Use of a small molecule inhibitor targeting vgrg protein in the preparation of a medicament for combating bacterial infection

Abstract

The application provides an application of a small-molecule inhibitor targeting VgrG protein in preparation of an antibacterial infection drug, and belongs to the technical field of biological medicine. The application provides an application of a compound shown in general formula (I) or a pharmaceutically acceptable salt thereof: preparation of a bacterial T6SS inhibitor, inhibition of a bacterial T6SS function, treatment and / or prevention of a disease caused by a gram-negative bacterial infection, preparation of a drug for preventing and treating a related disease, or surface disinfection. The compound targets and specifically combines with a bacterial VgrG protein, thereby inhibiting a T6SS related biological function, playing a broad-spectrum antibacterial virulence activity and not inhibiting bacterial growth. Meanwhile, the application also provides a method for screening an antibacterial infection drug based on a conserved region [(N / S / G / R / H / Q)XXXX(E / D)D] of the VgrG protein, and has important application value for developing an antibacterial infection drug with low drug resistance risk.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to the application of a small molecule inhibitor targeting the VgrG protein in the preparation of drugs for antibacterial infection. Background Technology

[0002] Bacterial infectious diseases are widespread and serious, with rapid progression often leading to critical complications such as sepsis and multiple organ failure. Effective antibiotic treatment is currently crucial for reducing mortality. Traditional antibiotic treatment strategies primarily rely on targeting conserved genes or metabolic processes essential for bacterial survival (such as cell wall synthesis, protein translation, or DNA replication). However, this mechanism of directly inhibiting bacterial growth inevitably exerts extremely high survival selection pressure on bacterial populations, forcing bacteria to rapidly evolve drug resistance through gene mutation or horizontal gene transfer. With the continuous emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria, clinicians face the severe challenge of "no available drugs." Developing anti-infective drugs with novel mechanisms of action that are less likely to induce resistance has become an urgent need in the global pharmaceutical field.

[0003] "Antivirulence therapy" has emerged as a novel intervention strategy with significant clinical application potential. This strategy aims to specifically target non-essential virulence factors in bacteria, effectively weakening their infectivity and pathogenicity without affecting normal bacterial growth and reproduction by inhibiting their pathogenic mechanisms or blocking the function of virulence proteins. Among numerous virulence factors, the Type VI Secretion System (T6SS) is widely present in various Gram-negative pathogens. T6SS is structurally and functionally similar to an inverted bacteriophage tail, enabling it to directly inject toxin effector proteins into neighboring target cells (including eukaryotic host cells and other bacteria). Studies have shown that loss of T6SS function puts pathogens at a disadvantage in interspecies competition, thus preventing them from establishing long-term infection within the host. Therefore, T6SS is an ideal target for developing broad-spectrum antiviral drugs. Although the structure of T6SS has been extensively studied, current technologies still lack small-molecule inhibitors that effectively block T6SS function. Summary of the Invention

[0004] The purpose of this invention is to provide the application of a small molecule inhibitor targeting VgrG protein (Valine-glycine repeat protein G) in the preparation of antibacterial drugs. The small molecule compound exerts broad-spectrum antibacterial virulence by targeting and binding VgrG protein and inhibiting T6SS-related biological functions.

[0005] This invention provides the use of a compound or a stereoisomer of the compound or a pharmaceutically acceptable salt or solvate that targets a conserved region of the interface between VgrG protein trimer subunits in the preparation of a medicament for the prevention and / or treatment of diseases caused by Gram-negative pathogen infection, an inhibitor of the type VI secretion system of Gram-negative pathogens, or an anti-Gram-negative pathogen preparation. The compound is at least one of the following: SRT1720, HY-X5613 and SRT 2183; The structural formula of the SRT1720 is shown in Formula II: Formula II The structural formula of HY-X5613 is shown in Formula III: Formula III The structural formula of the SRT 2183 is shown in Formula IV: Formula IV; The Gram-negative pathogens mentioned are Gram-negative pathogens with a type VI secretion system; The amino acid sequence of the conserved region at the intersubunit interface of the VgrG protein trimer is shown in X1XXXXX2D; where X1 is N, S, G, R, H or Q, X2 is E or D, and X is any amino acid residue.

[0006] Preferably, the Gram-negative bacteria having a type VI secretion system include at least one of the following: Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Salmonella ( Salmonella Yersinia spp. Yersinia ), Vibrio cholerae ( Vibrio cholerae Acinetobacter baumannii ( Acinetobacter baumannii Burkholderia ( ) Burkholderia ) and Proteus spp. ( Proteus ).

[0007] Preferably, the anti-Gram-negative pathogen infection includes at least one of the following: weakening the infectivity or pathogenicity of Gram-negative pathogens, reducing the load of Gram-negative pathogens in organs, alleviating pathological damage caused by Gram-negative pathogens, regulating the balance of intestinal flora, and reducing the expression level of type VI secretion system-related proteins.

[0008] Preferably, the drug has at least one of the following characteristics: 1) the drug targets the conserved region of the VgrG protein trimer subunit interface; the amino acid sequence of the conserved region of the VgrG protein trimer subunit interface is shown as X1XXXXX2D; wherein X1 is N, S, G, R, H or Q, X2 is E or D, and X is any amino acid residue; 2) The drug is a broad-spectrum antitoxic drug; 3) The drug does not inhibit bacterial growth in vitro at effective therapeutic doses; 4) The drug does not cause bacteria to develop drug resistance.

[0009] This invention provides the application of the conserved region of the VgrG protein trimer subunit interface in the development, screening, or evaluation of drugs against Gram-negative pathogen infections and those that do not induce drug resistance, or inhibitors of the type VI secretion system of Gram-negative pathogens. The amino acid sequence of the conserved region of the VgrG protein trimer subunit interface is shown as X1XXXXX2D, where X1 is N, S, G, R, H, or Q, X2 is E or D, and X is any amino acid residue. The Gram-negative pathogen is a Gram-negative pathogen with a type VI secretion system.

[0010] This invention provides the use of a compound, or a stereoisomer of the compound, or a pharmaceutically acceptable salt or solvate, targeting the conserved region of the interface between the VgrG protein trimer subunits in the preparation of medicaments for the prevention and / or treatment of diseases caused by Gram-negative pathogens, inhibitors of the type VI secretion system of Gram-negative pathogens, anti-Gram-negative pathogen preparations, or in vitro disinfection. The compound targets the bacterial VgrG protein and specifically binds to the conserved region of the interface between its trimer monomers, thereby inhibiting T6SS-related biological functions, thus exerting broad-spectrum antibacterial virulence without inhibiting bacterial growth. This results in a medicament with broad-spectrum antibacterial virulence without inducing bacterial resistance. The amino acid sequence of the conserved region of the interface between the bacterial VgrG protein trimer subunits is shown as X1XXXXX2D, where X1 is N, S, G, R, H, or Q, X2 is E or D, and X is any amino acid residue. This invention has significant application value for developing antibacterial drugs with low risk of resistance.

[0011] This invention also provides the application of conserved regions of the VgrG protein trimer subunit interface in the development, screening, or evaluation of drugs against Gram-negative pathogen infections and those that do not induce drug resistance, or inhibitors of the type VI secretion system of Gram-negative pathogens. This invention, by mining conserved functional regions of VgrG proteins from different bacterial pathogens, reveals that D489 and its conserved region in the VgrG protein possess evolutionarily conserved characteristics and have the potential to serve as broad-spectrum drug targets. Furthermore, intraspecific and interspecific growth competition experiments demonstrate that the D489 site and conserved functional regions of the VgrG protein are closely related to the intraspecific and interspecific growth competition ability mediated by the pathogen's T6SS, and that the conserved residues D489, N483, and E488 are related to the protein's thermal stability. This invention utilizes virtual screening technology to identify candidate small molecules that interact with the trimer structure of the VgrG1 protein. Intraspecific and interspecific growth competition experiments then validated the selection of compounds capable of efficiently inhibiting the intraspecific and interspecific killing effects mediated by *Pseudomonas aeruginosa* T6SS. These compounds exhibit good antiviral activity and do not affect VgrG1 protein expression; they achieve targeted inhibition of the T6SS virulence system solely by binding to the VgrG1 protein trimer structure. In vivo and in vitro experiments confirmed that the screened compounds possess inhibitory activity against T6SS virulence and effectively prevent and / or treat diseases caused by broad-spectrum bacterial pathogens. Therefore, the screening method provided by this invention can effectively obtain target drugs, and the method is simple, highly operable, and provides a new approach for drug development, screening, or evaluation. Attached Figure Description

[0012] Figure 1 The mass spectrum detection results for HY-X5613 are shown below. Figure 2 The results are the proton spectrum of HY-X5613. Figure 3 The flowchart shows the process for mining conserved functional regions of VgrG proteins; A shows the process for identifying conserved motifs in VgrG proteins: 750 representative VgrG sequences from 390 species were obtained from the CDD database, seed HMM profiles were constructed through multiple sequence alignment, and used to scan 130,825 VgrG proteins in the dbVgrG database. Note: Except for three conserved sites (N, E / D, D), the other four sites exhibit hydrophilic (blue) or hydrophobic (black) characteristics, respectively; B is a pie chart showing the distribution of conserved motifs in the 130,825 VgrG proteins; 94.6% of VgrG proteins encode this motif, of which 83.9% start with asparagine (N); Figure 4Multiple sequence alignments of VgrG proteins from 20 representative bacteria are shown; the yellow area represents the highly conserved [(N / S / G / R / H / Q)XXXX(E / D)D] motif; the terminal aspartate (D) remains unchanged in all aligned sequences, indicated by asterisks (...). ) mark; Figure 5 The effects of conserved functional regions of the VgrG1 protein on the growth competitive ability of the Pseudomonas aeruginosa type VI secretion system (T6SS); A shows the effect of VgrG1 and its mutants on T6SS-dependent intraspecific competition; B shows the effect of VgrG1 and its mutants on T6SS-dependent interspecific competition; the donor strains were Pseudomonas aeruginosa PAO1 and its derivatives, and the intraspecific competitive recipient strain was PAO1Δ. PA0092-0093 The interspecies competing receptor bacterium is *Pseudomonas putida* KT2440; p <0.05, p <0.01, p <0.001, p <0.0001, ns indicates no significant difference; Figure 6 The full-length nucleotide sequence (1932 bp) of the gene encoding the PAO1 VgrG1 protein of Pseudomonas aeruginosa is shown in the figure. The boxed area is the coding sequence NEIRMED corresponding to the conserved functional motif N4X(E / D)D. Three key mutation sites (N483, E488, D489) are highlighted in different colors in the figure. Figure 7 pETDuet1-VgrG1 WT Recombinant plasmid map; Note: VgrG1 WT The gene (whose nucleotide sequence is shown in SEQ ID NO:1) is transmitted through Sal I and Not The restriction endonuclease site was cloned into the multiple cloning site region of the pET-Duet1 vector; the plasmid map was marked with the 6×His tag coding sequence (6×His) and the restriction enzyme site ( Sal I, Not I) and gene insertion direction (ATG initiation direction); Figure 8 Results of thermostability analysis of wild-type and conserved site mutant VgrG1 protein; A represents VgrG1. D489A Mutant; B is VgrG1N483A Mutant; C is VgrG1 E488A Mutants; Figure 9 The flowchart shows the virtual screening process for small molecule compounds based on the conserved VgrG region; A represents the NEIRMED (boxed) conserved binding site of the VgrG1 protein as the target region for virtual screening; B shows the binding energy distribution of docking compounds; C is the flowchart for the virtual screening process for small molecule compounds. Figure 10 A shows the inhibitory effect of compound SRT1720 on the growth competition ability of *Pseudomonas aeruginosa*; A shows the effect of compound SRT1720 on the T6SS-dependent interspecific competition of *P. aeruginosa* PAO1; B shows the effect of compound SRT1720 on the T6SS-dependent intraspecific competition of *P. aeruginosa* PAO1; the donor strains were *P. aeruginosa* PAO1 and its derivatives, the interspecific competitive recipient strain was *P. putridae* KT2440, and the intraspecific competitive recipient strain was PAO1Δ. PA0092-0093 ; p <0.05, p <0.0001, ns indicates no significant difference; Figure 11 To investigate the effects of different concentrations of compound SRT1720 on the in vitro growth of Pseudomonas aeruginosa PAO1 and... vgrG Effects on gene transcription; A shows the effect of different concentrations of compound SRT1720 (10 nM, 10 μM, 50 μM) on the growth curves of PAO1, with each concentration treatment group largely overlapping with the DMSO control group; B shows the effect of compound SRT1720 (10 μM) on the growth curves of PAO1. vgrG Effects on gene transcription levels, after 3 h and 10 h of treatment vgrG No significant changes were observed in expression. Figure 12 The results show the broad-spectrum inhibitory activity of compound SRT1720 against the virulence of Salmonella and Yersinia pseudotuberculosis T6SS; A shows the inhibitory effect of compound SRT1720 on the interspecies competition between Salmonella SL1344 and Escherichia coli DH5α; B shows the inhibitory effect of compound SRT1720 on the interspecies competition between Yersinia pseudotuberculosis YPIII and Escherichia coli DH5α. p <0.05, p <0.01; Figure 13 For the Lip-MS detection of compound SRT1720 and VgrG1 protein N483 EIRMED 489 The integration of regions; Figure 14 The effect of the seven amino acids in the conserved NEIRMED region of the VgrG1 protein on the interaction between compound SRT1720 and this region; Note: The donor bacteria were Pseudomonas aeruginosa PAO1 and its derivative strains, and the recipient bacteria were Pseudomonas putida KT2440. p <0.05, p <0.0001, ns indicates no significant difference; Figure 15 The results show the effects of compound SRT1720 on bacterial load and histopathology in the intestines of Salmonella-infected mice; A is a schematic diagram of the mouse infection experiment; B-D are the bacterial loads in the liver (B), spleen (C), and ileum (D) of mice in each group on day 5 post-infection; tissue homogenates were plate-scraped and counted, and the results are expressed as Log. 10 CFU / g represents the number of mice, with each data point representing one mouse; E represents the histopathological analysis results of liver and ileum tissues; scale bar = 50 μm; data are expressed as mean ± SD (n = 8), and comparisons between two groups were performed using unpaired t-tests or Mann-Whitney tests. p <0.05, p <0.01, p <0.001, ns indicates no significant difference; Figure 16 The results show the effects of the metagenomic sequencing-based compound SRT1720 on the gut microbiota and T6SS virulence function in Salmonella-infected mice. A shows the principal coordinate analysis (PCoA) based on Bray-Curtis differences, with ellipses representing 95% confidence intervals, and the PERMANOVA test was used to compare differences between groups. B shows the abundance comparison of Salmonella enterica, with abundance quantified by RPKM, and the Mann-Whitney U test was used for comparisons between groups. C shows the Violin plot of the total abundance of T6SS-related proteins, and the Mann-Whitney U test was used for comparisons between groups. Figure 17 The structural formula of a derivative compound of compound SRT1720; Figure 18 The pharmacological results are for derivatives of compound SRT1720. Detailed Implementation

[0013] This invention provides a method for screening drugs against Gram-negative pathogen infections by targeting conserved regions at the interface between VgrG protein trimer subunits, comprising the following steps: Contact the candidate compound with the bacterial VgrG protein; Detect whether the conserved regions at the interface between the candidate compound and the bacterial VgrG protein trimer subunit have binding activity or whether there are changes in the thermal stability of the bacterial VgrG protein. If binding occurs or the thermal stability of the bacterial VgrG protein decreases, it indicates that the candidate compound has the property of targeting the bacterial VgrG protein and has the potential to prepare drugs against Gram-negative pathogen infections.

[0014] This invention involves contacting the candidate compound with the bacterial VgrG protein.

[0015] In this invention, to screen drugs against Gram-negative pathogen infections, a conserved region at the inter-subunit interface of the VgrG protein trimer, capable of effectively inhibiting the virulence function of T6SS, was first identified. This conserved region exhibits good conservation among various common clinical pathogens, including *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Bacteroides fragilis*, pathogenic *Escherichia coli*, *Salmonella*, *Yersinia*, and *Vibrio cholerae*. The VgrG protein plays a crucial role in the assembly and release of T6SS. VgrG forms a rigid trimer structure, constituting the "spike" of the injection device, which not only physically penetrates the target cell membrane but also serves as a core hub connecting the tail tube protein (Hcp) and effector proteins. The virulence function of T6SS is achieved through the precise assembly and physical connection of VgrG and Hcp.

[0016] In this invention, the amino acid sequence of the conserved region at the inter-subunit interface of the VgrG protein trimer is as shown in X. 483 XXXXX 488 As shown in D, X 483 For N, S, G, R, H or Q, X 488E or D, X is any amino acid residue, N, S, G, R, H, Q represent asparagine, serine, glycine, arginine, histidine, and glutamine respectively; E represents glutamic acid, and D represents aspartic acid; the 489th amino acid residue is the most critical site, and D represents aspartic acid. The conserved region of the VgrG protein trimer subunit interface is highly conserved in all subtypes of T6SS evolutionary subtype i, which is currently the most widely distributed subtype in the known bacterial genomes carrying T6SS, accounting for 98.6% in the dbVgrG database. This lays the foundation for using the conserved region of the VgrG protein trimer subunit interface as a broad-spectrum anti-infection target. In this embodiment of the invention, the conserved region of the VgrG protein trimer subunit interface can be N 483 EIRMED 489 As shown in the motif (SEQ ID NO:2).

[0017] In this invention, further verification through mutation and complementation at specific sites in the conserved region revealed that amino acid D489 is a key amino acid residue for the function of the VgrG protein and is closely related to the intraspecific and interspecific growth competition mediated by the pathogen T6SS. Simultaneously, conserved residues D489, N483, and E488 maintain the thermostability of the VgrG1 protein, but D489 contributes the most. This suggests that the conserved region at the VgrG protein trimer subunit interface participates in maintaining the stability of the VgrG1 trimer interface through thermodynamic contributions, which is crucial for the normal functioning of T6SS. Therefore, the potential of candidate compounds as drugs for inhibiting bacterial virulence can be evaluated by whether contact with the conserved region at the VgrG protein trimer subunit interface reduces the thermostability of the VgrG1 protein.

[0018] In this invention, the candidate compounds are preferably obtained through virtual screening. Virtual screening involves pre-treating the trimer structure of the VgrG1 protein, defining an active pocket centered on the conserved region containing the conserved residue D489, and performing high-throughput molecular docking on the compounds of interest. Compounds that can form hydrogen bonds or strong electrostatic interactions with key residues in the conserved region at the trimer-monomer interface are selected as candidate compounds. The pre-treatment method includes hydrogenation and / or energy minimization. During molecular docking, a binding energy below -7 kcal / mol is set as the initial screening threshold, and screening is achieved using binding mode checks. In one embodiment of this invention, 56 candidate compounds were obtained from 13,908 compounds of interest through virtual screening.

[0019] In this invention, the bacterial VgrG protein can be a recombinant VgrG protein expressed in vitro or a protein expressed under intracellular conditions. The protein expressed under intracellular conditions exists in the form of bacterial cells.

[0020] In this invention, the contact comprises mixing the candidate compound with the in vitro recombinantly expressed VgrG protein or with bacterial cells. This invention does not impose any particular limitation on the concentration of the candidate compound; concentrations of candidate compounds well known in the art can be used, such as 10–30 μM, or even 20 μM.

[0021] In this invention, during the contact, the in vitro recombinantly expressed VgrG protein and the candidate compound are mixed to obtain a mixed system.

[0022] After contact, the present invention detects whether the conserved region of the interface between the candidate compound and the bacterial VgrG protein trimer subunit has binding activity or changes in the thermal stability of the bacterial VgrG protein; if binding occurs or the thermal stability of the bacterial VgrG protein decreases, it indicates that the candidate compound has the characteristic of targeting the bacterial VgrG protein and has the potential to prepare drugs against Gram-negative pathogen infections.

[0023] In this invention, the detection of whether the candidate compound has binding activity with the conserved region at the interface between the bacterial VgrG protein trimer subunits preferably includes digesting the bacterial VgrG protein in a trypsin-digested mixture, removing undigested protein, collecting the digested peptides, and analyzing them using Lip-MS. When the candidate compound is present, if the mass spectrometry signal of the conserved region and its adjacent peptides decreases overall, it indicates that the candidate compound protects the conserved region of the VgrG protein from enzymatic cleavage by binding.

[0024] In this invention, the method for detecting changes in the thermostability of bacterial VgrG protein is preferably such that, compared with the control group with added candidate compound, the Tm value of the treatment group with added candidate compound is significantly reduced, indicating that the thermostability of bacterial VgrG protein is reduced.

[0025] In this invention, to verify that the screened compounds have excellent inhibitory activity against T6SS-mediated bacterial killing effects, under laboratory conditions, based on wild-type PAO1 (positive control) and T6SS-deficient strain Δ... clpV1 (Negative control) vgrG1 Gene (PA0091) deletion mutant Δ vgrG1 , replenishment plant Δ vgrG1 + vgrG1Intraspecific and interspecific competition experiments were conducted, and the inhibitory activity of the compounds was evaluated using the Competitive Index (CI). A significant decrease in the CI directly indicates that the survival or reproductive ability of the strain in a specific environment (such as the host or culture medium) is weakened. In a specific embodiment of this invention, compared with other candidate compounds, candidate compound SRT1720 exhibited the best inhibitory activity against the intraspecific killing effect mediated by T6SS, that is, SRT1720 had the best antiviral activity among all candidate molecules. Therefore, SRT1720 was selected as the core lead compound.

[0026] Traditional growth-inhibiting drugs often act as screening pressures, leading to the emergence of drug-resistant pathogens and posing a challenge to the development of subsequent antibacterial drugs. To screen for drugs that do not induce resistance, this invention also verifies the effects of SRT1720 on bacterial growth and... vgrG The study investigated the effects on gene transcription levels, showing that compound SRT1720, at doses above its effective antiviral concentration, did not inhibit the normal in vitro growth and reproduction of *Pseudomonas aeruginosa*, nor did it affect... vgrG Gene transcription level. Combined with the aforementioned experimental results, this suggests that compound SRT1720 exerts its effects by targeting the T6SS virulence system, ensuring that the screened target drug does not induce drug resistance.

[0027] This invention provides the use of a compound having the general formula I, or a stereoisomer of the compound, or a pharmaceutically acceptable salt or solvate, targeting a conserved region of the intersubunit interface of the VgrG protein trimer in the preparation of a medicament for the prevention and / or treatment of diseases caused by Gram-negative pathogens, an inhibitor of the type VI secretion system of Gram-negative pathogens, an anti-Gram-negative pathogen preparation, or an in vitro disinfectant. General Formula I R1 is any one of the following groups: , and ; R2 is or .

[0028] In this invention, the compound preferably includes at least one of the following: SRT1720, HY-X5613 and SRT 2183.

[0029] In the SRT1720, R1 is... R2 is ; In the HY-X5613, R1 is... R2 is ; In the SRT 2183, R1 is R2 is .

[0030] In this invention, the molecular formula of SRT1720 is C 25 H 23 N7OS, CAS number 925434-55-5, structural formula shown in Formula II, belongs to the polyfused aromatic heterocyclic carboxamide class of small molecules. In this embodiment of the invention, the SRT 1720 was purchased from MCE (MedChemExpress), catalog number HY-10532.

[0031] Formula II Formula III Formula IV.

[0032] In this invention, HY-X5613 and SRT 2183 are derivatives obtained through a cyclical screening process based on the "structure-property-activity" model, with SRT1720 as the lead compound. The molecular formula of HY-X5613 is C1. 25 H 22 N6OS, structural formula is shown in Formula III. In this embodiment of the invention, HY-X5613 was synthesized by MCE with a purity of 96.56%, mass spectrum is shown in Figure 1, and proton spectrum is shown in Figure 1. Figure 2 The molecular formula of SRT 2183 is C2. 27 H 24 N4O2S, CAS number 1001908-89-9, structural formula is shown in Formula IV. This invention does not impose any special restrictions on the source of the SRT 2183; any commercially available SRT 2183 known in the art may be used.

[0033] In one embodiment of the present invention, the antibacterial virulence activity of different structural derivatives of SRT1720 was evaluated by the change in the competition index. The results showed that HY-X5613, SRT 2183, and SRT1720 all exhibited good inhibitory activity compared with the competition index of other derivative groups.

[0034] In this invention, the term "pharmaceutically acceptable salt" refers to a salt suitable for contact with human and lower animal tissues without causing excessive toxicity, irritation, allergic reactions, etc., within the scope of reliable medical judgment, and with a reasonable effect / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in SM Berge, et al., J. Pharmaceutical Sciences, 1977, 66: 1. The term "pharmaceutically acceptable solvate" refers to a solid complex with a specific crystal structure formed after binding with one or more solvent molecules at the molecular level. The pharmaceutically acceptable salt of SRT1720 is preferably a hydrochloride salt. The pharmaceutically acceptable solvate of SRT1720 is preferably DMSO.

[0035] In this invention, the Gram-negative pathogens preferably include Gram-negative pathogens possessing a type VI secretion system (T6SS). Gram-negative pathogens generally possess a type VI secretion system (T6SS), which plays a role in infecting the host and evading immune clearance. When T6SS function is lost, the pathogen is at a disadvantage in interspecies competition, thus failing to establish a long-term infection in the host. The Gram-negative bacteria possessing a type VI secretion system preferably include at least one of the following: *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Salmonella ( Salmonella Yersinia spp. Yersinia ), Vibrio cholerae ( Vibrio cholerae Acinetobacter baumannii ( Acinetobacter baumannii Burkholderia ( ) Burkholderia ) and Proteus spp. ( Proteus Yersinia includes Yersinia pseudotuberculosis.

[0036] In this invention, the antibacterial infection preferably includes at least one of the following: weakening the infectivity or pathogenicity of pathogens, reducing the pathogen load in organs, alleviating pathological damage caused by pathogens, regulating intestinal flora balance, and reducing the expression level of type VI secretion system-related proteins. The compound inhibits T6SS function by targeting and binding to the conserved region of the VgrG protein trimer subunit interface, thereby reducing the infectivity and pathogenicity of pathogens, and consequently reducing the pathogen load in organs, alleviating pathological damage caused by pathogens, regulating intestinal flora balance, and reducing the expression level of type VI secretion system-related proteins.

[0037] In this invention, the drug preferably targets and binds to the conserved region of the VgrG protein trimer subunit interface. The amino acid sequence of the conserved region of the VgrG protein trimer subunit interface is shown as X483XXXXX488D, where X...483 For N, S, G, R, H or Q, X 488 Option E or D can be as shown in SEQ ID NO:2 (NEIRMED). Based on the fact that the active ingredient in the drug targets and binds to a conserved region at the interface between the VgrG protein trimer subunits, and that this region is a conserved sequence in various Gram-negative pathogens, the drug is a broad-spectrum antiviral agent. The compound in the drug targets and binds to the VgrG protein without affecting the transcriptional level of the gene encoding this protein, and growth curve results indicate that it does not affect bacterial growth. Therefore, the drug blocks the infection process, does not exert selection pressure on bacterial growth, does not cause bacterial resistance, and avoids the adverse consequences of developing superbugs.

[0038] In this invention, the drug preferably further includes pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients include carriers and / or excipients. The carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Using these materials, various dosage forms can be formulated, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. To formulate unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. To formulate unit-dose dosage forms into injectable formulations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be used for administration via injection, preferably including subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; cavity administration, such as rectal and vaginal administration; respiratory administration, such as nasal administration; and mucosal administration.

[0039] This invention provides a disinfection method for inhibiting the virulence of Gram-negative pathogenic bacteria, comprising the following steps: The application of the above technical solution involves contacting the object or environment to be disinfected with a compound represented by general formula I, or a stereoisomer of the compound, or a pharmaceutically acceptable salt or solvate.

[0040] In this invention, since Gram-negative pathogens can cause various diseases in humans, animals, and plants, the screened compounds can be used not only to prepare drugs for disease prevention and treatment but also to prepare in vitro disinfectant preparations for surface disinfection of medical devices, medical materials, implants, operating rooms, wards, medical staff, or patients. They can also be used for surface disinfection of plants and plant biomaterials, and for disinfecting aquaculture water, soil for planting, or cultivation substrates. The plant biomaterials include seeds, explants, tissues, and organs. Furthermore, given that Gram-negative pathogens can also easily infect through ingestion, the compounds can also be added to food, food additives, feed additives, or feed to prevent the proliferation of Gram-negative pathogens in food or feed, thus preventing infection in humans or animals.

[0041] In this invention, the working concentration of the compound under in vitro conditions is 10-30 μM, and can be 10-20 μM. The working concentration of the compound in animal experiments is 100 mg / kg. The preferred methods of contact include application, soaking, spraying, etc.

[0042] In this invention embodiment, antibacterial virulence verification experiments were conducted against Pseudomonas aeruginosa, Salmonella, and Yersinia. However, the VgrG protein in Klebsiella pneumoniae, Acinetobacter baumannii, Bacteroides fragilis, pathogenic Escherichia coli, and Vibrio cholerae all possess conserved functional regions. Therefore, based on the verification experiments against Pseudomonas aeruginosa, Salmonella, and Yersinia, the screened compounds can be found to possess broad-spectrum antibacterial properties.

[0043] The following examples illustrate the application of a small molecule inhibitor targeting the VgrG protein provided by the present invention in the preparation of drugs for antibacterial infection, but these examples should not be construed as limiting the scope of protection of the present invention.

[0044] The sequence information involved in the embodiments of this application is shown in Tables 1 and 2.

[0045] Table 1. Homologous arm sequences involved in recombinant strains

[0046] Table 2 Primer sequences involved in the embodiments of the present invention

[0047] Example 1 Discovery of conserved functional regions of VgrG protein This embodiment aims to identify conserved functional regions in VgrG proteins and analyze their evolutionary conservation across different bacterial VgrG proteins, providing a foundation for screening VgrG-targeting compounds with broad-spectrum potential. The study selected several representative T6SS-positive bacteria, including *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Bacteroides fragilis*, *Escherichia coli*, *Salmonella enterica*, *Yersinia pseudotuberculosis*, and *Vibrio cholerae*, for analysis.

[0048] 1. Experimental Methods By comparing multiple domains associated with VgrG proteins, it was found that only COG3501 (NCBI COG database ID, corresponding to the VgrG domain) was present in all 872 experimentally validated VgrG protein sequences ranging from 601 to 1,100 amino acids. The 2025 updated COG3501 multiple sequence alignment data contains 750 protein sequences from 390 bacterial species, demonstrating good representativeness. Preliminary analysis indicates that it contains a significantly conserved amino acid region.

[0049] To further analyze the evolutionary conservation of this region in large-scale VgrG sequences, a hidden Markov model (HMM) based on the COG3501 domain was constructed using HMMER software. A scan was performed on 130,825 VgrG protein sequences encoded by 45,041 bacterial genomes collected from the dbVgrG database (https: / / www.mgc.ac.cn / dbVgrG / ). Figure 3 (A). For VgrG proteins that can match the COG3501 domain, the sequence information of the corresponding region was extracted, and the composition and frequency of each amino acid site were statistically analyzed, with a focus on analyzing the conservation of the [(N / S / G / R / H / Q)XXXX(E / D)D] region. Figure 3 (B)

[0050] Based on the aforementioned large-scale sequence analysis, further multiple sequence alignment was performed on VgrG proteins from 20 representative bacteria, including Vibrio parahaemolyticus (V. parahaemolyticus). Vibrio parahaemolyticus ), Serratia marcescens ( Serratia marcescens Edwardsiella tarda ( ), Edwardsiella tarda Aeromonas hydrophila ( ) Aeromonas hydrophila Enterobacter cloacae () Enterobacter cloacae ), Vibrio cholerae ( Vibrio cholera e) Salmonella enterosus ( Salmonella enterica ), Citrobacter freundii ( Citrobacter freundii ), Escherichia coli ( Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Shigella sonnei ( Shigella sonnei Yersinia pseudotuberculosis ( Yersinia pseudotuberculosis Agrobacterium fabromii ( Agrobacterium fabrum ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), pear fire blight fungus ( Erwinia amylovora Xanthomonas oryzae ( ) Xanthomonas oryzae Acinetobacter baumannii ( Acinetobacter baumannii Burkholderia melioides ( ), Burkholderia pseudomallei Burkholderia cepacia (), Burkholderia cepaci a) and Bacteroides fragilis ( Bacteroides fragili (s) to further verify the conservation of the identified conserved regions in VgrG proteins of different bacteria.

[0051] 2. Experimental Results See results Figure 3 and Figure 4 Analysis showed that among the 130,825 VgrG protein sequences included in the dbVgrG database, 129,425 (98.9%) could identify the COG3501 domain, indicating that this domain is highly conserved in VgrG proteins.

[0052] Further sequence analysis of the COG3501 matching region revealed that the [(N / S / G / R / H / Q)XXXX(E / D)D] region exhibited significant evolutionary conservation. Among these, VgrG proteins with N or S as the first amino acid site accounted for 108,587 (83.9%) and 6,212 (4.8%) of these sites, respectively, representing the two most common types and totaling 88.7%. Besides N and S, the site could also be G, R, H, or Q, with proportions of approximately 3.3%, 1.4%, 0.6%, and 0.5%, respectively.

[0053] The terminal aspartate residue (D) in this region exhibits higher conservation, with 128,671 VgrG proteins retaining D at this site, accounting for 99.4% of the COG3501 matching sequences. Simultaneously, 122,436 VgrG proteins conforming to the aforementioned conservation characteristics, containing the sequence pattern [(N / S / G / R / H / Q)XXXX(E / D)D], account for 94.6% of the COG3501 matching sequences. Figure 3 (B)

[0054] Further, VgrG proteins from 20 representative bacteria were selected for multiple sequence alignment. The results showed that although there were some sequence differences in the amino acids flanking and in the middle of this region among different bacteria, the region corresponding to [(N / S / G / R / H / Q)XXXX(E / D)D] exhibited significant sequence conservation overall, especially the terminal aspartic acid (D), which remained unchanged in all 20 aligned representative VgrG protein sequences. Figure 4 This result is consistent with the results of large-scale VgrG sequence statistical analysis, further indicating that this region is not limited to a single bacterial species, but is widely present in VgrG proteins from different sources and species.

[0055] In summary, the terminal aspartate residue (D489 in the amino acid sequence of Pseudomonas aeruginosa PAO1 VgrG1) and the surrounding [(N / S / G / R / H / Q)XXXX(E / D)D] region constitute a highly evolutionarily conserved feature of the VgrG protein, with the terminal aspartate residue exhibiting particularly significant conservation. Therefore, this conserved region has the potential to serve as a target for VgrG functional studies and for developing broad-spectrum T6SS inhibitor drugs.

[0056] Example 2 Effects of conserved functional regions of VgrG1 protein on the growth competitive ability of Pseudomonas aeruginosa type VI secretion system (T6SS) This embodiment aims to study the effect of the conserved region discovered in Example 1 on T6SS through intraspecific and interspecific growth competition experiments.

[0057] 1. Experimental Methods (1) Construction of gene knockout mutant strains In this embodiment, the P. aeruginosa PAO1 gene knockout mutant strains were all constructed using the homologous recombination double exchange method based on the suicide vector pK18mobsacB. Using PAO1 genomic DNA as a template, the upstream and downstream homologous arms of the target gene were amplified by PCR (gene sequences are shown in Table 1), cloned into pK18mobsacB, and transformed into *E. coli* S17-1 λpir. PAO1 was then introduced via conjugation transfer. SacB-positive clones were screened on LB agar plates containing 12% (w / v) sucrose and 50 μg / mL kanamycin. Verification was performed by PCR (primers are shown in Table 2) and sequencing. vgrG1 Gene deletion mutant strain Δ vgrG1 and clpV1 Gene deletion mutant strain Δ clpV1 The pME6032 plasmid (tetracycline resistance) was electroporated into the above knockout strain for subsequent competition experiments. All primer sequences are shown in Table 2.

[0058] In addition, to construct a recipient bacterium highly sensitive to T6SS killing, the same method described above was used, with PAO1 genomic DNA as a template, for PCR amplification. PA0092 - PA0093 Upstream and downstream homologous arms of the gene region (gene sequences are shown in Table 1), knocked out in one go. PA0092 - PA0093 After verification by PCR (primers are shown in Table 2) and sequencing, the pBBR1-MCS-5 plasmid (gentamicin resistant) was electroporated into the knockout strain for subsequent competition experiments.

[0059] (2) Construction of replacement plants Using PAO1 genomic DNA as a template, PCR amplification vgrG1 Full-length gene (see) Figure 6 The middle sequence was cloned into the broad-host vector pME6032 (containing the tetracycline resistance gene), and the recombinant plasmid pME6032- was constructed. vgrG1 The recombinant plasmid was introduced into Δ by electroporation. vgrG1 The mutant strain was screened on LB plates containing tetracycline (50 μg / mL) and verified by PCR to obtain the complemented strain Δ. vgrG1 + vgrG1 .

[0060] Using PAO1 genomic DNA as a template, site-directed mutagenesis was performed using overlap extension PCR (primers are shown in Table 2). vgrG1 The aspartic acid (D) at position 489 of the gene was mutated to alanine (A), and the gene was cloned into the pME6032 vector and introduced into the vector. ΔvgrG1 The mutant strain was sequenced and verified to yield a point mutation complement strain.

[0061] All mutant and complemented strains were verified to be correct by PCR and DNA sequencing.

[0062] (3) Intraspecific growth competition The donor strains were *Pseudomonas aeruginosa* PAO1 and its derivatives, including: wild-type PAO1 (positive control) and T6SS-deficient strain Δ. clpV1 (Negative control) vgrG1 Gene (PA0091) deletion mutant Δ vgrG1 , replenishment plant Δ vgrG1 + vgrG1 and mutant complementation strain Δ vgrG1 + vgrG1 D489A All donor strains carried the pME6032 plasmid (tetracycline resistance), and the recipient strain was the P. aeruginosa PAO1 derivative PAO1Δ carrying the pBBR1-MCS-5 plasmid (gentamicin resistance). 0092-0093 This strain lacks the PA0092 gene encoding virulence protein and the PA0093 gene encoding antiviral protein, making it highly sensitive to T6SS killing and enabling it to compete with the donor strain within the species.

[0063] Donor bacteria were inoculated into LB broth containing tetracycline (50 μg / mL), and recipient bacteria were inoculated into LB broth containing gentamicin (50 μg / mL). Both cultures were incubated at 37°C with shaking at 220 rpm for 10 hours. After incubation, 1.5 mL of each culture was transferred to EP tubes, centrifuged at 4500 rpm for 5 minutes at room temperature, the supernatant was discarded, and the precipitate was washed once with 1 mL of LB broth. After centrifugation again and discarding the supernatant, the bacterial cells were resuspended in 1.2 mL of LB broth. 100 μL of the resuspended culture was diluted 10-fold, and the OD was measured using a cuvette. 600 Value. Based on the measurement results, the OD value of the donor and recipient bacteria was determined. 600 Adjust to 1.0. Perform serial dilutions on the adjusted bacterial culture (donor bacteria diluted to 10). -6 The recipient bacteria were diluted to 10 -6 The bacteria were spread on LB agar plates containing tetracycline (to count donor bacteria) and LB agar plates containing gentamicin (to count recipient bacteria), and the number of single colonies was counted to determine the initial ratio of donor to recipient.

[0064] Growth competition experiments were conducted on solid culture media, with donor and recipient bacteria mixed at a volume ratio of 5:1 (100 μL donor, 20 μL recipient). 5 μL of the mixed bacterial suspension was spotted onto a 0.22 μm nitrocellulose membrane, which was then placed on an LB agar plate containing 3% agar and incubated at 37°C for 16 hours. After incubation, the bacterial growth on the membrane was resuspended in 1 mL of LB broth using a sterile pipette tip, followed by serial dilutions (donor bacteria diluted to 10 μL). -6 / 2, recipient bacteria diluted to 10 -5 / 2) The colonies were then spread onto LB agar plates containing tetracycline (for selective counting of donor bacteria) and LB agar plates containing gentamicin (for selective counting of recipient bacteria). After single colonies grew, the colony count was recorded, and the competitive index was calculated using the following formula I: Competition index = (CFU of donor bacteria after co-culture / CFU of recipient bacteria) / (CFU of initial donor bacteria / CFU of initial recipient bacteria) Formula I.

[0065] The experiment was performed in three biological replicates. Data are expressed as mean ± SD. One-way ANOVA and Dunnett's multiple comparison test were used to analyze the significance of differences between groups.

[0066] (4) Interspecific growth competition The donor strains are *Pseudomonas aeruginosa* PAO1 and its derivatives, including: wild-type PAO1 and T6SS-deficient strain Δ. clpV1 , vgrG1 Gene deletion mutant strain Δ vgrG1 , replenishment plant Δ vgrG1 + vgrG1 and mutant complementation strain Δ vgrG1 + vgrG1 D489A All strains carried the pME6032 plasmid (tetracycline resistant). The recipient strain was *Pseudomonas putida* KT2440, which carried the pBBR1 MCS-5 plasmid (gentamicin resistant).

[0067] Donor bacteria were inoculated into LB broth containing tetracycline (50 μg / mL), and recipient bacteria were inoculated into LB broth containing gentamicin (50 μg / mL). Both cultures were incubated at 37°C with shaking at 220 rpm for 10 hours. After incubation, 1.5 mL of the bacterial culture was transferred to an EP tube and centrifuged at 4500 rpm for 5 minutes at room temperature. The supernatant was discarded. The precipitate was washed once with 1 mL of LB broth, centrifuged again, and the supernatant was discarded. The bacterial cells were resuspended in 1.2 mL of LB broth. 100 μL of the resuspended culture was diluted 10-fold, and the OD was measured using a cuvette. 600 Value. Based on the measurement results, the OD value of the donor bacteria was determined. 600 Adjusted to 1.0, recipient bacteria OD 600 Adjust to 2.0. Take the adjusted bacterial culture and perform serial dilutions, then spread it on LB agar plates containing the corresponding antibiotics. Count the number of single colonies to determine the initial donor-recipient ratio.

[0068] Growth competition experiments were conducted on solid culture media, with donor and recipient bacteria mixed at a volume ratio of 1:5 (20 μL donor, 100 μL recipient). 10 μL of the mixed bacterial suspension was spotted onto a 0.22 μm nitrocellulose membrane, which was then placed on a 3% LB-LS agar plate and co-cultured at 37°C for 16 hours. After incubation, the bacterial growth on the membrane was resuspended in 1 mL of LB liquid using a sterile pipette tip. After serial dilution, the fragments were spread onto LB agar plates containing tetracycline (for selective counting of donor bacteria) and LB agar plates containing gentamicin (for selective counting of recipient bacteria). Once single colonies appeared, the colony count was recorded, and the competitive index was calculated to assess the competitive ability of each donor strain. The competitive index was calculated as: (CFU of donor bacteria after co-culture / CFU of recipient bacteria) / (CFU of initial donor bacteria / CFU of initial recipient bacteria). The experiment was performed in six biological replicates. Data are expressed as mean ± SD. One-way ANOVA and Dunnett's multiple comparison test were used to analyze the significance of differences between groups.

[0069] 2. Experimental Results See results Figure 5 The results showed that, compared with wild-type Pseudomonas aeruginosa PAO1 (positive control), the T6SS-deficient strain (negative control) and vgrG1 The deletion strain showed significantly reduced intraspecific competitive ability (A) and interspecific growth competitive ability (B), and the wild-type was reinstated. vgrG1 It can restore its corresponding growth competitiveness, while replenishing D489A can only partially restore it.

[0070] 3. Conclusion The conserved functional regions of the VgrG protein discovered in Example 1, especially the last amino acid D489, are key amino acid residues for VgrG protein function and are closely related to the intraspecific and interspecific growth competition mediated by the pathogen T6SS.

[0071] Example 3 The effect of conserved regions D489, N483, and E488 of the VgrG1 protein on protein thermostability This embodiment aims to compare the conformational stability of wild-type VgrG1 protein and D489A, N483A, and E488A mutant proteins at different temperatures through thermal shift assay (TSA) to evaluate the influence of conserved residues of D489A, N483A, and E488A on the thermal stability of VgrG1 protein.

[0072] 1. Experimental Methods: (1) Protein expression and purification: Encode VgrG1 WT (Its nucleotide sequence is shown in the image) Figure 6 (SEQ ID NO:1) Sal I and Not I restriction endonuclease site cloned into pETDuet-1 expression vector (recombinant plasmid map see...) Figure 7 The D489A, N483A, and E488A mutations were introduced into the recombinant plasmid pETDuet-VgrG1 using site-directed PCR with overlap extension (primers shown in Table 2) based on this sequence. WT pETDuet-VgrG1 D489A pETDuet-VgrG1 N483A and pETDuet-VgrG1 E488A The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing ampicillin (100 μg / mL), and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin, and incubated overnight at 37°C with shaking at 180 rpm. The next day, a 1% inoculum was transferred to 500 mL of LB liquid medium containing ampicillin and incubated at 37°C with shaking at 180 rpm until OD500. 600 The concentration was approximately 0.4-0.6. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and expression was induced at 16°C and 180 rpm for 18-20 hours.

[0073] After induction, the bacterial culture was centrifuged at 4500 rpm for 20 minutes at 4°C to collect the bacterial cell pellet. The pellet was resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0) and sonicated on ice (500 bar, 5 min). The lysate was filtered through a 0.22 μm filter and centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was collected for later use.

[0074] Affinity chromatography purification was performed using the ÄKTA pure protein purification system. The supernatant was loaded into a pre-packed (5 mL) BabyBio NiMAC column equilibrated with lysis buffer at a flow rate of 0.5 mL / min. After loading, unbound contaminating proteins were washed with lysis buffer. A gradient elution method was then used, eluting the target protein stepwise with elution buffers containing different concentrations of imidazole (20 mM epes-free acid, 200 mM NaCl, 20–500 mM imidazole, pH 8.0), and the elution peaks were collected. The distribution of the target protein was analyzed by SDS-PAGE electrophoresis, and the elution fractions containing the target protein were combined.

[0075] The combined protein samples were transferred to concentration tubes (molecular weight cutoff 30 kDa) and concentrated to a volume of 500 μL by centrifugation at 2300 g at 4 °C. The concentrated protein samples were then centrifuged at 12000 rpm for 10 min at 4 °C to remove precipitate, and the supernatant was further purified by molecular sieve chromatography. A Superdex 200 Increase 10 / 300 GL gel filtration chromatography column was used with elution buffer (20 mM Hepes-Free Acid, 200 mM NaCl, pH 8.0) as the mobile phase at a flow rate of 0.2 mL / min, and each elution peak was collected. SDS-PAGE electrophoresis was used to detect the distribution of the target protein. The high-purity target protein fractions were combined, concentrated, and after determining the protein concentration, aliquots were dispensed, flash-frozen in liquid nitrogen, and stored at -80 °C for later use.

[0076] (2) Thermal Stability Assay (TSA): The purified recombinant protein (30 μg) was added to a PCR tube, and the volume was adjusted to 30 μL with buffer (20 mM Hepes Free Acid, 200 mM NaCl, pH 8.0). The tube was then heated for 200 s in a gradient PCR instrument at the specified temperature. After heating, the samples were centrifuged at 20,000 g for 30 min at 4 °C, and 10 μL of the supernatant from each sample was taken for SDS-PAGE electrophoresis. The protein was transferred to a PVDF membrane and incubated for 1 h with mouse anti-6×His tag monoclonal antibody (Abcam, ab18184) as the primary antibody. Subsequently, it was incubated at room temperature for 1 h with horseradish peroxidase-labeled goat anti-mouse IgG (H+L) antibody (Abcam, ab205719). The chemiluminescence signal was detected in a chemiluminescence imaging system using NcmECL Ultra ultrasensitive luminescent solution (NewSemi Biotechnology Co., Ltd., P10100), and quantitative analysis was performed using ImageJ software. Using the normalized band intensity of the lowest temperature control sample as a benchmark, the relative band intensity at each temperature point was calculated, and the protein melting curve was plotted.

[0077] 2. Experimental Results See results Figure 8 Thermal stability analysis showed that the band intensity of wild-type VgrG1 protein gradually decreased when the temperature rose above 50℃, exhibiting typical temperature-dependent instability characteristics; while the D489A mutant protein showed a significant decrease at 40℃, with the Tm value being about 14℃ lower than that of the wild type, indicating that the D489A mutation significantly reduced the thermal stability of VgrG1 protein. Figure 8 (A). Furthermore, the N483A and E488A mutants also exhibited varying degrees of Tm value shift, decreasing by approximately 9℃ and 2.5℃, respectively. Figure 8(B and C) indicates that N483 and E488 residues also participate in maintaining the thermal stability of the VgrG1 protein, but their contribution is less than that of D489.

[0078] 3. Conclusion The experimental results indicate that the conserved residues D489, N483, and E488 of the VgrG1 protein play a crucial role in maintaining its thermal stability, with D489 contributing the most significantly. Mutations in D489A, N483A, and E488A all lead to varying degrees of decrease in the protein's thermal stability, suggesting that the N4X(E / D)D conserved motif participates in maintaining the stability of the VgrG1 trimer interface through thermodynamic contributions, which is of great importance for the normal functioning of T6SS.

[0079] Example 4 Virtual screening of small molecule compounds based on VgrG conserved regions and the inhibitory effect of compound SRT1720 on the interspecies killing effect mediated by Pseudomonas aeruginosa T6SS. In this embodiment, 56 candidate compounds were initially screened from 13,908 small molecule compounds using structure-based virtual screening technology. The lead compound SRT1720 was further screened through intraspecies bacterial competition experiments, and its inhibitory effect on the interspecies killing effect mediated by Pseudomonas aeruginosa T6SS was evaluated.

[0080] 1. Experimental Methods (1) Structure-based virtual screening Based on the trimer structure of Pseudomonas aeruginosa PAO1 VgrG1 protein (PDB ID: [6H3L]), after protein pretreatment (hydrogenation, energy minimization), a virtual screening activity pocket was defined centered on the conserved region containing the conserved residue D489 identified in Example 1. The MCE small molecule database (L001 and L001P compound libraries, totaling 13908) was selected as the ligand library, and high-throughput molecular docking was performed using AutoDock Vina software. A binding energy below -7 kcal / mol was set as the initial screening threshold, and candidate small molecules (56 in total, see [link to relevant documentation]) that could embed into the trimer monomer interface and form hydrogen bonds or strong electrostatic interactions with key residues in the conserved region were selected through binding mode checks. Figure 9 ).

[0081] (2) Biological validation and compound identification of candidate compounds The candidate compounds selected through virtual screening were subjected to intraspecies bacterial growth competition experiments, using the same methods as in Example 2. The difference was that when the donor bacterium *Pseudomonas aeruginosa* PAO1 was inoculated into LB broth containing tetracycline, the experimental group received a final concentration of 10 μM of the candidate compound, while the control group received an equal volume of the candidate compound's solvent. When 100 μL of donor bacteria and 20 μL of recipient bacteria were mixed (volume ratio 5:1), 1.2 μL of the candidate compound (final concentration 10 μM) or an equal volume of solvent (control) was added simultaneously. Subsequent procedures were the same as in Example 2 to evaluate the effect of the candidate compounds on the growth competition activity of *Pseudomonas aeruginosa* PAO1. The experiment was performed in triplicate, and data are expressed as mean ± SD. Unpaired t-tests were used to analyze the significance of differences between groups.

[0082] (3) Inhibitory effect of compound SRT1720 on interspecies competition of Pseudomonas aeruginosa PAO1 To further verify the inhibitory effect of compound SRT1720 on T6SS function, an interspecies competition model was used to evaluate its inhibitory effect on the interaction between *Pseudomonas aeruginosa* and *Pseudomonas putida*. The interspecies competition experiment method was the same as in Example 2. The difference was that when the donor *Pseudomonas aeruginosa* PAO1 was inoculated into LB liquid medium containing tetracycline, the experimental group was given compound (SRT1720) at a final concentration of 10 μM, while the control group was given an equal volume of DMSO. When 20 μL of donor bacteria and 100 μL of recipient bacteria were mixed (volume ratio 1:5), 1.2 μL of compound SRT1720 (final concentration 10 μM) or an equal volume of DMSO (control) was added simultaneously, and subsequent operations were the same as in Example 2. The experiment was performed in six biological replicates, and the data are expressed as mean ± SD. Two-way ANOVA and Šidák multiple comparison test were used to analyze the significance of differences between groups.

[0083] 2. Experimental Results Intraspecific growth competition experiments showed that candidate compound SRT1720 exhibited optimal inhibitory activity against T6SS-mediated intraspecific killing effects. Figure 10 (B), and was therefore identified as the core lead compound of this invention. Interspecific growth competition experiments showed that compound SRT1720 significantly inhibited wild-type PAO1 and the supplementary plant (Δ). vgrG1 + vgrG1 Its growth competitiveness, but for vgrG1 Gene deletion strains showed no significant effect, indicating that compound SRT1720 exerts its effect by targeting the VgrG1 protein. Figure 10 (A)

[0084] 3. Conclusion Fifty-six candidate molecules were obtained through virtual screening, and the lead compound SRT1720 was selected from them after biological validation. Experimental results showed that compound SRT1720 could effectively inhibit the intraspecific and interspecific killing effects mediated by Pseudomonas aeruginosa T6SS, and had the best antiviral activity among all candidate molecules.

[0085] Example 5 Compound SRT1720 inhibits the in vitro growth of Pseudomonas aeruginosa and vgrG Evaluation of the impact of gene transcription This embodiment aims to evaluate the effect of compound SRT1720 at different concentrations on the growth of *Pseudomonas aeruginosa* by measuring bacterial growth curves and transcriptome sequencing, and to analyze its effect on... vgrG The influence of gene transcription level was investigated to confirm its non-bacterial properties as an antiviral drug.

[0086] 1. Experimental Methods (1) Effect of compound SRT1720 on the in vitro growth kinetics of Pseudomonas aeruginosa The tested strain was *Pseudomonas aeruginosa* PAO1 (PAO1 WT). 60 μL of glycerol-containing bacteria stored at -80℃ was inoculated at a 1:50 ratio into 3 mL of fresh LB medium containing tetracycline and cultured overnight at 37℃ with shaking at 220 rpm to fully activate the strain. The next day, the bacteria were transferred at a 1:50 ratio to 3 mL of fresh LB medium containing tetracycline and cultured at 37℃ with shaking at 220 rpm until OD (dimethylformaldehyde) was reached. 600 It is approximately 1.0.

[0087] The stock solutions of compound SRT1720 were prepared with DMSO at concentrations of 1 mM, 10 mM, and 50 mM. Before the experiment, the corresponding stock solutions were diluted with DMSO to prepare working solutions of 1 μM, 1 mM, and 5 mM. One volume of the corresponding working solution was added to 99 volumes of culture medium to achieve final SRT1720 concentrations of 10 nM, 10 μM, and 50 μM, respectively, with a final DMSO volume fraction of 1% in each treatment system. Specifically, the 1 μM working solution was obtained by diluting the 1 mM stock solution 1,000 times; the 1 mM working solution was obtained by diluting the 10 mM stock solution 10 times; and the 5 mM working solution was obtained by diluting the 50 mM stock solution 10 times. An equal volume of DMSO was added to the solvent control group.

[0088] Cultivate to OD 600The bacterial culture solution of approximately 1.0 was thoroughly shaken and mixed, then inoculated into 2 mL of LB liquid medium containing tetracycline at a ratio of 1:100. Corresponding volumes of compound SRT1720 were added according to the above concentrations, with a solvent control group containing 1% DMSO as the final concentration. The specific experimental groups are as follows: Solvent control group: PAO1 WT + DMSO (1% final DMSO concentration); Drug treatment group: PAO1 WT + SRT1720, with concentration gradients of 10 nM, 10 μM, and 50 μM.

[0089] The samples were placed in a growth curve analyzer and cultured at 37℃ and 220 rpm with shaking. The OD values ​​at each time point from 0 to 14 hours were monitored in real time. 600 Values ​​were calculated, growth curves were plotted, and the effect of compound SRT1720 on the growth kinetics of Pseudomonas aeruginosa PAO1 was analyzed.

[0090] (2) Compound SRT1720 on vgrG Effects of gene transcription levels Pseudomonas aeruginosa PAO1 was streaked onto LB agar plates and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing compound SRT1720 (10 μM) or DMSO, and incubated at 37°C with shaking at 220 rpm for 3 hours or 10 hours. After incubation, the bacterial cells were collected by centrifugation, washed with PBS, and the bacterial pellet was rapidly frozen and stored at -80°C for later use.

[0091] Total bacterial RNA was extracted using the TRIzol assay, and RNA purity was assessed using Nanodrop 2000. RNA integrity was assessed using agarose gel electrophoresis. rRNA was removed using a capture method to enrich mRNA. The enriched mRNA was fragmented to approximately 200 bp and reverse transcribed into first-strand cDNA. For second-strand synthesis, dUTP was used instead of dTTP. After adapter ligation, the second-strand cDNA was digested using UNG enzyme to generate a strand-specific library. After library quantification, paired-end 150 bp sequencing was performed on the DNBSEQ-T7 platform. Library construction and sequencing were performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0092] RNA-seq data included four conditions: SRT1720 treatment for 3 hours, SRT1720 treatment for 10 hours, DMSO control for 3 hours, and DMSO control for 10 hours, with five biological replicates for each condition. Raw sequencing reads were quality controlled using FastQC v0.12.1 and preprocessed using fastp v1.0.1 (including adapter removal, low-quality read filtering, and simple repetitive sequence filtering). Bowtie2 v2.5.4 was used to align the cleaned reads to the *Pseudomonas aeruginosa* PAO1 reference genome (NCBI accession: NC_002516.2), and SAMtools v1.6 was used to retain reads with an alignment quality ≥20 and correct pairing. FeatureCounts v2.1.1 was used for read counting, and DESeq2 v1.46 was used for differential expression analysis and TPM normalization. A corrected p-value <0.05 and |log2(fold change)|>1 were used as the screening threshold for differentially expressed genes. The visualization was done using an R script.

[0093] 2. Experimental Results See results Figure 11 Growth curve measurements showed that, during the 0–14 hour incubation period, the growth curves of the 10 nM, 10 μM, and 50 μM SRT1720 treatment groups largely overlapped with those of the DMSO solvent control group. Figure 11 (A). The time to enter the logarithmic growth phase, the growth rate during the logarithmic growth phase, and the bacterial density during the plateau phase were not significantly different between the treatment groups and the control group. The results indicate that compound SRT1720 had no significant inhibitory effect on the in vitro growth of *Pseudomonas aeruginosa* within the test concentration range of 10 nM to 50 μM. Transcriptome sequencing results showed that after treatment with compound SRT1720 (10 μM) for 3 and 10 hours, vgrG Gene expression levels were not significantly different from those in the DMSO control group (|log2FC|<1, p >0.05), indicating that compound SRT1720 does not affect transcription at the transcriptional level. vgrG Gene expression ( Figure 11 (B)

[0094] 3. Conclusion: Experimental data show that compound SRT1720, at doses higher than its effective antiviral concentration, does not inhibit the normal growth and reproduction of Pseudomonas aeruginosa in vitro, nor does it affect... vgrG Gene transcription level. Combined with the results of the foregoing examples, this suggests that compound SRT1720 exerts its effects by targeting the T6SS virulence system.

[0095] Example 6 Evaluation of the inhibitory activity of compound SRT1720 against the virulence of Salmonella and Yersinia T6SS Given that the bioinformatics analysis in Example 1 showed that the conserved region of the identified VgrG protein is widely present in a variety of Gram-negative pathogens, this example aims to further verify the broad-spectrum antiviral activity of compound SRT1720 against different species of pathogens (especially Salmonella and Yersinia) through interspecies growth competition experiments.

[0096] 1. Experimental Materials and Methods: (1) Interspecies competition between Salmonella SL1344 and Escherichia coli DH5α: The donor strain was wild-type Salmonella SL1344 (SL1344 WT), which was naturally resistant to streptomycin; the recipient strain was Escherichia coli DH5α, carrying the pBBR1-MCS-5 plasmid (gentamicin resistance). The donor strain was inoculated into LB liquid medium containing streptomycin (200 μg / mL) and the corresponding drug (10 μM compound SRT1720 or 1% DMSO control) and cultured at 37°C and 220 rpm for 10 hours with shaking; the recipient strain was inoculated into LB liquid medium containing gentamicin (20 μg / mL) and cultured at 37°C and 220 rpm for 10 hours with shaking.

[0097] After cultivation, the bacterial culture was washed once with LB medium to adjust the OD. 600 To 1.0. Mix 20 μL of the adjusted donor bacteria with 100 μL of the recipient bacteria (volume ratio 1:5), and add 1.2 μL of compound SRT1720 (final concentration 10 μM) or an equal volume of DMSO as a control. Take 100 μL of the mixed bacterial solution and perform serial dilutions, then spread it on LB agar plates containing the corresponding antibiotics (donor bacteria on plates containing streptomycin, recipient bacteria on plates containing gentamicin), and count the number of single colonies to determine the initial donor-recipient ratio.

[0098] 10 μL of the above mixed bacterial suspension was spotted onto a 0.22 μm nitrocellulose membrane. The membrane was then placed on a 3% LB agar plate containing 0.05% bile salts and incubated at 37°C for 20 hours. After incubation, the bacterial growth on the membrane was scraped off with a sterile pipette tip and resuspended in 1 mL of LB liquid. After serial dilution, the bacterial growth was spread onto LB plates containing the corresponding antibiotics. Once single colonies appeared, the colony count was recorded, and the competition index was calculated to evaluate the effect of compound SRT1720 on interspecific growth competition. The competition index was calculated using the following formula: Competition index = (CFU of donor bacteria after co-culture / CFU of recipient bacteria) / (CFU of initial donor bacteria / CFU of initial recipient bacteria).

[0099] The experiment was performed in six biological replicates. Data are expressed as mean ± SD. Unpaired t-tests were used to analyze the significance of differences between the DMSO group and the SRT1720 treatment group.

[0100] (2) Interspecies competition between Yersinia pseudotuberculosis YPIII and Escherichia coli DH5α: The donor bacterium was wild-type Yersinia pseudotuberculosis YPIII (YPIII WT) carrying the pKT100 plasmid (kanamycin resistance); the recipient bacterium was Escherichia coli DH5α carrying the pBBR1-MCS-5 plasmid (gentamicin resistance). The donor bacterium was inoculated into YLB liquid medium containing kanamycin (50 μg / mL) and the corresponding drug (10 μM compound SRT1720 or 1% DMSO control) and cultured at 30°C and 220 rpm for 8 hours with shaking; the recipient bacterium was inoculated into LB liquid medium containing gentamicin (20 μg / mL) and cultured at 37°C and 220 rpm for 8 hours with shaking.

[0101] After cultivation, the bacterial culture was washed once with LB medium to adjust the OD. 600 To 1.0. Mix 100 μL each of the adjusted donor and recipient bacteria (volume ratio 1:1), and add 2 μL of compound SRT1720 (final concentration 10 μM) or an equal volume of DMSO as a control. Take 100 μL of the mixed bacterial solution for serial dilution, spread it on LB agar plates containing the corresponding antibiotics, and count the number of single colonies to determine the initial ratio of donor to recipient.

[0102] 10 μL of the above mixed bacterial culture was spotted onto a 0.22 μm nitrocellulose membrane. The membrane was placed on an M9 agar plate containing 3% agar and incubated at 26°C for 48 hours. After incubation, the bacterial growth on the membrane was scraped off with a sterile pipette tip and resuspended in 1 mL LB liquid. After serial dilution, the bacterial growth was spread onto plates containing kanamycin (for selective counting of donor bacteria) and plates containing gentamicin (for selective counting of recipient bacteria). After single colonies grew, the colony count was counted, the competition index was calculated, and the effect of compound SRT1720 on interspecies competition was evaluated. The experiment was performed in six biological replicates. Data are expressed as mean ± SD. Unpaired t-tests were used to analyze the significance of differences between the DMSO group and the SRT1720 treatment group.

[0103] 3. Experimental Results See results Figure 12 Compound SRT1720 significantly inhibited the interspecies killing effect of Salmonella and Yersinia pseudotuberculosis on Escherichia coli. Figure 12 (A and B), confirming that its anti-T6SS virulence activity is applicable across strains.

[0104] 4. Conclusion This embodiment demonstrates that compound SRT1720 not only inhibits the virulence of Pseudomonas aeruginosa, but also effectively inhibits the T6SS function of Salmonella and Yersinia pseudotuberculosis. This indicates that compound SRT1720 has the potential to be developed into a broad-spectrum antibacterial drug for the prevention and treatment of infections caused by a variety of different pathogens.

[0105] Example 7 Lip-MS method for detecting compound SRT1720 and VgrG1 protein N 483 EIRMED 489 Regional integration This embodiment aims to detect the interaction between compound SRT1720 and VgrG1 protein N using ligand-binding mass spectrometry (Lip-MS) for protease protection. 483 EIRMED 489 The combination of regions (SEQ ID NO:2).

[0106] 1. Experimental Methods Purified VgrG1 WT Protein samples (including the SRT1720 treatment group and the DMSO control group) were diluted with ultrapure water to a final concentration of 5 μg / 20 μL. Trypsin was added for enzymatic digestion at a final concentration of 2% (w / w), and the digestion was carried out at 37℃ for 16 hours. After digestion, formic acid was added to a final concentration of 0.1% to precipitate undigested proteins. The mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant (digested peptides) was transferred to a dedicated mass spectrometry loading tube.

[0107] Peptides were pre-separated using a Thermo Fisher Scientific EASY-nLC 1200 nano-scale liquid chromatography system. The gradient program for mobile phase B (acetonitrile system) was set as follows: 0–3 min, B phase 3%→7%; 3–83 min, B phase 7%→35%; 85 min, B phase 90%; 95 min, B phase 90%. The chromatographically separated peptide fractions were then introduced online into an LTQ Orbitrap Velos Pro mass spectrometer via a nano-current electrospray ionization source. Data acquisition employed dynamic exclusion mode. Primary mass spectrometry was performed in the Orbitrap mass analyzer with scanning parameters set to: mass resolution 60,000, mass-to-charge ratio detection range 400–1200. The system automatically selected the top 10 most abundant precursor ions (charge state ≥ +2, isolation window 2 Da) according to preset conditions, and secondary mass spectrometry analysis was performed in the ion trap using collision-induced dissociation (CID, collision energy 35%). To reduce interference from repeated detections, the dynamic exclusion repeat duration was set to 30 s, and the exclusion duration was set to 15 s.

[0108] Raw mass spectrometry data (raw files) were analyzed using Mascot software for database matching, with the VgrG1 protein sequence as the reference database (FASTA format dataset obtained from UniProt). Peptides containing predicted sites were manually peaked. Non-standard quantitative analysis of peak areas was performed using MaxQuant software with the following parameters: enzyme digestion parameters were limited to trypsin-specific digestion (Trypsin / P), with a maximum allowed number of missed digestions of 2; primary mass spectrometry mass deviation range was ±20 ppm, and secondary fragment ion mass deviation range was ±0.8 Da; FDR was set to less than 1%. Missing values ​​in the non-standard quantitative peak areas were replaced with random numbers. The ion intensity peak diagrams of peptides were visualized using Thermo Xcalibur software to extract the mass-to-charge ratio of the corresponding ions.

[0109] 2. Experimental Results See results Figure 13 The figure shows the extracted ion chromatograms of six peptides: including the control peptide, the N-terminal flanking peptide, the three peptides spanning the motif, and the C-terminal flanking peptide. Lip-MS analysis results show that in VgrG1... WT In, containing N 483 EIRMED 489 The mass spectrometric signal of the motif and its adjacent peptides decreased by approximately 30% in the presence of compound SRT1720, indicating that SRT1720 exerts a protease protective effect on this region, with the binding site located at N. 483 EIRMED 489 area.

[0110] 3. Conclusion The above experimental results indicate that compound SRT1720 can bind to the VgrG1 protein N. 483 EIRMED 489 It binds to the region, producing a protease protective effect on that region.

[0111] Example 8 Compound SRT1720 affects the VgrG1 protein N of PAO1. 483 EIRMED 489 Evaluation of the binding of key amino acids in conserved regions This embodiment preliminarily determines N based on the aforementioned Lip-MS experiment. 483 EIRMED 489 Based on the regional importance, the influence of each amino acid residue in this motif on the binding of compound SRT1720 was further evaluated through in vitro competition experiments.

[0112] 1. Experimental Methods Using Pseudomonas aeruginosa PAO1 as the donor bacterium, a construct was built vgrG1 Gene deletion mutant strain Δ vgrG1 Based on this, they will be replenished with wild-type [products]. vgrG1 Genes and their single-point mutants (N483A, E484A, I485A, R486A, M487A, E488A, D489A) vgrG1 Gene replacement strains. To carry the wild-type gene. vgrG1 The gene-reintroduced strain served as a positive control, with Δ vgrG1 The deletion strains served as negative controls, and interspecific competition experiments were used to evaluate the drug response of each mutant complemented strain to compound SRT1720.

[0113] The interspecies competition experiment was conducted using the same method as in Example 2. The donor bacteria were the aforementioned complement strains, and the recipient bacteria was *Pseudomonas putida* KT2440 (carrying the pBBR1 MCS-5 plasmid, gentamicin resistant). The difference was that when inoculating the donor bacteria, the experimental group received a final concentration of 10 μM of compound SRT1720, while the control group received an equal volume of DMSO. When the donor and recipient bacteria were mixed (volume ratio 1:5), a final concentration of 10 μM of compound SRT1720 or an equal volume of DMSO was simultaneously added, and subsequent procedures were the same as in Example 2. The experiment was performed in six biological replicates. Data are expressed as mean ± SD, and two-way ANOVA and Šidák multiple comparison tests were used to analyze the significance of differences between groups.

[0114] 2. Experimental Results Interspecific competition experiments showed that, in the presence of compound SRT1720, complement plants carrying the wild-type vgrG1 gene (Δ) vgrG1 + vgrG1 WT It showed a drug response to compound SRT1720, with a significantly reduced competition index. (Regarding N...) 483 EIRMED 489 The detection results of single-point mutants within the motif are shown in [the table below]. Figure 14 The results showed that the competition index of the D489A mutant complementation strain did not change significantly in the presence of compound SRT1720, consistent with the expectation that D489 is a key binding residue. The E484A, I485A, and E488A mutant complementation strains also did not show obvious drug responses, while the N483A, R486A, and M487A mutant complementation strains retained their sensitivity to compound SRT1720, with a significantly reduced competition index, indicating that residues D489, E484, I485, and E488 are involved in the binding of compound SRT1720.

[0115] The above results indicate that N 483 EIRMED 489 Different amino acid residues in the motif contribute differently to the binding of compound SRT1720.

[0116] 3. Conclusion The above experimental results indicate that the VgrG1 protein N 483 EIRMED 489 A conserved motif (SEQ ID NO:2) is involved in the binding of compound SRT1720, with residues D489, E484, I485, and E488 making significant contributions to this binding, while residues N483, R486, and M487 have a smaller impact. These results further clarify the molecular basis of the interaction between compound SRT1720 and the VgrG1 protein, providing experimental evidence for understanding its targeting mechanism of T6SS.

[0117] Example 9 Evaluation of the inhibitory activity of compound SRT1720 on the virulence function of Salmonella T6SS in vivo This embodiment aims to evaluate the inhibitory activity and protective effect of compound SRT1720 on the virulence function of Salmonella T6SS in vivo using a Salmonella mouse infection model, and to analyze its effects on gut microbiota and T6SS virulence function through metagenomic sequencing.

[0118] 1. Experimental Materials and Methods (1) Establishment of animal infection model SPF-grade female C57BL / 6J mice (6-8 weeks old) were acclimatized for 7 days and then randomly divided into two groups: the Salmonella enteritidis strain SL1344 group (n=8) and the Salmonella enteritidis strain SL1344+SRT1720 group (n=8). All mice were pretreated with streptomycin (20 mg / mouse) by gavage. 24 hours later, each mouse was inoculated by gavage with 200 μL of streptomycin containing 5×10⁻⁶ mg / mouse. 5 CFU Salmonella strain SL1344 was administered as a PBS suspension. Following infection, the SL1344 group received sterile water daily by gavage, while the SL1344+ SRT1720 group received compound SRT1720 (100 mg / kg) daily by gavage until the experimental endpoint. The flowchart is shown below. Figure 15 A.

[0119] (2) Tissue bacterial load determination On day 5 post-infection (D5), all mice were euthanized, and liver, spleen, and ileum tissues were collected under aseptic conditions. After weighing, the tissues were homogenized, serially diluted, and spread onto LB agar plates containing streptomycin (200 μg / mL). The plates were incubated overnight at 37°C, and colony-forming units (CFU) were counted to calculate the bacterial load per gram of tissue.

[0120] (3) Histopathological analysis Mouse liver and ileum tissues were collected, fixed in 4% paraformaldehyde solution for 24 hours, embedded in paraffin, and prepared into paraffin sections for hematoxylin and eosin (H&E) staining. Histopathological scoring (0-4 points) was performed by three pathologists using a double-blind method based on the degree of tissue degeneration, necrosis, inflammatory cell infiltration, hemorrhage, erosion, and submucosal edema.

[0121] (4) Metagenomic sequencing and bioinformatics analysis On day 5 post-infection (D5), 0.2 g of small intestinal contents from mice were collected, and total DNA was extracted using the FastPure Stool DNAIsolation Kit. The DNA was fragmented to approximately 350 bp using Covaris M220, and paired-end libraries were constructed and sequenced on the DNBSEQ-T7 platform. Raw data underwent FastP quality control (removing reads <50 bp or with an average quality value <20), BWA alignment for host removal, MEGAHIT assembly (retaining contigs ≥300 bp), and Prodigal prediction of open reading frames (ORFs) ≥100 bp. CD-HIT was used to construct a non-redundant gene set with 90% sequence identity and 90% coverage, SOAPaligner was used for gene abundance assessment with 95% identity, and DIAMOND (e-value cutoff: 1e) was used. -5 Non-redundant genes were aligned to the NCBINR database for species classification and annotation.

[0122] (5) Identification and abundance analysis of T6SS-related proteins A custom reference database was constructed based on all experimentally validated T6SS proteins in the SecReT6 database. Predicted protein sequences were searched for in the custom database using DIAMOND, with high-confidence matches retained based on bit score ≥80, minimum query coverage ≥60%, and target coverage ≥60%. Identified T6SS proteins were further categorized into core structural components and effector proteins based on SecReT6 annotations. The abundance of T6SS-related proteins was quantified by aligning metagenomic reads to the identified protein sequences and normalized to per million transcripts (TPM). The taxonomic origin of T6SS-related proteins was inferred from the source genomes of the corresponding annotated genes, and contribution analysis was performed to explore bacterial taxa associated with T6SS composition.

[0123] 2. Experimental Results (1) Tissue bacterial load See results Figure 15On day 5 post-infection, compared with the SL1344 group, the Salmonella load in the liver (p=0.0037), spleen (p=0.0148), and small intestine (p=0.0070) of mice treated with compound SRT1720 (SL1344+ SRT1720) was significantly reduced, indicating that treatment with compound SRT1720 can effectively inhibit the colonization and proliferation of Salmonella in vivo.

[0124] (2) Histopathological analysis See results Figure 15 H&E staining results of liver and ileum tissue sections showed that the liver and ileum tissues of mice in the SL1344 group exhibited significant pathological damage, characterized by inflammatory cell infiltration, tissue necrosis, and mucosal injury; while the histopathological damage in the SL1344+SRT1720 group was significantly reduced. These results indicate that treatment with compound SRT1720 can effectively alleviate histopathological damage induced by Salmonella infection in mice.

[0125] (3) Analysis of gut microbiota diversity and species composition See results Figure 16 Groups A and B. PCoA analysis based on Bray-Curtis differences showed significant differences in microbial community structure between the two groups (PERMANOVA test). Further analysis revealed that Salmonella spp. (SRT1720 treatment group) were significantly different. Salmonella enterica The abundance of ) was significantly lower than that of the control group.

[0126] (3) Abundance analysis of T6SS-related genes See results Figure 16 The C. Violin plot showed that the total abundance of T6SS-related genes in the SRT1720-treated group was significantly lower than that in the control group, indicating that SRT1720 treatment can reduce the overall abundance of T6SS-related genes in the gut microbiota.

[0127] 3. Conclusion The above experimental results indicate that treatment with compound SRT1720 significantly reduced bacterial load in various tissues of mice infected with wild-type SL1344 strain, and alleviated pathological damage to the liver and intestines. Metagenomic sequencing analysis further showed that treatment with compound SRT1720 altered the intestinal flora structure of Salmonella-infected mice, significantly reduced the relative abundance of Salmonella, and downregulated the total abundance of T6SS-related genes, thus confirming at the molecular level the inhibitory effect of compound SRT1720 on the virulence function of T6SS in vivo.

[0128] Example 10 Evaluation of the inhibitory activity of SRT1720 derivatives against T6SS-mediated bacterial competition This embodiment evaluates the inhibitory activity of SRT1720 series derivatives against T6SS-mediated bacterial competition through interspecies competition experiments, in order to preliminarily clarify their structure-activity relationship.

[0129] 1. Experimental Methods To evaluate the structure-activity relationship of SRT1720-derived compounds, five representative analogs (a2–a6, structures shown in [link to sample]) were selected. Figure 17 The interspecies competition assay described in Example 2 was used to detect the inhibitory activity of each compound against T6SS-mediated bacterial competition. The donor strain was *Pseudomonas aeruginosa* PAO1 wild-type strain (carrying the PME6032 plasmid, tetracycline resistant), and the recipient strain was *Pseudomonas putida* KT2440 (carrying the pBBR1MCS-5 plasmid, gentamicin resistant). The donor and recipient strains were mixed at a volume ratio of 1:5. Each compound was added to the co-culture system at a final concentration of 10 μM, with an equal volume of DMSO as a solvent control. The competition index was calculated using the same method as in Example 2. The experiment was performed in six biological replicates. Data are expressed as mean ± SD. One-way ANOVA and Dunnett's multiple comparison test were used to analyze the significance of differences between the compound treatment groups and the control group.

[0130] 2. Experimental Results See results Figure 18 Interspecific competition experiments showed that, at a final concentration of 10 μM, compounds a3 and a4 both exhibited T6SS inhibitory activity comparable to that of the parent compound SRT1720, and their competition index was significantly lower than that of the DMSO control group. P <0.05). In contrast, the competition indices of the compound a2, a5, and a6 treatment groups were not significantly different from those of the DMSO control group, indicating that the above compounds lost their inhibitory activity against T6SS-mediated bacterial competition.

[0131] The above results indicate that the R2 site preferentially accommodates rigid fused bicyclic aromatic ring systems (such as quinoxaline in a1 and naphthalene ring in a4), and loses activity when replaced with larger or non-fused substituents (a2, a5); the R1 site can accommodate compact cyclic amines (such as 1-methylpyrrolidine in a3 and 3-hydroxypyrrolidine in a4), but excessively large steric hindrance groups (a6) lead to loss of activity.

[0132] 3. Conclusion The experimental results above indicate that the T6SS inhibitory activity of SRT1720 depends on the imidazo[2,1-b]thiazole core, the rigid fused bicyclic aromatic ring system at the R2 site, and the compact nitrogen-containing heterocycle at the R1 site. These structure-activity relationship results provide experimental basis for subsequent structural optimization of T6SS inhibitors targeting the conserved VgrG1 motif based on the SRT1720 backbone.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a compound or a stereoisomer of said compound or a pharmaceutically acceptable salt or solvate that targets a conserved region of the interface between VgrG protein trimer subunits in the preparation of a medicament for the prevention and / or treatment of diseases caused by Gram-negative pathogen infection, an inhibitor of the type VI secretion system of Gram-negative pathogens, or an anti-Gram-negative pathogen preparation; The compound is at least one of the following: SRT1720, HY-X5613 and SRT 2183; The structural formula of the SRT1720 is shown in Formula II: Formula II The structural formula of HY-X5613 is shown in Formula III: Formula III The structural formula of the SRT 2183 is shown in Formula IV: Formula IV; The Gram-negative pathogens mentioned are Gram-negative pathogens with a type VI secretion system; The amino acid sequence of the conserved region of the intersubunit interface of the VgrG protein trimer is shown as X1XXXXX2D; where X1 is N, S, G, R, H or Q, X2 is E or D, and X is any amino acid residue.

2. The application according to claim 1, characterized in that, The Gram-negative bacteria with a type VI secretion system include at least one of the following: Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Salmonella ( Salmonella Yersinia spp. Yersinia ), Vibrio cholerae ( Vibrio cholerae Acinetobacter baumannii ( Acinetobacter baumannii Burkholderia ( ) Burkholderia ) and Proteus spp. ( Proteus ).

3. The application according to claim 1, characterized in that, The anti-Gram-negative pathogen infection includes at least one of the following: weakening the infectivity or pathogenicity of Gram-negative pathogens, reducing the load of Gram-negative pathogens in organs, alleviating pathological damage caused by Gram-negative pathogens, regulating the balance of intestinal flora, and reducing the expression level of type VI secretion system-related proteins.

4. The application according to claim 1, characterized in that, The drug has at least one of the following characteristics: 1) The drug targets the conserved region of the intersubunit interface of the VgrG protein trimer; the amino acid sequence of the conserved region of the intersubunit interface of the VgrG protein trimer is shown as X1XXXXX2D; wherein X1 is N, S, G, R, H or Q, X2 is E or D, and X is any amino acid residue; 2) The drug is a broad-spectrum antitoxic drug; 3) The drug does not inhibit bacterial growth in vitro at effective therapeutic doses; 4) The drug does not cause bacteria to develop drug resistance.

5. The application of the conserved region of the VgrG protein trimer subunit interface in the development, screening, or evaluation of drugs against Gram-negative pathogen infections and those that do not induce drug resistance, or inhibitors of the type VI secretion system of Gram-negative pathogens, wherein the amino acid sequence of the conserved region of the VgrG protein trimer subunit interface is as shown in X1XXXXX2D, where X1 is N, S, G, R, H, or Q, X2 is E or D, and X is any amino acid residue; the Gram-negative pathogen is a Gram-negative pathogen with a type VI secretion system.

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