Application of GW4064 in preparation of anti-porcine reproductive and respiratory syndrome virus drugs

Abstract

The invention relates to the technical field of biological medicine, in particular to application of GW4064 in preparation of drugs for resisting porcine reproductive and respiratory syndrome virus. The invention discloses a novel application of a farnesyl ester X receptor stimulant GW4064 in preparation of a PRRSV (porcine reproductive and respiratory syndrome virus) resisting medicine. The GW4064 has strong antiviral activity on PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), and particularly has a remarkable inhibition effect on a high-pathogenicity strain (HP-PRRSV). The GW4064 interferes with the life cycle of the virus by targeting the NSP3 protein of the PRRSV, so that the replication and propagation of the virus are inhibited. The GW4064 has a remarkable antiviral effect on various variants of the PRRSV, and shows a broad-spectrum antiviral effect. Meanwhile, the GW4064 is small in molecular weight, can easily enter cells, and can efficiently play a role without carrier delivery. The invention also provides a pharmaceutical composition taking GW4064 as a main active component, and a theoretical basis and a practical basis are provided for development of PRRSV antiviral drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of GW4064 in the preparation of anti-polysporic reproductive virus drugs. Background Art

[0002] my country is the largest pig farming and pork consumption country, with both farming and consumption accounting for more than half of the world's total, and pork accounting for 60% of my country's meat consumption. Disease is an important factor affecting the healthy development of the pig industry. Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, first appeared in the United States in 1987 and was subsequently reported in Europe. In 1996, Guo Baoqing et al. first isolated PRRSV in my country and named the isolated strain CH-1a, confirming the existence of the disease in my country. At present, PRRS is one of the most threatening infectious diseases to the healthy development of the global pig industry. PRRSV mainly causes reproductive disorders in sows, respiratory symptoms in pigs of different ages, especially piglets, and secondary infections such as Haemophilus parasuis and Streptococcus infection, causing huge economic losses to the global pig industry. In particular, in 2006, porcine high fever syndrome (PHFS) broke out in most provinces in my country and Vietnam, causing extremely heavy losses. According to incomplete statistics, PRRS causes economic losses of up to $650 million per year in the United States, 1.5 billion euros per year in Europe, and 40 billion yuan per year in China. Currently, the pathogenic mechanism of the virus and the mechanism of inducing host immune response are still unclear.

[0003] The biggest difference between PRRSV and other viruses is that it mainly infects immune cells - porcine alveolar macrophages, destroying the immune system, similar to HIV. The virus has the following characteristics: ① immunosuppression, interfering with other vaccine immunity; ② highly variable, extremely easy to mutate and recombine, producing more pathogenic strains; ③ no / low neutralizing antibodies, the main antigenic epitope GP5 protein that baits the production of neutralizing antibodies is not fully exposed on the surface of the virus particles; ④ inflammatory storm, excessive induction of cytokine expression; ⑤ antibody-dependent enhancement (ADE), low\non-neutralizing antibodies promote virus invasion; ⑥ persistent infection, long viremia, intermittent detoxification; ⑦ secondary infection, secondary infection with Haemophilus parasuis, Streptococcus, etc.

[0004] Based on the above characteristics of PRRSV, combined with years of practical experience, the commercial vaccines currently developed, including attenuated and inactivated vaccines, are not ideal and cannot prevent the occurrence and spread of the disease. In addition, these vaccines have the following problems: slow production of neutralizing antibodies, poor cross-protection against heterologous strains, long vaccine virus viremia, and recombination with field strains to produce recombinant viruses with stronger pathogenicity. Therefore, the development of more and more efficient antiviral drugs is an important direction for the prevention and control of PRRS. Based on the principle of virus-host interaction, the research and development of antiviral drugs targets viral proteins and host proteins. At present, there are no reports of antiviral drugs targeting viral proteins for PRRSV. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides the use of GW4064 in the preparation of anti-polysporic reproductive virus drugs.

[0006] The present invention is achieved by: GW4064 compound, which is an effective farnesoid X receptor FXR agonist, EC 50 GW4064 inhibits the weight gain of C57BL / 6 mice induced by high-fat diet HFD or high-fat, high-cholesterol diet; its molecular weight is 542.84, molecular formula: C 28 H 22 Cl3NO4, the structural formula is:

[0007]

[0008] Another object of the present invention is to provide an application of the GW4064 compound, wherein the application is the following (a) and / or (b) and / or (c):

[0009] (a) Use of GW4064 compound in the preparation of drugs against PRRSV virus;

[0010] (b) Use of GW4064 compound in the preparation of products for preventing porcine blue ear disease;

[0011] (c) Use of GW4064 compound in drugs for treating porcine blue ear disease.

[0012] Furthermore, the drug inhibits viral infection by targeting the NSP3 protein of the PRRSV virus.

[0013] Furthermore, the PRRSV virus is highly pathogenic porcine blue ear disease virus HP-PRRSV.

[0014] Furthermore, the drug is antiviral by blocking the expression and replication of the virus.

[0015] Furthermore, the porcine blue ear disease is blue ear disease caused by highly pathogenic porcine blue ear disease virus HP-PRRSV or blue ear disease caused by a virus variant;

[0016] The virus variant strain is PRRSV Ch-1a strain, WuH3 strain, SD16 strain, Li11 strain, TA12 strain, NL1207 strain or NADC30 strain.

[0017] Another object of the present invention is to provide a drug for resisting PRRSV virus or treating blue ear disease in pigs, wherein the drug contains the GW4064 compound.

[0018] Furthermore, the concentration of the GW4064 compound in the drug is 1 to 50 μM.

[0019] Furthermore, the concentration of the GW4064 compound in the drug is 2 to 10 μM.

[0020] Furthermore, the dosage form of the drug can be prepared into different desired dosage forms, such as injection preparations or oral preparations, such as capsules, tablets, etc.

[0021] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0022] First, the present invention proves for the first time that GW4064 has anti-PRRSV activity, especially it still has a strong antiviral effect on the highly pathogenic strain HP-PRRSV. Studies have shown that GW4064 can target the NSP3 protein of the PRRSV, inhibit viral replication, and has significant antiviral activity against PRRSV at extremely low concentrations (10 μM). In addition, due to the small molecular weight of GW4064, it can easily pass through the cell membrane and enter the cell to exert an antiviral effect. Therefore, it does not require a carrier to be delivered into the cell, laying the foundation for the development of new drugs against PRRSV. The present invention provides a new use for the GW4064 compound, provides a basis for the development of PRRSV antiviral drugs, and provides more methods and means for the prevention and treatment of PRRSV.

[0023] Second, technical problems existing in the existing technology:

[0024] 1) Lack of effective drugs against PRRSV

[0025] Porcine reproductive and respiratory syndrome virus (PRRSV), especially highly pathogenic PRRSV (HP-PRRSV), is a major pathogen that causes huge economic losses in the pig farming industry. Existing antiviral drugs and vaccines have limited efficacy in practical applications and are difficult to effectively control the spread of the virus, especially in dealing with mutant strains.

[0026] 2) The treatment of blue ear disease is single and ineffective

[0027] Currently, the treatment of blue ear disease mainly focuses on symptomatic treatment and antibiotics to prevent secondary infection, but there is a lack of efficient treatment methods directly targeting the virus. At the same time, the rapid mutation of the virus makes it difficult for existing treatment options to maintain long-term effectiveness.

[0028] 3) Insufficient drug safety and targeting

[0029] Some antiviral drugs on the market lack specific targets and may cause side effects on the animal body, reducing the application value of the drugs in actual breeding industry.

[0030] Third, the technical problems solved by the present invention are:

[0031] 1) Develop highly effective antiviral drugs by targeting NSP3 protein

[0032] Based on the characteristics of the GW4064 compound, the present invention found that it can significantly inhibit viral infection by targeting the NSP3 protein of the PRRSV virus. This technical path has high targeting and selectivity, and provides a new solution for the development of new anti-PRRSV drugs.

[0033] 2) Broad-spectrum inhibitory effect against PRRSV variants

[0034] The present invention proves that the GW4064 compound has an inhibitory effect on multiple PRRSV variants (such as SD16 strain and NADC30 strain), solves the problem that existing drugs have poor efficacy on variants, and improves the applicability of drugs.

[0035] 3) Provide a variety of preparation forms to improve the convenience of clinical application

[0036] The present invention develops a pharmaceutical composition in various dosage forms including oral tablets, injection preparations, etc., so that the drug is easier to use in actual breeding environments and meets different treatment needs.

[0037] Fourth, the significant technological advancement brought about by the present invention:

[0038] 1) Highly effective anti-virus mechanism

[0039] The present invention discloses for the first time that the GW4064 compound achieves antiviral effects by dual action of targeting farnesoid X receptor (FXR) and NSP3 protein of PRRSV, and shows higher antiviral efficiency and lower side effects compared with existing drugs.

[0040] 2) Broad-spectrum antiviral applications

[0041] The present invention shows that GW4064 is not only effective against HP-PRRSV, but also can be widely applied to the prevention and treatment of various PRRSV variants, thus filling the gap in the prior art in coping with virus mutations.

[0042] 3) Improve disease prevention and control in the aquaculture industry

[0043] The present invention provides a new drug for treating blue ear disease, provides a more reliable prevention and control means for the pig breeding industry, reduces economic losses, and promotes the healthy development of the industry.

[0044] 4) Cross-domain technology integration

[0045] The present invention expands the role of FXR agonists from metabolic regulation to the antiviral field, realizes the innovative application of the drug action mechanism, and provides a new idea for research and development in related fields.

[0046] The technical solution of the present invention breaks through the limitations of the existing treatment methods, and through the development of efficient, safe and broad-spectrum antiviral drugs, provides a more effective disease prevention and control method for large-scale aquaculture, with significant economic value and social benefits. At the same time, the wide applicability and low side effect characteristics of the present invention lay a solid foundation for its promotion in actual aquaculture environments.

[0047] Currently, there is no effective drug for the prevention and treatment of PRRSV in clinical practice. The present invention shows that GW4064 has a very strong antiviral effect on PRRSV, and is expected to fill this gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a simulation diagram of the interaction between GW4064 compound and porcine reproductive and respiratory syndrome virus NSP3 protein.

[0049] Figure 2 This is a test of the toxicity of GW4064 compound to Marc-145 cells.

[0050] Figure 3 After PRRSV was treated with different concentrations of GW4064, the expression level of PRRSV-N protein was detected by western-blot. Rib: Ribavirin, ribavirin, positive control.

[0051] Figure 4 After PRRSV was treated with different concentrations of GW4064, the expression levels of PRRSV-ORF7 and NSP3 protein mRNA were detected by qRT-PCR. Rib: Ribavirin, ribavirin, positive control.

[0052] Figure 5After PRRSV was treated with different concentrations of GW4064, the expression level of PRRSV-N protein was detected by immunofluorescence (IFA). Rib: Ribavirin, ribavirin, positive control.

[0053] Figure 6 After PRRSV was treated with different concentrations of GW4064, the cell supernatant was collected and the virus titer was detected by plaque assay. Rib: Ribavirin, ribavirin, positive control.

[0054] Figure 7 is the antiviral effect of GW4064 against different strains of PRRSV. WuH3, SD16, Li11, TA12, NL1207 and NADC30 are different strains of PRRSV. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0056] The present invention provides a compound GW4064, which is an effective farnesoid X receptor FXR agonist, EC 50 (The concentration of GW4064 required to inhibit 50% viral replication) is 65nM. GW4064 inhibits the weight gain of C57BL / 6 mice induced by high-fat diet HFD or high-fat, high-cholesterol diet; its molecular weight is 542.84, molecular formula: C 28 H 22 Cl3NO4, the structural formula is:

[0057]

[0058] The present invention provides an application of the GW4064 compound, wherein the application is the following (a) and / or (b) and / or (c):

[0059] (a) Use of GW4064 compound in the preparation of drugs against PRRSV virus;

[0060] (b) Use of GW4064 compound in the preparation of products for preventing porcine blue ear disease;

[0061] (c) Use of GW4064 compound in drugs for treating porcine blue ear disease.

[0062] The drug inhibits viral infection by targeting the NSP3 protein of the PRRSV virus.

[0063] The PRRSV virus is highly pathogenic porcine blue ear disease virus HP-PRRSV.

[0064] The drug works against viruses by blocking the expression and replication of the virus.

[0065] The porcine blue ear disease is a blue ear disease caused by the highly pathogenic porcine blue ear disease virus HP-PRRSV or a blue ear disease caused by a virus variant;

[0066] The virus variant strain is PRRSV Ch-1a strain, WuH3 strain, SD16 strain, Li11 strain, TA12 strain, NL1207 strain or NADC30 strain.

[0067] An embodiment of the present invention provides a drug for resisting PRRSV virus or treating blue ear disease in pigs, wherein the drug contains the GW4064 compound.

[0068] The concentration of the GW4064 compound in the drug is 1 to 50 μM.

[0069] The concentration of the GW4064 compound in the drug is 2 to 10 μM.

[0070] The dosage form of the drug can be prepared into different desired dosage forms, such as injection preparations or oral preparations, such as capsules, tablets, etc.

[0071] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the present technical field.

[0072] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0073] Statistical analysis of the following examples of the present invention: All experiments were repeated at least 3 times independently, and the results were expressed as mean values ​​and standard errors, and analyzed using one-way analysis of variance and T test. All statistical analyses used P<0.05 as the test standard for significant statistical differences, and the analysis software was SPSS16.0 and GraphPad Prism 5.

[0074] The cells and different strains of PRRSV used in the following examples were all sourced and stored in the laboratory of the research group of the present invention. GW4064 was provided by MedChemExpress (MCE), CAS No.: 278779-30-9.

[0075] Example 1: Screening of small molecule compounds targeting porcine reproductive and respiratory syndrome virus NSP3 protein

[0076] The present invention first uses computer virtual screening of small molecule compounds targeting viral NSP3 protein to block the life cycle of PRRSV and thus inhibit the virus. The software used for virtual screening is Maestro11.4, 3D drawing software is PyMol.

[0077] 1. Protein preparation

[0078] The crystal structure of Sus scrofa CD163 was downloaded from the RCSB PDB database (PDB ID: 5HRJ), and the protein was hydrogenated using the Protein Preparation Wizard module. Energy optimization was then performed (OPLS2005 force field, RMSD ), use the Receptor Grid Generation module to create a grid file for the processed protein, and generate a grid file with CYS486 / CYS515 / CYS520 / CYS576 as the center. The box size is set to

[0079] 2. Compound preparation

[0080] The 2D format of the MCE Bioactive Compound Library Plus (containing 16.7K compounds) was converted to The software LigPrep Module performs hydrogenation, energy optimization and other processing, and outputs 3D structures for virtual screening.

[0081] 3. Molecular docking

[0082] The Virtual Screening Workflow module was used for virtual screening, the prepared compounds were imported, and the Glide module was used for molecular docking, that is, the receptor and ligand molecules were docked with each other through geometric matching and energy matching. First, the high-throughput screening (HTVS) mode in the Glide module was used to screen the small molecule compounds of the MCE Bioactive Compound Library Plus, and the small molecule compounds with the top 15% of the scores were selected for the second round of screening using the standard (SP) mode; then the top 15% of the scores were selected for the third round of screening using the high-precision (XP) mode to obtain the ranking of small molecule compounds. Finally, the binding force between the target and the compound, the compound structure, etc. were manually reviewed, and the top 200 compounds in the MCE Bioactive Compound Library Plus were selected for output and delivered as the final result. The binding mode of the top 5 compounds in the MCEBioactive Compound Library Plus with the Sus scrofa CD163 protein was selected for 2D and 3D mapping.

[0083] 4. Analysis of screening results

[0084] The higher the absolute value of the molecular docking score (docking_score), the stronger the binding force between the compound and the protein. According to the ranking of the intermolecular interaction force between the small molecule compound and the porcine reproductive and respiratory syndrome virus NSP3 protein, the GW4064 compound was finally locked. Figure 1 This is a simulation diagram of the interaction between GW4064 and viral NSP3 protein.

[0085] Example 2: GW4064 cytotoxicity test

[0086] 1. Materials

[0087] GW4064 powder (purchased from MedChemExpress (MCE)) and CCK-8 (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) were used to detect cell activity (vitality).

[0088] 2. Test methods

[0089] Marc-145 cells (or PAMs cells) were cultured with DMEM culture medium containing 10% fetal bovine serum until 60-70%, the culture medium was discarded, and nutrient solutions containing GW4064 diluted in multiples (the concentrations diluted with DMSO were: 0.5, 1, 1.5, 2, 2.5, and 3 μM, respectively) were added for 36 hours, a DMSO control group was set up, and then 10% (v / v) CCK-8 was added and cultured for 3 hours; a multifunctional microplate reader was used to read the fluorescence values ​​of 540nm excitation light and 590nm emission light, and a GW4064 cytotoxicity graph was drawn. The cell activity of the DMSO control group was taken as 100%, and the fluorescence value of the cells treated with the multiple dilutions of GW4064 was divided by the fluorescence value of the PBS control group, which was the relative cell activity of GW4064 at different concentrations.

[0090] 3. Results

[0091] The cytotoxicity results of GW4064 are as follows Figure 2 As shown, CC50 is 145.7 μM, indicating that GW4064 has little cytotoxicity. CC50 (Concentration of cytotoxicity 50%): the concentration that causes 50% cytotoxicity, that is, the concentration that reduces the number of viable cells by 50% compared with the control.

[0092] Example 3, GW4064 antiviral test

[0093] 1. Western blot detection

[0094] Marc-145 cells were cultured in a 6-well plate in DMEM culture medium containing 10% fetal bovine serum until the cell confluence was 70%, then the culture medium was discarded, the cells were washed three times with PBS, and HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 1.2. At the same time, different concentrations of GW4064 compound were added, and a control group without GW4064 was set up. Then, the cells were cultured in a 37°C, 5% CO2 incubator, washed three times with PBS after 36 hours, digested with 0.25% trypsin, and the cells were lysed. The protein concentration was measured, and the expression level of PRRSV-N protein was detected by Western-Blot.

[0095] The results are as follows Figure 3 As shown, GW4064 significantly inhibited the expression of PRRSV-N protein, and as the concentration of GW4064 increased, the inhibitory effect became more obvious, with a dose-dependent effect, and the antiviral effect at a concentration of 20 μM was equivalent to 200 μM ribavirin (Rib, positive control). The above experimental results show that GW4064 can significantly inhibit the expression of PRRSV-N protein and has a strong antiviral effect on PRRSV.

[0096] 2. Fluorescence quantitative PCR (qRT-PCR) detection

[0097] First, the total RNA of Marc-145 cells was extracted by Trizol method, and RNA was used as template to synthesize cDNA by reverse transcription. Then, the mRNA expression of PRRSV-ORF7 and PRRSV-NSP3 proteins was detected by fluorescence quantitative PCR using SYBR Green method. The fluorescence quantitative PCR reaction system included: upstream and downstream primers of PRRSV-ORF7 and PRRSV-NSP3 mRNA, cDNA template and polymerase mix (purchased from Nanjing Novozyme Biotechnology Co., Ltd., catalog number: Q711), and the reaction program was set as follows: 95℃300s, 40 cycles (95℃20s; 55℃20s; 72℃20s).

[0098] The nucleotide sequences of qRT-PCR quantitative primers and internal reference primers are as follows:

[0099] PRRSV-ORF7-F: 5′-AAAACCAGTCCAGAGGCAAG-3′ (see SEQ IDNO.1);

[0100] PRRSV-ORF7-R: 5′-CGGATCAGACGCACAGTATG-3′ (see SEQ ID NO. 2);

[0101] PRRSV-NSP3-F: 5′-GCACATGCTTGCTGGGATTT-3′ (see SEQ ID NO. 3);

[0102] PRRSV-NSP3-R: 5′-AGAGAGAGCCAGGTCCGTAG-3′ (see SEQ ID NO. 4);

[0103] GAPDH-F: 5′-TGACAACAGCCTCAAGATCG-3′ (see SEQ ID NO.5);

[0104] GAPDH-R: 5′-GTCTTCTGGGTGGCAGTGAT-3′ (see SEQ ID NO. 6);

[0105] The results are as follows Figure 4 As shown, GW4064 significantly inhibited the expression of PRRSV-ORF7 and NSP3 protein mRNA, and as the concentration of GW4064 increased, the inhibitory effect became more obvious, with a dose-dependent effect, and the antiviral effect at a concentration of 20 μM was equivalent to 200 μM ribavirin (Rib, positive control). The above experimental results show that GW4064 can significantly inhibit PRRSV replication.

[0106] 3. Immunofluorescence (IFA) detection

[0107] Marc-145 cells were cultured in a 6-well plate in DMEM culture medium containing 10% fetal bovine serum until the cell confluence was 70%, the culture medium was discarded, the cells were washed 3 times with PBS, HP-PRRSV was inoculated with an infection multiplicity MOI = 1.2, and different concentrations of GW4064 compound were added at the same time. A control group without GW4064 was set up, and then cultured in a 37°C, 5% CO2 incubator. After 36 hours, the cells were washed 3 times with PBS, and the cells were fixed with 4% paraformaldehyde. Immunofluorescence (IFA) was used to detect the expression level of PRRSV-N protein.

[0108] The results are as follows Figure 5 As shown, GW4064 significantly inhibited the expression of PRRSV-N protein, and as the concentration of GW4064 increased, the inhibitory effect became more obvious and dose-dependent. The above experimental results show that GW4064 has strong antiviral activity against PRRSV.

[0109] 4. Plaque assay (PFU) detection

[0110] Marc-145 cells were cultured in a 6-well plate in DMEM medium containing 10% fetal bovine serum until the cell confluence reached 70%, the culture medium was discarded, the cells were washed three times with PBS, and HP-PRRSV was inoculated at a multiplicity of infection MOI = 1.2. At the same time, different concentrations of GW4064 compound were added, and a control group without GW4064 was set up. The cells were then cultured in a 37°C, 5% CO2 incubator. After 36 hours, the cell supernatant was collected and the virus titer was detected by plaque assay.

[0111] The results are as follows Figure 6 As shown, GW4064 significantly inhibited PRRSV replication (the number of plaques was significantly reduced), and as the concentration of GW4064 increased, the inhibitory effect became more obvious and dose-dependent. The above experimental results show that GW4064 can significantly inhibit the titer of PRRSV and has strong antiviral activity against PRRSV.

[0112] In addition, the present invention also tested the antiviral effect of GW4064 on different variants WuH3, SD16, Li11, TA12, NL1207 and NADC30 according to the above method. Figure 7 As shown, GW4064 has a significant inhibitory effect on different strains of PRRSV, indicating that GW4064 has a broad spectrum of inhibition on different strains of PRRSV.

[0113] In summary, the present invention has proved for the first time that GW4064 has strong resistance to porcine blue ear virus, especially has significant antiviral effect on highly pathogenic strain HP-PRRSV; GW4064 can target porcine blue ear virus NSP3 protein, block the PRRSV life cycle, and then inhibit the virus, and has strong antiviral activity against different mutants. At the same time, due to the small molecular weight of GW4064, it is very easy to pass through the cell membrane into the cell and exert an antiviral effect, so there is no need for a carrier to deliver it into the cell, which lays the foundation for the development of new drugs against porcine blue ear disease. The present invention provides a new use of the GW4064 compound, provides a basis for the development of PRRSV antiviral drugs, and provides more strategies for the prevention and treatment of porcine blue ear disease.

[0114] Example 4: Experiment on the inhibitory effect of GW4064 on PRRSV virus infection

[0115] PRRSV virus was inoculated into porcine alveolar macrophages (PAMs) cultured in vitro and divided into GW4064 treatment group and control group. The treatment group was added with 1μM, 5μM and 10μM concentrations of GW4064, and the control group was only added with an equal amount of solvent. After 48 hours of culture, the PRRSV viral RNA level was detected by real-time fluorescence quantitative PCR. The results showed that compared with the control group, GW4064 significantly reduced the expression level of viral RNA, and its inhibitory effect was dose-dependent.

[0116] Example 5: In vivo protective effect of GW4064 against highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV)

[0117] C57BL / 6 mice were divided into three groups: GW4064-treated group (10 mg / kg), positive control group (antiviral drug treatment) and negative control group. Each group of mice was inoculated with HP-PRRSV virus by intraperitoneal injection, and the corresponding drug treatment was given every day after virus inoculation. The weight changes and survival rates of mice were recorded during the experiment, and the levels of viral RNA in lung tissue were detected after the experiment. The results showed that the GW4064-treated group significantly inhibited the weight loss caused by viral infection and significantly reduced the levels of viral RNA in lung tissue.

[0118] Example 6: Broad-spectrum antiviral effect of GW4064 on PRRSV mutants

[0119] PRRSV variants (SD16, NL1207, and NADC30) were selected to infect porcine alveolar macrophages (PAMs), and GW4064 (5 μM) or control treatment was given, and the expression of viral proteins was detected by immunofluorescence technology after 48 hours of culture. The results showed that GW4064 significantly inhibited the expression of viral proteins in each variant infection model, proving that it has a broad-spectrum antiviral effect on a variety of PRRSV variants.

[0120] Example 7: Preparation and clinical application of GW4064 oral preparation

[0121] Oral tablets containing GW4064 were prepared, with a dose of 50 mg per tablet. Ten pigs infected with HP-PRRSV were selected and randomly divided into a treatment group (GW4064 tablets, once a day for 10 consecutive days) and a control group. The pigs' body temperature, appetite and activity status were recorded during the treatment, and the viral load in the blood was detected by RT-PCR after the treatment. The results showed that GW4064 significantly reduced the viral load, and the clinical symptoms of the pigs (such as fever and loss of appetite) were significantly improved.

[0122] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A GW4064 compound, characterized in that This compound is a potent farnesoid X receptor FXR agonist with an EC50 of 65nM and a molecular formula of C8H 22 Cl3NO4, molecular weight is 542.84, structural formula is:

2. Use of the GW4064 compound according to claim 1 in the preparation of a drug for resisting weight gain caused by a high-fat diet or a high-fat, high-cholesterol diet, characterized in that: The drug regulates metabolic function by targeting the farnesoid X receptor FXR, thereby achieving weight control.

3. A pharmaceutical composition containing the GW4064 compound according to claim 1, characterized in that: The pharmaceutical composition comprises the following components: (a) active ingredient GW4064 compound, at a concentration of 1 to 50 μM; (b) pharmaceutically acceptable carriers or excipients; (c) The pharmaceutical composition can be prepared in the form of an injection preparation, an oral preparation or a transdermal preparation.

4. Use of the GW4064 compound in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus (PRRSV) infection, characterized in that: The drug inhibits viral replication and spread by targeting the NSP3 protein of the PRRSV virus.

5. The use according to claim 4, characterized in that The GW4064 compound is used to prepare a drug for preventing and / or treating blue ear disease caused by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), wherein the virus includes HP-PRRSV and its variants, and the variants include PRRSV Ch-1a strain, WuH3 strain, SD16 strain, Li11 strain, TA12 strain, NL1207 strain or NADC30 strain.

6. The use according to claim 4, characterized in that The drug blocks the expression and replication of PRRSV virus and regulates the signaling pathway of farnesoid X receptor (FXR), thereby exerting antiviral effects and improving host immune function.

7. A drug for preventing porcine reproductive and respiratory syndrome virus (PRRSV) infection or treating blue ear disease in pigs, characterized in that: The drug contains the active ingredient GW4064 compound, which is a farnesoid X receptor (FXR) agonist that can inhibit viral infection by targeting the NSP3 protein of the PRRSV virus.

8. The drug according to claim 1, characterized in that The concentration of the GW4064 compound in the drug is 1 to 50 μM, preferably 2 to 10 μM.

9. The drug according to claim 1, characterized in that The dosage form of the drug is an injection preparation or an oral preparation, including but not limited to tablets, capsules, oral solutions or suspensions.

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