Cyclic peptide for inhibiting T3SS activity by targeting SctV and application of cyclic peptide

Abstract

The invention belongs to the technical field of cyclic peptide synthesis and biological medicine, and particularly relates to a cyclic peptide for inhibiting T3SS activity by targeting SctV and application of the cyclic peptide. Specifically, a series of macrocyclic peptides targeting InvA (derived from SctV protein of salmonella T3SS) are screened from a cyclic peptide library, and the macrocyclic peptides are proved to have the function of inhibiting the T3SS function, so that the optimal cyclic peptide CP-L1 is subjected to mutation optimization treatment, and a mutant peptide with a broad-spectrum antibacterial effect is obtained. As a promising antibacterial agent, the cyclic peptide provided by the technical scheme can be used for preventing and treating related diseases mediated by gram-negative pathogenic bacteria, so that the cyclic peptide has a good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cyclic peptide synthesis and biomedicine, and particularly relates to a cyclic peptide targeting SctV to inhibit the activity of T3SS and its application. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Antimicrobial resistance (AMR) is one of the public health and development threats faced by all mankind. It is estimated that more than 1 million people die from microbial resistance globally every year. The list of priority bacterial pathogens released by the World Health Organization in 2024 shows that the disease burden caused by multidrug-resistant Gram-negative bacteria is increasing. Data from the National Antimicrobial Resistance Monitoring Network in China also indicate that there is an urgent need for new antibiotics against drug-resistant Gram-negative pathogens in clinical practice. However, the evolution and spread of pathogen resistance are faster than the development of new antibiotics, and the treatment strategy of directly inhibiting the virulence of pathogens provides a new option. The anti-virulence treatment strategy targets the ability of pathogens to initiate and maintain infections to prevent or slow down the progression of infections. Compared with antibiotics, anti-virulence drugs may minimize the selection pressure and have the least impact on the healthy microbiota, thereby reducing the development rate of drug resistance.

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

[0005] SctV is one of the most conserved components in 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 linker region (SctV L ), and a 40 kDa cytoplasmic domain (SctV C ). SctV TM acts as a proton pump to provide energy for T3SS, SctV CIt is divided into four subdomains (SD1 - SD4), where SD3 is located on the inner surface of the nonameric ring (pocket 1). The channel formed thereby changes with the environmental pH value, allowing the secretion of effector proteins ( Figure 1 b). SctV C - The structure of SctO shows that SctO binds in the gap between SD4 - SD4 of adjacent monomers of SctV C and connects the ATPase SctN of the T3SS with SctV (pocket 2) to facilitate the transfer of effector proteins ( Figure 1 b). In addition, SctV C the conformational change between SD2 - SD4 within the monomer molecule directly affects its selection and binding of 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 pathogenic bacteria and alleviating the occurrence of diseases. Summary of the Invention

[0006] In view of the above - mentioned prior art, the present invention provides a cyclic peptide targeting SctV to inhibit the activity of T3SS and its application. Specifically, the present invention screens a series of macrocyclic peptides targeting InvA (the SctV protein derived from the T3SS of Salmonella) from a cyclic peptide library to study their potential to disrupt the interaction between SctV and substrate proteins; and performs mutation optimization on the best - performing cyclic peptide CP - L1 among them, and then obtains a mutant peptide with a broad - spectrum antibacterial effect. Based on the above research results, the present invention is completed.

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

[0008] In the first aspect of the present invention, there is provided a cyclic peptide targeting SctV to inhibit the activity of T3SS, and the cyclic peptide has any one of the following structures:

[0009]

[0010] Furthermore, the cyclic peptide further includes mutants of cyclic peptide L1, and the mutants of cyclic peptide L1 include:

[0011]

[0012]

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

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

[0015] In the second aspect of the present invention, there is provided a method for preparing the above cyclic peptide, and the preparation method includes: synthesizing a linear peptide chain by solid-phase synthesis, and constructing the above cyclic peptide through a thioether structure.

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

[0017] In the third aspect of the present invention, there is provided the use of the above cyclic peptide in inhibiting the function of T3SS or in preparing a T3SS inhibitor.

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

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

[0020] In the fourth aspect of the present invention, there is provided a T3SS inhibitor, and the T3SS inhibitor at least contains the above cyclic peptide.

[0021] In the fifth aspect of the present invention, there is provided the use of the above cyclic peptide or T3SS inhibitor as or in preparing an antibacterial agent.

[0022] Specifically, the antibacterial agent exhibits a broad-spectrum antibacterial effect against Gram-negative pathogenic bacteria, and the Gram-negative pathogenic bacteria can be pathogenic bacteria containing T3SS, including but not limited to EPEC, Pseudomonas aeruginosa, and pathogenic bacteria of the genera Salmonella, Shigella, Vibrio cholerae, and Yersinia.

[0023] Therefore, in the present invention, the antibacterial agent can be used for environmental disinfection and as or in preparing a drug for diseases mediated by the above Gram-negative pathogenic bacteria.

[0024] The drug can be used for preventing and / or treating diseases mediated by the above Gram-negative pathogenic bacteria, and no specific limitation is made here.

[0025] Therefore, in the sixth aspect of the present invention, there is provided a drug, and the active ingredient of the drug at least contains the above cyclic peptide or T3SS inhibitor.

[0026] Similarly, the drug can be used to inhibit the above Gram-negative pathogenic bacteria, and thus can be used for preventing and / or treating diseases mediated by the above Gram-negative pathogenic bacteria.

[0027] Further, the drug may further comprise at least one pharmaceutically inactive ingredient. The pharmaceutically inactive ingredient may be a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a conditioner, a surfactant, a dispersant or an antifoaming agent.

[0028] The pharmaceutically acceptable carrier may also be a virus, a microcapsule, a liposome, an exosome, a nanoparticle or a polymer and any combination thereof. The delivery vehicle of the pharmaceutically acceptable carrier may be a liposome, an exosome, a biocompatible polymer, a lipoprotein, a lipopolysaccharide, an artificial virus envelope, an inorganic particle, and a bacterial or viral, cosmid or plasmid vector, etc. This will not be elaborated here.

[0029] The drug may also be administered as a separate composition or in a dosage form different from the main active ingredient to other prophylactic and / or therapeutic compounds. A partial dose of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. During the treatment, the dose of the drug of the present invention may be adjusted according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

[0030] The drug of the present invention can be administered into the body by known methods. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, it is administered via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, the biological type or its tissue, the general condition of the subject to be treated, the administration route, the administration mode, and so on.

[0031] The subject to which the drug is administered may be a human or a non-human animal, and no specific limitation is made here.

[0032] In the seventh aspect of the present invention, a method for preventing and / or treating a disease mediated by Gram-negative pathogenic bacteria is provided, the method comprising administering the above-mentioned cyclic peptide, T3SS inhibitor or drug to a subject.

[0033] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. It must be recognized that the optimal dosage and interval of the active ingredient described in the present invention are determined by its nature and external conditions such as the form, route and site of administration and the specific animal to be treated, and this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dose of the compound within a rated time, can be determined by methods well known in the art.

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

[0035] The above technical scheme screened a series of InvA-targeting peptides from the cyclic peptide library. C The cyclic peptides were identified and proved to have the function of inhibiting T3SS function, and then the best performing cyclic peptide CP-L1 was subjected to mutation optimization treatment to obtain mutant peptides with the same broad-spectrum antibacterial effect. The cyclic peptides provided by the above technical scheme are promising antibacterial drugs that can be used for the prevention and treatment of related diseases mediated by Gram-negative pathogens, and therefore have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 :Possible drug binding pocket of SctV protein; a. T3SS in situ structure; bc. Three key functional sites of SctV protein.

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

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

[0040] Figure 4 :Antibacterial effect of cyclic peptide CP-L1 on Shigella and EPEC; ab. Inhibitory effect of CP-L1 on Shigella and EPEC3SS function; 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 5:Antibacterial effect of cyclic peptide CP-L1 against 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 the T3SS function of multidrug-resistant Salmonella; d. Inhibitory effect of CP-L1 on the invasion of host cells by multidrug-resistant Salmonella.

[0042] Figure 6 :Optimization results of cyclic peptide CP-L1; a. Mutant peptide library; b. Statistical results of the affinity between mutant peptides and InvA C ; c. Inhibitory effect of mutant peptides on the T3SS function; d-e. Inhibitory effect of mutant peptides on the invasion of host cells by Salmonella.

[0043] Figure 7 :Antibacterial effect of mutant peptides M2 and M17 against Shigella and EPEC; a-b. Inhibitory effect of M2 on the invasion of host cells by Shigella; c-d. Inhibitory effect of M17 on the adhesion of EPEC to host cells.

[0044] Figure 8 :Mass spectrometry and chromatogram 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 mode

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

[0046] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention. If the experimental methods are not specified in the following specific embodiments, they are generally carried out according to the conventional methods and conditions of molecular biology in 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 further explains and illustrates the present invention through examples, but does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the materials, reagents, carriers, strains, etc. used are obtained from commercial sources unless otherwise specified.

[0048] Example

[0049] 1. Research method

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

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

[0052] For the coupling of the FAM group, first, the resin was shaken with a DMF solution containing 1% trifluoroacetic acid (TFA) at room temperature for 30 minutes to selectively remove the monomethoxytrityl (Mmt) protecting group on the peptide chain. After thorough washing, the resin was reacted with a DMF solution of 5-carboxyfluorescein N-hydroxysuccinimide ester (FAM-NHS) for 2 hours. After the peptide chain synthesis was completed, it was cleaved from the resin, 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 slowly rotated at room temperature for 1 hour to form a thioether macrocycle structure. Subsequently, the pH was adjusted to 3 - 4 with TFA, and the product was purified by reverse-phase high-performance liquid chromatography (HPLC) (the mobile phase was an aqueous solution containing 0.1% TFA and acetonitrile (MeCN) containing 0.1% TFA, with linear gradient elution). The purity of the final product was verified by an LC-2020 system (Shimadzu), and mass spectrometry (MS) analysis was performed using an LCMS-2020 (Shimadzu) to confirm the successful synthesis. The chromatograms and mass spectra of each cyclic peptide are as Figure 8 shown.

[0053] 1.2 Salmonella T3SS effector protein secretion experiment

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

[0055] 1. Streak-activate the S. Typhimurium UK-1x8956 strain on an LB antibiotic-free plate.

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

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

[0058] 4. Take 1 mL of the bacterial solution, centrifuge at 14000 g and 4 °C 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] 6. Centrifuge at 14000 g and 4 °C for 5 min, discard the supernatant, and invert the EP tube to remove residual TCA.

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

[0062] 8. Centrifuge at 14000 g and 4 °C for 5 min, discard the supernatant, and dry the residual acetone in a laminar flow hood.

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

[0064] 10. Continuing from step 4, resuspend the centrifuged bacterial pellet in 50 μL of lysis buffer. Take 20 μL and add 20 μL of 2× loading buffer, heat at 99 °C for 5 min to lyse the bacteria and release intracellular proteins. Determine the loading volume according to the OD600nm of the bacterial solution and perform Western blot to detect the expression of intracellular InvA.

[0065] 1.3 Detection of InvA protein expression level by Western blot

[0066] 1. Load the prepared samples onto a 10% SDS-PAGE gel. First, set the voltage to 80 V and perform electrophoresis for 20 min to allow all samples to enter the stacking gel; then set the voltage to 200 V and continue electrophoresis for 25 min to separate the samples.

[0067] 2. Cut off the excess part on the gel. Select a PVDF membrane slightly larger than the gel according to the size of the gel, soak it in absolute ethanol for later use, and transfer it to the wet transfer buffer before transfer.

[0068] 3. Soak the gel and filter paper in the wet transfer buffer in advance. According to the order of "black - gel - membrane", place the black side of the sandwich clip at the bottom, and then lay the filter paper - gel - PVDF membrane - filter paper flat in sequence upwards, paying attention not to have air bubbles.

[0069] 4. Put the prepared sandwich clip into the electrophoresis tank, set the transfer at 180V for 40 min (InvA, 76 kDa), and perform the whole process in a cold room.

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

[0071] 6. Block with 5% non-fat milk powder at room temperature for 1 h.

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

[0073] 8. Take out the incubated membrane, and rinse it three times with TBST, 5 min each time.

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

[0075] 10. Take out the incubated membrane, and rinse it three times with TBST, 5 min each time.

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

[0077] 1.4 Isothermal Titration Calorimetry

[0078] Isothermal Titration Calorimetry (ITC) is a technique used to quantitatively study various biomolecular interactions. It can directly measure the heat released or absorbed during the binding process of biomolecules. The experimental data is presented in the form of a thermogram, providing data on the amount of substances in the reaction (titration endpoint) and the characteristics of the reacting substances (enthalpy change). Using ITC detection, the complete thermodynamic information of biomolecular interactions can be accurately obtained: binding constant (Kd), reaction stoichiometry (n), enthalpy (ΔH), and entropy (ΔS). It is widely used in detecting interactions between proteins - proteins, proteins - small molecules, proteins - nucleic acids, nucleic acids - nucleic acids, etc.

[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: Dissolved in DMSO and gradually diluted to 500 μM, 100 μL with protein buffer. Observe carefully for precipitation.

[0082] Blank control: Protein buffer.

[0083] 2. Before the experiment, centrifuge the samples at 14,000 rpm for 5 min to remove air bubbles. Protein samples need to be pre - placed at room temperature to equilibrate the temperature and avoid generating air bubbles during the experiment.

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

[0085] 4. Wash the instrument in wash mode, titrate ddH2O with ddH2O until the baseline is stable to ensure the instrument is clean.

[0086] 5. Titrate cyclic peptides or small molecule compounds with protein buffer. (Blank control)

[0087] 6. Titrate cyclic peptides or small molecule compounds with protein. (Experimental group)

[0088] 7. Use the MicroCal PEAQ - ITC Analysis Software to subtract the blank control, analyze the fitting curve, and calculate the Kd value.

[0089] 1.5 Determination of bacterial growth curve

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

[0091] 2. Transfer the overnight culture 1:100 to 1.3 mL of LB liquid medium, add arabinose at a final concentration of 0.02% to induce T3SS expression; add cyclic peptides or small molecule inhibitors to the experimental group, add an equal volume of DMSO to the control group, mix well, and dispense 400 μL / well into a 48 - well plate. Set three replicates for each sample.

[0092] 3. Place the 48-well plate in a microplate reader, set the temperature to 37 °C, and incubate with shaking at 450 rpm. Measure the absorbance at OD600nm every 15 minutes for a total of 6 hours.

[0093] 4. Use GraphPad prism 6 to plot and analyze the measurement results.

[0094] 2.5 Detection of the invasion ability of Salmonella enterica serovar Typhimurium to host cells

[0095] 1.5.1 Cell resuscitation and passage

[0096] Cell resuscitation:

[0097] Take out the cryopreserved cells from the liquid nitrogen tank and quickly thaw the cells at 37 °C. Centrifuge at 850 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 10 mL of RPMI1640 medium + 10% FBS. Transfer the resuspended cells to a 10 cm culture dish and culture in an incubator at 37 °C and 5% CO2 for 2 days.

[0098] Cell passage:

[0099] 1. Use a pipette pump to aspirate the cell culture medium, add 5 mL of pre-warmed PBS to wash once, aspirate the PBS, and add 3 mL of 0.5% trypsin. Digest at 37 °C for 3 minutes.

[0100] 2. Add 3 mL of RPMI1640 medium (10% FBS) to terminate the digestion. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 850 rpm for 5 minutes to collect the cells.

[0101] 3. Discard the supernatant, add 6 mL of RPMI1640 medium to resuspend the cells. Take 2 mL of the cell suspension and add it to a 10 cm culture dish, and supplement with about 8 mL of RPMI1640 medium (10% FBS). Mix well and culture in an incubator at 37 °C and 5% CO2 for 2 - 3 days.

[0102] 1.5.2 Gentamicin protection assay

[0103] CCK8 toxicity detection

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

[0105] 2. Cell density (number of cells / mL) = (total number of cells in the four squares / 4) × 10^4 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 culture overnight at 37 °C and 5% CO2.

[0107] 4. Dilute to obtain small molecule inhibitors or cyclic peptides with different concentrations using RPMI1640 medium (10% FBS), ensuring that the DMSO content in each gradient is 0.1%. Set three replicates for each concentration, and the blank control is 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 the 96-well plate, and culture at 37 °C and 5% CO2 for 4 h.

[0109] 6. Measure the absorbance at OD450nm using a microplate reader.

[0110] 7. Calculate the survival rate of the compound on cells according to the following formula:

[0111] Survival rate = (OD450nm of experimental group - OD450nm of blank group) ÷ (OD450nm of negative group - OD450nm of blank group) × 100%.

[0112] Gentamicin protection assay

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

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

[0115] 3. Inoculate the overnight culture of S. Typhimurium UK-1 at 1:100 into 3 mL of LB medium containing 0.02% arabinose, and at the same time add the corresponding small molecule inhibitor or cyclic peptide, and culture at 37 °C for about 4 hours until 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. When OD600nm reaches 0.9, the bacterial suspension concentration is approximately 1×109 CFU / mL. Add 0.5 mL of the diluted bacterial suspension to the 12-well plate according to MOI = 50, mix well, and place it in an incubator at 37 °C and 5% CO2 for 1 h to allow the bacteria to invade the cells.

[0117] 5. Wash the cells three times with pre-warmed 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 h to further remove unbound bacteria.

[0118] 6. Wash the cells 3 times with pre-warmed 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 medium.

[0120] 8. Take 20 μL of the cell lysate and transfer it to a 1.5 mL EP tube. Add 180 μL of PBS and dilute it to a concentration of 1. Then take 20 μL from the bacterial solution of concentration 1, add 180 μL of PBS and dilute it. The diluted solution is designated as concentration 2.

[0121] 9. Take 100 μL of the bacterial solutions of concentration 1 and concentration 2 respectively and spread them on LB plates. Incubate the plates upside down at 37 °C overnight. Count the monoclonal colonies on the plates. Finally, multiply the number of monoclonal colonies of concentration 1 by 100 and the number of monoclonal colonies of concentration 2 by 1000. The number of bacteria invading the cells in each well can be obtained.

[0122] 2. Results and Analysis

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

[0124] Screening of the cyclic peptide library was carried out, targeting InvA C Five cyclic peptides (L1, D1, D2, D7 and D9) were enriched ( Figure 2 a). To determine whether these cyclic peptides can inhibit T3SS function by targeting InvA, first use isothermal titration calorimetry (ITC) to evaluate the binding affinity of these cyclic peptides to InvA C The ITC results showed that cyclic peptides CP-D1, CP-D2 and CP-D7 did not show binding affinity to InvA C while cyclic peptides CP-L1 and CP-D9 were both able to bind InvA C , K D values were 614 ± 323 nM (CP-L1) and 2.93 ± 0.395 μM (CP-D9) respectively ( Figure 2b-c). Next, CP-L1 and CP-D9 were selected to detect their effects on the S. Typhimurium T3SS. CP-L1 and CP-D9 were co-cultured with S. Typhimurium separately, and the culture supernatants were collected for SDS-PAGE analysis. Without affecting bacterial growth, 50 μM CP-L1 could significantly reduce the secretion levels of T3SS effector proteins (SipA, SipB, SipC, and SipD) and inhibit the secretion of T3SS effector proteins in a dose-dependent manner ( Figure 3 a-b). Although the secretion levels of S. Typhimurium T3SS effector proteins decreased after CP-L1 treatment, the expression levels of effector proteins and InvA were not affected. Even at a concentration of 100 μM, CP-D9 did not affect the secretion of S. Typhimurium T3SS effector proteins. According to the ITC results and the results of the in vitro secretion system assay of T3SS effector proteins, the activity of CP-L1 was superior to that of other cyclic peptides (CP-D9, CP-D1, CP-D2, and CP-D7).

[0125] The invasion of pathogenic bacteria into host cells strictly depends on the function of T3SS. Therefore, the inhibitory effect of CP-L1 on the invasion of S. Typhimurium into NCM460 cells (human normal colon epithelial cells) and Raw cells (macrophages) was evaluated. Before the invasion experiment, the cytotoxicity of CP-L1 was determined by the CCK-8 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, through the gentamicin protection assay, it was found that compared with the untreated bacteria (DMSO group), CP-L1 significantly reduced the invasion of S. Typhimurium into NCM460 cells and Raw cells. In the 50 μM CP-L1 treatment group, the invasion rates of S. Typhimurium into NCM460 cells and Raw cells were reduced to 69% and 62% respectively, and in the 100 μM CP-L1 treatment group, the invasion rates of S. Typhimurium into NCM460 cells and Raw cells were reduced 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 pathogenic bacteria

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

[0128] We evaluated the inhibitory effect of CP-L1 on the T3SS of Shigella and EPEC to determine the potential of CP-L1 as a therapeutic / inhibitor 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 (Figures a-d). Subsequently, we evaluated the inhibitory activity of CP-L1 against Shigella and EPEC at the cellular level, showing that CP-L1 significantly reduced the invasion rate of Shigella into NCM460 cells and Raw cells and could inhibit the adhesion of EPEC to NCM460 and Raw cells ( Figure 4 e-f). These results indicate that CP-L1 exhibits potential antivirulence activity against other Gram-negative pathogenic bacteria.

[0129] 2.3 Cyclic peptide CP-L1 effectively inhibits the invasion of multidrug-resistant Salmonella into host cells

[0130] We isolated a multidrug-resistant Salmonella strain from a clinical sample. This strain was insensitive to a variety of antibiotics, including sulfamethoxazole, ampicillin, nalidixic acid, and cefotaxime, etc. ( Figure 5 a). To determine whether CP-L1 could inhibit the activity of the T3SS of this multidrug-resistant Salmonella strain, we co-cultured CP-L1 with the resistant Salmonella in LB containing 0.3 M NaCl and collected the supernatant of the culture fluid for western blot detection of the secretion level of the T3SS effector protein SipC. The results showed that SipC could not be detected in the 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 b-c). 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 invasion rate of the resistant Salmonella into NCM460 cells and Raw cells decreased to 61% ( Figure 5d). These results indicate that the cyclic peptide CP-L1 effectively inhibits the invasion of multi-drug resistant Salmonella into host cells by inhibiting the activity of T3SS.

[0131] 2.4 Optimization of cyclic peptide CP-L1

[0132] To further enhance the effect of CP-L1, we constructed a mutant library of CP-L1 and synthesized 13 mutants according to sequence conservation ( Figure 6 a). By ITC measurement, the binding affinities of mutants M1, M2, M3, M4, M5, M8 and M17 to InvA C were enhanced ( Figure 6 b).

[0133] To further quantitatively evaluate the activities of these mutants on T3SS, we constructed a NanoLuc-based reporter gene system to detect the T3SS activity of S. Typhimurium. According to the results of the reporter gene system, the secretion levels of T3SS effector proteins of all mutants were decreased. Compared with CP-L1, the inhibitory effects of mutants M1 and M8 on T3SS function were reduced, M2 and M17 showed enhanced inhibitory effects, and the inhibitory effects of other mutants (M2, M3, M4 and M5) on T3SS function were not significantly different from those of CP-L1 ( Figure 6 c).

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

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

[0136] 2.5 The mutant peptides exhibit broad-spectrum antibacterial effects against Gram-negative pathogenic bacteria

[0137] By comparative analysis of the amino acid compositions of M2 and M17, we observed that Phe6 of CP-L1 was replaced by Leu6 to generate M2, and Val12 of CP-L1 was replaced by Asp12 to generate M17. These two single-point mutations enhanced the binding affinity of the cyclic peptide to InvA C Considering that the SctV protein is highly conserved in Gram-negative pathogenic bacteria, we speculated that M2 and M17 had antibacterial effects against Shigella and EPEC. The results showed that, compared with CP-L1, M2 had a better inhibitory effect on Shigella infecting NCM460 cells and Raw cells (*P<0.05)( Figure 7 a-b). There was no significant difference in the inhibitory effect of M2 on EPEC adhesion to host cells compared with CP-L1( Figure 7 c-d). M17 had inhibitory effects on the virulence of both Shigella and EPEC, and there was no significant difference in its effect compared with CP-L1( Figure 7 a-d).

[0138] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill 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 targeting SctV to inhibit T3SS activity, characterized in that: The cyclic peptide has any of the following structures:

2. The cyclic peptide according to claim 1, characterized in that The cyclic peptide further comprises a mutant of the cyclic peptide L1, and the mutant of the cyclic peptide L1 comprises:

3. The method for preparing the cyclic peptide according to claim 1 or 2, characterized in that: The preparation method comprises: synthesizing a linear peptide chain by solid phase synthesis, and constructing the cyclic peptide by a thioether structure.

4. Use of the cyclic peptide according to claim 1 or 2 in inhibiting T3SS function or preparing a T3SS inhibitor.

5. The use according to claim 4, characterized in that: The inhibition of T3SS function is achieved by targeting SctV; further, the SctV is InvA.

6. A T3SS inhibitor, characterized in that The T3SS inhibitor comprises the cyclic peptide according to claim 1 or 2.

7. Use of the cyclic peptide according to claim 1 or 2 or the T3SS inhibitor according to claim 6 as or in the preparation of an antibacterial agent; Furthermore, the antibacterial agent exhibits a broad-spectrum antibacterial effect on Gram-negative pathogens, which are pathogens containing T3SS, including EPEC, Pseudomonas aeruginosa, and pathogens of the genera Salmonella, Shigella, Vibrio cholerae, and Yersinia.

8. The use according to claim 7, characterized in that The antibacterial agent is used for environmental disinfection and as or in the preparation of a drug for the diseases mediated by the above-mentioned Gram-negative pathogens; The medicine is used for preventing and / or treating diseases mediated by the Gram-negative pathogens.

9. A drug, characterized in that The active ingredient of the drug comprises the cyclic peptide or T3SS inhibitor according to any one of claims 1 to 2; The drug can be used to inhibit Gram-negative pathogens, and further used to prevent and / or treat diseases mediated by the Gram-negative pathogens; The Gram-negative pathogens are pathogens containing T3SS, including EPEC, Pseudomonas aeruginosa, and pathogens of the genera Salmonella, Shigella, Vibrio cholerae, and Yersinia.

10. The drug according to claim 9, characterized in that The medicament may also include at least one pharmaceutically inactive ingredient.

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