A cyclic peptide targeting sctv to inhibit t3ss activity and application thereof

Abstract

This invention belongs to the field of cyclic peptide synthesis and biomedical technology, specifically relating to a cyclic peptide that targets SctV to inhibit T3SS activity and its applications. Specifically, this invention screens a series of macrocyclic peptides targeting InvA (the SctV protein derived from Salmonella T3SS) from a cyclic peptide library and demonstrates their inhibitory effect on T3SS function. Furthermore, the best-performing cyclic peptide, CP-L1, is mutated and optimized to obtain a mutant peptide with the same broad-spectrum antibacterial effect. The cyclic peptide provided by the above technical solution, as a promising antibacterial drug, can be used for the prevention and treatment of diseases mediated by Gram-negative pathogens, thus possessing significant practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of cyclic peptide synthesis and biomedicine, specifically relating to a cyclic peptide that targets SctV to inhibit T3SS activity and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Antimicrobial resistance (AMR) is one of the public health and development threats facing all of humanity. It is estimated that more than one million people die globally each year from drug-resistant microorganisms. The World Health Organization's 2024 list of priority bacterial pathogens shows that the disease burden caused by multidrug-resistant Gram-negative bacteria is increasing. Data from my country's antimicrobial resistance surveillance network also indicates that the clinical need for novel antibiotics for drug-resistant Gram-negative pathogens is particularly urgent. However, the rate of evolution and spread of pathogen resistance exceeds the rate of new antibiotic development. Treatment strategies that directly inhibit the virulence of pathogens offer a new option. Antiviral therapy targets the pathogen's ability to initiate and maintain infection, aiming to stop or slow the progression of infection. Compared to antibiotics, antiviral drugs may minimize selective pressure and have the least impact on the healthy microbiome, thereby slowing the rate of resistance development.

[0004] The type III secretion system (T3SS) is the virulence apparatus of many important Gram-negative bacterial pathogens, such as *Epidermophyton floccosum*, *Pseudomonas aeruginosa*, *Salmonella*, *Shigella*, *Vibrio cholerae*, and *Yersinia*. These pathogens manipulate the physiological activities of host cells during bacterial infection through effector proteins secreted by the T3SS, causing related symptoms. Due to the high conservation of the T3SS among different pathogens, most bacteria lose their pathogenicity when the T3SS is inactivated, making the T3SS an attractive antiviral target.

[0005] SctV is one of the most conserved components of T3SS and plays a crucial role in the pathogenesis mediated by T3SS. Figure 1 a). Studies have shown that SctV forms a nonameric ring structure on the bacterial inner membrane, and the monomeric protein consists of an N-terminal transmembrane domain (SctV). TM ), a connection region (SctV) L ) and a 40kDa cytoplasmic domain (SctV) C Composed of ) SctV TM SctV acts as a proton pump to provide energy to T3SS. CIt is divided into four subdomains (SD1-SD4), with SD3 located on the inner surface of the nonameric ring (pocket 1). The resulting channel changes with environmental pH, thereby allowing the secretion of effector proteins. Figure 1 b). SctV C The structure of -SctO indicates that SctO binds to SctV. C In the gap between SD4-SD4 of adjacent monomers, the ATPase SctN of T3SS is linked to SctV (pocket 2) to promote the delivery of effector proteins. Figure 1 b). In addition, SctV C Conformational changes between SD2 and SD4 within monomer molecules directly affect their selection and binding to effector proteins (pocket 3) Figure 1 c). Therefore, disrupting these key protein interaction sites can block the secretion of T3SS effector proteins, thereby weakening the virulence of pathogens and alleviating the occurrence of diseases. Summary of the Invention

[0006] To address the aforementioned limitations of existing technologies, this invention provides a cyclic peptide that targets SctV to inhibit T3SS activity and its applications. Specifically, this invention screens a series of macrocyclic peptides targeting InvA (the SctV protein derived from Salmonella T3SS) from a cyclic peptide library to investigate their potential to disrupt the interaction between SctV and substrate proteins; and further optimizes the performance of the cyclic peptide CP-L1 by mutation to obtain a mutant peptide with broad-spectrum antibacterial effects. Based on the above research results, this invention is thus completed.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a cyclic peptide that targets SctV to inhibit T3SS activity, said cyclic peptide having any of the following structures:

[0009]

[0010] Furthermore, the cyclic peptide also includes a mutant of cyclic peptide L1, wherein the mutant of cyclic peptide L1 includes:

[0011]

[0012]

[0013] Furthermore, the mutants of the cyclic peptide L1 are L1-M2 and L1-M17.

[0014] It should be noted that, among the above cyclic peptides, except for Tyr(Y) in D1, D2, D7, and D9 which is a D-type amino acid, all other amino acids in each cyclic peptide are L-type amino acids.

[0015] A second aspect of the present invention provides a method for preparing the above-mentioned cyclic peptide, the method comprising: synthesizing a linear peptide chain using a solid-phase synthesis method, and constructing the above-mentioned cyclic peptide through a thioether structure.

[0016] Furthermore, the thioether structure is formed between Tyr(Y) and Cys(C) to obtain the above-mentioned cyclic peptide.

[0017] A third aspect of the present invention provides the use of the above-mentioned cyclic peptide in inhibiting T3SS function or in preparing T3SS inhibitors.

[0018] In this invention, the T3SS inhibition function is achieved by targeting SctV.

[0019] More specifically, in this invention, SctV is InvA, which is the SctV protein of Salmonella T3SS.

[0020] In a fourth aspect, the present invention provides a T3SS inhibitor, wherein the T3SS inhibitor comprises at least the above-mentioned cyclic peptide.

[0021] In a fifth aspect, the invention provides the use of the above-mentioned cyclic peptide or T3SS inhibitor as or in the preparation of antibacterial agents.

[0022] Specifically, the antibacterial agent exhibits broad-spectrum inhibitory effects against Gram-negative pathogens, which may be pathogens containing T3SS, including but not limited to EPEC, Pseudomonas aeruginosa, and pathogens of Salmonella, Shigella, Vibrio cholerae, and Yersinia.

[0023] Therefore, in this invention, the antibacterial agent can be used for environmental disinfection and as a drug for the treatment or preparation of diseases mediated by the aforementioned Gram-negative pathogens.

[0024] The drug can be used to prevent and / or treat diseases mediated by the aforementioned Gram-negative pathogens, without specific limitations.

[0025] Therefore, in a sixth aspect of the present invention, a medicament is provided, wherein the active ingredient of the medicament comprises at least the above-mentioned cyclic peptide or T3SS inhibitor.

[0026] As above, the drug can be used to inhibit the aforementioned Gram-negative pathogens, and thus can be used to prevent and / or treat diseases mediated by the aforementioned Gram-negative pathogens.

[0027] Furthermore, the drug may also include at least one inactive pharmaceutical ingredient. The inactive pharmaceutical ingredient may be a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, emulsifier, suspending agent, stabilizer, preservative, excipient, filler, coagulant and blending agent, surfactant, dispersant, or defoamer.

[0028] The pharmaceutically viable carrier can also be a virus, microcapsule, liposome, exosome, nanoparticle, or polymer, or any combination thereof. The delivery carrier of the pharmaceutically viable carrier can be a liposome, exosome, biocompatible polymer, lipoprotein, lipopolysaccharide, artificial viral envelope, inorganic particles, or bacterial or viral, granular, or plasmid carrier, etc. Further details will not be elaborated here.

[0029] The drug can also be administered alone as a single composition or in a dosage form different from the main active ingredient, along with other preventative and / or therapeutic compounds. A portion of the main ingredient may be administered concurrently with other therapeutic compounds, while other doses may be administered alone. During treatment, the dosage of the drug can be adjusted according to the severity of symptoms, the frequency of relapses, and the physiological response to the treatment regimen.

[0030] The medicament of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. It will be understood by those skilled in the art that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0031] The drug can be administered to humans or non-human animals, without any specific limitation.

[0032] A seventh aspect of the present invention provides a method for preventing and / or treating diseases mediated by Gram-negative pathogens, the method comprising administering to a subject the application of the aforementioned cyclic peptide, T3SS inhibitor, or medicament.

[0033] The subject refers to an animal that is already being treated, observed, or experimented on, preferably a mammal, and most preferably a human. It must be recognized that the optimal dosage and interval of the active ingredient described in this invention are determined by its properties and external conditions such as the form, route, and site of administration, as well as the specific animal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dose of the compound within a specified time period, can be determined using methods known in the art.

[0034] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0035] The above technical solution screens a series of InvA-targeting peptides from a cyclic peptide library. C The macrocyclic peptides were identified and demonstrated to inhibit T3SS function. Furthermore, the best-performing macrocyclic peptide, CP-L1, was mutated and optimized to obtain a mutant peptide with the same broad-spectrum antibacterial effect. The cyclic peptides provided by the above technical solution, as promising antibacterial drugs, can be used for the prevention and treatment of diseases mediated by Gram-negative pathogens, thus possessing significant practical application value. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1 : Possible drug-binding pockets of the SctV protein; a. In situ structure of T3SS; b. Three key functional sites of the SctV protein.

[0038] Figure 2 Screening results of cyclic peptide libraries; a. Cyclic peptide sequences; b. Cyclic peptides CP-L1 and CP-D9 and InvA C Affinity assay (ITC); c. Cyclic peptides CP-D1, CP-D2 and CP-D7 with InvA C Affinity testing.

[0039] Figure 3 a. Screening results of CP-L1 and CP-D9; b. Effects of CP-L1 and CP-D9 on the secretion of T3SS effector proteins; c. Effects of CP-L1 and CP-D9 on the growth of S. Typhimurium; d. CP-L1 cytotoxicity test results; d. Inhibitory effect of CP-L1 on S. Typhimurium infection of NCM460 cells and Raw cells.

[0040] Figure 4 The antibacterial effect of cyclic peptide CP-L1 on Shigella and EPEC; ab. Inhibitory effect of CP-L1 on the function of Shigella and EPEC3SS; cd. Effect of CP-L1 on the growth of Shigella and EPEC; e. Inhibitory effect of CP-L1 on Shigella infection of host cells; f. Inhibitory effect of CP-L1 on EPEC adhesion to host cells.

[0041] Figure 5The inhibitory effect of cyclic peptide CP-L1 on multidrug-resistant Salmonella; a. Antibacterial spectrum of multidrug-resistant Salmonella; b. Effect of CP-L1 on the growth of multidrug-resistant Salmonella; c. Inhibitory effect of CP-L1 on T3SS function of multidrug-resistant Salmonella; d. Inhibitory effect of CP-L1 on infection of host cells by multidrug-resistant Salmonella.

[0042] Figure 6 Optimization results of cyclic peptide CP-L1; a. Mutant peptide library; b. Mutant peptide and InvA C a. Affinity statistics; c. Inhibitory effect of mutant peptide on T3SS function; de. Inhibitory effect of mutant peptide on Salmonella infection of host cells.

[0043] Figure 7 Antibacterial effects of mutant peptides M2 and M17 on Shigella and EPEC; inhibitory effect of ab.M2 on Shigella infection of host cells; inhibitory effect of cd.M17 on EPEC adhesion to host cells.

[0044] Figure 8 Mass spectra and chromatograms of each cyclic peptide; a. L1; b. D1; c. D2; d. D7; e. D9; f. L1-M1; g. L1-M2; h. L1-M3; i. L1-M4; j. L1-M5; k. L1-M6; l. L1-M8; m. L1-M11; n. L1-M13; o. L1-M15; p. L1-M17; q. L1-M19; r. L1-M21. Detailed Implementation

[0045] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments of the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the invention. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0047] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials, reagents, carriers, strains, etc., used in the following examples are commercially available.

[0048] Example.

[0049] 1. Research Methods

[0050] 1.1 Solid-phase synthesis and modification of cyclic peptides

[0051] The series of cyclic peptides in this embodiment were obtained from a cyclic peptide library and synthesized by Anhui Guoping Pharmaceutical Co., Ltd. In short, the cyclic peptides were constructed using the following method: The synthesis of the cyclic peptides was performed on Rink Amide MBHA resin using standard fluoromethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) technology. 0.5 g of the resin sample was suspended in 8 ml of freshly prepared dimethylformamide (DMF) solution containing 0.3 mmol Fmoc-Gly-OH (or Fmoc-Lys(Mmt)-OH for fluorescein (FAM)-coupled peptides), 0.3 mmol 1-hydroxybenzotriazole (HOBT), and 0.5 ml N,N'-diisopropylcarbodiimide (DIC). The reaction mixture was stirred under nitrogen bubbling for 1.5 hours. Subsequently, the resin was treated with a 20% piperidine / DMF solution to remove the Fmoc protecting groups. Subsequent amino acid coupling was performed by adding 10 ml of freshly prepared DMF solution (containing 0.9 mmol Fmoc-protected amino acid (Fmoc-AA-OH), 0.9 mmol HOBT, and 1 ml DIC) to the system and reacting continuously under a nitrogen atmosphere for 1 hour. By repeating the deprotection and coupling steps, peptide chains of the target length were finally synthesized.

[0052] For the coupling of the FAM group, the resin was first shaken in a DMF solution containing 1% trifluoroacetic acid (TFA) at room temperature for 30 minutes to selectively remove the monomethoxytriphenylmethyl (Mmt) protecting group on the peptide chain. After thorough washing, the resin was reacted with a DMF solution of 5-carboxyfluorescein N-hydroxysuccinimide (FAM-NHS) for 2 hours. After peptide chain synthesis, the resin was cleaved, precipitated with diethyl ether (Et2O), and then redissolved in dimethyl sulfoxide (DMSO). The pH of the solution was adjusted to 8.0 with triethylamine (Et3N), and the reaction was carried out slowly at room temperature for 1 hour to form a thioether macrocyclic structure. Subsequently, the pH was adjusted to 3-4 with TFA, and the product was purified by reversed-phase high-performance liquid chromatography (HPLC) (mobile phase: aqueous solution containing 0.1% TFA and acetonitrile (MeCN) containing 0.1% TFA, linear gradient elution). The purity of the final product was verified using an LC-2020 system (Shimadzu), and mass spectrometry (MS) analysis using an LCMS-2020 (Shimadzu) confirmed successful synthesis. The chromatograms and mass spectra of each cyclic peptide are shown below. Figure 8 As shown.

[0053] 1.2 Salmonella T3SS effector protein secretion assay

[0054] An arabinose promoter was inserted before the T3SS regulatory gene in strain S. Typhimurium UK-1x8956, which led to high expression of T3SS-related genes through arabinose induction.

[0055] 1. Streaking activation of S. Typhimurium UK-1x8956 on LB antibiotic-free plates.

[0056] 2. Pick a single clone and inoculate it into 3 mL of LB liquid, and incubate overnight at 25°C and 220 rpm.

[0057] 3. Transfer the culture to 3 mL of LB liquid at a ratio of 1:10, add 0.02% arabinose to a final concentration, and induce culture at 37°C and 220 rpm for 4 h.

[0058] 4. Take 1 mL of bacterial culture at 14000 g, centrifuge at 4℃ for 5 min, and transfer 700 μL of the supernatant to a new EP tube.

[0059] 5. Add 150 μL of 100% TCA to the supernatant from the previous step, mix well, and place on ice for half an hour.

[0060] Centrifuge at 14000g, 4℃ for 5 minutes, discard the supernatant, and invert the EP tube to remove residual TCA.

[0061] 7. Add 400 μL of pre-cooled acetone to the centrifuged precipitate, shake to mix, and place on ice for 10 min.

[0062] Centrifuge at 14000g, 4℃ for 5min, remove the supernatant, and dry the residual acetone in a clean bench.

[0063] 9. Add 20 μL of 2×loading buffer to the EP tube, heat at 99℃ for 5 min, homogenize the sample loading volume according to OD600nm, and detect the secretion of T3SS effector protein by 10% SDS-PAGE gel.

[0064] 10. Following step 4, add 50 μL of lysis buffer to the centrifuged bacterial pellet to resuspend the bacterial cells. Take 20 μL of the pellet and add 20 μL of 2×loading buffer. Incubate at 99°C for 5 min to lyse the bacterial cells and release intracellular proteins. Determine the loading amount based on the bacterial OD600nm and perform Western blot to detect the expression of intracellular InvA.

[0065] 1.3 Western blot detection of InvA protein expression level

[0066] 1. Load the prepared sample into a 10% SDS-PAGE gel. First, set the voltage to 80V and perform electrophoresis for 20 minutes to ensure all the sample enters the stacking gel. Then, set the voltage to 200V and continue electrophoresis for 25 minutes to separate the samples.

[0067] 2. Remove excess material from the gel, select a PVDF membrane slightly larger than the gel, soak it in anhydrous ethanol for later use, and transfer it to a wet transfer buffer before transfer.

[0068] 3. Soak the gel and filter paper in a wet transfer buffer beforehand. Following the order of "black-gel-membrane", place the sandwich with the black side at the bottom, and then lay the filter paper-gel-PVDF membrane-filter paper upwards in that order, being careful to avoid air bubbles.

[0069] 4. Place the prepared sandwich clamps into the electrophoresis tank, set the voltage to 180V for 40 minutes (InvA, 76kDa), and perform the entire process in a cold chamber.

[0070] 5. After the transfer is complete, rinse once with TBST to remove the ethanol on the surface.

[0071] 6.5% skim milk powder sealed at room temperature for 1 hour.

[0072] 7. Add the primary antibody (anti-flag) at a ratio of 1:10,000 and incubate at room temperature for 1.5 hours or at 4°C overnight.

[0073] 8. Remove the incubated membrane and rinse it three times with TBST for 5 minutes each time.

[0074] 9. Add secondary antibody (anti-mouse) at a ratio of 1:10,000 and incubate at room temperature for 1 hour.

[0075] 10. Remove the incubated membrane and rinse it three times with TBST for 5 minutes each time.

[0076] 11. Prepare the developing solution and perform chemiluminescence imaging.

[0077] 1.4 Isothermal titration calorimetry

[0078] Isothermal titration calorimetry (ITC) is a technique used to quantitatively study various biomolecular interactions. It directly measures the heat released or absorbed during biomolecular binding, and the experimental data is presented as a thermogram, providing information on the amount of substance in the reaction (titer endpoint) and the properties of the reactants (enthalpy change). ITC detection can accurately obtain complete thermodynamic information about biomolecular interactions: binding constant (Kd), reaction stoichiometry (n), enthalpy (ΔH), and entropy (ΔS). It is widely used to detect interactions between proteins, small molecules, nucleic acids, and other molecules.

[0079] 1. Sample preparation:

[0080] InvAC Protein: 50 μM, 400 μL / tube, buffer 20 mM Hepes 8.0, 150 mM NaCl.

[0081] Cyclic peptides and small molecule compounds: Dissolve in DMSO, and gradually dilute with protein buffer to 500 μM and 100 μL, carefully observing for any precipitation.

[0082] Blank control: protein buffer.

[0083] 2. Before conducting the experiment, centrifuge the sample at 14,000 rpm for 5 minutes to remove air bubbles. The protein sample needs to be placed at room temperature in advance to balance the temperature of the protein sample and avoid the generation of air bubbles during the experiment.

[0084] 3. Set the instrument parameters: total injection 19 drops, titration temperature 25℃, reference power 10, feedback high, stir speed (rpm) 750, initial delay 60s, set the first drop to 0.4μL, the second drop onwards to 2μL each, injection spacing 150s, injection duration 4s.

[0085] 4. Clean the instrument in WATER mode, and titrate ddH2O until the baseline is stable to ensure the instrument is thoroughly cleaned.

[0086] 5. Protein titration buffer for cyclic peptides or small molecule compounds. (Blank control)

[0087] 6. Titration of proteins with cyclic peptides or small molecule compounds. (Experimental group)

[0088] 7. Using MicroCal PEAQ-ITC Analysis Software, the blank control was removed, the fitted curve was analyzed, and the Kd value was calculated.

[0089] 1.5 Determination of bacterial growth curve

[0090] 1. Select a single S. Typhimurium UK-1 colony and inoculate it into 3 mL of LB liquid medium. Incubate overnight at 25°C and 220 rpm.

[0091] 2. Overnight culture was transferred 1:100 to 1.3 mL LB liquid medium, and arabinose was added to a final concentration of 0.02% to induce T3SS expression. Cyclic peptides or small molecule inhibitors were added to the experimental group, and an equal volume of DMSO was added to the control group. After mixing, 400 μL / well was aliquoted into 48-well plates. Three replicates were set up for each sample.

[0092] 3. Place the 48-well plate in a microplate reader, set the temperature to 37℃ and shake at 450 rpm, and measure the absorbance at OD600 nm every 15 min for a total of 6 h.

[0093] 4. The measurement results were plotted and analyzed using GraphPad Prism 6.

[0094] 2.5 Detection of the invasive ability of Salmonella rat to host cells

[0095] 1.5.1 Cell resuscitation and passage

[0096] Cell resuscitation:

[0097] Remove the frozen cells from the liquid nitrogen container and thaw them rapidly at 37°C. Centrifuge at 850 rpm for 5 min, discard the supernatant, and resuspend the cells in 10 mL of RPMI 1640 medium + 10% FBS. Transfer the resuspended cells to a 10 cm culture dish and incubate at 37°C with 5% CO2 for 2 days.

[0098] Cell passage:

[0099] 1. Use a suction pump to remove the cell culture medium, add 5 mL of preheated PBS to wash once, remove the PBS, add 3 mL of 0.5% trypsin, and digest at 37°C for 3 min.

[0100] 2. Add 3 mL of RPMI 1640 medium (10% FBS) to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 850 rpm for 5 min, and collect the cells.

[0101] 3. Discard the supernatant, add 6 mL of RPMI 1640 medium to resuspend the cells, take 2 mL of the cell suspension and add 2 mL of RPMI 1640 medium (10% FBS) to a 10 cm culture dish, add about 8 mL of RPMI 1640 medium (10% FBS), mix well and incubate at 37℃ in a 5% CO2 incubator for 2-3 days.

[0102] 1.5.2 Gentamicin Protection Experiment

[0103] CCK8 Toxicity Testing

[0104] 1. Cell counting: Take an appropriate amount of digested and resuspended cell suspension and add it to a hemocytometer. Count the cells in the four squares at the four corners of the counting plate under a microscope. For cells that are pressed against the lines, only count the cells at the top and left lines.

[0105] 2. Cell density (number of cells / mL) = (total number of cells in four squares / 4) × 10⁴ cells / mL.

[0106] 3. Plating: Add the diluted cell suspension to a 96-well cell culture plate, 100 μL per well (approximately 5000 cells), and incubate overnight at 37°C with 5% CO2.

[0107] 4. Dilute with RPMI 1640 medium (10% FBS) to obtain different concentrations of small molecule inhibitors or cyclic peptides, ensuring that the DMSO content in each gradient is 0.1%, and set up three replicates for each concentration, with a blank control of 0.1% DMSO.

[0108] 5. Add 20 μL of the small molecule inhibitor or cyclic peptide diluted in the previous step and 10 μL of CCK-8 reagent to a 96-well plate, and incubate at 37°C and 5% CO2 for 4 h.

[0109] 6. Measure the absorbance at OD450nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0110] 7. Calculate the cell survival rate of the compound using the following formula:

[0111] Survival rate = (Experimental group OD450nm - Blank group OD450nm) ÷ (Negative group OD450nm - Blank group OD450nm) × 100%.

[0112] Gentamicin protection experiment

[0113] 1. Cell count is the same as above.

[0114] 2. Plating: Add 1 mL of diluted cell suspension to each well of a 24-well cell culture plate at a cell density of 2.5 × 10⁴ / mL, and incubate overnight at 37°C with 5% CO₂. Simultaneously, pick a single S. Typhimurium UK-1 colony from the plate and inoculate it into 3 mL of LB liquid medium, and incubate overnight at 25°C with 220 rpm.

[0115] 3. Inoculate 1:100 of the overnight culture of S. Typhimurium UK-1 into 3 mL of LB medium containing 0.02% arabinose, and add the corresponding small molecule inhibitor or cyclic peptide. Incubate at 37°C for about 4 hours until the OD600nm reaches 0.9.

[0116] 4. Wash NCM460 cells twice with pre-warmed PBS, and add 0.5 mL of Hanks buffer to each well. The bacterial concentration when the OD600nm reaches 0.9 is approximately 1 × 10⁹ CFU / mL. Add 0.5 mL of diluted bacterial suspension to each well of a 12-well plate at MOI = 50, mix well, and incubate at 37°C in a 5% CO₂ incubator for 1 hour to allow bacteria to invade the cells.

[0117] 5. Wash the cells three times with preheated PBS to remove unbound bacteria. Add RPMI 1640 medium (10% FBS) containing 100 μg / mL gentamicin to the cells and incubate in a cell culture incubator for 1 hour to further remove unbound bacteria.

[0118] 6. Wash the cells three times with preheated PBS, and add 0.5 mL of 1% Triton X-100 solution to each well to lyse the cell membrane.

[0119] 7. Transfer the cell lysate to a 1.5 mL EP tube and add 0.5 mL of LB liquid culture medium.

[0120] 8. Take 20 μL of cell lysis buffer into a 1.5 mL EP tube, add 180 μL of PBS, dilute to concentration 1, and then take 20 μL of the bacterial solution from concentration 1, add 180 μL of PBS, dilute to concentration 2.

[0121] 9. Spread 100 μL of bacterial suspensions of concentration 1 and concentration 2 onto LB plates, incubate overnight at 37°C upside down, and count the single colonies on the plates. The final number of single colonies of concentration 1 × 100 and the number of single colonies of concentration 2 × 1000 can be used to obtain the number of bacteria invading cells in each well.

[0122] 2. Results and Analysis

[0123] 2.1 Cyclic peptide CP-L1 inhibits Salmonella infection of host cells by targeting InvA.

[0124] A cyclic peptide library was screened, targeting InvA. C Five cyclic peptides (L1, D1, D2, D7, and D9) were obtained through enrichment. Figure 2 a). To determine whether these cyclic peptides can inhibit T3SS function by targeting InvA, the interaction between these cyclic peptides and InvA was first assessed using isothermal titration calorimetry (ITC). C The binding affinity of cyclic peptides CP-D1, CP-D2, and CP-D7 to InvA was shown by ITC results. C It did not exhibit binding affinity, while cyclic peptides CP-L1 and CP-D9 were both able to bind to InvA. C K D The values ​​were 614±323 nM (CP-L1) and 2.93±0.395 μM (CP-D9), respectively. Figure 2bc). Next, CP-L1 and CP-D9 were selected to detect their effects on S. Typhimurium T3SS. CP-L1 and CP-D9 were co-cultured with S. Typhimurium, and the culture supernatant was collected for SDS-PAGE analysis. 50 μM CP-L1 significantly reduced the secretion levels of T3SS effector proteins (SipA, SipB, SipC, and SipD) without affecting bacterial growth, and inhibited the secretion of T3SS effector proteins in a dose-dependent manner. Figure 3 (ab). Although CP-L1 treatment decreased the secretion level of S. Typhimurium T3SS effector protein, the expression levels of effector protein and InvA were not affected. CP-D9, even at a concentration of 100 μM, did not affect the secretion of S. Typhimurium T3SS effector protein. Based on ITC results and in vitro secretion system assays of T3SS effector protein, CP-L1 activity was superior to other cyclic peptides (CP-D9, CP-D1, CP-D2, and CP-D7).

[0125] The infection of host cells by pathogens is strictly dependent on the function of T3SS. Therefore, the inhibitory effect of CP-L1 on the infection of NCM460 cells (normal human colonic epithelial cells) and raw cells (macrophages) by *S. typhimurium* was evaluated. Before the invasion assay, the cytotoxicity of CP-L1 was determined using a CCK-8 assay kit. Different concentrations of CP-L1 were co-incubated with NCM460 cells and raw cells, respectively. The results showed that CP-L1 had no cytotoxicity in the concentration range of 0–100 μM. Figure 3 c). Next, using gentamicin protection assays, it was found that CP-L1 significantly reduced the infection rate of *S. typhimurium* in NCM460 cells and raw cells compared to the untreated bacteria (DMSO group). In the 50 μM CP-L1 treatment group, the infection rates of *S. typhimurium* in NCM460 cells and raw cells decreased to 69% and 62%, respectively, while in the 100 μM CP-L1 treatment group, the infection rates of *S. typhimurium* in NCM460 cells and raw cells decreased to 25% and 27%, respectively. Figure 3 d). These results indicate that CP-L1 inhibits the activity of T3SS by targeting InvA, thereby inhibiting the invasion of S. Typhimurium into NCM460 cells and Raw cells.

[0126] 2.2 Cyclic peptide CP-L1 exhibits broad-spectrum antibacterial activity against Gram-negative pathogens.

[0127] Since the SctV protein is highly conserved among Gram-negative pathogens, we hypothesized that CP-L1 also has an inhibitory effect on other T3SS-dependent pathogens. To test this hypothesis, we selected two common pathogens—Shigella and enteropathogenic Escherichia coli (EPEC)—to examine the effect of CP-L1. Shigella is the pathogen of infectious diarrhea, infecting host cells using an invasion mechanism similar to that of S. Typhimurium. Enteropathogenic Escherichia coli secretes virulence proteins into intestinal epithelial cells via T3SS, attaches to the intestine, and causes diarrhea in the host.

[0128] We evaluated the inhibitory effect of CP-L1 on Shigella and EPEC T3SS to determine its potential as a therapeutic / inhibitory agent for Gram-negative pathogen infections. Western blot results showed that CP-L1 at 50 μM inhibited the secretion of T3SS effector proteins (OspC3 in Shigella and Cif in EPEC) without affecting bacterial growth (Figure ad). Subsequently, we evaluated the inhibitory activity of CP-L1 on Shigella and EPEC at the cellular level, demonstrating that CP-L1 significantly reduced the infection rate of Shigella on NCM460 and Raw cells and inhibited the adhesion of EPEC to NCM460 and Raw cells. Figure 4 These results indicate that CP-L1 exhibits potential antiviral activity against other Gram-negative pathogens.

[0129] 2.3 Cyclic peptide CP-L1 effectively inhibits the infection of host cells by multidrug-resistant Salmonella.

[0130] We isolated a multidrug-resistant Salmonella strain from a clinical setting. This strain was resistant to multiple antibiotics, including sulfamethoxazole, ampicillin, nalidixic acid, and cefotaxime. Figure 5 a). To determine whether CP-L1 could inhibit the activity of T3SS in this multidrug-resistant Salmonella strain, we co-cultured CP-L1 and the drug-resistant Salmonella in LB broth containing 0.3M NaCl. The supernatant was collected and Western blot was used to detect the secretion level of the T3SS effector protein SipC. The results showed that SipC was undetectable in the secretion supernatant of the 50 μM CP-L1 treatment group, indicating that CP-L1 could inhibit the activity of T3SS without affecting bacterial growth. Figure 5 bc). We further evaluated the inhibitory effect of CP-L1 on T3SS-mediated bacterial invasion. The results showed that in the 50 μM CP-L1 treatment group, the infection rate of drug-resistant Salmonella in NCM460 cells and Raw cells decreased to 61% (bc). Figure 5d). These results indicate that the cyclic peptide CP-L1 effectively inhibits the infection of host cells by multidrug-resistant Salmonella by inhibiting the activity of T3SS.

[0131] 2.4 Optimization of cyclic peptide CP-L1

[0132] To further enhance the efficacy of CP-L1, we constructed a CP-L1 mutant library and synthesized 13 mutants based on sequence conservation. Figure 6 a) By ITC measurements, mutants M1, M2, M3, M4, M5, M8, and M17 were associated with InvA. C Enhanced binding affinity ( Figure 6 b).

[0133] To further quantitatively assess the activity of these mutants on T3SS, we constructed a NanoLuc-based reporter gene system to detect T3SS activity in *S. typhimurium*. According to the reporter gene system results, the secretion levels of T3SS effector proteins were reduced in all mutants. Compared to CP-L1, mutants M1 and M8 showed decreased inhibitory effects on T3SS function, while M2 and M17 exhibited enhanced inhibitory effects. Other mutants (M2, M3, M4, and M5) showed no significant difference in inhibitory effects on T3SS function compared to CP-L1. Figure 6 c).

[0134] The activity of T3SS is directly related to the ability of bacteria to invade and establish infection. Therefore, we evaluated the inhibitory effects of mutants (M2, M3, M4, M5, and M17) on the invasion of NCM460 and Raw cells. The invasion rates of M2 and M17-treated *S. typhimurium* in NCM460 cells were 37% and 40%, respectively, comparable to CP-L1 (40%). The invasion rates of the other mutants (M3, M4, and M5) were 70%, 57%, and 58%, respectively. Figure 6 d). It can be seen that M2 and M17 showed relatively strong inhibitory effects on S. Typhimurium infection of NCM460 cells.

[0135] For Raw cells, the inhibitory effects of mutants M2 and M3 on S. typhimurium invasion were comparable to those of CP-L1 (59%). The infection rates of S. typhimurium treated with M4, M5, and M17 were 24%, 40%, and 49%, respectively. Figure 6 e). Overall, the M2 and M17 treatment groups showed significantly reduced invasiveness of S. Typhimurium to NCM460 and Raw cells.

[0136] The 2,5-mutant peptide exhibited broad-spectrum antibacterial activity against Gram-negative pathogens.

[0137] By comparing the amino acid composition of M2 and M17, we observed that the substitution of Phe6 with Leu6 in CP-L1 produces M2, and the substitution of Val12 with Asp12 in CP-L1 produces M17. These two single-point mutations enhance the interaction between the cyclic peptide and InvA. C Binding affinity. Considering that SCTV proteins are highly conserved among Gram-negative pathogens, we hypothesized that M2 and M17 have antibacterial effects against Shigella and EPEC. The results showed that M2 had a better inhibitory effect on Shigella infection of NCM460 cells and Raw cells compared to CP-L1 (*P<0.05). Figure 7 ab). The inhibitory effect of M2 on EPEC adhesion to host cells was not significantly different from that of CP-L1. Figure 7 M17 inhibits the toxicity of both Shigella and EPEC, and its effect is not significantly different from that of CP-L1. Figure 7 ad).

[0138] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A cyclic peptide that targets SctV to inhibit T3SS activity, characterized in that, The cyclic peptide is a mutant of cyclic peptide L1, and the mutant of cyclic peptide L1 is named L1-M2, with the following structure: 。 2. The method for preparing the cyclic peptide according to claim 1, characterized in that, The preparation method includes: synthesizing a linear peptide chain using a solid-phase synthesis method, and constructing the cyclic peptide through a thioether structure.

3. A T3SS inhibitor, characterized in that, The T3SS inhibitor comprises the cyclic peptide of claim 1.

4. The use of the cyclic peptide of claim 1 or the T3SS inhibitor of claim 3 in the preparation of antibacterial agents; The antibacterial agent exhibits broad-spectrum inhibitory effects against Gram-negative pathogens, which are pathogens containing T3SS, including EPEC, Pseudomonas aeruginosa, Salmonella spp., and Shigella spp.

5. The application as described in claim 4, characterized in that, The antibacterial agent is used for environmental disinfection and as a drug for the above-mentioned Gram-negative pathogens that mediate diseases. The drug is used to treat diseases mediated by the Gram-negative pathogens.

6. A drug, characterized in that, The active ingredient of the drug comprises the cyclic peptide of claim 1 or the T3SS inhibitor of claim 3; The drug can be used to inhibit Gram-negative pathogens, and thus to treat diseases mediated by the Gram-negative pathogens; The Gram-negative pathogens mentioned are pathogens containing T3SS, including EPEC, Pseudomonas aeruginosa, Salmonella spp., and Shigella spp.

7. The drug as described in claim 6, characterized in that, The drug may also include at least one inactive pharmaceutical ingredient.

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