG n -CA 1 H-NMR spectrum as follows Figure 2 As shown, AG n -The mass spectrum of CA is as follows Figure 3 As shown.

[0073] Example 2: Preparation and characterization of Cβ-AMPs with n=12 and R1 representing different functional groups. First, a CEM peptide synthesizer was used with MBHA resin as the solid support and Fmoc-L-allylglycine as the amino acid monomer. The coupling reaction used N,N′-diisopropylcarbodiimide (DIC) and ethyl oxime cyanoacetate (Oxyma) as catalysts, and dimethylformamide (DMF) as the solvent. During the synthesis, the Fmoc protecting group was removed using a 10% piperidine / DMF solution. The target peptide had 12 amino acid residues, and in the final synthesis step, C6 and C7 were modified at the N-terminus of the peptide. 12 The MBHA resin was swollen with DMF before the reaction. The amide coupling reaction was carried out at 90 °C under microwave assistance for 4 min, with one coupling cycle. After the reaction, the resin was washed three times with DMF. The above steps yielded polypeptides R1-AG modified with different alkyl chains or aromatic groups. 12 .

[0074] Weigh 2.73 g of mercaptoethylamine hydrochloride and dissolve it in 10 mL of DMF. Place the solution in a 25 mL round-bottom flask, then add 400 mg of R1-AG-loaded solution. 12 MBHA resin (loading 0.5 mmol / g) was stirred for 15 min under a nitrogen atmosphere. 2,2-Dimethoxy-2-phenylethyl ketone (DMPA, 5 mg) was added, and stirring continued for 15 min under light-protected and nitrogen atmosphere. Then, a click reaction was carried out under a 365 nm UV point light source for 2 h. After the reaction, the resin was washed three times with DMF and methanol, respectively. After soaking in methanol for 15 min, the solvent was removed under vacuum, and the resin was collected and dried for later use.

[0075] Subsequently, a peptide lysis buffer (volume ratio: 95% trifluoroacetic acid, 2.5% triisopropylsilane, 2.5% deionized water) was prepared, and the dried resin was soaked for 2 h to cleave the peptides on the resin. The resulting lysis buffer was transferred to a 50 mL centrifuge tube, and a mixture of diethyl ether and n-hexane (volume ratio 1:1) was added. The mixture was centrifuged (4 ℃, 3000 rpm), and the supernatant was discarded. This operation was repeated 3 times, and the precipitate was collected. The solvent was removed under vacuum.

[0076] The precipitate was collected and dissolved in DMSO (15 mL) with mercaptoethylamine hydrochloride (10 mmol), and the click reaction was repeated as described above. After the reaction was completed, the product was purified by dialyzing in deionized water using a dialysis bag with a molecular weight cutoff of 1000 Da, and then lyophilized to obtain the final product R1-AG. 12 -CA. The prepared R1-AG 12 -CA 1 H-NMR spectrum as follows Figure 4 As shown, R1-AG 12 -The mass spectrum of CA is as follows Figure 5 As shown.

[0077] Example 3: n=12, R1 is phenylpropionyl Cβ-AMPs (Bn-AG) 12 Secondary structure characterization of -CA) Liposomes mimicking bacterial cell membranes (PG content 15% to 90%) or mammalian cell membranes (PS content 15% to 90%) were constructed, and Bn-AG was characterized by circular dichroism spectroscopy (CDs). 12 - Changes in the secondary structure of CA when bound to different liposomes. Synthetically synthesized phospholipids DPPG, DPPS, DOPC, and DOPE were dissolved in deionized water by sonication to obtain a phospholipid stock solution (10 mg / mL). Different ratios of DPPG and DOPE (phospholipid mass ratio DPPG:DOPE = 15:85, 30:70, 45:55, 60:40, 75:25, 90:10) (stock solution volume ratio V...) were then used to further investigate the effects of different ratios of DPPG and DOPE (phospholipid mass ratio DPPG:DOPE = 15:85, 30:70, 45:55, 60:40, 75:25, 90:10) (stock solution volume ratio V...). DPPG :V DOPE = 15:85, 30:70, 45:55, 60:40, 75:25, 90:10, total volume 100 μL) were mixed and diluted with deionized water (final volume 1.3 mL, final concentration 0.96 mM (total phospholipid concentration)). DPPS and DOPC (phospholipid mass ratio DPPS:DOPC = 15:85, 30:70, 45:55, 60:40, 75:25, 90:10) were mixed and diluted with deionized water to prepare a solution (final volume 1.3 mL, final concentration 0.96 mM (total phospholipid concentration)). The above liposome solutions were sonicated before use. Antimicrobial peptide Bn-AG was prepared using deionized water. 12 -CA solution (0.048mM), Bn-AG 12 -CA solution (100 μL) was mixed with equal volumes of different liposome solutions, and then added to a quartz dish with an optical path of 0.5 mm to test CDs. The experimental results are as follows. Figure 6 As shown.

[0078] Example 4: n=12, Cβ-AMPs (R1-AG) modified with R1 12-CA) Antibacterial activity and toxicity evaluation (1) Minimum inhibitory concentration test Selected Staphylococcus aureus ( Staphylococcus aureus After three generations of subculturing in LB medium (ATCC 29213), the supernatant was discarded by centrifugation, and the bacteria were resuspended and washed with PBS three times to obtain a ready-to-use bacterial suspension. Subsequently, bacterial suspensions were prepared using Mueller-Hinton Broth (MHB) medium at a concentration of 2 × 10⁻⁶. 7 CFU / mL; five antimicrobial peptides R1-AG were prepared separately. 12 -CA(C6, C) 12 Gradient concentration solutions of (F, W, Bn) with concentration ranges of 200, 100, 50, 25, 12, 6 and 3 μM.

[0079] In a sterile 96-well plate without cell culture pretreatment, 100 μL of the above bacterial suspension and an equal volume of antimicrobial peptide R1-AG were added to each well. 12 -CA solution. 200 μL MHB medium was used as a blank control (negative control), and a mixture of 100 μL MHB medium and an equal volume of bacterial suspension was used as a positive control. The 96-well plate was incubated at 37 ℃ for 24 h. After incubation, the optical density (OD) value of each well was measured at 600 nm using a microplate reader. Figure 7 As shown in a, Figure 7 b shows the minimum inhibitory concentrations (MICs) of the five antimicrobial peptides. Figure 7 It can be seen that Bn-AG with end group modified to phenylpropionyl group 12 -CA has the lowest minimum inhibitory concentration, meaning it has the highest antibacterial activity.

[0080] (2) Clinical isolation of drug-resistant bacteria killing test Clinical isolates VREfm, MRSE, MRSC, XDR-Sca, and XDR-Sag were passaged three times, centrifuged, and the supernatant was discarded. The isolates were then resuspended in PBS and washed three times to obtain a bacterial suspension for later use. Subsequently, a bacterial suspension with a concentration of 2 × 10⁻⁶ was prepared using MHB medium. 6 CFU / mL; simultaneously prepare 25 μM Bn-AG. 12 -CA in PBS solution. The bacterial suspension was mixed with the peptide solution and incubated at 37 °C for 4 h. Subsequently, plates were plated and incubated at 37 °C for 24 h, and the bacterial count was performed. Experimental results are as follows: Figure 8 As shown.

[0081] (3) Hemolysis test Take 1 mL of anticoagulated sheep blood into a 50 mL centrifuge tube, slowly add 20 mL of physiological saline and shake to wash, centrifuge (3000 rpm, 5 min), discard the supernatant, and repeat washing and centrifugation until the supernatant no longer shows obvious blood red. Prepare a 4% sheep blood cell suspension by resuspending the blood in physiological saline (24 mL). Prepare gradient concentrations (800, 400, 200, 100, 50, 25, 12, and 6 μM) of the antimicrobial peptide Bn-AG using physiological saline. 12 -CA solution, containing the antimicrobial peptide Bn-AG 12 -CA solution (100 μL) and an equal volume of sheep blood cell suspension were mixed and added to a 1.5 mL centrifuge tube. Physiological saline and 0.2% (v / v) Triton X-100 were used as negative and positive controls, respectively. The mixture was incubated at 37 ℃ for 1 h, then centrifuged (2000 rpm, 10 min). The supernatant was collected into a 96-well plate, and the OD value at 576 nm was measured using a microplate reader. The experimental results are as follows: Figure 9 As shown in a.

[0082] (4) MTT experiment Antimicrobial peptide R1-AG was characterized by the MTT assay. 12 -Cytotoxicity of CA. NIH / 3T3 mouse fibroblasts were digested and counted, and cell suspensions (1.2 × 10⁻⁶) were prepared using serum-containing complete culture medium. 5 Cell suspension (100 μL / well) was added to sterile 96-well plates prepared for tissue culture and incubated at 37 °C for 24 h. Gradual concentrations (200, 100, 50, 25, and 12 μM) of the antimicrobial peptide Bn-AG were prepared using serum-containing complete culture medium. 12 -CA solution. Aspirate the culture medium from the 96-well plate and add the antimicrobial peptide Bn-AG. 12 Complete culture medium solution of CA was used, with complete culture medium as a negative control. Three parallel controls were set up for each experimental group. The cells were incubated at 37 ℃ for 24 h. The supernatant was aspirated and MTT solution (0.5 mg / mL, 100 μL) prepared with complete culture medium was added. The cells were incubated at 37 ℃ for 2 h. Then, the supernatant was aspirated and DMSO (100 μL) was added. The OD value at 490 nm was detected using a microplate reader, and the IC50 was measured simultaneously. 50 The drug concentration at which the survival rate of NIH / 3T3 mouse fibroblasts is inhibited by 50% and the MIC (minimum inhibitory concentration) values ​​(the lowest drug concentration, i.e., the minimum inhibitory concentration, that can significantly inhibit the survival of NIH / 3T3 mouse fibroblasts after 24 hours of incubation under specific conditions). Experimental results are as follows: Figure 9 bc shows Bn-AG 12 -CA showed increased antibacterial activity without significant changes in cytotoxicity, and exhibited significantly enhanced antibacterial selectivity.

[0083] Example 5: n=12, R1 is phenylpropionyl Cβ-AMPs (Bn-AG) 12 Phospholipid selectivity studies of -CA Bn-AG was evaluated using isothermal titration calorimetry (ITC). 12 - The interaction between CA and phospholipids. Both peptide (0.383 mM) and phospholipid (DPPG or DPPS, 0.792 mM) solutions were prepared using PBS. The phospholipid solution was sonicated with a probe (sonic power 30%, 5 s intervals followed by a 5 s pause, then sonication for 1 min) before use. Both peptide and phospholipid solutions were degassed under vacuum (25 inHg, 10 mins). 50 μL of peptide solution was injected into the ITC syringe, and 300 μL of phospholipid solution was added to the sample cell. The ITC experiment was performed at 25 °C, with 25 injections of 2 μL each, spaced 300 s apart, and a stirring speed of 300 rpm. The heat of dilution (i.e., titration of peptide into PBS) was subtracted, and the resulting binding isotherms were fitted using an independent model. The experimental results are as follows: Figure 10 As shown, Bn-AG 12 -CA interacts strongly with DPPG but no significant interaction was detected with DPPS, indicating that Bn-AG 12 -CA can selectively bind to bacterial phospholipid PG.

[0084] Example 6: n=12, R1 is phenylpropionyl Cβ-AMPs (Bn-AG) 12 Selective membrane-breaking activity study of -CA) Buffer A (10 mM Na₂HPO₄, pH 7.0) and Buffer B (10 mM Na₂HPO₄, 90 mM NaCl, pH 7.0) were prepared using deionized water. A mixed solution of 1,1'-[1,4-phenylenebis(methylene)]bis(1-pyridinium) dibromide (ANTS, 12.5 mM) and disodium 8-amino-1,3,6-naphthalenetrisulfonic acid (DPX, 45 mM) was prepared using Buffer A. Liposomes with different phospholipid compositions were prepared by thin-film hydration with mass ratios of DPPG:DOPE = 15:85, 30:70, 45:55, 60:40, 75:25, and 90:10, DPPS:DOPC = 15:85, and DOPC / DOPE = 85:15. The specific method is as follows: A phospholipid-chloroform mixed solution (total concentration 25 mg / mL, total volume 160 μL) was placed in a round-bottom flask, and chloroform was added to bring the volume to 2 mL. The solution was evaporated under reduced pressure in a rotary evaporator at 37 ℃ in a water bath to form a uniform lipid film, and residual solvent was further removed under vacuum. Then, 2 mL of a fluorescent dye and quencher mixed solution was added to the flask, and the solution was hydrated by rotary evaporation at 65 ℃ and 500 rpm for 1 h. The hydrated liposome solution was subjected to repeated freeze-thaw cycles in liquid nitrogen and a 65 ℃ water bath 10 times. Then, using buffer B as the mobile phase, free ANTS and DPX were removed by passing the solution through a G-50 dextran gel column to obtain a liposome solution encapsulated with the dye. Different concentrations of the antimicrobial peptide Bn-AG were prepared using buffer B. 12 -CA solutions (100, 50, 25, and 12 μM). In a 96-well black flat-bottom plate, equal volumes of liposome solution and the antimicrobial peptide Bn-AG were added sequentially. 12 -CA solution. The negative control was buffer B, and the positive control was 0.2% (v / v) Triton X-100 solution. After incubation at 37 ℃ for 1 h, fluorescence intensity was detected using a microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 530 nm. Dye leakage rate was calculated using the following formula: Dye Leakage (%) = (F sample -F Negative ) / (F positive -F Negative )Calculation. Experimental results are as follows Figure 11 As shown.

[0085] Example 7: n=12, R1 is phenylpropionyl Cβ-AMPs (Bn-AG) 12 -CA) In vitro anti-biofilm effect characterization (1) Inhibits biofilm formation Dissolve glucose in MHB medium to prepare a 1% (g / mL) glucose MHB solution, and aseptically filter through a 0.22 μm filter membrane for later use. Add Staphylococcus aureus (… S. aureus After centrifugation (ATCC 29213), the supernatant was discarded, and the sample was resuspended in PBS. This process was repeated three times, and the solution was diluted with MHB medium containing 1% (g / mL) glucose to prepare a concentration of 2 × 10⁻⁶ g / mL. 7 CFU / mL bacterial suspension. Bn-AG was prepared using 1% (g / mL) glucose MHB solution. 12 -CA antimicrobial peptide Bn-AG 12 -CA solution (100, 50, 25, 12, 6, 3 μM) was mixed with an equal volume of bacterial suspension (V / V = 50 / 50 μL) and added to 96-well plates treated with tissue culture. The negative control was a mixture of bacterial suspension and 1% (g / mL) glucose MHB solution, and the positive control was a gradient concentration of ampicillin (100, 50, 25, 12 μM). Three replicates were set up for each concentration. After incubation at 37 ℃ for 24 h, the plates were carefully washed three times with sterile PBS to remove airborne bacteria. Then, 100 μL of methanol was added to each well for fixation for 15 min, the methanol was discarded, and the plates were air-dried. Staining was performed with 1% (g / mL) crystal violet PBS solution (100 μL) for 15 min, the supernatant was discarded, and the plates were washed three times with PBS. The crystal violet was dissolved in 33% (v / v) glacial acetic acid (200 μL), and the absorbance at 535 nm was measured using a microplate reader. The biomass content (%) was calculated using the formula: Abs sample / Abs Negtive *100%. Experimental results are as follows: Figure 12 As shown in a.

[0086] (2) Removal of mature biofilm A bacterial suspension (1 × 10⁻⁶) was prepared using a 1% (g / mL) glucose MHB solution. 7 Bacterial suspension (100 μL per well) was added to a high-adhesion 96-well plate and incubated at 37 °C for 48 h to obtain Staphylococcus aureus biofilm. After incubation, the bacterial suspension was carefully aspirated with a sterile insulin syringe, and the biofilm was washed three times with sterile PBS to remove planktonic bacterial cells. A gradient concentration (100, 50, 25, 12, 6, 3 μM) of Bn-AG was prepared using 1% (g / mL) glucose MHB solution. 12-CA and ampicillin solutions were added to 96-well plates (100 μL per well) and incubated with the biofilm at 37 ℃ for 24 h. A negative control was prepared by incubation with 1% (g / mL) glucose in MHB solution and bacterial biofilm. Three parallel groups were set up for each experimental concentration. After incubation, each sample well was carefully washed three times with sterile PBS to remove airborne bacteria. The washed sample wells were fixed with methanol (100 μL) for 15 min, the methanol was removed, and the samples were air-dried. 1% (g / mL) crystal violet in PBS solution (100 μL) was added to each well for staining for 15 min, the crystal violet solution was removed, and the samples were slowly washed three times with PBS. 33% (v / v) glacial acetic acid (200 μL) was added to each well to completely dissolve the crystal violet. The absorption signal at 535 nm was measured using a microplate reader, and the biomass content was calculated. The experimental results are as follows: Figure 12 As shown in b.

[0087] A bacterial suspension (1 × 10⁻⁶) was prepared using a 1% (g / mL) glucose MHB solution. 7 Bacterial suspension (800 μL per compartment) was added to a four-compartment confocal microsphere and incubated at 37 °C for 48 h to obtain Staphylococcus aureus biofilm. After incubation, the bacterial suspension was carefully aspirated with a sterile insulin syringe, and the biofilm was washed three times with sterile PBS to remove airborne bacterial cells. A solution of Bn-AG12-CA and ampicillin (50 μM) was prepared using 1% (g / mL) glucose MHB solution and added to a confocal microsphere (800 μL per compartment) for co-incubation with the biofilm at 37 °C for 24 h. Incubation with the bacterial biofilm using 1% (g / mL) glucose MHB solution served as a negative control. After incubation, each sample well was carefully washed three times with sterile PBS to remove airborne bacteria. A solution containing 3 μL SYTO9 (3.34 mM) and 3 μL propidium iodide PI (20 μL) per milliliter was prepared using sterile PBS. A 750 μL solution of dye (mM) was added to each dish. After staining in the dark for 15 min, the dye was gently aspirated, and 1% (g / mL) low-melting-point agarose solution was added. The dishes were allowed to cool and solidify. The samples were then photographed using a rotating confocal microscope at excitation wavelengths of 488 nm and 561 nm, with a Z-axis imaging interval of 0.4 μm. 115 frames were captured for each sample. The images were then merged using Imaris software to obtain a three-dimensional image of the biofilm. The experimental results are as follows: Figure 13 As shown.

[0088] A bacterial suspension (1 × 10⁷ CFU / mL) was prepared using 1% (g / mL) glucose MHB solution. 500 μL of the bacterial suspension was added to each well of a high-adhesion 24-well plate and incubated at 37 °C for 48 h to obtain Staphylococcus aureus biofilm. After incubation, the bacterial suspension was carefully aspirated using a sterile insulin syringe, and the biofilm was washed three times with sterile PBS to remove planktonic bacterial cells. Bn-AG was prepared using 1% (g / mL) glucose MHB solution. 12 CA and ampicillin solution (50 μM) were added to 24-well plates (500 μL per well) and incubated with the biofilm at 37 ℃ for 24 h. A negative control was prepared by incubating the bacterial biofilm with 1% (g / mL) glucose MHB solution. Three parallel control groups were set up for each group. After incubation, each sample well was carefully washed three times with sterile PBS to remove airborne bacteria. 500 μL of sterile PBS was added to each sample well, and the plates were sonicated for 10 min to disperse the bacteria in the biofilm. The bacterial suspension was then aspirated and diluted 1000-fold with PBS. 10 μL of the diluted suspension was spread onto LB agar plates and incubated at 37 ℃ for 24 h before colony counting. Experimental results are as follows: Figure 14 As shown.

[0089] Example 8: n=12, R1 is phenylpropionyl Cβ-AMPs (Bn-AG) 12 -CA) safety and efficacy against bacterial infectious diseases evaluation (1) Blood biochemistry Six-week-old female C57BL / 6J mice were randomly divided into three groups of three. Each group received either PBS or Bn-AG via tail vein injection. 12 -CA (dose of 1 mg / kg and 2 mg / kg, respectively). Whole blood was collected 24 h after administration, and serum was separated by centrifugation at 2000 rpm for 5 min for the determination of blood urea nitrogen (UREA) and alanine aminotransferase (ALT) concentrations. Experimental results are as follows: Figure 15 As shown.

[0090] (2) Treatment of acute bacterial cystitis in mice Bacterial suspensions were prepared using sterile PBS (7 × 10⁻⁶). 8 CFU / mL). Anesthetize C57BL / 6J mice (female, 6 weeks old, approximately 15 g, SPF grade), and inject 70 μL of bacterial suspension (1.34 × 10⁻⁶ CFU / mL) into the bladder via catheter. 7 (CFU / mL), with infection remaining for 2 h. Six h after infection, instillation was performed: after anesthetizing mice, 70 μL of the antimicrobial peptide Bn-AG was instilled into the bladder via a catheter.12 -CA solution (25 μM, 1 mg / kg or 2 mg / kg), drug retention for 2 h; control group perfused with PBS. Three mice per group. Mice were sacrificed 24 h after infection, and bladder tissue was collected. 300 μL of sterile ultrapure water and 5 steel balls (2 mm diameter) were added, and the mixture was homogenized at 80 Hz for 120 s, paused for 80 s, and repeated 3 times. The resulting tissue fluid was diluted 10-fold, 100-fold, and 1000-fold with PBS, and 10 μL of each dilution was plated on LB agar plates and incubated at 37 ℃ for 24 h before colony counting. Experimental results are as follows: Figure 16 As shown.

[0091] (3) Treatment of bacterial biofilm-related infectious diseases in mice For a bacterial biofilm-associated bladder infection model, female C57BL / 6 mice (6 weeks old, n=5 per group) were pretreated by intravesical injection of 0.1 mM HCl and 0.1 M NaOH (residual for 20 s each), followed by treatment with Staphylococcus aureus (S. aureus). S. aureus ATCC 29213, 1 × 10 7 CFU / bladder infection for 48 h. Mice were then administered PBS, ampicillin, or Bn-AG. 12 -CA (2 mg / kg) was administered for 24 h. Mice were sacrificed 24 h post-infection, and their bladders were harvested, homogenized in sterile PBS, serially diluted, and plated onto LB agar plates for colony counting. Experimental results are as follows: Figure 17 As shown.

[0092] For a subcutaneous implant-associated bacterial biofilm infection model, the catheter was cut into 10 mm segments, sterilized with 75% (v / v) ethanol, and then placed in a solution containing 1 × 10⁻⁶ ethanol. 7 Incubate 1 mL of LB medium containing CFU / mL Staphylococcus aureus at 37 °C for 24 h. After washing three times with PBS, insert the catheter subcutaneously into the medial thigh of female C57BL / 6 mice. 24 h later, mice (6 weeks old, n = 5 per group) are subcutaneously injected with PBS, ampicillin, or Bn-AG. 12 -CA (2 mg / kg). Mice were sacrificed after 24 h of treatment, and the ducts and surrounding skin tissue were collected. The ducts were sonicated in PBS, and the tissue was homogenized in PBS, serially diluted, and plated onto LB agar plates for colony counting. Experimental results are as follows: Figure 18 As shown, Bn-AG12-CA can effectively treat acute bacterial infections and infectious diseases caused by bacterial biofilms in mice.

[0093] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects: the solid-phase synthesis and click chemistry method produces accurate peptide structures and the method is simple; the present invention can significantly reduce the toxicity of antimicrobial peptides to normal cells while maintaining high antimicrobial activity.

Claims

1. A bacterial phospholipid-induced random-β-sheet structure that can be converted into an antimicrobial peptide, characterized in that, The bacterial phospholipid-induced random-β-sheet structure can be transformed into the general formula of the antimicrobial peptide structure, as shown below: （I） Wherein, n represents the number of amino acid residues, and its value ranges from 5 to 15; R1 is selected from: amino acid residues, short peptides, alkyl groups, aromatic groups, or carbonyl groups substituted with polyether chains.

2. The antimicrobial peptide with a bacterial phospholipid-induced random-β-sheet structure as described in claim 1, characterized in that, R1 is selected from: , , , .

3. The antimicrobial peptide with a bacterial phospholipid-induced random-β-sheet structure as described in claim 1, characterized in that, R1 is selected from: , , , , , , , One of them.

4. The method for preparing antimicrobial peptides with a bacterial phospholipid-induced random-β-sheet structure as described in claim 1, characterized in that, Includes the following steps: (1) Fmoc-allyl glycine, terminal molecules, N,N′-diisopropylcarbodiimide DIC, and ethyl oxime cyanoacetate Oxyma were dissolved in dimethylformamide DMF and installed in the corresponding positions of the CEM peptide synthesizer. MBHA resin was added to the reaction tank. (2) After setting the microwave heating time, number of coupling cycles and number of amino acid residues of the target polypeptide, start the reaction; (3) After the reaction is complete, the resin carrying the polypeptide is mixed with mercaptoethylamine molecules and added to DMF. After purging with nitrogen, 2,2-dimethoxy-2-phenylacetophenone (DMPA) is added and click reaction is carried out under ultraviolet light to modify the mercaptoethylamine part to the side chain of the polypeptide. (4) Wash the resin with DMF and methanol, and then soak it in methanol and dry it under vacuum. (5) The peptide was cleaved from the resin using a lysis buffer obtained by mixing trifluoroacetic acid, triisopropylsilane and water, precipitated in a mixed solution of diethyl ether and n-hexane, and then dried under vacuum. (6) The obtained product was mixed with mercaptoethylamine and added to dimethyl sulfoxide (DMSO). 2,2-Dimethoxy-2-phenylacetophenone was added, and a click reaction was performed under ultraviolet light to completely modify the side chain of the polypeptide with mercaptoethylamine, thereby obtaining the bacterial phospholipid-induced random-β-sheet structure convertible antimicrobial peptide R1-AGn-CA.

5. The preparation method according to claim 4, characterized in that, In step (2), the allyl glycine polypeptide has 5-15 residues and the terminal group is one of n-hexyl, dodecyl, phenylpropyl, tryptophan, and phenylalanine.

6. The preparation method according to claim 4, characterized in that, In step (2), the microwave heating time is 1 to 4 minutes and the number of coupling cycles is 1 to 3.

7. The preparation method according to claim 4, characterized in that, In step (3), the molar ratio of carbon-carbon double bonds in allyl glycine to mercaptoethylamine is 0.1:1, and the ultraviolet irradiation time is 1-4 h.

8. The preparation method according to claim 4, characterized in that, In step (4), the methanol soaking time is 5-20 min.

9. The preparation method according to claim 4, characterized in that, In step (5), the volume ratio of trifluoroacetic acid, triisopropylsilane and water is 95:2.5:2.5, and the volume ratio of diethyl ether to n-hexane is 1:

1.

10. The application of the antimicrobial peptide with a bacterial phospholipid-induced random-β-sheet structure as described in claim 1 in antimicrobial activity.

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