Application of swine STING molecular agonist SR-717 in preparation of pseudorabies virus infection resisting medicine

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of a swine STING molecular agonist SR-717 in preparation of a pseudorabies virus infection resisting medicine. The swine STING molecular agonist SR-717 can be stably combined with swine STING protein, the promoter activity of a downstream signal molecule interferon regulatory factor 3 (IRF3) and I-type interferon (IFN beta) is enhanced, PRV replication can be remarkably inhibited, the virus load is reduced, and pathological changes of infected mouse lung, brain and other tissues are reduced. The SR-717 provided by the invention has good targeting property and immune activation ability, and is suitable for intervention treatment of porcine pseudorabies virus infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a porcine STING molecule agonist SR-717 in the preparation of a drug for resisting pseudorabies virus infection. Background Art

[0002] Pseudorabies virus (PRV) is a highly contagious alphaherpesvirus that is clinically susceptible to a wide range of mammals. Pigs are the natural reservoir for PRV, and infection can cause damage to the nervous and respiratory systems, with a particularly high mortality rate in piglets, posing a serious threat to the healthy development of the swine industry. Although vaccines are currently in use, the frequent emergence of variants in recent years has significantly reduced the effectiveness of vaccines, posing a significant challenge to prevention and control. Therefore, it is necessary to explore new approaches to prevent and treat PRV infection. Summary of the Invention

[0003] To develop new approaches for preventing and treating pseudorabies virus (PRV) infection, the present invention provides the use of the STING agonist SR-717 in the preparation of a drug for preventing and treating PRV infection. SR-717 stably binds to porcine STING protein, enhancing the promoter activity of downstream signaling molecules interferon regulatory factor 3 (IRF3) and type I interferon (IFNβ). This agonist significantly inhibits PRV replication, reduces viral load, and reduces pathological changes in tissues such as the lungs and brain of infected mice. SR-717, provided by the present invention, possesses excellent targeting and immune-stimulating properties, making it suitable for the interventional treatment of PRV infection in pigs.

[0004] The present invention provides the use of a porcine STING molecule agonist SR-717 in the preparation of an anti-pseudorabies virus infection drug. The porcine STING molecule agonist SR-717 is a small molecule compound of 4,5-difluoro-2-[(6-imidazol-1-ylpyridazine-3-carbonyl)amino]benzoate, and its structure conforms to the following molecular characteristics: .

[0005] The porcine STING agonist SR-717 stably binds to the porcine STING protein, enhancing the promoter activity of the downstream signaling molecules interferon regulatory factor 3 (IRF3) and type I interferon (IFNβ). This significantly inhibits PRV replication, reduces viral load, and reduces lesions in the lungs and brain of infected mice. The SR-717 provided by the present invention possesses excellent targeting and immune activation capabilities, making it suitable for interventional treatment of pseudorabies virus infection in pigs.

[0006] Furthermore, the medicine is an injection.

[0007] Furthermore, the injection also includes excipients.

[0008] Furthermore, the auxiliary materials include polyethylene glycol, Tween 80 and physiological saline.

[0009] Furthermore, the injection is prepared from 8% to 10% by mass of SR-717, 30% to 40% by mass of polyethylene glycol, 4% to 5% by mass of Tween 80, and the balance of normal saline.

[0010] Furthermore, the drug is used to activate antiviral innate immune signaling pathways and inhibit viral replication.

[0011] Furthermore, the drug is used to improve the integrity of alveolar structure, reduce neutrophil infiltration and alleviate brain tissue neuronal damage.

[0012] The present invention also provides the use of a porcine STING molecule agonist SR-717 in the preparation of a drug for treating or preventing a disease caused by pseudorabies virus infection, wherein the disease is viral pneumonia or viral encephalitis caused by PRV; The porcine STING agonist SR-717 is a small molecule compound, 4,5-difluoro-2-[(6-imidazol-1-ylpyridazine-3-carbonyl)amino]benzoate, whose structure conforms to the following molecular characteristics: .

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides information on the anti-pseudorabies virus (PRV) activity of the porcine STING agonist SR-717. SR-717 inhibits PRV replication by activating antiviral innate immune signaling pathways. It is used to improve alveolar structural integrity, reduce neutrophil infiltration, and alleviate brain neuronal damage, thereby combating PRV infection.

[0014] The porcine STING agonist SR-717 stably binds to the porcine STING protein, enhancing the promoter activity of the downstream signaling molecules interferon regulatory factor 3 (IRF3) and type I interferon (IFNβ). This significantly inhibits PRV replication, reduces viral load, and reduces pathological changes in lung and brain tissues in infected mice. The SR-717 provided by the present invention possesses excellent targeting and immune-stimulating properties, making it suitable for interventional treatment of pseudorabies virus infection in pigs. This invention overcomes the limitations of traditional vaccines that rely on antigen recognition and instead adopts a "targeted host immune activation" approach. It encompasses a comprehensive drug development process from target selection to molecular screening, cell-based validation, and animal testing. It has broad application prospects, not only for PRV prevention and control but also for other DNA virus infections. Experimental results confirm its stable efficacy from multiple perspectives, including cytotoxicity, viral load, and tissue pathology. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Schematic diagram of molecular docking and intermolecular forces between SR-717 and the STING protein binding pocket; A is the three-dimensional structural model of SR-717 binding to STING protein; B is an enlarged view of the active pocket region of SR-717 and STING protein; C is a schematic diagram of the intermolecular forces between SR-717 and the STING protein binding pocket.

[0017] Figure 2 This is the RMSD trend diagram of the molecular dynamics simulation of the SR-717 and STING protein complex; A is the overall structural stability of the SR-717 and STING complex (RMSD trend); B is the global compactness of the SR-717 and STING complex (Rg analysis); C is the change in protein solvent accessible surface area (SASA); D is the dynamic change of the number of hydrogen bonds; E is the dynamic fluctuation of STING-sr717-Chain-A residue level; F shows the dynamic fluctuations at the residue level of STING-sr717-Chain-B.

[0018] Figure 3 The results of cytotoxicity evaluation of SR-717 in PK-15 cells (CC 50 ).

[0019] Figure 4 The inhibitory effect of SR-717 on pseudorabies virus (PRV) infection (IC 50 ).

[0020] Figure 5 This figure shows the effect of SR-717 treatment on virus-induced cytopathic effect and fluorescence signal intensity in PRV-infected cells; A is the observation of cell morphological changes under a microscope; B is the intensity and distribution of the fluorescence signal under the microscope.

[0021] Figure 6The qPCR detection results of the viral gB gene copy number after SR-717 treatment.

[0022] Figure 7 The results of dual luciferase activity assay of IRF3 and IFNβ after SR-717 treatment; A is the dual luciferase activity assay result of IRF3 after SR-717 treatment; B is the dual luciferase activity detection result of IFNβ after SR-717 treatment.

[0023] Figure 8 This is a Western blot detection image of key STING pathway proteins p-STING, p-TBK1 and p-IRF3 after SR-717 treatment.

[0024] Figure 9 This is a visual record of the "strange itching" behavior and death symptoms of PRV-infected mice; A is the characterization of itch behavior on the face of mice; B shows the signs of severe scratching of the mouse's legs; C: The mouse has reduced activity and stiff body, showing a weak state; D shows that the skin on the head and legs of the mouse was bitten, damaged and bleeding, and the mouse eventually died of collapse.

[0025] Figure 10 These are the detection results of viral loads in various tissues of mice after infection with PRV.

[0026] Figure 11 HE staining pathological analysis of mouse lung tissue after PRV infection and SR-717 treatment; A is the HE staining image of lung tissue in the PRV infection control group; B is the HE staining image of the lung tissue of the PRV infection control group; C shows the bronchial and interstitial structures at all levels in the PRV-infected control group; D is the HE staining image of the alveolar cavity in the SR-717 treatment group; E is the HE staining image of the alveolar cavity of the parallel sample of the SR-717 treatment group; F is the HE staining image of lung tissue structure in the SR-717-treated group.

[0027] Figure 12 HE staining after PRV infection and SR-717 treatment; In the figure, A to D are HE staining images of brain tissue of mice in the virus control group; the arrow in C indicates that many vascular dilations can be seen in the thalamus of the mouse brain tissue; the arrow in D indicates a small amount of neuronal degeneration and cytoplasmic vacuolation; E~HE staining of brain tissue of mice in the HSR-717 treatment group; the arrow in E indicates the shrinkage of granule cells in the DG region of the hippocampus; the arrow in F indicates the vacuolation of neurons in brain tissue; the arrow in G indicates the shrinkage of neurons; the arrow in H indicates the dilation of a small number of blood vessels in the thalamus and hippocampus.

[0028] Figure 13 Different tissues of infected mice STING qPCR analysis results of gene expression levels.

[0029] Figure 14 Different tissues of infected mice IRF3 qPCR analysis results of gene expression levels.

[0030] Figure 15 Different tissues of infected mice IFNβ qPCR analysis results of gene expression levels.

[0031] Figure 16 Different tissues of infected mice ISG15 qPCR analysis results of gene expression levels. DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0033] The present invention screened for agonist molecules that effectively activated porcine STING and systematically validated their anti-PRV activity in cell and animal models, clarifying their potential role in preventing pseudorabies virus infection in pigs. The following methods were used:

[0034] 1. Drug screening and molecular simulation stage: a. Using the Schrödinger platform, the porcine STING protein crystal structure (PDB ID: 6A03) was subjected to hydrogenation, dehydration, protonation optimization, and energy minimization to construct a docking model of the ligand binding pocket. b. Import approximately 3 million "drug-like" small molecules from the ZINC15 database. After constructing their 3D structures using LigPrep, molecular docking was performed using the Glide module. Pharmacophore analysis and MM-GBSA energy scoring were combined to screen for potential STING agonists. c. GROMACS molecular dynamics simulations were used to analyze the structural stability and free energy changes of candidate molecules in complex with the STING protein at the nanosecond scale, and lead compounds with strong binding ability and stable conformation were preliminarily screened.

[0035] 2. Cell-level activity verification stage: a. Evaluation of the cytotoxicity of candidate compounds in PK-15 cells (CC 50 ); b. Construct a PRV-infected cell model and test the inhibitory effects of different concentrations of candidate compounds on viral replication; c. qPCR detection of STING pathway-related genes in PRV-infected PK-15 cells after drug treatment ( STING 、 IRF3 、 IFNβ 、 ISG15 ) expression changes to evaluate the activation trend of immune pathways; d. Further Western blot analysis was performed to observe changes in the phosphorylation levels of key proteins (such as p-STING, p-TBK1, and p-IRF3) in PRV-infected PK-15 cells to preliminarily determine the pathway activation effect.

[0036] 3. Animal experiment stage: a. Establish a PRV infection mouse model and administer candidate compounds in vivo; b. Monitor changes in viral load in target organs such as the lungs and brain; c. Evaluate the inflammatory response and improvement of damage in different organs through histopathology (HE staining); d. Combined with qPCR to detect the expression levels of immune genes in tissues, further verify the immune-enhancing effect of the compound.

[0037] The specific embodiments include the following.

[0038] Example 1: Use of the STING molecular agonist SR-717 in the preparation of drugs for preventing and treating pseudorabies virus infection in pigs.

[0039] This example uses computer-assisted drug screening (CADD) to screen for small molecule agonists based on the three-dimensional structure of the porcine STING protein. First, the solved crystal structure of the porcine STING ligand-binding domain (PDB ID: 6A03) was selected from the Protein Data Bank. Three million potential small molecules were obtained from the ZINC compound library. Preprocessing was performed using the Protein Preparation Wizard module in the Schrödinger 2021 software suite, which includes adding hydrogen atoms, removing water molecules, optimizing protonation states, and minimizing energy.

[0040] The Receptor Grid Generation module was then used to construct grid regions around known ligand binding pockets and perform high-throughput molecular docking. Initial screening was performed using the SP mode, with the primary criteria being minimum binding free energy (Glide Score ≤ -8.0 kcal / mol), formation of ≥ 2 hydrogen bonds (donor-receptor distance ≤ 3.0 Å), and a hydrophobic contact area ≥ 50 Ų. The top 500 molecules were further rearranged and scored using the XP mode.

[0041] The Prime MM-GBSA method was used to calculate the binding free energy of the top 20 small molecules. Taking into account indicators such as binding conformational stability, pharmacophore distribution and binding energy, three candidate compounds were finally selected as subsequent research objects, namely SR-717, compound 2 and compound 3.

[0042] The structure of the SR-717 is: .

[0043] like Figure 1 As shown, SR-717 can stably bind to the active pocket of STING protein and form multiple interactions.

[0044] Example 2: Molecular dynamics simulation of candidate compounds and STING complexes The three candidate compounds screened in Example 1 were respectively used to form complexes with the STING protein, and molecular dynamics simulation was performed using GROMACS2021.3 software to verify their conformational stability.

[0045] A 100 ns molecular dynamics (MD) simulation of the complex was performed using Gromacs 2022. The protein was simulated using the CHARMM 36 force field parameters, and the ligand topology was constructed using the GAFF2 force field parameters. The protein-ligand complex was placed in a cubic box with periodic boundary conditions. Water molecules were packed into the box using the TIP3P water model, resulting in a 1.2 nm periodic boundary. Electrostatic interactions were addressed using the particle mesh Ewald (PME) and Verlet algorithms, respectively. Subsequently, 100,000 steps of isothermal, isochoric, and isothermal, isobaric ensemble equilibrium were performed with a coupling constant of 0.1 ps and a simulation duration of 100 ps. A cutoff of 1.0 nm was used for both van der Waals and Coulomb interactions. Finally, a molecular dynamics simulation was performed using Gromacs 2022 at constant temperature (310 K) and pressure (1 bar) for a total of 100 ns.

[0046] During the simulation, metrics such as protein backbone RMSD, the number of ligand-protein hydrogen bonds, and the total system energy were recorded. The results showed that the SR-717-STING complex remained stable throughout the simulation, with RMSD fluctuations less than 2.5 Å and stable binding energy, indicating that the STING protein is bound in a stable conformation and warrants further experimental verification.

[0047] Example 3: Detection of cytotoxicity and antiviral activity of the compounds.

[0048] In this example, the cytotoxicity and inhibitory effect of candidate compounds on the replication of pseudorabies virus (PRV) were evaluated in PK-15 cells.

[0049] Cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM complete medium containing 10% FBS at 37°C and 5% CO2. The initial compound was a solid powder, which was dissolved in dimethyl sulfoxide (DMSO) to prepare a 71.19 mM (25 mg / mL) stock solution. After gradient dilution with DMEM medium, the concentration gradient was set to 0.05 μM, 0.5 μM, 5 μM, 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, and 500 μM, with the final DMSO concentration always ≤ 0.5%. Cells were seeded in 96-well plates (1×10 cells per well) when they were in the logarithmic growth phase. 5 After cells adhered to the wall, different concentrations of compounds were added, and 3 replicate wells were set up for each group.

[0050] After 24 h of treatment, CCK-8 reagent was added and the absorbance was measured at 450 nm. CC 50 value.

[0051] The results are as follows Figure 3 As shown in Figure 2, SR-717 had no significant toxicity to cells within 100 μM. 50 is 330.4 μM.

[0052] To evaluate its anti-PRV activity, PK-15 cells were infected with PRV at an MOI of 0.1. After virus adsorption for 1 hour, the cells were washed and treated with different concentrations of SR-717. After 48 hours of culture, total RNA was extracted and the expression of PRV gB gene was detected. The results showed that SR-717 had a dose-dependent inhibitory effect on PRV replication, with an IC 50 About 50.62 μM ( Figure 4 ).

[0053] Further fluorescence microscopy was used to observe the pathological changes of infected cells and found that SR-717 could alleviate PRV-induced cell damage and reduce the fluorescence signal intensity ( Figure 5At the same time, the PRV viral load in the SR-717 treated group was significantly lower than that in the infected group ( Figure 6 ).

[0054] Example 4: Detection of cGAS-STING pathway-related gene and protein expression To verify the activation effect of SR-717 on the cGAS-STING signaling pathway, dual-luciferase reporter assays and related molecular detection were performed in PRV-infected PK-15 cells.

[0055] The IRF3 response element and IFNβ promoter sequences were synthesized and cloned into the pGL4.23 basic vector, respectively, to construct the luciferase reporter plasmids pGL4-IRF3-Luc and pGL4-IFNβ-Luc. They were co-transfected into PK-15 cells with the pRL-TK (Renilla internal reference) plasmid, and 0.1 μg of total plasmid per well was used using Lipofectamine 3000 transfection reagent (ThermoFisher). 24 hours after transfection, a mock group, a PRV infection group (MOI=5), and an SR-717 treatment group (the maximum safe concentration of the drug was added 1 hour before infection) were set up. Cells were collected 24 hours after infection and transfected using Dual-Glo ® The system detected the activity of firefly and Renilla luciferase, calculated the relative fluorescence intensity (Fold change), and standardized it with the Mock group as the benchmark. Figure 7 As shown, SR-717 significantly enhanced the transcriptional activities of IRF3 and IFNβ.

[0056] Protein detection was performed by Western blot. Total protein was extracted and lysed using RIPA buffer. Protein concentration was determined by the BCA method. 20 μg of protein per sample was separated by SDS-PAGE electrophoresis and transferred to the membrane. Primary antibodies: anti-pSTING, anti-pTBK1, and anti-pIRF3 (anti-pSTING purchased from ThermoFisher Scientific, anti-pTBK1 and anti-pIRF3 purchased from Proteintech Group) were used. HRP-conjugated goat anti-rabbit IgG secondary antibody was used for color development, and ECL development was performed for 3 minutes. The results showed ( Figure 8 ), SR-717 can enhance the PRV-induced expression levels of p-STING, p-TBK1 and p-IRF3, indicating that it can activate the cGAS-STING downstream signaling pathway.

[0057] Example 5: Construction of PRV infection mouse model and drug efficacy evaluation Fifteen 6-week-old SPF female Kunming mice (Experimental Animal Center, Anhui Agricultural University) weighing approximately 30 g were randomly divided into three groups: control group, PRV infection group, and SR-717 treatment group (n = 5 / group). The PRV infection group and SR-717 treatment group were treated with 1×10 5 TCID 50 The model was established by intraperitoneal injection of PRV at different doses, and the control group was injected with an equal volume of PBS.

[0058] One hour after modeling, SR-717 was administered intraperitoneally at a dose of 20 mg / kg / day. The formulation consisted of 10% SR-717 solution (SR-717 stock solution diluted in DMSO) + 40% PEG300 (polyethylene glycol) + 5% Tween 80 (Tween 80) + 45% saline for 5 consecutive days. The control and PRV-infected groups received an equal volume of saline.

[0059] During the experiment, it was observed that mice in the infection group showed severe itching and abnormal behavior, e.g. Figure 9 shown.

[0060] After the treatment, the mice were killed, and the heart, brain, lung, liver, spleen, kidney and other tissues were quickly dissected. Some of them were frozen in liquid nitrogen for nucleic acid extraction and detection of PRV gB gene, and some were fixed in 4% neutral formalin and then paraffin sectioned for HE staining to observe histopathological changes.

[0061] The results showed that the viral load in the lungs and brain of the SR-717 group was significantly reduced ( Figure 10 ), HE sections showed that the integrity of alveolar structure was improved, neutrophil infiltration was reduced, and brain tissue neuronal damage was alleviated, showing excellent in vivo antiviral effect and tissue protection effect ( Figure 11 、 Figure 12 After PRV infection, alveoli of varying sizes were observed, with eosinophilic floccules visible within the alveolar cavity. No significant abnormalities were observed in the bronchial structure or interstitial tissue. After SR-717 treatment, eosinophilic release was observed within the alveolar cavity, and inflammatory cell infiltration within the alveolar cavity was significantly reduced. The inflammatory response in lung tissue was significantly ameliorated, and the tissue structure became clearer.

[0062] Example 6: Effects of SR-717 on the Expression of Immune-Related Genes in Multiple Organs of Mice To further evaluate the immunomodulatory effects of SR-717, RNA was extracted from mouse lung, brain, liver, spleen, and kidney tissues, and cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). ChamQ SYBR qPCR Master Mix (Vazyme) was used with primers including STING, IRF3, IFNβ, and ISG15, and the internal reference gene GAPDH. Detection was performed using ABIQuantStudio 5. The results showed ( Figures 13 to 15 ), the expression levels of related genes in various tissues were significantly upregulated after SR-717 treatment, especially in the lung and brain tissues, suggesting that it can activate the systemic antiviral immune response.

[0063] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0064] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. The use of the porcine STING molecule agonist SR-717 in the preparation of a drug for preventing pseudorabies virus infection, characterized in that: The porcine STING agonist SR-717 is a small molecule compound, 4,5-difluoro-2-[(6-imidazol-1-ylpyridazine-3-carbonyl)amino]benzoate, whose structure conforms to the following molecular characteristics: 。 2. The use of the porcine STING molecule agonist SR-717 according to claim 1 in the preparation of an anti-pseudorabies virus infection drug, characterized in that: The medicine is an injection.

3. The use of the porcine STING molecule agonist SR-717 in the preparation of an anti-pseudorabies virus infection drug according to claim 2, characterized in that: The injection further comprises excipients.

4. The use of the porcine STING molecule agonist SR-717 in the preparation of an anti-pseudorabies virus infection drug according to claim 3, characterized in that: The auxiliary materials include polyethylene glycol, Tween 80 and normal saline.

5. The use of the porcine STING molecule agonist SR-717 in the preparation of an anti-pseudorabies virus infection drug according to claim 4, characterized in that: The injection is prepared from 8% to 10% by mass of SR-717, 30% to 40% by mass of polyethylene glycol, 4% to 5% by mass of Tween 80 and the balance of normal saline.

6. The use of the porcine STING molecule agonist SR-717 in the preparation of an anti-pseudorabies virus infection drug according to claim 1, characterized in that: The drug is used to activate antiviral innate immune signaling pathways and inhibit viral replication.

7. The use of the porcine STING molecule agonist SR-717 according to claim 1 in the preparation of an anti-pseudorabies virus infection drug, characterized in that: The drug is used to improve the integrity of alveolar structure, reduce neutrophil infiltration and alleviate brain tissue neuron damage.

8. Use of the porcine STING molecule agonist SR-717 in the preparation of a drug for treating or preventing diseases caused by pseudorabies virus infection, characterized in that: The disease is viral pneumonia or viral encephalitis caused by PRV; the porcine STING molecule agonist SR-717 is 4,5-difluoro-2-[(6-imidazole-1-ylpyridazine-3-carbonyl)amino]benzoate in claim 1.

Images