Application of compounds targeting 3CLpro in inhibiting porcine reproductive and respiratory syndrome virus

Abstract

This invention relates to the field of viral inhibitor technology, and more particularly to the application of compounds targeting 3CLpro in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV). Using 3CLpro as a target, this invention comprehensively screened various compounds against PRRSV and coronaviruses, discovering that compounds targeting 3CLpro exhibit varying degrees of inhibitory effects on both PRRSV and coronaviruses. In particular, cimetidine, LLY-507, and flupentixol hydrochloride all demonstrate significant anti-PRSV activity. Furthermore, in addition to targeting 3CLpro, LLY-507 and flupentixol hydrochloride may also act on other cellular targets to inhibit PRRSV replication. The three compounds provided by this invention show promising application prospects in the development of anti-PRRSV drugs.

Description

Technical Field

[0001] This invention relates to the field of virus inhibitor technology, and more particularly to the application of compounds targeting 3CLpro in inhibiting porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Its main clinical features include reproductive disorders in pregnant sows and respiratory diseases in pigs at all production stages. Outbreaks of this disease can severely impact the economic benefits of pig farming. Furthermore, research indicates that currently available commercial vaccines are not closely related to NADC30-like viruses, resulting in unsatisfactory cross-protection. There is still a lack of commercial vaccines that provide broad-spectrum protection against various field strains. In addition, the viral titer, or the ability of PRRSV to proliferate in the body, is closely related to its pathogenic damage. PRRSV infection can also cause severe immunosuppression, leading to secondary infections of other diseases and reduced vaccine efficacy.

[0003] Currently, commercially available attenuated live vaccines remain the primary means of PRRSV prevention and control. However, due to PRRSV's tendency to mutate and recombine, vaccines are unlikely to provide complete protection against heterologous strains, and there are also risks of virulence reversion and recombination with other strains. Therefore, finding broad-spectrum drugs that effectively combat various PRRSV strains is crucial for reducing PRRSV viremia, alleviating clinical symptoms, and increasing the survival rate of infected animals.

[0004] PRRSV is a member of the order Nidovirales, family Arteriviridae, and genus Betaarterivirus. Based on differences in the viral genome sequence, PRRSV is further divided into two independent species: Betaarterivirus suid 1 and Betaarterivirus suid 2. The representative strain of the former is Lelystad virus (LV), while the representative strain of the latter is VR-2332. The genome of this virus is a single-stranded positive-sense RNA without segmentation, approximately 15 kb in length, encoding at least 10 open reading frames (ORFs). Among them, the two overlapping reading frames at the 5' end, ORF1a and ORF1b, occupy about 3 / 4 of the viral genome, encoding the polyproteins pp1a and pp1ab, the latter of which requires frameshift translation by type-1 ribosomes. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides the application of compounds targeting 3CLpro in inhibiting porcine reproductive and respiratory syndrome virus.

[0006] In a first aspect, the present invention provides the use of compounds targeting 3CLpro in inhibiting porcine reproductive and respiratory syndrome virus.

[0007] The present invention further provides the use of compounds targeting 3CLpro in the preparation of medicaments for inhibiting porcine reproductive and respiratory syndrome virus.

[0008] Furthermore, the compounds targeting 3CLpro include one or more of cimetidine, LLY-507, or flupentixol hydrochloride.

[0009] LLY-507 is 5-cyano-2'-(4-(2-(3-methyl-1H-indol-1-yl)ethyl)piperazin-1-yl)-N-(3-(pyrrolidine-1-yl)propyl)-[1,1'-biphenyl]-3-amide.

[0010] This invention reveals that although the coronavirus 3CLpro and arteritis virus 3CLpro have low sequence homology, they share certain similarities in the enzyme's active site region. Furthermore, almost all known 3CLpro structures contain a conserved histidine residue at the bottom of their substrate-binding pocket. This suggests that small-molecule inhibitors designed targeting PRRSV 3CLpro may also have inhibitory effects on coronaviruses; furthermore, this indicates that drugs designed targeting coronavirus 3CLpro may also serve as indicators for screening PRRSV inhibitors.

[0011] To this end, this invention utilized the Surflex molecular docking technology of Sybyl-X2.0 to virtually screen 48 compounds targeting PRRSV 3CLpro. To further increase the success rate of screening, a virtual screening compound library of SARS-CoV-2 3CLpro compounds (161 compounds) was purchased. Then, using a previously constructed complementary reporter gene system as a high-throughput screening platform, the anti-PRRSV efficacy of 209 small molecule compounds was evaluated at the cellular level. After two rounds of screening, cimetidine, LLY-507, and flupentixol dihydrochloride (FD) were identified as having the best inhibitory effect on PRRSV, and these drugs were selected for further research.

[0012] Furthermore, the inhibition of porcine reproductive and respiratory syndrome virus (PRRSV) is achieved by inhibiting the enzyme activity of 3CLpro.

[0013] Furthermore, the inhibition of porcine reproductive and respiratory syndrome virus (PRRSV) is to inhibit the replication, adsorption, or internalization of PRSV.

[0014] Secondly, the present invention provides a method for screening inhibitors of porcine reproductive and respiratory syndrome virus, comprising:

[0015] The inhibitor that targets porcine reproductive and respiratory syndrome virus 3CLpro is an inhibitor of porcine reproductive and respiratory syndrome virus.

[0016] Thirdly, the present invention provides an inhibitor of porcine reproductive and respiratory syndrome, including one or more of cimetidine, LLY-507 or flupentixol hydrochloride.

[0017] Further, the dosage of cimetidine is 1.75-10 μM; and / or the dosage of LLY-507 is 5.35-10 μM; and / or the dosage of flupentixol hydrochloride is 5.78-20 μM.

[0018] Fourthly, the present invention provides a method for screening compounds targeting PRRSV 3CLpro, comprising:

[0019] By sequence alignment and structural comparison between PRRSV 3CLpro and coronavirus 3CLpro, compounds were selected from a drug library targeting coronavirus 3CLpro based on the three-dimensional structure of the target protein and the enzyme active site. Compounds that inhibit PRRSV were screened using high-throughput PRRSV recombinant virus tagged with nanoLuc.

[0020] The present invention has the following beneficial effects:

[0021] This invention screened three compounds—cimetidine, LLY-507, and flupentixol hydrochloride—targeting 3CLpro to obtain high levels of inhibition against porcine reproductive and respiratory syndrome virus (PRRSV). All three compounds exhibited low cytotoxicity. 50 The concentrations were 22.78 μM, 33.71 μM, and 46.40 μM, respectively. Furthermore, experiments verified that all three compounds could act on the viral replication phase. Additionally, LLY-507 and flupentixol hydrochloride also exhibited anti-PRRSV effects by acting on cells. This indicates that cimetidine, LLY-507, and flupentixol hydrochloride possess good anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity, and are of significant pharmaceutical value for the development of drugs to inhibit PRRSV. Attached Figure Description

[0022] Figure 1This diagram illustrates the genetic evolution analysis results of arteritis virus and coronavirus 3CLpro provided in Example 1 of this invention; where A is the phylogenetic tree of arteritis virus and coronavirus 3CLpro, B is a schematic diagram of the amino acid homology analysis results between PRRSV and intra- and inter-species 3CLpro, and C is the amino acid sequence alignment of arteritis virus and coronavirus 3CLpro. * represents the catalytic site and some substrate-binding residues of arteritis virus and coronavirus 3CLpro.

[0023] Figure 2 This is a schematic diagram of the screening of 3CLpro targeted inhibitors provided in Embodiment 1 of the present invention; wherein, in A, the upper figure is a structural simulation schematic diagram of SARS-CoV-2 3CLpro and PRRSV 3CLpro, and the lower figure is a schematic diagram of drug screening; B is a flowchart of drug screening and a schematic diagram of screening results.

[0024] Figure 3 CC of Simeprevir, LLY-507 and FD provided in Embodiment 1 of the present invention 50 and IC 50 Schematic diagram of detection results; where A is a schematic diagram of the molecular structures of Simeprevir, LLY-507, and FD; B is a schematic diagram of the CC of Simeprevir, LLY-507, and FD. 50 A schematic diagram of the test results; C represents the IC50 of Simeprevir, LLY-507, and FD. 50 A schematic diagram of the test results.

[0025] Figure 4 This is a schematic diagram of the anti-PRRSV activity detection results of Simeprevir, LLY-507, and FD provided in Example 1 of the present invention; wherein A is the result of indirect immunofluorescence assay of the anti-PRRSV activity of Simeprevir, LLY-507, and FD; B is the result of Western Blot assay of the anti-PRRSV activity of Simeprevir, LLY-507, and FD; C is the result of luciferase activity assay of Simeprevir, LLY-507, and FD; and D is the result of limiting dilution assay of the anti-PRRSV activity of Simeprevir, LLY-507, and FD.

[0026] Figure 5 This is a comparative schematic diagram of the action stages of Simeprevir, LLY-507 and FD against PRRSV provided in Embodiment 1 of the present invention; wherein A is a schematic diagram of the action stages of Simeprevir against PRRSV, B is a schematic diagram of the action stages of LLY-507 against PRRSV, and C is a schematic diagram of the action stages of FD against PRRSV. Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0028] Example 1

[0029] 1. Experimental Materials

[0030] 1.1 Viruses and Cells

[0031] The PRRSV rescue virus RvJX-Nsp2325-HiBiT was previously constructed by the laboratory of this invention (Key Laboratory of Animal Epidemiology, Ministry of Agriculture, China Agricultural University). It is a recombinant virus carrying a complementary luciferase small subunit and has been patented, application number 202110369372.1.

[0032] MARC-145 cells are a monkey-derived kidney cell line, preserved in our laboratory, and used for PRRSV proliferation culture.

[0033] 1.2 Reagents

[0034] DMEM medium and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific.

[0035] The MTT assay cell viability test kit (M1020) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0036] The HiBiT Lytic Detection System reagents were purchased from Promega Corporation (Wisconsin, USA).

[0037] The SARS-CoV-2 3CLpro Targeted compound library (161 species) and the PRRSV 3CLpro Targeted compound library (48 species) were both purchased from Shanghai Taosu Biotechnology Co., Ltd.

[0038] The PRRSV N protein monoclonal antibody N35, with accession number CGMCC NO.3238, was prepared by our laboratory and is disclosed in Chinese patent CN101661042B.

[0039] FITC-labeled goat anti-mouse IgG was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China).

[0040] 1.3 Cell Culture Reagents

[0041] DMEM cell culture medium: Dissolve DMEM powder (Invitrogen, CA, USA) in water, adjust the pH to about 7.3-7.4 with HEPES buffer and NaHCO3, filter sterilize with a 0.22μM filter, and store at 4℃ for later use.

[0042] Fetal bovine serum (FBS): a product of Invitrogen (Invitrogen, CA, USA).

[0043] Preparation of 1000× bispecific antibody: penicillin (1×10) 7 Dissolve U / mL and streptomycin (10g / mL) in 100mL of ultrapure water, filter sterilize using a 0.22 filter, aliquot and store at -20℃ for later use.

[0044] Cell culture medium (10%): Add 10% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) to DMEM cell culture medium and store at 4°C.

[0045] Cell maintenance medium (2%): DMEM cell culture medium was supplemented with 2% fetal bovine serum, penicillin (100 μU / ml) and streptomycin (100 μg / ml) and stored at 4°C.

[0046] 0.25% trypsin: Weigh 17.80g NaCl, 6.4g Na2HPO4·12H2O, 0.45g Na2EDTA, 0.45g KCl, and 0.45g KH2PO4 and dissolve them in 2L of sterile deionized water. Then add 5.56g of trypsin at a 1:250 ratio. After it is completely dissolved, add ultrapure water to make up to 2.5L. Filter and dispense into containers and store at -20℃ for later use.

[0047] PBS: Weigh out 8.00g NaCl, 1.44g Na2HPO4, 0.24g KH2PO4 and 0.20g KCl respectively. After they are completely dissolved, add ultrapure water to make up to 1000mL. Autoclave at 121℃ for 15min and store at 4℃ for later use.

[0048] 1.4 Main Instruments and Equipment

[0049] PB-21 pH meter: Sartorius GmbH, Germany

[0050] ULTRA GENETIC Ultrapure Water System: ELGA (UK)

[0051] Nikon Eclipse Ti-U Inverted Fluorescence Microscope: Nikon Corporation, Japan

[0052] Thermo Scientific Forma 905 refrigerator: ThermoFisher, USA

[0053] HERAcell i CO2 Cell Incubator: ThermoFisher, USA

[0054] SterilGARDIII Advnance Biosafety Cabinet: Baker Company, USA

[0055] Multifunctional microplate reader: TECAN, Switzerland

[0056] Protein electrophoresis apparatus and transfer apparatus: Bio-Rad Systems, USA

[0057] FlourChemE Imager: ProteinSimple, USA

[0058] 2. Experimental Procedure

[0059] 2.1 Sequence Analysis

[0060] The 3CLpro sequences of representative strains from the order Oscillatoriformes were downloaded from NCBI. MEGAX software was used for phylogenetic tree analysis, Megalign software was used for homology analysis, and https: / / swissmodel.expasy.org / and https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi were used for predicting protein secondary structure and aligning amino acid multiple sequences, respectively.

[0061] The cleavage function of 3CLpro is a common feature of nested viruses and plays a crucial role in viral replication. To analyze the possibility of developing broad-spectrum inhibitors targeting 3CLpro, this invention first selected 3CLpro, an important pathogen of the Arteriviridae and Coronaviridae families, for genetic evolution analysis. The results showed ( Figure 1The fact that 3CLpro from the same genus of virus belongs to the same evolutionary branch indicates that this protein is highly conserved across species. However, amino acid homology results show that the amino acid sequence homology of 3CLpro is very low across different species. Taking PRRSV as an example, the amino acid sequence of PRRSV 3CLpro has more than 90% homology within its species, but only about 20% homology with members of the Coronaviridae family. The cleavage function of 3CLpro is largely determined by its enzyme active site and substrate binding pocket. To further investigate the similarity of 3CLpro between arteritis virus and coronavirus in enzymatic reactions, this invention continued with multiple sequence alignment. The sequence alignment results showed that arteritis virus and coronavirus are conserved in the enzyme reaction center region, including the enzyme active sites His39, Asp64, and Ser118 of arteritis virus 3CLpro, and the enzyme active sites His39 and Cys144 of coronavirus 3CLpro. Furthermore, the substrate binding pocket residue His133 is also highly conserved across species. This indicates that drug screening targeting PRRSV 3CLpro is beneficial for the development of broad-spectrum anti-PRRSV inhibitors.

[0062] 2.2 Screening of PRRSV 3CLpro-targeting inhibitors

[0063] This invention is based on the three-dimensional structure of target proteins and utilizes molecular docking technology to rapidly simulate and screen small molecule compounds with high affinity, which are then validated at the cellular level. The specific process is as follows:

[0064] This invention first used the Surflex molecular docking technology of Sybyl-X2.0 to simulate and virtually screen out the first 48 compounds with strong affinity for PRRSV3CLpro. Then, a virtual compound library targeting SARS-CoV-2 3CLpro was purchased to increase the chance of successful screening, resulting in a total of 209 compounds. The results showed that these compounds exhibited varying degrees of inhibitory effects on porcine reproductive and respiratory syndrome virus.

[0065] The present invention further performs cell-level screening on the above 209 according to the following procedure:

[0066] 209 drugs were pretreated at a concentration of 10 μM / L in 96-well plates confluent with a monolayer of MARC-145 cells. The plates were incubated at 37°C for 2 hours, then the solutions were discarded, and the cells were washed twice with PBS. Recombinant virus RvJX-Nsp2 325 HiBiT cells were seeded at an MOI of 0.1, with the drug maintained at a concentration of 10 μM / L. After 1.5 h of infection, the culture medium was discarded, and the cells were washed twice with PBS. Fresh maintenance medium containing 10 μM / L of the drug was then added, and the cells were cultured for 36 h. The cell culture plates were collected, and after two freeze-thaw cycles at -80°C, 50 μL of the virus solution was added dropwise to each well of a black 96-well plate, followed by an equal volume of [unspecified ingredient]. HiBiT Lytic Reagent, after thorough shaking and mixing for 10 minutes at room temperature, Readings were taken and recorded using a multi-functional microplate reader. Drugs that showed a decrease in fluorescence intensity of more than 80% were selected for a second round of screening. The second round of screening used a decrease in fluorescence intensity of more than 95% as the standard, and cell status was observed under a microscope to screen candidate drugs.

[0067] The results are as follows Figure 2 As shown in B, in the first round of screening, this invention identified 20 drugs that could reduce fluorescence by 80%, and further screening was conducted. In the second round of screening, this invention found five small molecule compounds with the most significant anti-PRRSV activity, and simultaneously conducted preliminary observations of cell activity. It was found that compounds M127 and M131 were too toxic, and finally, three drugs, M123 (cimetidine), M138 (LLY-507), and M140 (flupentixoldihydrochloride, FD), were selected for further investigation.

[0068] 2.3CC 50 and IC 50 Measurement

[0069] To further investigate the toxicity of Simeprevir, LLY-507, and FD in MARC-145 cells, this invention examined the effects of different drug concentrations on cell viability. The specific procedure is as follows:

[0070] CC 50 MARC-145 cells were seeded in 96-well plates. After confluent monolayers or complete cell sedimentation, different concentrations of the natural drug monomer (200 μM / L, 100 μM / L, 50 μM / L, 25 μM / L, 12.5 M / L, 6.25 μM / L, 3.125 μM / L) were added and incubated for 48 h. Then, MTT solution (10 μL per well) was added, and the plates were incubated at 37°C for 4 h. The supernatant was discarded, and 200 μL of DMSO was added to each well. The plates were then shaken at room temperature for 10 min to dissolve the drug. Finally, the absorbance at 490 nm was measured. Zeroing wells (2% maintenance solution) and control wells (DMSO with the same drug concentration) were also included. After data collection, the concentration of the natural drug monomer at which 50% cell death was induced (50% cytotoxic concentration, CC) was calculated using GraphPad Prism software. 50 The curve was fitted using nonlinear regression.

[0071] IC 50 :RvJX-Nsp2 325HiBiT was inoculated at an MOI of 0.1. The drug was started at the highest non-cytotoxic concentration and serially diluted ten times (2-fold) to maintain the corresponding concentration throughout the entire viral infection process, including pretreatment of cells for 2 hours, adsorption phase for 1.5 hours, and replication phase for 36 hours. Subsequently, the virus culture was collected after two freeze-thaw cycles at -80°C. Fluorescence was measured using the HiBiT Lytic Reagent kit. Data were collected, and the antiviral drug concentration (IC50) at which 50% viral replication was inhibited was calculated using GraphPad Prism software. 50 The curve was fitted using nonlinear regression.

[0072] The results are as follows Figure 3 As shown, Simeprevir, LLY-507, and FD all exhibited low cytotoxicity, while CC... 50 The concentrations were 22.78 μM / L, 33.71 μM / L, and 46.40 μM / L, respectively, indicating that the inhibitory effect of these three compounds on viral proliferation was not caused by affecting cell viability. Simultaneously, this invention determined the inhibitory activities of Simeprevir, LLY-507, and FD on PRRSV based on the fluorescence intensity of the recombinant virus. The results showed that the IC50 values ​​of these three drugs were... 50 All concentrations were below 10 μM / L, with Simeprevir showing the best inhibitory effect (IC50). 50 It is 1.75 μM / L.

[0073] 2.4 Determination of the anti-PRRSV activity of the 3CLpro targeted inhibitor

[0074] To further verify the anti-PRRSV efficacy of Simeprevir, LLY-507, and FD, this invention further conducted four experiments: indirect immunofluorescence, protein immunoblotting, luciferase activity assay, and limiting dilution. The specific procedures are as follows:

[0075] Indirect immunofluorescence: RvJX-Nsp2 in 96-well plates confluent with a monolayer of MARC-145 cells. 325 HiBiT was inoculated at an MOI of 0.1, maintaining the drug concentration throughout the entire viral infection process. After 36 hours of culture, the culture was discarded, and then 200 μL of PRRSV N protein monoclonal antibody (1:4000 dilution) was added to each well. The mixture was incubated at 37°C for 60 min. The cells were then washed three times with PBS. Next, 200 μL of FITC-labeled goat anti-mouse IgG (1:200 dilution) was added to each well. The mixture was incubated at 37°C for 60 min, discarded, and then washed three times with PBS for 3 min each time. Finally, the specific yellow-green fluorescence of the N protein was observed under an inverted fluorescence microscope.

[0076] Western Blot: RvJX-Nsp2 in a 6-well plate containing a monolayer of MARC-145 cells 325 HiBiT was inoculated at an MOI of 0.1, maintaining the drug concentration throughout the entire viral infection phase. After 36 hours of incubation, the culture was discarded, and the sample was washed twice with pre-chilled PBS. 100 μL of protein lysis buffer NP40 containing 1 mM PMSF was added to each well. Lysis was performed on ice for 30 minutes, and the supernatant was collected in a 1.5 EP tube by centrifugation. Protein concentration was determined using the BCA method. 5× loading buffer was added according to the calculated volume, and the sample was boiled for 10 minutes before SDS-PAGE electrophoresis and Western blotting. SDS-PAGE: Separating and stacking gels were prepared according to protein size. 20 μg of sample was loaded into each well, and electrophoresis buffer was added. The voltage was adjusted: 80V for 30 minutes for the stacking gel and 120V for 90 minutes for the separating gel. Electrophoresis was stopped when the bromophenol blue indicator appeared. Western blotting: Before transfer, a PVDF membrane of similar size to the protein gel was prepared and activated with methanol for 2 minutes. Filter paper and a sponge pad were pre-soaked in pre-chilled transfer buffer. Subsequently, the membrane was arranged in the following order: negative electrode clamp - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - positive electrode clamp. After removing air bubbles, it was placed in the transfer tank and transferred at 100V for 2 hours. The PVDF membrane was then removed and blocked overnight at 4°C with 5% skim milk or at room temperature for 1 hour. The blocked PVDF membrane was washed with PBST for 5 minutes, and the corresponding primary antibody was diluted and incubated overnight at 4°C. The PVDF membrane bound to the primary antibody was then removed and washed 5 times with PBST for 5 minutes each time. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 1 hour, followed by washing 5 times with PBST for 5 minutes each time. An appropriate amount of exposure solution was prepared by mixing equal volumes of solution A and solution B, and then evenly dropped onto the PVDF membrane. The membrane was developed in the dark for 2 minutes, and then chemically exposed in a gel imaging system. The images were saved and the results were analyzed.

[0077] Fluorescence intensity and viral titer: RvJX-Nsp2 in 24-well plates confluent with a monolayer of MARC-145 cells 325 HiBiT was inoculated at an MOI of 0.1, maintaining the drug concentration throughout the entire viral infection process. After 36 hours of culture, the cell plates were collected and cooled to -80°C. After two freeze-thaw cycles, the viral fluid was collected and serially diluted 10-fold with 2% maintenance medium. 10 cells were then collected. -1 ~10 -7 Dilutes were inoculated into 96-well plates containing a confluent monolayer of MARC-145, 100 μL per well, with each dilution inoculated in quadruplicate. After incubation at 37°C for 48 h, indirect immunofluorescence was performed, and the results were observed and recorded under a fluorescence microscope. The viral titer was calculated using the Reed-Muench method. Simultaneously, the fluorescence intensity of the collected viral fluid was directly measured. The viral titer and fluorescence intensity of each sample were measured at least three times.

[0078] The results are as follows Figure 4 As shown, indirect immunofluorescence results revealed a significant decrease in fluorescence intensity after treatment with different drug concentrations, exhibiting a dose-dependent effect. Protein immunoblotting results also showed the same trend; treatment with 5 μM / L, 2.5 μM / L Simeprevir, 5 μM / L LLY-507, and 10 μM / L FD resulted in almost undetectable viral N protein expression. Similarly, treatment with these drug concentrations significantly reduced luciferase activity and viral titer (10 μM / L). 2 ~10 4 (times), and with IC 50 Consistent with the results, Simeprevir exhibited the best anti-PRRSV activity. These results demonstrate that Simeprevir, LLY-507, and FD are promising candidates for inhibiting PRRSV.

[0079] 2.5 Stages of Action of 3CLpro Inhibitors Against PRRSV

[0080] Although small molecules targeting PRRSV 3CLpro were obtained through drug library simulation screening, these compounds have their own functions. This invention further explores the stages of these three molecules' inhibition of viral replication to determine whether these drugs have other targets against PRRSV. The specific process is as follows:

[0081] Cells under treatment (pre): MARC-145 cells with a confluent monolayer were pretreated with the inhibitor for 2 h, the culture medium was discarded, and the cells were inoculated with virus solution at MOI = 0.1. After 1.5 h of incubation, the supernatant was discarded, and fresh maintenance medium containing 2% FBS was added. After 36 h of incubation, the virus solution was collected and the fluorescence intensity was measured (see steps 2.2.4.3 and 2.2.1). The control was the addition of the same volume of DMSO.

[0082] Adsorption and entry phase (during): The virus was diluted to MOI=0.1 with 2% maintenance medium, and the inhibitor was diluted with the diluted virus solution. The two were then inoculated into cells and allowed to incubate for 1.5 h. The supernatant was discarded, and fresh maintenance medium containing 2% FBS was added. After culturing for 36 h, the sample was collected and the fluorescence intensity was measured. The control was the addition of the same volume of DMSO.

[0083] Replication phase (post): The virus was inoculated into a confluent monolayer of MARC-145 cells at MOI=0.1 and allowed to incubate for 1.5 h. The supernatant was discarded, and fresh maintenance medium containing a certain concentration of inhibitor was added. After culturing for 36 h, the sample was collected and the fluorescence intensity was measured. The control was the addition of the same volume of DMSO.

[0084] The above experimental inhibitor drug concentrations were Simeprevir (5 μM / L), LLY-507 (5 μM / L), and FD (10 μM / L).

[0085] The results are as follows Figure 5 As shown, only Simeprevir acts on the viral replication phase, while LLY-507 and FD can both act on cells to antagonize viral replication, indicating that the antiviral activity of these two drugs has other mechanisms.

[0086] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Use of a compound targeting 3CLpro in the manufacture of a medicament for inhibiting porcine reproductive and respiratory syndrome virus; The compound targeting 3CLpro includes one or more of simeprevir, LLY-507, or flupenthixol hydrochloride.

2. Use according to claim 1, characterized in that, The inhibition of porcine reproductive and respiratory syndrome virus is by inhibiting the activity of 3CLpro.

3. Use according to claim 1 or 2, characterized in that, The inhibition of porcine reproductive and respiratory syndrome virus is inhibiting the replication, adsorption, or internalization infection of porcine reproductive and respiratory syndrome virus.

4. A method of screening for a compound targeting 3CLpro of porcine reproductive and respiratory syndrome virus, characterized in that, Comprise: Based on the sequence alignment and structure comparison of porcine reproductive and respiratory syndrome virus 3CLpro and coronavirus 3CLpro, the compounds are selected from the drug library targeting coronavirus 3CLpro based on the three-dimensional structure of the target protein and the active site of the enzyme, and the compounds inhibiting porcine reproductive and respiratory syndrome virus are screened by high-throughput screening of porcine reproductive and respiratory syndrome virus recombinant virus with nanoLuc label.

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