Application of Secoemestrin C in the Preparation of Anti-QPseudomonas aeruginosa Quorum Sensing Inhibitors

Abstract

This invention discloses the application of secoemestrin C in the preparation of quorum sensing inhibitors for *Pseudomonas aeruginosa*, belonging to the fields of pharmaceutical and natural product chemistry. This invention demonstrates that secoemestrin C can inhibit the activation of the LAS, RHL, and PQS quorum sensing pathways without significantly affecting the growth of *P. aeruginosa*, reducing the production of multiple virulence factors in carbapenem-resistant *P. aeruginosa*, and enhancing the in vitro efficacy of antibiotics such as imipenem and ceftazidime. Mechanistic studies show that secoemestrin C can interfere with the PqsE-RhlR quorum sensing regulatory axis and the interaction between the PqsE-RhlR complex and... rhl This compound binds to box DNA. It can be used to prepare Pseudomonas aeruginosa quorum sensing inhibitors, antiviral agents, adjuvant drugs against Pseudomonas aeruginosa infections, or antibiotic combination therapies.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical and natural product chemistry, and in particular to the application of secoemestrin C in the preparation of Pseudomonas aeruginosa quorum sensing inhibitors. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa Pseudomonas aeruginosa is an important opportunistic pathogen closely associated with various acute and chronic infections, particularly vulnerable to infecting immunocompromised individuals such as patients with pneumonia, cystic fibrosis, burns, cancer, and HIV / AIDS. As one of the most threatening ESKAPE pathogens, Pseudomonas aeruginosa infection can lead to high morbidity and mortality, posing a significant challenge to clinical anti-infective therapy. In recent years, resistance to carbapenems and other antibiotics in Pseudomonas aeruginosa has been steadily increasing, significantly increasing the difficulty of clinical treatment, prolonging treatment cycles, and raising healthcare costs. Traditional antibacterial drugs primarily work by inhibiting bacterial growth or killing bacteria, which can exert strong selective pressure on bacteria, thus promoting the emergence of drug-resistant strains. Therefore, developing novel anti-infective strategies with mechanisms different from traditional bactericidal or bacteriostatic methods is of great importance.

[0003] Antivirulence therapy is an intervention strategy that targets the pathogenic process of pathogenic bacteria rather than their basic growth process. This strategy weakens the pathogenicity of pathogens by inhibiting the production of virulence factors, biofilm formation, or regulation of bacterial community behavior, and is expected to reduce direct selective pressure on the basic bacterial growth process. *Pseudomonas aeruginosa* can produce a variety of virulence factors, including rhamnolipids, proteases, elastases, pyocyanin, fluorescein, and hemolysin, which play important roles in host tissue damage, immune escape, and infection progression. Furthermore, the biofilm formed by *P. aeruginosa* can significantly increase its tolerance to antimicrobial drugs and host immune clearance, which is one of the important reasons for persistent and difficult-to-cure infections.

[0004] Quorum sensing is a key cell communication mechanism in *Pseudomonas aeruginosa* that regulates the production of virulence factors, biofilm formation, and adaptive infection behaviors. *P. aeruginosa* can sense changes in bacterial population density through its quorum sensing system and coordinate the expression of various infection-related genes. The LAS, RHL, and PQS systems together constitute a complex quorum sensing regulatory network, participating in the regulation of various virulence factor synthesis, biofilm formation, and host adaptive responses. The PqsE-RhlR axis is an important regulatory module in the *P. aeruginosa* quorum sensing network, participating in the transcriptional regulation of various virulence-related genes. Therefore, screening and developing quorum sensing inhibitors that can intervene in PqsE-RhlR-related regulatory processes holds promise for providing new drug candidates and adjuvant therapy strategies for the treatment of *P. aeruginosa* infection, especially drug-resistant *P. aeruginosa* infection.

[0005] Natural products are an important source for innovative drug discovery. Fungal secondary metabolites, with their diverse structural types and bioactivities, are a vital resource for discovering active lead compounds. Secoemestrin C (SC) is a fungal-derived tetrathiodikepiperazine compound with a unique sulfur-containing dikepiperazine structural skeleton. Current technologies have not disclosed the applications of SC in inhibiting the quorum sensing system of *Pseudomonas aeruginosa*, reducing the production of virulence factors, intervening in the PqsE-RhlR quorum sensing regulatory axis, enhancing antibiotic efficacy, or as adjunctive therapy against *Pseudomonas aeruginosa* infections. Therefore, developing novel uses for SC in inhibiting *Pseudomonas aeruginosa* quorum sensing and in antiviral therapy is of great significance for the intervention of drug-resistant *Pseudomonas aeruginosa* infections. Summary of the Invention

[0006] The purpose of this invention is to provide the application of SC in the preparation of Pseudomonas aeruginosa quorum sensing inhibitors, so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of SC in the preparation of Pseudomonas aeruginosa quorum sensing inhibitors.

[0008] The second technical solution of this invention is the application of SC in the preparation of drugs against Pseudomonas aeruginosa infection.

[0009] The third technical solution of this invention is the application of SC in the preparation of drug sensitizers against Pseudomonas aeruginosa infection.

[0010] The fourth technical solution of the present invention is the application of SC in the preparation of an antiviral drug for Pseudomonas aeruginosa, wherein the antiviral drug is used to reduce the production of virulence factors in Pseudomonas aeruginosa; the virulence factors are selected from at least one of protease, elastase, hemolysin, and Pseudomonas aeruginosa fluorescein.

[0011] The fifth technical solution of the present invention is a pharmaceutical composition for treating Pseudomonas aeruginosa infection, comprising SC and antibiotics.

[0012] Based on the above technical solution, the present invention has the following technical effects: (1) The present invention found that SC has quorum sensing inhibitory activity against Pseudomonas aeruginosa and can inhibit the activation of the las, rhl and pqs quorum sensing pathways without significantly affecting the growth of Pseudomonas aeruginosa; (2) The present invention found that SC can reduce the production of a variety of virulence factors in carbapenem-resistant Pseudomonas aeruginosa, including protease, elastase, hemolysin and fluorescein, supporting its use as a candidate compound for antiviral activity. (3) The present invention found that SC can enhance the effect of imipenem and ceftazidime on carbapenem-resistant Pseudomonas aeruginosa, and exhibits synergistic effect in vitro at specific concentration combinations; (4) The present invention found that the combined use of SC and imipenem can reduce the bacterial load of wounds, alleviate the inflammatory response and promote wound repair in a mouse model of carbapenem-resistant Pseudomonas aeruginosa infection. (5) The mechanism study of the present invention shows that SC may exert quorum sensing inhibition and antiviral effects by intervening in the PqsE-RhlR quorum sensing regulatory axis, affecting the function of PqsE, PqsE-RhlR complex and PqsE-RhlR-DNA transcriptional regulatory complex, and reducing the binding ability between PqsE-RhlR complex and rhl box DNA. (6) This invention provides new natural product candidate molecules and experimental evidence for the antiviral-imipenem and / or ceftazidime combination therapy strategy for drug-resistant Pseudomonas aeruginosa infection, and has good application prospects. Attached Figure Description

[0013] Figure 1 The effects of SC on the quorum sensing reporter system and virulence factors of Pseudomonas aeruginosa; where A represents the effect of SC on the GFP-labeled quorum sensing reporter strain PAO1- lasB - gfp PAO1- rhlA - gfp and PAO1- pqsA - gfp The impact of the reported signal; B represents the effect of SC on the levels of protease, elastase, hemolysin, and fluorescein in clinically isolated carbapenem-resistant Pseudomonas aeruginosa (CRPA).

[0014] Figure 2 The study investigated the in vitro effects of SC combined with antibiotics and its influence on CRPA surface adhesion and biofilm-related structures. Specifically, A represents the dose-response relationship, heatmap matrix of combined effects, and synergistic effect score when SC is used in combination with imipenem; B represents the dose-response relationship, heatmap matrix of combined effects, and synergistic effect score when SC is used in combination with ceftazidime; and C represents the effect of SC combined with imipenem on CRPA surface adhesion and biofilm-related structures. The synergistic effect score was evaluated using Bliss, ZIP, and HSA models.

[0015] Figure 3The therapeutic effect of SC combined with imipenem on a mouse CRPA-infected wound model is shown in the following figures: A is a flowchart of the establishment and administration of the mouse skin infection model; B shows representative images and comparative analysis results of wound healing under different administration regimens; C shows H&E staining results of wound tissue inflammation under different administration regimens; D shows immunohistochemical analysis results of MPO, HIF-1α, CD86, and CD206 in wound tissue under different administration regimens; E shows wound tissue remodeling under different administration regimens; and F shows the expression levels of VEGF, CD31, and α-SMA in wound tissue under different administration regimens.

[0016] Figure 4 The results show the preliminary in vivo tolerability evaluation of SC; where A is the flowchart of the in vivo tolerability evaluation experiment; B is the weight change of mice in the control group and the SC-treated group; C is the major organ index of mice in the control group and the SC-treated group; and D is the representative H&E staining images of the major organs of mice in the control group and the SC-treated group.

[0017] Figure 5 The results of SC treatment are as follows: A represents the overall effect of SC on the protein expression profile of CRPA; B represents the functional enrichment analysis results of differentially expressed proteins; C represents the changes in transport-related processes and related protein expression affected by SC; and D represents the gene set enrichment analysis (GSEA) results of differentially expressed proteins.

[0018] Figure 6 The effects of SC on the expression of quorum sensing-related virulence proteins and related genes are shown in Figure 1. A represents the effect of SC on the expression level of PqsE-RhlR-regulated network-related virulence proteins; B represents the effect of SC on the transcription level of PqsE-RhlR-regulated network-related virulence genes; and C represents a schematic diagram of the PqsE-RhlR-DNA complex structure.

[0019] Figure 7 The results show the interaction and potential binding region analysis between SC and PqsE. A represents the molecular dynamics simulation grouping and the trajectory of SC relative to PqsE; B represents the effect of SC on the triaxial component of the PqsE gyroscope radius; C represents the effect of SC on the SC-PqsE binding energy, the interchain interaction energy of PqsE dimers, and the average interchain distance; D represents the binding ability analysis of SC and PqsE, and the effect of the active site inhibitor benzoic acid (BA) on their binding ability; E represents the effect of SC on the thermal stability of PqsE; F represents the key amino acids and their spatial positions in the interaction between SC and PqsE, and the thermal stability analysis of related site mutants; G is a schematic diagram of the mechanism by which SC acts on the potential binding region of PqsE.

[0020] Figure 8This diagram illustrates the effect of SC on the interaction and DNA binding ability of the PqsE-RhlR-DNA complex. A shows the molecular dynamics simulation groupings and the trajectory of SC relative to the PqsE-RhlR-DNA complex; B shows the RMSD changes of SC in the molecular dynamics simulation; C shows the effect of SC on the RMSF of each RhlR residue and the spatial location of the affected residues, with red indicating increased volatility after SC binding and dark blue indicating decreased volatility; D shows the effect of SC binding on the triaxial component of the gyrotropy radius of the PqsE-RhlR-DNA complex; and E shows the effect of SC on its interaction with PqsE-RhlR-DNA. The effects of SC on the binding energy of the PqsE-RhlR complex, the interaction energy between components within the complex, and the average distance; F shows the binding ability of SC to the PqsE-RhlR complex and the effect of endogenous ligand C4-HSL on their binding ability; G shows the thermal migration curves and thermal transition temperatures of the PqsE-RhlR complex; H shows the effect of SC on the thermal stability of the PqsE-RhlR complex; I shows the key amino acids and their spatial positions in the interaction between SC and the PqsE-RhlR complex, and the thermal stability analysis of related site mutants; J shows the effect of SC on the interaction between the PqsE-RhlR complex and... rhl The effect of box DNA interaction; K is a schematic diagram of the mechanism by which SC interferes with the function of the PqsE-RhlR-DNA complex. Detailed Implementation

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0022] This invention provides the application of SC in the preparation of Pseudomonas aeruginosa quorum sensing inhibitors.

[0023] In some specific implementations, the Pseudomonas aeruginosa quorum sensing inhibitor is used to inhibit at least one of the Pseudomonas aeruginosa las, rhl and pqs quorum sensing pathways; The Pseudomonas aeruginosa quorum sensing inhibitor inhibits the activation of the Pseudomonas aeruginosa quorum sensing pathway without significantly affecting the growth of Pseudomonas aeruginosa.

[0024] This invention also provides the application of SC in the preparation of drugs against Pseudomonas aeruginosa infection.

[0025] In some specific implementations, the drug also includes imipenem and / or ceftazidime.

[0026] This invention also provides the application of SC in the preparation of drug sensitizers against Pseudomonas aeruginosa infection.

[0027] In some specific implementations, the sensitizer is used to enhance the inhibitory effect of antibiotics against Pseudomonas aeruginosa.

[0028] In some specific implementations, the sensitizer is used to enhance the inhibitory effect of imipenem and / or ceftazidime on Pseudomonas aeruginosa.

[0029] This invention also provides the application of SC in the preparation of an antiviral drug for Pseudomonas aeruginosa, wherein the antiviral drug is used to reduce the production of virulence factors in Pseudomonas aeruginosa; wherein the virulence factors are selected from at least one of protease, elastase, hemolysin, and Pseudomonas aeruginosa fluorescein.

[0030] This invention also provides a pharmaceutical composition for treating Pseudomonas aeruginosa infection, comprising SC and an antibiotic.

[0031] In some specific implementations, the antibiotic is imipenem and / or ceftazidime.

[0032] In some specific embodiments, the pharmaceutical composition is used to reduce the bacterial load at the site of Pseudomonas aeruginosa infection, alleviate the inflammatory response at the site of infection, and / or promote the repair of infected wounds.

[0033] In some specific implementations, the *Pseudomonas aeruginosa* is a carbapenem-resistant *Pseudomonas aeruginosa*.

[0034] This invention also provides the application of SC in intervening in the function of the PqsE-RhlR quorum sensing regulatory axis in Pseudomonas aeruginosa, particularly in reducing the interaction between the PqsE-RhlR complex and... rhl Applications of box DNA binding ability.

[0035] This invention also provides the application of SC in the preparation of drugs or reagents that interfere with the function of the PqsE-RhlR quorum sensing regulatory axis of Pseudomonas aeruginosa.

[0036] In some specific implementations, the drug or reagent is used to interfere with the function of PqsE, the PqsE-RhlR complex, and / or the PqsE-RhlR-DNA transcriptional regulatory complex.

[0037] In some specific embodiments, the drug or reagent is used to reduce the interaction between the PqsE-RhlR complex and... rhl The binding affinity between box DNA molecules.

[0038] In some specific implementations, the drug or reagent is used to inhibit PqsE-RhlR-mediated quorum sensing transcriptional regulation.

[0039] The compound of this invention is SC, which belongs to the tetrathiodione piperazine class of fungal-derived compounds, and its structural formula is shown in formula (1): Equation (1).

[0040] The *Pseudomonas aeruginosa* strains include standard strains, quorum-sensing reporter strains, clinical isolates, and carbapenem-resistant *Pseudomonas aeruginosa*. The quorum-sensing inhibitory activity includes inhibition of one or more pathways in the LAS, RHL, and PQS quorum-sensing pathways. The antiviral effects include reducing the production of *P. aeruginosa* virulence factors, weakening surface adhesion or biofilm-related structure formation, and reducing the expression of quorum-sensing-related virulence genes or virulence proteins. The virulence factors include, but are not limited to, proteases, elastases, hemolysins, and pyocyanin.

[0041] The following examples illustrate the technical solution of this invention from the aspects of SC's in vitro quorum sensing inhibition and antiviral activity, in vitro combined antibiotic efficacy, in vivo wound treatment effect, preliminary in vivo tolerability evaluation, and the related mechanisms of the PqsE-RhlR quorum sensing regulatory axis. Related results show that SC can inhibit quorum sensing system activity without significantly affecting the basal growth of *Pseudomonas aeruginosa*, reduce various virulence-related phenotypes of carbapenem-resistant *Pseudomonas aeruginosa*, and enhance the in vitro effects of antibiotics such as imipenem and ceftazidime. In a mouse wound infection model, combined treatment with SC and imipenem can reduce the bacterial load of the wound, alleviate the inflammatory response, and promote wound repair. Mechanistic studies further support that SC may intervene in the PqsE-RhlR quorum sensing regulatory axis and the interaction between the PqsE-RhlR complex and... rhl The binding process of box DNA plays a supporting role in antiviral activity and anti-infection.

[0042] Example 1 Evaluation of SC's in vitro quorum sensing inhibition and antiviral activity This example illustrates the effects of the SC obtained in Example 1 on the quorum sensing system and virulence-related phenotypes of *Pseudomonas aeruginosa*. Experimental results show that, without significantly affecting the growth of *P. aeruginosa*, SC can inhibit the activity of the quorum sensing system and reduce the production of virulence-related factors such as protease, elastase, hemolysin, and fluorescein in carbapenem-resistant *P. aeruginosa*.

[0043] 1. The impact of SC on the quorum sensing reporter system of Pseudomonas aeruginosa Three Pseudomonas aeruginosa quorum sensing GFP reporter strains PAO1- were used. lasB - gfp PAO1- rhlA - gfpand PAO1- pqsA - gfp The reporter strains were used to evaluate the effects of SC on the LASIK, RHL, and PQS quorum sensing pathways. The reporter strains were inoculated into ABTGC liquid medium and treated with different concentrations (200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM) of SC (a DMSO control group was also included). After cultivation, the GFP fluorescence signal intensity and OD of each group were measured. 600 Value, and expressed as GFP fluorescence intensity via OD 600 The normalized results were used as an indicator of quorum sensing pathway activity. The final solvent concentration was kept consistent across all treatment groups.

[0044] The results showed that, compared with the solvent control group, PAO1- was significantly reduced after SC treatment. lasB - gfp PAO1- rhlA - gfp and PAO1- pqsA - gfp The normalized GFP fluorescence signal of the reporter strains was reduced in all cases; meanwhile, at the corresponding treatment concentrations, SC had no significant effect on the growth of the reporter strains. These results indicate that SC can inhibit the activation of the three quorum sensing pathways (las, rhl, and pqs) without significantly affecting the growth of *Pseudomonas aeruginosa*. Figure 1 (A)

[0045] 2. Effects of SC on virulence-related phenotypes of carbapenem-resistant Pseudomonas aeruginosa Further, carbapenem-resistant Pseudomonas aeruginosa isolated clinically was used. Pseudomonas aeruginosa The effect of carbapenem-resistant Pseudomonas aeruginosa (CRPA) on virulence-related phenotypes was evaluated (the carbapenem-resistant Pseudomonas aeruginosa used in this example is a publicly available strain, DOI: 10.1016 / j.ejmech.2023.115665). CRPA was inoculated into ABTGC medium and treated with SC at concentrations of 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM (a DMSO control group was also included). After culture, the levels of protease, elastase, hemolysin, and Pseudomonas aeruginosa luciferin were measured.

[0046] The results showed that, compared with the solvent control group, the production levels of protease, elastase, hemolysin, and Pseudomonas aeruginosa fluorescein in CRPA were reduced after SC treatment. Figure 1 (B)

[0047] The above results indicate that SC can inhibit the quorum sensing-related virulence phenotype of *Pseudomonas aeruginosa* and reduce the production of multiple pathogenic factors in CRPA. Therefore, SC can be used as a *P. aeruginosa* quorum sensing inhibitor or antiviral candidate compound for the preparation of adjuvant drugs, quorum sensing inhibitors, or antiviral drugs against *P. aeruginosa* infection.

[0048] Example 2 The in vitro effects of SC combined with antibiotics and its influence on CRPA surface adhesion and biofilm-related structures. This example illustrates the in vitro effects of SC in combination with imipenem or ceftazidime on CRPA, and further evaluates the effects of SC alone and in combination with imipenem on CRPA surface adhesion and biofilm-related structures. Experimental results show that SC can enhance the in vitro inhibitory effects of imipenem and ceftazidime on CRPA, and exhibits a synergistic effect at specific concentration combinations.

[0049] 1. In vitro effects of SC combined with imipenem To evaluate the potential effects of combined use of SC and imipenem, this example investigated the in vitro effects of SC and imipenem on CRPA. CRPA was exposed to different concentrations of imipenem, SC, and combinations thereof. Bacterial growth was assessed after culturing, and the dose-response relationship and IC50 were calculated for imipenem alone and in combination with SC. 50 change.

[0050] The processing for each group is as follows: Single-drug group: Imipenem: Treatments were administered at 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM, respectively; a DMSO control group (0 µM) was also included. SC: 100 µM, 50 µM, and 25 µM were used for treatment; there was also a DMSO control group (0 µM).

[0051] Combined use group: Imipenem (+25 µM SC): 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM were synergistically treated with 25 µM SC; a DMSO control group (0 µM) was also included. Imipenem (+50 µM SC): 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM were synergistically treated with 50 µM SC; a DMSO control group (0 µM) was also included. Imipenem (+100 µM SC): 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM were synergistically treated with 100 µM SC; a DMSO control group (0 µM) was also included.

[0052] The results showed that in the presence of SC, the concentration-response curve of imipenem against CRPA shifted to the left overall. For example, with 100 μM SC, the IC50 of imipenem at 24 h... 50 The concentration decreased from 12.97 ± 0.29 μM to 7.20 ± 0.31 μM, a decrease of approximately 44.5%, indicating that SC can enhance the in vitro inhibitory effect of imipenem on CRPA. Figure 2 (A). The above results suggest that there is a combined sensitizing effect between SC and imipenem.

[0053] To further evaluate the synergistic effect of SC and imipenem, this example uses the Bliss model, the zero interaction potency (ZIP) model, and the highest single agent (HSA) model to quantitatively analyze the dose-response data of the combined administration. The results show that after 24 hours of combined administration of 100 μM SC and 12.5 μM imipenem, the synergistic scores under the Bliss, ZIP, and HSA models were 24.55, 25.20, and 30.75, respectively, all higher than the synergistic judgment threshold of 10 (…). Figure 2 (A). The above results indicate that, under the experimental conditions described, SC and imipenem have a synergistic effect in vitro.

[0054] 2. In vitro effects of SC combined with ceftazidime Using a similar method as described above, the in vitro effects of SC combined with ceftazidime on CRPA were further evaluated. CRPA was exposed to different concentrations of ceftazidime, SC, and combinations thereof. After incubation, bacterial growth was assessed, and the dose-response relationship and IC50 values ​​of ceftazidime alone and in combination with SC were calculated. 50 change.

[0055] The processing for each group is as follows: Single-drug group: Ceftazidime: 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM were used for treatment; a DMSO control group (0 µM) was also included. SC: 100 µM, 50 µM, and 25 µM were used for treatment; there was also a DMSO control group (0 µM).

[0056] Combined use group: Ceftazidime (+25 µM SC): 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM were synergistically treated with 25 µM SC; a DMSO control group (0 µM) was also included. Ceftazidime (+50 µM SC): 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM were synergistically treated with 50 µM SC; a DMSO control group (0 µM) was also included. Ceftazidime (+100 µM SC): 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, and 3.125 µM were synergistically treated with 100 µM SC; there was also a DMSO control group (0 µM).

[0057] The results showed that ceftazidime's in vitro inhibitory effect on CRPA was enhanced in the presence of SC. For example, with 100 μM SC, the IC50 of ceftazidime at 24 h was... 50 The concentration decreased from 25.65 ± 1.35 μM to 12.65 ± 0.50 μM, a decrease of approximately 50.7%, suggesting that SC can enhance the in vitro inhibitory effect of ceftazidime on CRPA. Figure 2 (B)

[0058] The synergistic effect of SC and ceftazidime was further evaluated using Bliss, ZIP, and HSA models. The results showed that after 24 h of combined administration of 50 μMSC and 12.5 μM ceftazidime, the synergistic scores under the Bliss, ZIP, and HSA models were 40.36, 39.19, and 44.00, respectively, all higher than the synergistic threshold of 10 (…). Figure 2 (B). The above results indicate that, under the experimental conditions described, SC and ceftazidime have a synergistic effect in vitro.

[0059] 3. Effects of SC on CRPA surface adhesion and biofilm-related structures To investigate the effects of SC alone and in combination with imipenem on CRPA surface adhesion and biofilm-related structures, this example uses microscopic observation to evaluate changes in CRPA cell morphology, surface attachment status, and biofilm-related structures under different treatment conditions. Figure 2 (C)

[0060] The results showed that, compared with the control group, imipenem treatment altered the morphology of CRPA cells, with some bacteria changing from typical rod-shaped morphology to irregular morphology. SC treatment alone had little effect on bacterial cell morphology but reduced the number of surface-attached bacteria, suggesting that SC may interfere with the surface adhesion and biofilm-related structure formation process of CRPA. When SC was combined with imipenem treatment, the changes in CRPA cell morphology were more pronounced, the number of surface-attached bacteria was further reduced, and biofilm-related extracellular matrix-like structures were also reduced.

[0061] The results suggest that SC can weaken CRPA surface adhesion and biofilm-related structure formation, and can be used in combination with imipenem to further enhance the intervention effect on CRPA biofilm-related defense structures.

[0062] In summary, the results of this embodiment demonstrate that SC can enhance the in vitro inhibitory effect of imipenem and ceftazidime on CRPA, and exhibits synergistic effects with imipenem or ceftazidime at specific concentration combinations. Simultaneously, SC can reduce the number of bacteria attached to the CRPA surface and affect the formation of biofilm-related structures. Therefore, SC can be considered as a candidate compound for adjuvant antibiotic therapy in the preparation of adjuvant drugs or pharmaceutical compositions for use in combination with imipenem and / or ceftazidime against Pseudomonas aeruginosa infections.

[0063] Example 3 Evaluation of the in vivo therapeutic effect of SC combined with imipenem on infected wounds This embodiment uses a mouse model of carbapenem-resistant Pseudomonas aeruginosa infection to evaluate the adjuvant therapeutic effect and wound repair effect of the combined use of SC and imipenem on carbapenem-resistant Pseudomonas aeruginosa (CRPA) infection.

[0064] 1. Establishment of a mouse model of CRPA-infected wounds and drug administration regimen Male BALB / c mice aged 6–8 weeks were selected, and a full-thickness skin defect with a diameter of approximately 8 mm was constructed on their backs after anesthesia. Subsequently, CRPA bacterial suspension (50 μL, 2 × 10⁻⁶ mcg) was instilled into the wound. 8 (CFU / mL) was used to establish a CRPA-infected wound model. CRPA was detected by combining local redness, swelling, exudation, inflammatory response, and delayed healing with wound tissue homogenate culture, which served as the criteria for successfully establishing the CRPA-infected wound model.

[0065] After CRPA inoculation, mice were randomly divided into a control group, an imipenem group, and a combination therapy group, with five mice in each group. The control group received PBS carrier solution with the same solvent composition as the treatment group; the imipenem group received 12.5 μM imipenem; and the combination therapy group received a combination of 12.5 μM imipenem and 100 μM SC. Administration was via local application to the wound, 100 μL once daily for 14 consecutive days.

[0066] Wound photographs were taken on days 4, 7, 10, and 14 during the drug administration period to evaluate wound closure. On day 14, mice were treated and skin tissue was collected from the wounds for histopathological analysis, analysis of inflammation-related markers, and analysis of tissue repair-related indicators. Figure 3 (A)

[0067] 2. Effect of combined treatment with SC and imipenem on wound healing Wound healing results showed that, compared with the control group and the imipenem monotherapy group, the combined treatment group exhibited faster wound closure. Wound photographs showed a more significant reduction in wound area in the combined treatment group, suggesting that combined treatment with SC and imipenem can improve the repair process of CRPA-infected wounds. Figure 3 (B)

[0068] The above results indicate that, under the experimental conditions of this embodiment, the combined treatment of SC and imipenem can enhance the in vivo adjuvant therapeutic effect of imipenem on CRPA-infected wounds and promote wound closure.

[0069] 3. Effects of combined treatment with SC and imipenem on wound inflammation-related biomarkers. To further evaluate the effect of combined treatment with SC and imipenem on the inflammatory response of infected wounds, wound tissue collected on day 14 was subjected to hematoxylin and eosin staining (H&E staining) and the detection of inflammatory markers.

[0070] H&E staining results showed that the control group exhibited significant inflammatory cell infiltration, tissue edema, and inflammatory exudation in the wound tissue. While the imipenem-only treatment group showed some reduction in inflammation, a certain degree of inflammatory cell infiltration was still observed in the wound area. In contrast, the combined treatment group showed reduced inflammatory cell infiltration and better tissue structure recovery. Figure 3 (C). The above results suggest that combined treatment with SC and imipenem can reduce the local inflammatory response in CRPA-infected wounds.

[0071] Immunohistochemistry was further used to detect inflammation and macrophage phenotype-related markers in the wound tissue. Results showed that, compared with the control group and the imipenem monotherapy group, the expression levels of myeloperoxidase (MPO) and hypoxia-inducible factor-1α (HIF-1α) were decreased in the combination therapy group; simultaneously, the expression level of CD86, a marker associated with M1 macrophages, decreased, while the expression level of CD206, a marker associated with M2 macrophages, increased. Figure 3 (D).

[0072] The above results suggest that combined treatment with SC and imipenem can reduce neutrophil-related inflammatory response, decrease the expression of pro-inflammatory macrophage-related markers, and increase the expression of repair-related macrophage markers, indicating that the local inflammatory state of the wound is changing in a direction conducive to tissue repair.

[0073] 4. Effects of combined treatment with SC and imipenem on wound tissue remodeling and vascular-related parameters. To evaluate the effect of combined treatment with SC and imipenem on tissue remodeling of infected wounds, this embodiment further performed Masson trichrome staining and immunohistochemical detection of vascular-related markers on the wound tissue.

[0074] Masson trichrome staining results showed that, compared with the control group and the imipenem monotherapy group, the combination therapy group had increased tissue remodeling in the wound area, manifested as reduced wound gaps, increased skin tissue thickness, and increased collagen fiber deposition. Figure 3 (E). The above results indicate that combined treatment with SC and imipenem can promote collagen deposition and tissue remodeling in infected wounds.

[0075] Immunohistochemistry was further used to detect angiogenesis and vascular structure-related markers. Results showed that, compared with the control group and the imipenem monotherapy group, the expression levels of vascular endothelial growth factor (VEGF) and α-smooth muscle actin (α-SMA) were increased in the combination therapy group, while the expression level of platelet endothelial cell adhesion molecule-1 (PECAM-1) was relatively decreased. Figure 3 (Middle F). Among them, elevated VEGF expression indicates enhanced angiogenesis-related responses in the wound area, and elevated α-SMA expression indicates increased formation of vascular wall support structures; combined with changes in PECAM-1 expression, it suggests that the combined treatment group showed changes in wound angiogenesis and vascular wall support structure-related indicators that are conducive to tissue repair.

[0076] The above results indicate that combined treatment with SC and imipenem can promote collagen deposition, tissue remodeling, and improvement of vascular-related structures in CRPA-infected wounds, thereby facilitating wound repair.

[0077] In summary, the results of this embodiment indicate that in a mouse CRPA-infected wound model, combined treatment with SC and imipenem can accelerate wound closure, reduce local inflammatory response, regulate the expression of inflammation-related markers and tissue repair-related markers, promote collagen deposition and improve vascular-related structures, and promote the repair of infected wounds.

[0078] The above results indicate that SC can be used as a candidate compound for adjuvant therapy with imipenem, and can be used to prepare adjuvant drugs for anti-Pseudomonas aeruginosa infections, adjuvant drugs for wound infection treatment, or drug compositions for use in combination with imipenem, especially suitable for adjuvant treatment of wounds related to carbapenem-resistant Pseudomonas aeruginosa infections.

[0079] Example 4 Preliminary in vivo tolerability evaluation of SC To preliminarily evaluate the in vivo tolerability of SC under systemic administration conditions, this example uses 6–8 week old BALB / c mice for continuous gavage administration observation experiment.

[0080] In the experiment, mice were randomly divided into a control group and an SC treatment group. The SC treatment group was administered 10 mg / kg once daily by gavage for 7 consecutive days; the control group was given an equal volume of PBS carrier solution. During the administration period, the general condition of the mice was observed daily, including activity, food intake, fur condition, and mortality, and weight changes were recorded.

[0081] After drug administration, mice were processed, and major organs, including the heart, liver, spleen, lungs, and kidneys, were collected and weighed. The organ index was calculated as "organ weight = organ weight / body weight". Subsequently, each major organ was stained with H&E to observe morphological changes. Figure 4 (A)

[0082] Weight monitoring results showed that during continuous administration, the mice in the SC-treated group were in good overall condition, with no obvious abnormal activity, reduced food intake, abnormal hair condition, or death observed; compared with the control group, the weight change trend of the mice in the SC-treated group was not significantly abnormal. Figure 4 (B)

[0083] Organ weight analysis results showed that SC treatment did not significantly affect the relative weight of the major organs in mice, and no significant abnormalities were observed in the indices of the major organs compared with the control group. Figure 4 (C)

[0084] Further histopathological analysis showed that after 7 days of continuous administration of SC, the heart, liver, spleen, lungs, and kidneys of mice remained structurally intact, with no obvious tissue damage, inflammatory infiltration, necrosis, or other pathological abnormalities observed. Figure 4 (D).

[0085] In summary, the results of this embodiment indicate that under the observation condition of continuous gavage administration at a dose of 10 mg / kg for 7 days, SC did not cause significant acute toxicity, suggesting that it possesses preliminary in vivo tolerability required for further development. These results provide preliminary in vivo tolerability evidence for the further development of SC as a Pseudomonas aeruginosa quorum sensing inhibitor, an antiviral candidate compound, and an adjunct antibiotic candidate.

[0086] Example 5 Effects of SC on CRPA protein expression profile and quorum sensing-related toxicity expression This embodiment illustrates the changes in the CRPA protein expression profile after SC treatment and the effects of SC on the expression of quorum sensing-related virulence proteins and related genes. The results show that SC treatment alters the CRPA protein expression profile, including changes in the expression of various transmembrane transport, metabolic regulation, and cell membrane-related proteins. Simultaneously, SC reduces the expression levels of hcn and phz pathway-related virulence proteins and related genes. These results suggest that SC may affect the regulation of quorum sensing-related virulence in *Pseudomonas aeruginosa*, particularly possibly related to the PqsE-RhlR regulatory network, and provide a basis for further investigation into the mechanisms of PqsE, the PqsE-RhlR complex, and the PqsE-RhlR-DNA transcriptional regulatory complex.

[0087] 1. Effects of SC on CRPA protein expression profile Changes in protein expression in CRPA after SC treatment were analyzed using proteomics. CRPA cells were treated with either a solvent control or 100 µM SC for 24 h. Cell samples were collected under identical culture conditions, total protein was extracted, and detected using data-independent acquisition (DIA) proteomics. Culture conditions, treatment time, and final solvent concentration were kept consistent across groups. Results were expressed as |log2FC|≥0.263 after correction. P A value <0.05 was used as the screening criterion for differentially expressed proteins, and the differences in protein expression between the SC treatment group and the control group were analyzed.

[0088] The results showed that, under the established differential screening criteria, a total of 354 upregulated proteins and 559 downregulated proteins were identified in the SC treatment group. Figure 5 (A) indicates that SC treatment can cause extensive changes in the protein expression profile of CRPA.

[0089] Further gene ontology (GO) enrichment analysis was performed on the differentially expressed proteins. The results showed that the differentially expressed proteins were mainly enriched in functional categories such as transmembrane transport, oxidoreductase activity, and amino acid catabolism. Figure 5 (B) Among the transport-related functional items, SC treatment resulted in decreased expression of several proteins related to dipeptide transport, ATP-binding cassette transporter complex, type VI secretion system, and amino acid transport. Figure 5 (C). In addition, SC also affects the expression of various proteins located on the cell membrane that are involved in potassium ion transport and other transmembrane transport processes.

[0090] Further analysis of proteomics data was performed using Gene Set Enrichment Analysis (GSEA). The results showed that after SC treatment, the protein sets involved in amino acid transport and those located in the periplasmic space defined by the outer membrane were downregulated; the protein sets involved in lipopolysaccharide (LPS) biosynthesis also showed a downregulated trend. Figure 5 (D). The above results suggest that SC can affect the functional status of CRPA, including transmembrane transport, cell membrane-related processes, amino acid metabolism, and redox reactions, which is supported by the in vitro results showing that SC reduces virulence phenotype and affects surface adhesion and biomembrane-related structures.

[0091] The above results indicate that SC treatment can affect the expression of various transport, metabolic regulation, and cell membrane-related proteins in CRPA, providing proteomics clues to explain why SC reduces virulence phenotype, affects surface structure, and has an impact on the efficacy of combined antibiotic use.

[0092] 2. Effects of SC on the expression of quorum sensing-related virulence proteins and related genes In the proteomics results, further analysis was conducted on the expression changes of quorum sensing and virulence-related proteins. The results showed that SC treatment reduced the expression levels of several virulence proteins involved in the Pseudomonas aeruginosa quorum sensing network and PqsE-RhlR-related regulatory processes, including HcnB, HcnC, PhzB1, PhzB2, PhzD1, PhzF1, PhzG1, PhzH, PhzS, and PhzM. Figure 6 (A). The proteins mentioned above are mainly involved in the synthesis of hydrogen cyanide and phenazine virulence factors, both of which are important virulence-related factors in Pseudomonas aeruginosa infection and tissue damage.

[0093] To further validate the proteomics results, qRT-PCR was used for detection. hcn and phzTranscription levels of related genes. Results showed that after SC treatment, most... hcn and phz The transcriptional levels of related genes showed a downward trend or decreased, except for phzB1 and phzB2 In addition, the rest hcn and phz The transcription levels of related genes decreased. Figure 6 (B). The results generally support SC's view. hcn / phz The suppression trend of related virulence gene expression is corroborated by the results of reduced expression of related virulence proteins in proteomics.

[0094] The above results indicate that SC can reduce the expression of multiple quorum sensing-related virulence proteins in CRPA and affect... hcn and phz Transcriptional levels of related virulence genes. Since the hcn and phz pathways are closely related to the Pseudomonas aeruginosa quorum sensing regulatory network, especially to PqsE-RhlR-related transcriptional regulation, the above results suggest that SC may affect the PqsE-RhlR-related quorum sensing regulatory network. Figure 6 (C)

[0095] 3. Summary of Results in this Example and Basis for Subsequent Target Validation In summary, the results of this embodiment indicate that SC treatment can alter the protein expression profile of CRPA and reduce the expression of various quorum sensing-related virulence proteins and related genes, particularly showing a decrease in the expression levels of hcn and phz pathway-related proteins and genes. These results demonstrate that SC can not only reduce the production of Pseudomonas aeruginosa virulence factors at the phenotypic level, but also influence the quorum sensing-related virulence regulatory network at the proteomic and transcriptional levels.

[0096] Since the hcn and phz pathways are closely related to the quorum sensing system of *Pseudomonas aeruginosa* and are associated with PqsE-RhlR-mediated transcriptional regulation of virulence genes, the above results suggest that the quorum sensing inhibition and antiviral effects of SC may be related to PqsE-RhlR-related regulatory processes. Therefore, this embodiment provides proteomic and transcriptional evidence for SC as a candidate compound for *Pseudomonas aeruginosa* quorum sensing inhibitors and antiviral activity.

[0097] Based on the above proteomics and qRT-PCR validation results, this application further analyzes the interactions between SC and PqsE, the PqsE-RhlR complex, and the PqsE-RhlR-DNA transcriptional regulatory complex. Subsequent embodiments will further evaluate whether SC can intervene in the PqsE-RhlR-related quorum sensing regulatory process and analyze the possible molecular mechanisms by which it reduces quorum sensing-related toxicity expression through methods such as molecular docking, molecular dynamics simulations, surface plasmon resonance, thermal migration experiments, mutant validation, and DNA binding experiments.

[0098] Example 6 Analysis of the binding affinity and potential binding region between SC and PqsE To analyze the potential impact of SC on the quorum sensing regulatory axis of PqsE-RhlR, this embodiment employs methods such as molecular docking, molecular dynamics simulations, binding energy calculations, surface plasmon resonance, thermal migration experiments, and mutant verification to study the interaction between SC and PqsE and the potential binding region. The results indicate a detectable direct interaction between SC and PqsE. Computational simulations, thermal migration experiments, and mutant verification results further support the possibility that SC may act on the region near the PqsE dimer interface and influence the interaction state between PqsE dimer chains.

[0099] 1. Molecular docking and molecular dynamics simulations predict potential binding regions between SC and PqsE. First, molecular docking and molecular dynamics simulations were used to analyze the binding mode of SC and PqsE. The initial PqsE structure was obtained from the PqsE-RhlR-DNA complex structure 8DQ1 in the PDB database. Based on this structure, PqsE dimers were extracted, and molecular docking was used to predict the potential binding conformation between SC and PqsE. Molecular docking results suggested that SC may bind in the region near the PqsE dimer interface.

[0100] Molecular dynamics simulations were further employed to evaluate the stability of the SC-PqsE complex conformation and its impact on the PqsE dimer conformation. Molecular dynamics simulations were performed using CUDA-accelerated GROMACS 2022.2 software. The protein and ligand were parameterized using the Amber14sb_OL15_corrected-Na-cation-params force field and the GAFF force field, respectively. The ligand topology file was generated using Sobtop and Multiwfn. The initial conformation of the complex was derived from molecular docking results and placed within a cubic periodic boundary condition box filled with a TIP3P water model; subsequently, Na+ was added. + and Cl - The system charge was neutralized, and the ionic strength was adjusted to 0.15 M.

[0101] The system first undergoes energy minimization. The conjugate gradient method is used in the energy minimization phase, with a maximum step size of 5000 steps and a convergence threshold of 100 kJ·mol⁻¹. -1 ·nm -1 The time step was 0.02 nm. After energy minimization, the system underwent a 200 ps confined kinetic equilibrium with a time step of 2 fs, and positional constraints were imposed on the initial conformation. Subsequently, formal molecular dynamics simulations were performed, also with a time step of 2 fs, accumulating 200 ns production trajectories for subsequent analysis.

[0102] All simulations were conducted under periodic boundary conditions, employing the Verlet cutoff scheme. Long-range electrostatic interactions were handled using the particle mesh Ewald method, with cutoff radii for both electrostatic and van der Waals interactions set to 1.0 nm. DispCorr was used for long-range dispersion correction of energy and pressure. Temperature coupling was achieved using the V-rescale method in both the confined kinetic equilibrium stage and the formal molecular dynamics simulation stage, with a reference temperature of 298.15 K. Pressure control in the confined kinetic equilibrium stage employed Berendsen isotropic coupling, while the formal molecular dynamics simulation stage used Parrinello-Rahman isotropic coupling. The reference pressure was set to 1 bar, and the compressibility was 4.5 × 10⁻⁶. -5 bar -1 Both the restricted kinetic equilibrium stage and the formal molecular dynamics simulation stage impose constraints on hydrogen bonds.

[0103] Trajectory analysis was performed using GROMACS' built-in tools, and result processing and visualization were performed using Origin 2021, PyMOL 3.1, and VMD 1.9.3 software. System equilibrium was primarily determined based on the convergence of the root mean square deviation (RMSD) in the later stages of the simulation. Considering that different simulated systems do not reach equilibrium at entirely the same time, subsequent conformational analysis and interaction characteristic statistics were all based on the stable trajectory segments after each system reached equilibrium.

[0104] Molecular dynamics simulations showed that SCs did not significantly detach from the predicted binding region during the simulation, and their RMSD changes were relatively small, suggesting that the predicted binding conformation had a certain degree of stability during the simulation. Further analysis of the SC trajectory after protein structure alignment revealed that during the simulation, SCs were mainly distributed near the PqsE dimer interface. Figure 7 (A)

[0105] 2. Effects of SC on the conformation of PqsE dimer and interchain interactions Further analysis of the conformational changes in the PqsE dimer revealed that the kinetic behavior of the two chains of the PqsE dimer changed after SC binding, manifested as a decrease in localized oscillations in chain A, while a slight increase in oscillations in certain regions of chain B. Simultaneously, the gyration radius of the PqsE dimer increased in the Y-axis direction after SC treatment. Figure 7 (B) This suggests that SC binding may alter the relative conformation between PqsE dimer chains and cause a certain degree of spatial separation between the two chains.

[0106] The binding free energy between SC and PqsE dimers was further calculated using the gmx_MMPBSA 1.6.4 tool, and energy analysis was performed on the interchain interactions of PqsE dimers. The binding energy calculation and energy decomposition were completed based on the stable trajectory segments after each simulation system reached equilibrium, with a frame interval of 20 ps, ​​and other parameters kept at default settings.

[0107] The results showed that the binding free energy of the SC-PqsE complex was -29.63 ± 4.45 kcal / mol, suggesting that this binding conformation has a certain energy advantage in the simulated system. Further analysis of the interchain interactions of the PqsE dimer revealed that the interchain interaction energy of the PqsE dimer weakened after SC binding, with an energy change of 4.85 kcal / mol, accompanied by an increase in the average interchain distance. Figure 7 (C). The above calculation results suggest that SC may affect the interchain interactions and conformational stability of PqsE dimers by binding to the region near the PqsE dimer interface. It should be noted that the gmx_MMPBSA calculation results are mainly used to reflect the relative energy changes in the simulated system and cannot be equated solely with the experimental affinity strength.

[0108] 3. Analysis of SPR binding ability of SC and PqsE and BA competition experiment To further verify whether there is a direct interaction between SC and PqsE, surface plasmon resonance (SPR) technology was used for detection. The experiment was performed using a Biacore 1K surface plasmon resonance instrument. Recombinant PqsE protein was immobilized on the surface of an S-series CM5 chip via amino-coupling, with an immobilization volume of approximately 12,000 RU. The running buffer was PBS buffer, containing 137 mM sodium chloride, 2.7 mM potassium chloride, 2 mM potassium dihydrogen phosphate, and 8 mM disodium hydrogen phosphate, with a pH of 7.2–7.4, and containing 5% (v / v) DMSO.

[0109] Before formal testing, five start-up cycles were performed to stabilize the chip surface and baseline. SC was diluted at eight concentration gradients and injected using a multi-cycle kinetics mode at a flow rate of 30 μL / min, with binding and dissociation times both set to 90 s. The final DMSO concentration was kept consistent across all groups. A blank reference channel was included in the experiment, and blank subtraction and DMSO solvent correction were performed. Each concentration was repeated at least three times. The resulting sensor chromatograms were fitted using a 1:1 binding model, and the binding rate constant, dissociation rate constant, and equilibrium dissociation constant were calculated.

[0110] The results show that there is a detectable direct interaction between SC and PqsE, and the equilibrium dissociation constant (K0) is within the range of 1 / 2 Ω. D The concentration was approximately 190 μM, indicating a relatively weak but detectable binding affinity between SC and PqsE. Further addition of benzoic acid (BA), an inhibitor of the PqsE active site, did not significantly affect the interaction between SC and PqsE. Figure 7 The results (D) suggest that under the experimental conditions described, SC and BA did not exhibit a significant competitive binding relationship, and SC may not primarily occupy the active site of the PqsE enzyme acted upon by BA.

[0111] 4. The effect of SC on the thermal expansion behavior of PqsE The effect of SC on the thermal expansion behavior of PqsE was further evaluated using a thermal shift assay (TSA). Recombinant PqsE protein was diluted to 0.5 mg / mL with PBS buffer and incubated with serially diluted SC or an equal volume of DMSO at room temperature for 30 min; the final concentration of DMSO was kept consistent across groups. The reaction was performed in 384-well plates, with a total reaction volume of 10 μL per well, including 9 μL of protein-ligand incubation mixture and 1 μL of SYPRO Orange protein dye. After brief centrifugation, melting curves were acquired using a real-time quantitative PCR system with excitation at 520 nm and emission at 558 nm. The program was set to incubate at 10 °C for 2 min, followed by a temperature ramp-up from 10 °C to 99 °C at a rate of 0.05 °C / s. The melting temperature (T0) was recorded. m The fitting calculation was performed using Protein Thermal Shift 1.4 software.

[0112] Two thermal transition temperatures can be observed in the PqsE thermal migration curve, denoted as T. m1 and T m2 The results showed that the T of PqsE after SC treatment m1 and Tm2 All showed a decline ( Figure 7 The results (E) suggest that SC can alter the thermal stability parameters or thermal unfolding behavior of PqsE. This indicates that the interaction between SC and PqsE may affect the conformational stability, interfacial state, or thermal unfolding process of PqsE. Furthermore, the different modes of influence of SC and BA on the thermal stability of PqsE further support the possibility that SC may not primarily bind to the PqsE enzyme active site represented by BA.

[0113] 5. Validation of PqsE dimer interface-related mutants To verify the potential key binding regions predicted by molecular dynamics simulations, alanine scanning mutations were performed on amino acid residues near the PqsE dimer interface predicted to be involved in SC interaction, and thermostability analysis was used to evaluate the interaction between SC and the mutants. Based on the molecular docking and molecular dynamics simulation results, residues near the PqsE dimer interface were selected as mutation sites, including Leu202, Leu242, Arg246 of the B chain, Asp232 of the A chain, and Ser236 of the A chain.

[0114] Protein expression purification and basal thermal stability assays confirmed that the relevant mutants still maintained detectable thermal development curves. Under the same SC treatment conditions, compared with wild-type PqsE, the effect of SC on the thermal stability parameters of the relevant mutants was weakened ( Figure 7 The results suggest that one or more residues in chain B (Leu202, Leu242, Arg246, Asp232, and Ser236) may be involved in the interaction between SC and PqsE, or in maintaining the SC-PqsE binding conformation. This result further supports the involvement of residues near the PqsE dimer interface in SC interactions.

[0115] 6. Summary of Results of This Example In summary, the SPR results indicate a detectable direct interaction between SC and PqsE. Molecular docking, molecular dynamics simulations, gmx_MMPBSA energy calculations, TSA, and mutant validation results further support the possibility that SC may act on the region near the PqsE dimer interface. The interaction between SC and the region near the PqsE dimer interface may alter the relative conformation between PqsE dimer chains, affecting the stability of the PqsE dimer interface and weakening the interaction between the two PqsE chains.

[0116] Based on the above results, it is speculated that SC may affect the conformational state or interfacial stability of PqsE by acting on the region near the PqsE dimer interface, thereby further influencing the interaction between PqsE and RhlR and interfering with the PqsE-RhlR-related quorum sensing transcriptional regulation process. These results provide molecular-level mechanistic clues for SC's inhibition of quorum sensing-related virulence expression in *Pseudomonas aeruginosa*, and may provide partial mechanistic basis for its adjuvant antibiotic efficacy.

[0117] Example 7 Effects of SC on PqsE-RhlR complex interaction and DNA binding ability To further analyze the effect of SC on the quorum sensing regulatory axis of PqsE-RhlR, this embodiment employs molecular docking, molecular dynamics simulations, surface plasmon resonance, thermal migration experiments, alanine scanning mutagenesis, and DNA binding experiments to evaluate the interaction between SC and the PqsE-RhlR complex, and to analyze the effect of SC on the interaction between the PqsE-RhlR complex and... rhl The effect of SC on box DNA binding ability. Unless otherwise specified, the basic experimental conditions for molecular dynamics simulation, SPR detection, and TSA detection in this embodiment are the same as those described in Example 7; the difference is that the research object in this embodiment is the PqsE-RhlR complex or the PqsE-RhlR-DNA complex, and the effect of SC on the binding ability of the PqsE-RhlR complex to DNA is further detected. rhl The influence of box DNA binding ability.

[0118] 1. Molecular docking and molecular dynamics simulations predict the potential interaction region of SC in the PqsE-RhlR-DNA complex. First, molecular docking and molecular dynamics simulations were used to analyze the potential binding region of SC in the PqsE-RhlR-DNA complex. The initial structure of the PqsE-RhlR-DNA complex was obtained from the 8DQ1 complex structure in the PDB database. Molecular dynamics simulation parameters were performed according to the method described in Example 7. The formal molecular dynamics simulations yielded a cumulative production trajectory of 200 ns, which was used for subsequent conformational and interaction analyses.

[0119] Building upon the aforementioned potential interaction regions near the PqsE dimer interface, molecular docking and molecular dynamics simulations further suggest that SC may also bind to the linker region between the RhlR B chain ligand-binding domain (LBD) and the DNA-binding domain (DBD) in the PqsE-RhlR-DNA complex. Figure 8(A, B). This result suggests that SC may alter the overall conformation of the PqsE-RhlR-DNA complex and its DNA-binding related functions by affecting the interdomain linker region of RhlR.

[0120] Molecular dynamics simulations further revealed that, upon SC binding, the conformational dynamics of multiple components in the PqsE-RhlR-DNA complex changed. Figure 8 (C and D in the original text). Compared with the control system, SC treatment caused changes in the relative conformation between PqsE dimer chains, rearrangement of the spatial relationship between RhlR LBD and DBD, and alteration of the relative positioning of DNA in the complex. These changes suggest that SC may induce multi-level conformational rearrangement of the PqsE-RhlR-DNA complex, including changes in the stability of the PqsE dimer interface, alterations in communication between RhlR domains, and changes in the relative positioning of DNA.

[0121] Further analysis revealed that upon SC binding, the conformational stability of the ligand mBTL in the RhlR structure changed, exhibiting a weakening interaction between mBTL and the RhlR ligand-binding domain. This suggests that SC may affect the local conformational state of the RhlR LBD region. Simultaneously, simulation results showed that SC can alter the RhlR DBD region and its relative spatial relationship with DNA, suggesting that SC may affect the PqsE-RhlR complex's response to DNA. rhl The ability of box DNA to recognize and bind ( Figure 8 (E).

[0122] 2. SPR binding ability analysis and C4-HSL competition experiment of SC-PqsE-RhlR complex. To verify whether a direct interaction exists between the SC and the PqsE-RhlR complex, SPR technology was used for detection. The recombinant PqsE-RhlR complex was immobilized on the surface of the S-series CM5 chip via amino coupling, with an immobilization amount of approximately 12000 RU. The running buffer, injection method, flow rate, binding / dissociation time, DMSO correction, number of repetitions, and fitting model for SPR detection were performed according to the conditions described in Example 7.

[0123] The results showed a detectable but relatively weak direct interaction between SC and the PqsE-RhlR complex, with its K... D Approximately 292 μM ( Figure 8 (F). After the addition of the RhlR signaling molecule C4-HSL, the interaction between SC and the PqsE-RhlR complex did not change significantly, suggesting that under the experimental conditions, SC and C4-HSL did not exhibit a significant competitive binding relationship, and SC may not primarily occupy the C4-HSL binding site.

[0124] 3. Effect of SC on the thermal development behavior of the PqsE-RhlR composite The effect of SC on the thermal development behavior of the PqsE-RhlR complex was further evaluated using TSA. The recombinant PqsE-RhlR complex was diluted to 0.5 mg / mL with PBS buffer and incubated with serially diluted SC or an equal volume of DMSO at room temperature for 30 min.

[0125] The results showed that the thermal transition temperature of the PqsE-RhlR complex decreased after SC treatment. Figure 8 The presence of G and H in the data suggests that SC can alter the thermal stability parameters or thermal expansion behavior of the PqsE-RhlR complex. The addition of C4-HSL did not significantly change the thermal stability parameters of the PqsE-RhlR complex, further supporting the possibility that SC may act on the PqsE-RhlR complex through a non-C4-HSL competitive mechanism.

[0126] 4. Validation of mutants related to the RhlR LBD-DBD linker region To verify the potential binding regions predicted by molecular dynamics simulations, alanine-based mutations were performed on the amino acid residues predicted to be involved in SC interaction in the connection region between RhlR LBD and DBD. Thermal stability analysis was then used to evaluate the interaction between SC and the mutant complex. Based on the molecular docking and molecular dynamics simulation results, residues near the RhlR LBD-DBD connection region were selected as mutation sites, including Asn128, Met175, Pro178, Leu238, and Leu240.

[0127] Protein expression purification and basal thermal stability testing confirmed that the relevant mutants still maintained detectable thermal expansion curves. Figure 8 (I). Under the same SC treatment conditions, compared with the wild-type PqsE-RhlR complex, SC had a weaker effect on the thermal stability parameters of the relevant mutants, especially in terms of T. m0 The magnitude of the change decreased. This result suggests that one or more residues among Asn128, Met175, Pro178, Leu238, and Leu240 may be involved in the interaction between SC and the PqsE-RhlR complex, or in maintaining the SC-PqsE-RhlR binding conformation, thus supporting the involvement of the RhlR LBD and DBD linker region in the SC interaction.

[0128] 5. SC reaction to the PqsE-RhlR complex and rhl Effect of box DNA binding ability Further evaluation of the SC reaction between the PqsE-RhlR complex and SPR technology was conducted using SPR technology. rhlThe effect of box DNA binding ability. In the experiment, 5′-Biotin-tetraethylene glycol (5′-Biotin TEG) modified... rhl Box DNA was immobilized on the surface of the SA chip, and the PqsE-RhlR complex was detected. rhl The binding between box DNAs. rhl The box DNA is double-stranded DNA, with its positive and reverse strand sequences identical to the DNA sequence in the 8DQ1 complex structure in the PDB database. It was annealed to form a double-stranded structure before use. After fixation, the chip surface was equilibrated using running buffer. The PqsE-RhlR complex was injected in a concentration gradient to detect its interaction with the chip surface. rhl Interactions between box DNAs.

[0129] The results showed that the PqsE-RhlR complex could interact with rhl box DNA binding, its K D The value was approximately 3.39 μM. The addition of free, unmodified [molecules] to the solution... rhl Following the box DNA, this binding signal is competitively inhibited, indicating that the PqsE-RhlR complex binds to... rhl Interactions between box DNAs are DNA sequence binding specific.

[0130] After joining SC, rhl The reduced binding affinity between box DNA and the PqsE-RhlR complex is manifested as a decrease in SPR response signal and epigenetic K. D Gradually increase ( Figure 8 (J). Specifically, in the presence of 50 μM SC, the PqsE-RhlR complex with rhl Epigenetic K between box DNAs D Increased to approximately 197 μM; in the presence of 100 μM SC, apparent K D Further increased to approximately 13.5 mM; in the presence of 200 μM SC, the PqsE-RhlR complex with rhl No effective binding signal was detected between the box DNAs. These results indicate that SC can concentration-dependently weaken the binding between the PqsE-RhlR complex and... rhl The interaction between box DNAs provides important experimental evidence to support the intervention of SC in the PqsE-RhlR-DNA transcriptional regulatory complex. This experimental result is supported by the findings in molecular dynamics simulations that SC induces changes in the relative positioning of DNA and affects the RhlR-DNA interaction pattern.

[0131] 6. Summary of Results of This Example In summary, the results of this embodiment demonstrate that SC can undergo detectable interactions with the PqsE-RhlR complex; molecular docking, molecular dynamics simulations, TSA, and mutant validation results support the possibility that the potential region of action of SC may involve the linker region between the RhlR LBD and DBD; DNA binding experiments further indicate that SC can reduce the interaction between the PqsE-RhlR complex and the PqsE-RhlR complex. rhl The binding ability between box DNAs was significantly weakened or eliminated at higher concentrations. These results collectively support the view that SC can influence PqsE-RhlR-DNA transcriptional regulatory complexes by interfering with their DNA binding ability, thereby affecting PqsE-RhlR-mediated quorum sensing transcriptional regulation. Figure 8 (Middle K).

[0132] Therefore, SC can be used as a candidate compound to intervene in the function of the PqsE-RhlR-DNA transcriptional regulatory complex, and can be used to prepare Pseudomonas aeruginosa quorum sensing inhibitors, antiviral drugs, adjuvant drugs against Pseudomonas aeruginosa infection, or adjuvant drugs or drug compositions against Pseudomonas aeruginosa infection used in combination with imipenem and / or ceftazidime.

[0133] Example 8 Summary of the technical effects of this invention The results of the above examples demonstrate that SC possesses quorum sensing inhibitory activity and antiviral activity against *Pseudomonas aeruginosa*. SC can inhibit the activation of the *LAS*, *RHL*, and *PQS* quorum sensing pathways in *P. aeruginosa* at effective concentrations that do not significantly affect bacterial growth, and reduce the production of multiple virulence-related phenotypes in CRPA, including proteases, elastases, hemolysins, and fluorescein. These results support the use of SC as a *P. aeruginosa* quorum sensing inhibitor and antiviral candidate compound.

[0134] Further in vitro combination therapy experiments showed that SC enhanced the in vitro inhibitory effects of imipenem and ceftazidime against CRPA, achieving the synergistic criteria under the Bliss, ZIP, and HSA models at specific concentration combinations. Simultaneously, SC reduced the number of bacteria attached to the CRPA surface and affected biofilm-related structures; this effect was further enhanced when used in combination with imipenem. These results suggest that SC could be a candidate compound for adjuvant antibiotic therapy to improve the efficacy of imipenem and / or ceftazidime against drug-resistant Pseudomonas aeruginosa.

[0135] In vivo pharmacodynamic experiments further demonstrated that, in a mouse model of CRPA-infected wounds, the combined use of SC and imipenem accelerated wound closure, reduced inflammatory cell infiltration, modulated the expression of inflammation-related markers and tissue repair-related markers, and promoted collagen deposition and improved vascular-related structures. Preliminary in vivo tolerability evaluation results showed that, within the stated dosage and observation period, SC did not cause significant abnormal changes in mouse body weight, major organ indices, or major organ tissue morphology, suggesting that it did not exhibit significant acute toxicity under these dosage and period conditions and possesses the preliminary in vivo tolerability required for further development.

[0136] Regarding the mechanism of action, proteomics results showed that SC treatment altered the expression of various transmembrane transport, metabolic regulation, and quorum sensing-related virulence proteins in CRPA, with decreased expression of multiple hcn and phz pathway-related virulence proteins. Combined with molecular docking, molecular dynamics simulations, surface plasmon resonance, thermal migration experiments, and mutant validation results, it is supported that SC may intervene in the PqsE-RhlR quorum sensing regulatory axis by affecting processes related to PqsE, the PqsE-RhlR complex, and the PqsE-RhlR-DNA transcriptional regulatory complex. On one hand, SC may act on the region near the PqsE dimer interface, affecting the conformational state and interchain interactions of the PqsE dimer, and potentially influencing the functional state of the PqsE-RhlR complex. On the other hand, the potential action region of SC may involve the connection region between the RhlR ligand-binding domain and the DNA-binding domain in the PqsE-RhlR-DNA complex, thereby altering the complex's conformational dynamics and thermal unfolding behavior, and weakening the interaction between the PqsE-RhlR complex and the DNA-binding domain. rhl The binding ability between box DNAs. These mechanistic results support the view that SC exerts quorum sensing inhibition, antiviral activity, and adjunctive anti-infection effects by intervening in PqsE-RhlR-mediated quorum sensing transcriptional regulation.

[0137] In summary, SC can serve as a candidate compound for intervening in the PqsE-RhlR quorum sensing regulatory axis function of *Pseudomonas aeruginosa*, and can be used to prepare *P. aeruginosa* quorum sensing inhibitors, antiviral drugs, adjuvant drugs against *P. aeruginosa* infection, or pharmaceutical compositions used in combination with imipenem and / or ceftazidime. This invention provides new candidate compounds and experimental evidence for developing adjuvant therapy strategies combining antiviral agents—imipenem and / or ceftazidime—against drug-resistant *P. aeruginosa* infection, and has promising application prospects.

[0138] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of secoemestrin C in the preparation of Pseudomonas aeruginosa quorum sensing inhibitors.

2. Use according to claim 1, characterized in that, The Pseudomonas aeruginosa quorum sensing inhibitor is used to inhibit at least one of the Pseudomonas aeruginosa las, rhl and pqs quorum sensing pathways. The Pseudomonas aeruginosa quorum sensing inhibitor inhibits the activation of the Pseudomonas aeruginosa quorum sensing pathway without significantly affecting the growth of Pseudomonas aeruginosa.

3. Application of secoemestrin C in the preparation of drugs against Pseudomonas aeruginosa infection.

4. Use according to claim 3, characterized in that, The drug also includes imipenem and / or ceftazidime.

5. Application of secoemestrin C in the preparation of drug sensitizers against Pseudomonas aeruginosa infection.

6. The drug according to claim 5, characterized in that, The sensitizer is used to enhance the inhibitory effect of antibiotics on Pseudomonas aeruginosa.

7. The application of secoemestrin C in the preparation of antiviral drugs for Pseudomonas aeruginosa, characterized in that the antiviral drug is used to reduce the production of virulence factors in Pseudomonas aeruginosa; the virulence factors are selected from at least one of protease, elastase, hemolysin, and Pseudomonas aeruginosa fluorescein.

8. A pharmaceutical composition for treating Pseudomonas aeruginosa infection, characterized in that, This includes secoemestrin C and antibiotics.

9. The pharmaceutical composition according to claim 8, characterized in that, The antibiotics are imipenem and / or ceftazidime.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that, The pharmaceutical composition is used to reduce the bacterial load at the site of Pseudomonas aeruginosa infection, alleviate the inflammatory response at the site of infection, and / or promote the repair of infected wounds.

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