New use of a sparteine derivative for inhibiting salmonella

Abstract

The application discloses a new use of sparteine derivatives for inhibiting salmonella, relates to the field of pharmacy, and discloses a screening method of a drug for inhibiting salmonella: obtaining a three-dimensional structure of salmonella PhoP protein and a natural small molecule compound database through homology modeling; screening and evaluating the binding capacity of a target salmonella PhoP protein ZINC02139758; analyzing the target inhibition of PhoP protein activity and the bacteriostatic capacity; analyzing the antibacterial capacity of ZINC02139758 and colistin combined treatment on salmonella; and the application of ZINC02139758 in inhibiting salmonella. The application combines structure-based virtual screening and dynamics simulation, discovers a compound derived from a natural product derivative, has the advantages of potential low toxicity and high specificity, and provides a new idea and candidate substance for developing and preventing and controlling multiple drug-resistant enteritis salmonella and other related serotype infections.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more particularly to a novel use of a cytisine derivative for inhibiting Salmonella. Background Technology

[0002] Salmonella spp. is a major foodborne pathogen, causing hundreds of millions of infections and hundreds of thousands of deaths worldwide each year. Antibiotics are the primary treatment, but their widespread use, misuse, and environmental residues have led to increasingly serious drug resistance problems. Currently isolated Salmonella strains commonly exhibit multidrug resistance, even to polymyxin E, which is considered a "last line of defense," posing a serious threat to public health and necessitating the development of novel control strategies.

[0003] Salmonella antibiotic resistance is determined by resistance genes and their regulatory factors. The two-component system (TCS) plays a crucial role in this process. A typical TCS consists of a membrane-bound histidine kinase (HK) and a cytoplasmic response regulatory protein (RR). Under environmental stress, activated HK autophosphorylates and transmits signals to RR. The phosphorylated RR then binds to DNA to regulate gene expression in response to stress. Therefore, targeting and inhibiting the TCS signaling pathway is a potential approach to overcome antibiotic resistance.

[0004] Studies have shown that PhoP, a response regulatory protein in the PhoPQ two-component system, is a core factor regulating colistin resistance in Salmonella. Deletion of its encoding gene significantly reduces the minimum inhibitory concentration (MIC) of Salmonella against colistin by 16-fold, highlighting PhoP's potential as a key drug target. Screening for natural small-molecule inhibitors with synergistic antibacterial activity targeting PhoP holds significant promise for future applications.

[0005] Computer-aided drug design (CADD), especially structure-based virtual screening, has become a powerful tool for the efficient discovery of targeted antimicrobial compounds. High-throughput molecular docking can be used to predict the binding affinity of targets (such as PhoP) to a compound library, enabling rapid identification of lead molecules. Although the complete structure of Salmonella PhoQ has not yet been resolved, the crystal structure of its homologous protein PhoP has been reported in related bacterial species (such as Escherichia coli). PhoP exists as a dimer, and its N-terminal receptor domain contains a conserved α4-β4-α5 motif, forming a hydrophobic / charged residue interface that mediates dimerization and the van der Waals forces and salt bridge reactions required for signal transduction. The high conservation of this interface allows for reliable homology modeling of Salmonella PhoP using the E. coli PhoP structure as a template. Therefore, virtual screening targeting the α4-β4-α5 dimerization interface of PhoP holds promise for obtaining highly specific natural inhibitors.

[0006] Cytisine is a natural quinolone alkaloid isolated from various legumes. Cytisine and its derivatives possess a wide range of biological activities, including antitumor, antiviral, antiparasitic, hypoglycemic, and neuroprotective effects.

[0007] Those skilled in the art are dedicated to developing a computer-aided drug screening method for drugs that inhibit Salmonella, and to screening out a natural compound that inhibits Salmonella. Summary of the Invention

[0008] The technical problem to be solved by this invention is how to develop a computer-aided drug screening method for drugs that inhibit Salmonella, and to screen out a natural compound that inhibits Salmonella.

[0009] To achieve the above objectives, the present invention provides a method for screening drugs that inhibit Salmonella, comprising the following steps: Step 1: Obtain the three-dimensional structure of Salmonella PhoP protein through homology modeling; Step 2: Construct a database of natural small molecule compounds; Step 3: Using the three-dimensional structure of Salmonella PhoP protein obtained in Step 1 as the receptor target protein, and the database of natural small molecule compounds obtained in Step 2 as the ligand library, batch molecular docking was performed using virtual screening software. Based on the binding energy threshold, the first natural small molecule compound was obtained after the first screening. The first natural small molecule compound was subjected to pharmacokinetic (Absorption, Distribution, Metabolism, Excretion and Toxicity, ADMET) property description, and screening directions were determined. A second screening was conducted, and the second natural small molecule compound was obtained according to the set screening conditions. The screening directions were: solubility, blood-brain barrier permeability, hepatotoxicity, cytochrome P450 2D6 enzyme inhibition ability, human intestinal absorption, and hemoglobin binding ability. The binding stability of PhoP protein and the second natural small molecule compound was evaluated using molecular dynamics simulation software. After the third screening, the third natural compound was obtained. The targeting binding mode of the third natural compound to PhoP protein was evaluated. After the fourth screening, the cytisine derivative ZINC02139758 was obtained. Step 4: Analyze the targeted inhibition of PhoP protein activity and antibacterial ability of the cytisine derivative ZINC02139758 obtained in Step 3.

[0010] Furthermore, step 1 also includes: using the crystal structure of the Klebsiella pneumoniae PmrA protein-DNA binding complex as a template, the homologous construction of the PhoP active receptor structure was completed using Modeller, while the PhoP inhibitory receptor structure was constructed using AlphaFold 2.

[0011] Furthermore, step 2 also includes: obtaining information on natural small molecule compounds from the ZINC Natural Products, TCMSP, and AnalytiConDiscovery Natural Derivatives databases, performing structural standardization on the compounds, including conformational isomers and stereoisomers, and assigning them a unified number; and calculating a series of descriptive information on the properties and structure of the compounds, including molecular weight, lipid-water partition coefficient, and polar surface area.

[0012] Furthermore, the binding energy threshold in step 3 is -8.0 kcal / mol. From energy scoring to specific recognition, -8.0 kcal / mol is a recognized "empirical threshold" for molecular docking screening. This threshold not only indicates excellent spatial geometric complementarity between the ligand and the acceptor, but also usually means that at least two strong hydrogen bonds or key π-π stacking interactions have formed within the complex.

[0013] Furthermore, the second screening condition in step 3 is Ammens teratogenicity, based on the Lipinski five rules, namely: molecular weight not exceeding 500 Da; oil / water partition coefficient (logP) of the candidate compound less than 5; hydrogen bond acceptors of the candidate compound not exceeding 5; number of hydrogen bond donors not exceeding 5; total number of hydrogen bond donors and acceptors of the candidate compound not exceeding 10; and number of rotatable bonds of the candidate compound not exceeding 10.

[0014] Furthermore, step 4 also includes: using micro-thermophoresis to determine the binding affinity of the cytisine derivative ZINC02139758 to the PhoP protein; and using RT-qPCR to analyze the targeted inhibition of PhoP activity by the cytisine derivative ZINC02139758.

[0015] Furthermore, step 4 of the analysis of antibacterial ability also includes: determination of Salmonella survival rate; analysis of the antibacterial effect of cytisine derivative ZINC02139758 on Salmonella; and analysis of the antibacterial ability of cytisine derivative ZINC02139758 combined with colistin on Salmonella.

[0016] This invention also provides the application of the cytisine derivative ZINC02139758 in inhibiting Salmonella.

[0017] Furthermore, it also includes the combined inhibition of Salmonella by the cytisine derivative ZINC02139758 and colistin.

[0018] Furthermore, Salmonella includes Salmonella enteritidis, Salmonella mensterae, Salmonella Kentuckyis, Salmonella typhimurium, and Salmonella Thompson.

[0019] In a preferred embodiment 1 of the present invention, the three-dimensional structure of Salmonella PhoP protein and the process of constructing a database of natural small molecule compounds are described in detail. In another preferred embodiment 2 of the present invention, the screening and binding ability evaluation process of targeting Salmonella PhoP protein ZINC02139758 is described in detail. In another preferred embodiment 3 of the present invention, the analysis process of ZINC02139758 targeting and inhibiting PhoP protein activity and antibacterial ability is described in detail. In another preferred embodiment 4 of the present invention, the process of analyzing the antibacterial ability of ZINC02139758 combined with colistin against Salmonella is described in detail.

[0020] The beneficial technical effects of this invention are as follows: This invention features precise and crucial target selection (PhoP), and the lead compound (ZINC02139758) combines the potential of natural low toxicity with the advantages of "drug repurposing." Its drug development strategy (CADD) is highly efficient and low-risk, and its core performance indicator (significantly reducing colistin MIC) directly addresses clinical pain points and has been experimentally validated. The production pathway is clear, the raw materials and processes are feasible, and the application scenarios (human / veterinary treatment, source control) are broad. The invention has a high level of technological maturity (target validation, lead compound discovery, and in vitro mechanism studies have been completed), a clear industrialization path, relatively controllable risks, and extremely high transformation potential and market prospects. It is the first to explicitly propose and validate that the response regulatory protein PhoP (especially its conserved α4-β4-α5 structural region) in the Salmonella PhoPQ two-component system is a key drug target for screening inhibitors to overcome the drug resistance problem of Salmonella (especially colistin-resistant strains). The method combines structure-based virtual screening with kinetic simulation. The discovered compound ZINC02139758, derived from a natural product, possesses the potential advantages of low toxicity and high specificity, providing a novel approach and candidate substance for developing methods to control multidrug-resistant (especially colistin-resistant) Salmonella enteritidis and other related serotype infections. Specifically: 1. Target innovation and precision: This study innovatively targets the PhoP protein (especially its conserved dimer interface α4-β4-α5), a core regulator of Salmonella resistance. This target plays a decisive role in colistin resistance (gene deletion reduces MIC by 16-fold), and intervention on this target can directly address the root cause of resistance.

[0021] Advantages: Compared to traditional broad-spectrum antibacterial agents, targeted PhoP has high specificity, reduces the impact on the host or symbiotic flora, reduces the risk of drug resistance development, and is in line with the trend of precision medicine.

[0022] Lead compounds have significant advantages: Naturally derived with high safety potential: ZINC02139758 is a derivative of cytisine (a known medicinal natural product). Compared with newly synthesized compounds, its core structure is known, and its potential toxic side effects are lower, which may give it an advantage in subsequent toxicological studies and registration applications.

[0023] This compound is not a completely new molecular entity. Its basic physicochemical properties and some metabolic data (based on the parent nucleus) may already be referenced, which can significantly shorten the preclinical research cycle and reduce the risk and cost of research and development failure.

[0024] Experiments have shown that it not only directly inhibits the PhoP target, but also disrupts the bacterial cell wall / membrane structure and redox homeostasis, with synergistic effects through multiple pathways, making it less likely to induce single-target drug resistance.

[0025] Advanced CADD technology application: Computer-aided drug design (CADD) based on homology modeling, high-throughput virtual screening, and molecular dynamics simulation has successfully discovered lead compounds. This method significantly shortens the traditional "trial and error" screening cycle (from several years to several months), reduces screening costs (saving significant costs on compound procurement and phenotyping), and improves R&D efficiency and success rate.

[0026] Mature in vitro validation system: The in vitro experimental system for MIC determination, target effect validation (such as molecular interaction, reporter gene, etc.), cell structure / function detection, etc. is complete, cost-controllable, and the results are reliable, providing solid data support for subsequent development.

[0027] 2. Core antibacterial properties: Significantly reduces colistin MIC values: In multiple Salmonella serotypes, ZINC02139758 significantly reduces the minimum inhibitory concentration (MIC) of colistin. The specific reduction is a key quantitative indicator for measuring its sensitizing effect; this invention reduces it by 4-16 times. Effectively reverses or overcomes Salmonella's resistance to colistin, the "last line of defense" antibiotic, enabling ineffective / inefficient antibiotics to regain their effectiveness.

[0028] 3. Compound sources and basic synthesis: ZINC02139758 is a known derivative with a well-defined chemical structure. The synthetic route can be found by referring to established methods for cytisine and its analogues.

[0029] The source of raw material (cytisine) is relatively stable: Cytisine can be extracted from legumes or obtained through chemical synthesis, so the supply of raw materials is guaranteed.

[0030] Process development risks are controllable: The synthesis and purification processes can draw on mature experience, have high feasibility for scale-up production, and have controllable costs.

[0031] Diverse and high-demand application scenarios: Human pharmaceuticals: Develop colistin sensitizers (combination preparations) for injection or oral administration to treat serious infections (such as sepsis) caused by multidrug-resistant (MDR) Salmonella. There is a clear and urgent market demand.

[0032] In the veterinary field: Developed as veterinary therapeutic drugs or feed additives for the prevention and treatment of salmonellosis in livestock and poultry, reducing the spread of drug-resistant bacteria at the source and ensuring food safety. The approval process for veterinary drugs is generally shorter than that for human drugs.

[0033] Disinfection / preservative products: Develop disinfectant / preservative ingredients for use in food processing environments or medical devices to control drug-resistant Salmonella contamination in the environment.

[0034] Platform technology scalability: The "PhoP-targeted virtual screening and validation" platform established in this solution can be rapidly applied to the development of inhibitors targeting PhopP homologous targets of other pathogens (such as Escherichia coli and Klebsiella pneumoniae), expanding the scope of application and enhancing the technological value.

[0035] The CADD process can be replicated for screening inhibitors against other key bacterial resistance targets (such as other two-component systems, efflux pumps, etc.).

[0036] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0037] Figure 1 This is a preferred embodiment 1 of the present invention, showing the three-dimensional structure of Salmonella Phop protein and the construction diagram of a natural small molecule compound database; Figure 2 This is a schematic diagram of the natural compound targeting and binding to the PhoP protein, which is a preferred embodiment 2 of the present invention. Figure 3 This is the result of the ZINC02139758 targeted inhibition of PhoP protein activity assay in a preferred embodiment 3 of the present invention; Figure 4 The results of the analysis of the antibacterial ability of ZINC02139758 and colistin in combination treatment against Salmonella are from a preferred embodiment 4 of the present invention. Detailed Implementation

[0038] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0039] In this invention, the three-dimensional structure modeling software for Salmonella Phop protein was Modeller and AlphaFold 2, the virtual screening software was AutoDock Vina, and the molecular dynamics simulation software was Gromacs 2022.3. Furthermore, natural small molecule compounds such as ZINC02139758 were purchased from VitasM, France.

[0040] Example 1: Three-dimensional structure of Salmonella Phop protein and construction of a database of natural small molecule compounds

[0041] The three-dimensional structure of the Salmonella PhoP protein (receptor) was obtained through homology modeling. Using the crystal structure of the Klebsiella pneumoniae PmrA protein-DNA binding complex (PDB ID: 4S04) as a template, the homology construction of the PhoP activated dimer was completed using Modeller, while the PhoP inactivated dimer was constructed using AlphaFold 2. Information on natural small molecule compounds from natural product databases such as ZINC Natural Products, TCMSP, and AnalytiCon Discovery Natural Derivatives was obtained. The compounds underwent structural standardization, including conformational and stereoisomerization, and were uniformly numbered. A series of descriptive information on compound properties and structures, such as molecular weight, lipid-water partition coefficient (LogP), and polar surface area, were calculated. The structures and related information of 262,229 processed natural small molecule compounds were stored in the NSMDB database. The molecular formulas and structural formulas of the compounds in NSMDB were visualized using the Zplus visualization tool.

[0042] The process of constructing a database of three-dimensional structures of Salmonella PhoP proteins and natural small molecule compounds is as follows: Figure 1 As shown, part A is the three-dimensional structure of the activated Salmonella PhoP protein, and part B is the three-dimensional structure of the inhibited Salmonella PhoP protein.

[0043] Example 2: Screening and Binding Ability Evaluation of Salmonella PhoP Protein ZINC02139758

[0044] 1. Virtual Filtering

[0045] The experiment used the three-dimensional structures of the activated and inhibited states of Salmonella PhoP protein obtained through homology modeling as receptor target proteins. The proteins were pretreated using AutoDock Vina, including protein hydrogenation, water molecule removal, and the addition of charge and force fields. The NSMDB database of natural small molecule compounds was used as a ligand library. Using the N-terminus of the PhoP protein as the receptor domain, batch molecular docking was performed using AutoDock Vina. Based on the three-dimensional structures of the activated and inhibited states of the PhoP protein, the binding energy threshold between the receptor and ligand was set to -8.0 kcal / mol, resulting in the screening of 12,380 and 3,828 natural small molecule compounds, respectively.

[0046] 2. ADMET property description, prediction, and screening

[0047] (1) Sort the candidate natural small molecule compounds according to docking binding energy and integrate them into a library, then import them into Discovery Studio 2019 (DS). (2) In DS, launch the Small Molecules toolbar and call the Prepare ligands function under the Prepare or Filter Ligands module to perform molecular optimization preprocessing; (3) Call the ADMET Descriptors function under the Calculate Molecular Properties module to perform ADMET property descriptions on natural compounds.

[0048] ADME screening was conducted using six criteria: solubility level (SL), blood-brain barrier permeability (BBB), hepatotoxicity (HP), cytochrome P450 2D6 enzyme inhibition (CYP2D6), human intestinal absorption (HIA), and plasma protein binding (PPB). The Toxicity Prediction module was used, and the screening criteria were set to Ames Mutagenicity (AM). Based on the Lipinski five principles (molecular weight not exceeding 500 Da; oil / water partition coefficient (logP) less than 5; no more than 5 hydrogen bond acceptors; no more than 5 hydrogen bond donors; a total of no more than 10 hydrogen bond donors and acceptors; and no more than 10 rotatable bonds), candidate compounds were filtered and screened. Three and seven natural small molecule compounds, respectively, bound to the inhibited and activated states of PhoP protein, were selected as the subjects for further research.

[0049] 3. Molecular dynamics simulation

[0050] Molecular dynamics simulations were performed using Gromacs 2022.3 software to evaluate the binding stability of PhoP protein to natural compounds.

[0051] (1) Natural compound pretreatment: AmberTools22 was used to add GAFF force fields to the natural compounds. At the same time, Gaussian 16W was used to perform hydrogenation and calculate RESP potentials on the natural compounds. The potential data will be added to the molecular dynamics system topology file. (2) The simulation was conducted at a static temperature of 300 K and a normal pressure (1 Bar). The force field was Amber99sb-ildn, the solvent was water molecules (Tip3p water model), and the total charge of the simulation system was neutralized by adding an appropriate amount of Na+ ions. (3) The molecular dynamics simulation system first uses the steepest descent method to minimize energy, followed by 100,000 steps of isothermal and isochoric ensemble equilibrium and isothermal and isobaric ensemble equilibrium, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, free molecular dynamics simulation is run, which takes 10,000,000 steps with a step size of 2 fs and a total duration of 200 ns. (4) After completing the 200 ns molecular dynamics simulation, analyze the simulated trajectory of the protein forming a complex with the natural small molecule compound, and calculate the root mean square deviation (RMSD), hydrogen bond number (H-Bond, HB), root mean square fluctuation (RMSF), and the radius of gyration (Rg) of the protein for each amino acid. Use the molecular mechanics Poisson-Boltzmann surface area (MM / PBSA) method to obtain the binding sites of important amino acid residues and draw the surface free energy map.

[0052] The results showed that five natural compounds, including ZINC02139758, could stably bind to the activated state of PhoP protein to form a complex structure.

[0053] 4. Evaluation of the pattern of natural compounds targeting and binding to PhoP protein

[0054] The Receptor-Ligand Interactions module in Discovery Studio 2019 software and Pymol software were used to analyze the binding characteristics of candidate natural compounds to protein PhoP, and two-dimensional (2D) and three-dimensional (3D) plots were performed.

[0055] Natural compounds target and bind to PhoP proteins in a manner such as Figure 2 As shown in the figure, part A is the docking and binding mode of ZINC02139758 with the activated PhoP protein; part B is the RMSD diagram of the molecular dynamics simulation of the ZINC02139758-PhoP activated protein binding complex; part C is the RMSF diagram of the molecular dynamics simulation of the ZINC02139758-PhoP activated protein binding complex; part D is the SASA diagram of the molecular dynamics simulation of the ZINC02139758-PhoP activated protein binding complex; and part E is the hydrogen bond count diagram of the molecular dynamics simulation of the ZINC02139758-PhoP activated protein binding complex. The results show that the cytisine derivative ZINC02139758 can bind to the PhoP protein via hydrogen bonds, with ARG163 and GLN177 being important amino acid residues.

[0056] In molecular docking virtual screening, the cytisine derivative ZINC02139758 exhibited significant binding potential (-8.3 kcal / mol) in the initial screening. Its selection logic was subsequently validated in multiple dimensions through molecular dynamics simulations: Regarding stability, 200 ns molecular dynamics simulations showed that the ligand maintained an extremely low RMSD level in binding to the Phop protein, and maintained at least three stable hydrogen bonds throughout the process, demonstrating its excellent conformational stability and solvent shielding effect. In terms of mechanism of action, ZINC02139758 precisely occupies key residues such as ARG163 and GLN177, physically blocking the DNA-binding domain or dimerization interface of the Phop protein, thereby regulating its downstream signaling pathways. Regarding skeletal advantages, leveraging the high bioactivity and structural plasticity of the natural alkaloid cytisine skeletal framework, this compound shows great potential for development into a novel Phop inhibitor.

[0057] Example 3: Analysis of the targeted inhibition of PhoP protein activity and antibacterial ability of ZINC02139758

[0058] 1. Microthermophoresis was used to determine the binding affinity of ZINC02139758 to PhoP protein.

[0059] Phopeptide-tagged PhoP proteins were fluorescently labeled using the Monolith His-Tag Labeling Kit RED-tris-NTA 2nd Generation. The binding affinity between Phop proteins and five screened natural compounds, as well as the positive control azomycin, was determined using a NanoTemper instrument (Monolith NT.115). The results showed that only ZINC02139758 and azomycin formed stable complexes with Phop proteins, with dissociation constants Kd of 168.33 ± 14.98 μM and 117.00 ± 14.12 μM, respectively. To further confirm the binding efficiency, azomycin, a PhoPQ system inhibitor, was introduced as a positive control, moving from known benchmarks to original innovation. In the micro-thermophoresis (MST) experimental system, the introduction of azomycin has dual significance: firstly, by determining its binding affinity constant with PhoP protein, false negative interference caused by protein inactivation is effectively eliminated; secondly, it sets a stringent affinity "pass threshold" for newly discovered natural compounds. Experimental results show that the dissociation constant of ZINC02139758 (Kd = 168.33 μm) is on the same order of magnitude as the positive control (Kd = 117.00 μm). For the initial screening of unoptimized natural products, this micromolar-level specific binding demonstrates the effectiveness of this compound.

[0060] 2. RT-qPCR analysis of the targeted inhibition of Phop activity by ZINC02139758

[0061] (1) RNA sample extraction and reverse transcription

[0062] Salmonella bacteria were cultured in LB broth to the logarithmic growth phase, and then treated with different concentrations of the natural compound ZINC02139758 for 6 h. Immediately afterwards, total RNA was extracted from the strain using an RNA extraction kit (Novizan, R403) and stored at -80°C. RNA concentration and integrity were determined using Nanodrop 2000c and agarose gel electrophoresis. The RNA extracted in this invention exhibited intact structure, with clear and bright 23S, 16S, and 5S bands, suitable for subsequent experiments. The reverse transcription kit HiScript® Ⅲ RT SuperMix for Qpcr (+ gDNA wiper) was used to remove gDNA from the RNA sample and reverse transcribe it into cDNA. The reaction product was stored at -20°C and used as soon as possible, avoiding repeated freeze-thaw cycles.

[0063] (2) Primer design and specificity verification

[0064] Genes ompF, pmrD, slyB, pagC, vanX, rstA, ssrB, and acnA, which are reportedly directly regulated by Phop-mediated gene expression, were selected as targets. Using the genome sequence of *Salmonella enteritidis* SJUTF 12367 as a template, RT-qPCR primers were designed using Primer Premier 5.0 software to detect the relative expression levels of these genes under ZINC02139758 treatment conditions. The following principles were followed in primer design: the length of the amplified product fragment was controlled between 100-250 bp; the primer length was controlled between 18-22 bp; the primer Tm value was between 55-60℃; and the primer GC content was maintained at 40-60%. Specific primer sequences are shown in Table 1. The primers designed in this invention can specifically amplify the target genes, indicating that these primers can be used for subsequent experiments. The primer sequences ompF-F, ompF-R, pmrD-F, pmrD-R, slyB-F, slyB-R, pagC-F, pagC-R, vanX-F, vanX-R, rstA-F, rstA-R, ssrB-F, ssrB-R, acnA-F, acnA-R, 16S rRNA-F, and 16S rRNA-R are shown in SEQ ID NO. 1-SEQ ID NO. 18, respectively.

[0065] Table 1 Primer sequences and corresponding gene function classifications used in RT-qPCR

[0066] (3) Determination of relative gene expression levels

[0067] The RT-qPCR reaction system is shown in Table 2, where 16S rDNA was used as the internal reference gene. Reaction conditions: 95℃ for 30 s; 95℃ for 5 s, 58℃ for 30 s, 68℃ for 20 s, 40 cycles; each group was set up in triplicate, and the experiment was repeated 3 times. Salmonella untreated with the natural compound ZINC02139758 was used as a control, and the relative expression level of genes directly regulated by PhoP was calculated using the formula (2-ΔΔCt).

[0068] Table 2 RT-qPCR reaction system

[0069] The results of the ZINC02139758 targeted inhibition of Phop protein activity assay are as follows: Figure 3 As shown, Part A is a schematic diagram of the dissociation constant of PhoP protein and the positive reference azotomycin; Part B is a schematic diagram of the dissociation constant of PhoP protein and the positive reference ZINC02139758; Part C is the determination of the relative expression level of PhoP target genes; Part D is the determination of Salmonella cell membrane permeability under ZINC02139758 pressure; and Part E is the determination of Salmonella intracellular redox homeostasis related indicators under ZINC02139758 pressure. The results showed that under ZINC02139758 treatment at molar concentrations of 1×Kd and 2×Kd, the expression level of the outer membrane pore protein gene ompF in Salmonella increased, while the relative expression levels of virulence genes ssrB, cis-aconitase gene acnA, and peptidoglycan and lipopolysaccharide synthesis-related genes vanX, pmrD, and bssR decreased. This is consistent with the previously reported negative regulation of the ompF gene by Phop and positive regulation of genes such as ssrB, acnA, vanX, pmrD, and bssR. In other words, ZINC02139758 can bind well to Phop protein and weaken the regulatory function of Phop protein on downstream genes.

[0070] like Figure 3 The results showed that the natural compound ZINC02139758 can target and bind to the PhoP protein to form a stable complex, and further inhibit the activity of the PhoP protein, thereby weakening its regulation of gene expression.

[0071] 3. Analysis of the antibacterial effect of ZINC02139758 on Salmonella

[0072] (1) Salmonella survival rate determination

[0073] A stock solution of 10 mg / mL ZINC02139758 and azotomycin was prepared. The prepared Salmonella suspension (approximately 10⁶ CFU / mL) was added to 96-well plates. Different concentrations of ZINC02139758 were added as treatment groups. A solution containing 0.1% DMSO without ZINC02139758 served as a negative control, and wells containing azotomycin served as positive controls. The 96-well plates were incubated at 25°C for 8 h. The survival rate of Salmonella treated with the cytisine derivative ZINC02139758 was determined using a tenfold serial dilution and plate count method.

[0074] The results showed that, with increasing concentration, both ZINC02139758 and azotomycin significantly inhibited Salmonella survival, with their half-maximal effective concentrations (EC50) values ​​of 111.90±0.56 µM and 71.22±0.64 µM, respectively.

[0075] (2) Analysis of the antibacterial effect of ZINC02139758 on Salmonella

[0076] Salmonella was treated with ZINC02139758 at concentrations of 1 / 2×EC50, 1×EC50, and 2×EC50. Cell membrane permeability of the treated strains was measured using N-phenyl-1-naphthylamine (NPN) fluorescent reagent. Intracellular reactive oxygen species (ROS) accumulation was measured using a reactive oxygen species detection kit (Beyotime, S0033S). Intracellular antioxidant enzyme activities were measured using a catalase detection kit (Beyotime, S0033S), a superoxide dismutase detection kit (Beyotime, S0033S), and an NAD+ / NADH detection kit to assess intracellular redox homeostasis imbalance.

[0077] like Figure 3As shown in Part B, after treatment with ZINC02139758 at concentrations of 1 / 2×EC50 and 1×EC50, a large amount of fluorescent probes infiltrated into the cells, indicating increased cell membrane permeability, leading to extensive antibiotic infiltration and cell death. Simultaneously, the accumulation of ROS in Salmonella cells increased after ZINC02139758 treatment, and the ROS accumulation under the 1×EC50 ZINC02139758 treatment was significantly higher than that under the 1 / 2×EC50 treatment group. Under the 1 / 2×EC50 ZINC02139758 treatment, the activities of catalase and superoxide dismutase in Salmonella cells significantly increased, but under the 1×EC50 treatment, the activities of these two enzymes decreased. The reason for the initial increase followed by a decrease in the above physiological and biochemical indicators may be due to the massive cell death of Salmonella under excessive ZINC02139758 treatment. This phenomenon can be explained by the NAD+ / NADH ratio in Salmonella cells. The results of this invention show that as the concentration of ZINC02139758 treatment increases, the NAD+ / NADH ratio gradually decreases, indicating that ZINC02139758 treatment causes an irreversible imbalance in the redox homeostasis of Salmonella cells, ultimately leading to bacterial death.

[0078] Example 4: Analysis of the antibacterial activity of ZINC02139758 combined with colistin against Salmonella.

[0079] Eight Salmonella strains resistant to colistin were selected as experimental strains: Salmonella enteritidis (SJTUF12367, SJTUF13076 and SJTUF16008), Salmonella mensterae (SJTUF16003), Salmonella Kentuckyis (SJTUF16006 and SJTUF16009), Salmonella typhimurium (SJTUF16002) and Salmonella Thompson (SJTUF16001). Their minimum inhibitory concentration (MIC) values ​​against colistin were all greater than or equal to 8 μg / mL. Using a checkerboard dilution method, colistin was added horizontally to 96-well plates at final concentration gradients (8, 4, 2, 1, 0.5, 0.25, 0 μg / mL), while ZINC02139758 was added vertically to 96-well plates at final concentration gradients (100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0 μg / mL). 198 μL of MH medium and 1 μL of Salmonella suspension (final concentration approximately 10⁶ CFU / mL) were added to each well. The plates were then incubated at 37°C, and growth was observed. After overnight incubation, OD600 was measured, and the effects of combined treatment with ZINC02139758 and colistin on Salmonella cells were observed using scanning electron microscopy.

[0080] The results of the analysis of the antibacterial activity of ZINC02139758 combined with colistin against Salmonella are as follows: Figure 4 As shown, exogenous addition of ZINC02139758 can reduce the minimum inhibitory concentration (MIC) of colistin in Salmonella enteritidis SJTUF12367, SJTUF13076, and SJTUF16008 by 4-fold, in Salmonella mentelist SJTUF16003, Salmonella Kentuckyis SJTUF16006, and SJTUF16009 by 4-fold, and in Salmonella typhimurium SJTUF16002 and Salmonella Thompson SJTUF16001 by 2-fold. Furthermore, under combined treatment with low concentrations of ZINC02139758 and colistin (1 / 4×EC50 ZINC02139758 treatment concentration and 1 / 4×MIC colistin treatment concentration), scanning electron microscopy revealed cell membrane damage and lysis in multiple Salmonella cells. This indicates that the natural compound ZINC02139758 can enhance the bactericidal efficacy of colistin and possesses synergistic antibacterial capabilities.

[0081] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for screening drugs that inhibit Salmonella, characterized in that, The method includes the following steps: Step 1: Obtain the three-dimensional structure of Salmonella PhoP protein through homology modeling; Step 2: Construct a database of natural small molecule compounds; Step 3: Using the three-dimensional structure of Salmonella PhoP protein obtained in Step 1 as the receptor target protein, and the natural small molecule compound database obtained in Step 2 as the ligand library, batch molecular docking is performed using virtual screening software. Based on the binding energy threshold, the first natural small molecule compound is obtained after the first screening. The pharmacokinetic properties of the first natural small molecule compound are described, and a screening direction is determined. A second screening is performed, and a second natural small molecule compound is obtained according to the set screening conditions. The screening direction is: selecting solubility, blood-brain barrier permeability, hepatotoxicity, cytochrome P450 2D6 enzyme inhibition ability, human intestinal absorption, and hemoglobin binding ability. The binding stability of the PhoP protein and the second natural small molecule compound is evaluated using molecular dynamics simulation software. After a third screening, a third natural compound is obtained. The targeting binding mode of the third natural compound to the PhoP protein is evaluated. After a fourth screening, the cytisine derivative ZINC02139758 is obtained. Step 4: Analyze the targeted inhibition of PhoP protein activity and antibacterial ability of the cytisine derivative ZINC02139758 obtained in Step 3.

2. The method as described in claim 1, characterized in that, Step 1 further includes: using the crystal structure of the Klebsiella pneumoniae PmrA protein-DNA binding complex as a template, the homologous construction of the PhoP active receptor structure was completed using Modeller, while the PhoP inhibitory receptor structure was constructed using AlphaFold 2.

3. The method as described in claim 1, characterized in that, Step 2 further includes: obtaining information on natural small molecule compounds from the ZINC Natural Products, TCMSP, and AnalytiCon Discovery Natural Derivatives databases, performing structural standardization on the compounds, including conformational isomers and stereoisomers, assigning them a unified number, and calculating a series of descriptive information on the properties and structure of the compounds, including molecular weight, lipid-water partition coefficient, and polar surface area.

4. The method as described in claim 1, characterized in that, The binding energy threshold in step 3 is -8.

0. kcal / mol.

5. The method as described in claim 1, characterized in that, The second screening condition in step 3 is Ammens teratogenicity, according to the Lipinski five rules, namely, the molecular weight does not exceed 500 Da; the oil / water partition coefficient logP of the candidate compound is less than 5; the number of hydrogen bond acceptors of the candidate compound does not exceed 5; the number of hydrogen bond donors does not exceed 5; and the total number of hydrogen bond donors and acceptors of the candidate compound does not exceed 10. The number of rotatable bonds in the candidate compounds does not exceed 10.

6. The method as described in claim 1, characterized in that, Step 4 further includes: using micro-thermophoresis to determine the binding affinity of the cytisine derivative ZINC02139758 to the PhoP protein; and using RT-qPCR to analyze the targeted inhibition of PhoP activity by the cytisine derivative ZINC02139758.

7. The method as described in claim 1, characterized in that, The analysis of the antibacterial ability in step 4 also includes: determining the survival rate of Salmonella; analyzing the antibacterial effect of the cytisine derivative ZINC02139758 on Salmonella; and analyzing the antibacterial ability of the combined treatment of the cytisine derivative ZINC02139758 and colistin on Salmonella.

8. The application of a cytisine derivative, ZINC02139758, in inhibiting Salmonella.

9. The application as described in claim 8, characterized in that, The application also includes the combined inhibition of Salmonella by the cytisine derivative ZINC02139758 and colistin.

10. The application as described in claim 9, characterized in that, The Salmonella species mentioned are Salmonella enteritidis, Salmonella misterii, Salmonella Kentuckyis, Salmonella typhimurium, and Salmonella Thompson.

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