Compound Talnetant as well as acquisition method and application thereof

Abstract

The invention relates to the technical field of new application of a compound Talnetant, in particular to a compound Talnetant as well as an acquisition method and application thereof.According to the compound Talnetant and the acquisition method and application thereof, a high-throughput screening technology is applied, 113 types of compound libraries possibly having an inhibition effect on PEDV are screened, the compound Talnetant with a good antiviral effect is obtained, a test result shows that Talnetant has a good inhibition effect on PEDV and PDCoV, and the compound Talnetant can be used for preparing the compound Talnetant with a good antiviral effect on PEDV and PDCoV. The compound has a broad-spectrum antiviral capability on coronavirus, and a delayed administration experiment result shows that the Talnetant has more than 80% of inhibition effect on PEDV (porcine epidemic diarrhea virus) when the compound is applied within 7 hours after virus infection, and in addition, the compound also has inhibition effect on common animal pathogenic bacteria such as escherichia coli, staphylococcus aureus and shigella freundii, which indicates that the compound has a broad-spectrum antiviral capability on the coronavirus. The compound Talnetant disclosed by the invention is good in antiviral and antibacterial effects.

Description

Technical field

[0001] The present invention relates to the technical field of new applications of the compound Talnetant, and in particular to the compound Talnetant and a method for obtaining and applying the compound. [Background Technology]

[0002] Talnetant is a small molecule chemical drug, now generally considered to be a neurokinin-3 (NK3) receptor antagonist. It is often used in the treatment of schizophrenia. There are no relevant studies on its inhibition of viruses, bacteria, and anti-inflammation.

[0003] Porcine epidemic diarrhea (PED) is an infectious disease of animals caused by porcine epidemic diarrhea virus (PEDV). PEDV is an enveloped, single-stranded, positive-sense RNA virus belonging to the genus Alphacoronavirus in the family Coronaviridae. The PEDV gene is approximately 28 kb long, with seven open reading frames encoding four structural proteins and 16 nonstructural proteins. The main clinical symptoms of PED include acute diarrhea, vomiting, and dehydration. Pigs of all ages are susceptible to PED, with symptoms ranging from severe diarrhea and high mortality in piglets to mild diarrhea and low mortality in adult pigs. The mortality rate of infected piglets under seven days of age can reach 100%. Since its first report in 1971, PEDV has impacted the global swine industry. The emergence of PEDV variants since 2010 has caused significant economic losses to the global swine industry and become a major problem for pig farms. The situation with porcine epidemic diarrhea in China is very complex. Clinically, it is difficult to distinguish whether TGEV, PoRV, PDCoV, SADS-CoV, or PEDV are single infections or mixed infections. Therefore, the focus of prevention and control is accurate detection and timely isolation. Vaccine prevention and control is also a major means of preventing and controlling PEDV. However, the rapid mutation of the virus poses significant challenges to vaccine development and updating. In addition to PEDV, some bacteria, such as Escherichia coli and Staphylococcus aureus, can also contaminate the pig intestine and cause diarrhea. Therefore, in order to effectively prevent and control porcine epidemic diarrhea, it is necessary to conduct drug research and develop related compounds that can effectively inhibit PEDV coronaviruses and effectively prevent and control Escherichia coli, Staphylococcus aureus, and other bacteria with a relatively broad spectrum. [Summary of the invention]

[0004] In view of the above, it is necessary to develop related compounds that can effectively inhibit PEDV coronavirus and effectively prevent and control Escherichia coli, Staphylococcus aureus and other broad-spectrum bacteria for porcine epidemic diarrhea.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The use of the compound Talnetant in the preparation of anti-coronavirus drugs, the structural formula of the compound Talnetant is as follows:

[0007]

[0008] Furthermore, the coronavirus is porcine deltacoronavirus and / or porcine epidemic diarrhea virus.

[0009] Furthermore, the half-maximal inhibitory concentration of the talnetant against porcine epidemic diarrhea virus is 2.57 μM, and the selectivity index is 25.92.

[0010] Furthermore, the half-maximal inhibitory concentration of the Talnetant against Porcine deltacoronavirus is 6.4 μM.

[0011] Furthermore, the talnetant inhibits the viral 3C-like protease of PEDV.

[0012] A method for screening the compound talnetant, the method comprising: screening out compounds that have an inhibitory effect on porcine epidemic diarrhea virus from 113 compounds in a compound library, treating cells infected with 0.1 MOI porcine epidemic diarrhea virus at a compound concentration of 10 μM, collecting samples 12 hours later, and measuring TCID 50 .

[0013] The use of the compound Talnetant in the preparation of antibacterial drugs, the structural formula of the compound Talnetant is as follows:

[0014]

[0015] Furthermore, the bacteria inhibited by the antibacterial drug are Escherichia coli, Staphylococcus aureus and / or Shigella flexneri.

[0016] The present invention has the following beneficial effects:

[0017] 1. The present invention uses high-throughput screening technology to screen a library of 113 compounds that may have inhibitory effects on PEDV, and obtains a compound with good antiviral effect, Talnetant. The test results show that Talnetant has a good inhibitory effect on PEDV and PDCoV, indicating that the compound Talnetant has a broad-spectrum antiviral ability against coronaviruses. After research, the half-maximal inhibitory concentration (IC50) of the compound against PEDV is 50 ) was 2.57 μM, the selectivity index (SI) was 25.92, and the IC 50 The results of delayed dosing experiments showed that Talnetant's inhibitory effect on PEDV gradually decreased with delayed dosing. However, when administered within 7 hours of infection, Talnetant exhibited over 80% inhibition of PEDV. Furthermore, the present application investigated the compound's inhibitory effects on PEDV adsorption, invasion, and replication, finding that Talnetant's inhibition of PEDV likely occurred during the replication phase. Furthermore, the compound exhibited inhibitory effects against common animal pathogens: Escherichia coli, Staphylococcus aureus, and Shigella flexneri, demonstrating the compound's excellent antiviral and antibacterial properties.

Brief Description of the Drawings

[0018] Figure 1 is the structural formula of the compound Talnetant.

[0019] Figure 2 This is the PEDV virus proliferation curve.

[0020] Figure 3 The antiviral activity of 113 compounds was screened and the inhibition rate against PEDV was calculated according to the compound number.

[0021] Figure 4 This is a cytotoxicity test of the compounds on Vero CCL-81; Figure A shows the compound numbered 4, Figure B shows the compound numbered 43, Figure C shows the compound numbered 51, Figure D shows the compound numbered 57, and Figure E shows the compound numbered 112.

[0022] Figure 5 Figure 2 shows the inhibitory effect of different concentrations of the compound Talnetant on PEDV; Figure A shows the inhibitory effect of different concentrations of Talnetant on the ability of PEDV to infect cells; Figure B shows the effect of different concentrations of Talnetant on the expression ability of PEDVN protein.

[0023] Figure 6 The effect of different administration times on PEDV inhibition.

[0024] Figure 7 This is the effect of compound Talnetant on the adsorption stage of PEDV.

[0025] Figure 8 This is the effect of the compound Talnetant on the invasion stage of PEDV.

[0026] Figure 9 The effect of the compound Talnetant on the PEDV replication stage; A and B are the virus titers 12h and 24h after virus incubation; C and D are the Western Blot results of N protein 12h and 24h after virus incubation.

[0027] Figure 10 The left graph shows the mRNA expression levels of PEDV M protein at 12 h and 24 h after virus incubation, with the left graph showing 12 h and the right graph showing 24 h.

[0028] Figure 11 This is the cytotoxicity test of compound Talnetant on LLC-PK1 cells.

[0029] Figure 12 The inhibitory effect of compound Talnetant at different concentrations on PDCoV.

[0030] Figure 13 Compound Talnetant and PEDV3CL pro Molecular docking diagram; A is Talnetant and PEDV 3CL pro Figure B shows the molecular docking 2D diagram of Talnetant and PEDV 3CL. pro Molecular docking 3D diagram. [Specific implementation method]

[0031] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Example 1:

[0033] This embodiment is a method for screening compounds, which is as follows:

[0034] This study first reviewed the literature and selected 113 compounds with antiviral potential as screening targets. Using an in vitro viral proliferation model, compounds with inhibitory activity against PEDV and high drug development potential were screened. The main screening methods are as follows:

[0035] 1. Reagents: Fetal bovine serum (FBS) was purchased from ExCell; DMEM high-glucose medium, 1× PBS buffer (sterile), trypsin-EDTA digestion solution (containing phenol red), RIPA tissue / cell lysis buffer, penicillin-streptomycin mixture (100×), tris(hydroxymethyl)methane (TRIS), glycine, sodium dodecyl sulfate (SDS), and Tween-20 were all purchased from Solarbio; ultrasensitive cell proliferation assay reagent (CCK-8) was purchased from Abbkine and protease inhibitor tablets (A32965) were purchased from Thermo Fisher; WB protein-free blocking buffer (1% in 1× PBST) and sensitive ECL luminescent solution were purchased from Shanghai Shenger Biotechnology Co., Ltd.; SDS-PAGE gel preparation kit was purchased from Sevier Biotechnology Co., Ltd.; HRP-conjugated Goat Anti-Mouse IgG (H+L) was purchased from Proteintech; TransZol was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; isopropanol, anhydrous methanol and anhydrous ethanol were all purchased from Xilong Science Co., Ltd.

[0036] 2. Main instruments and equipment: Protein electrophoresis apparatus was purchased from Bio-Rad Life Sciences (Shanghai) Co., Ltd.; Bio-Rad multifunctional imaging system was purchased from Bio-rad, USA; high-speed freezer (Sorvall ST16R), CO2 incubator, real-time fluorescence quantitative PCR system QuanStudio7Flex, and NanoDrop One were all purchased from Thermo Fisher Scientific, USA; stainless steel electric heated redistilled water device was purchased from Shanghai Sanshen Medical Instrument Co., Ltd.; ultra-low temperature freezer storage box was purchased from Zhongke Meiling Cryogenic Technology Co., Ltd.; CKX-41 inverted biological microscope was purchased from OLYMPUS, USA; NX-S vortex mixer was purchased from Scikogex, USA; vertical high-pressure steam sterilizer was purchased from Shanghai Shen'an Medical Instrument Factory; VD-850 desktop clean workbench was purchased from Suzhou Purification Equipment Co., Ltd.; frost-free refrigerator and freezer was purchased from Changhong Meiling Co., Ltd.; FA1204N electronic balance was purchased from Shanghai Jinghai Instrument Co., Ltd.; electronic constant temperature stainless steel water bath was purchased from Shanghai Yichang Instrument Screen Factory; multifunctional microplate reader was purchased from Guangzhou Radio and Television Metrology and Testing Co., Ltd.

[0037] 3. Cells, strains, and compound libraries: Vero CCL-81 cells and LLC-PK1 cell lines were purchased from the ATCC cell bank. The PEDV strain (CH / JX / 01) was isolated in our laboratory, and the compound library was purchased from Med Chemexpress Biotechnology, Inc., USA.

[0038] 4. Main reagent formula:

[0039] Complete culture medium: 50 mL FBS, 10 mL penicillin-streptomycin mixture, 5 mL glutamine, and 450 mL DMEM were mixed and placed at 4°C for later use.

[0040] PEDV virus maintenance solution: 100 mL of DMEM, 400 μL of 0.25% trypsin-EDTA digestion solution (containing phenol red), and 2 mL of penicillin-streptomycin mixture were mixed evenly.

[0041] PDCoV (Porcine deltacoronavirus, PDCoV) virus maintenance solution: 100 mL DMEM, 200 μL 0.25% trypsin-EDTA digestion solution (containing phenol red), and 2 mL penicillin-streptomycin mixture were mixed.

[0042] Tris-glycine running buffer (10×): Tris base 30.3 g, glycine 144 g, SDS 10 g, dilute to 1 L with double-distilled water, store at room temperature, and dilute to 1× before electrophoresis.

[0043] Transfer buffer: 2.9 g Tris base, 1.45 g glycine, dilute to 400 mL with double-distilled water, add 100 mL of anhydrous methanol before use, mix thoroughly and keep at low temperature for later use.

[0044] PBS buffer (10×): KH2PO4 2.7 g, Na2HPO4 14.2 g, NaCl 80 g, KCl 2 g, dilute to 1 L with double-distilled water.

[0045] PBST: Mix 500 mL of 1× PBS buffer and 250 μL of Tween-20.

[0046] Protein lysis buffer: 800 μL RIPA, 200 μL Loding Buffer, 10 μL 100× PIC, 10 μL 100× NaV3O4, 10 μL 0.1M NaF, 10 μL 100× PMSF.

[0047] 4. Test methods

[0048] (1) Cell culture: Vero CCL-81 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 2% penicillin-streptomycin, 1% glutamine, and the culture environment was a 37°C constant temperature cell incubator containing 5% carbon dioxide. After the cells in a T75 cell culture flask grew to a single layer covering the bottom, they were washed twice with 0.25% trypsin 1-2 mL, then an appropriate amount of trypsin was added to the 37°C incubator for digestion until the cells rounded, the rounded cells were discarded and continued to digest for 1-2 min, then the cells were blown apart with complete medium and subcultured according to the desired ratio.

[0049] (2) Virus propagation: After Vero CCL-81 cells were cultured to a single layer covering the bottom of a T75 culture flask, the cells were washed three times with PBS, then the virus was diluted with virus maintenance solution and incubated in the incubator for 2 h, after incubation, the virus solution was discarded and virus maintenance solution was added and returned to the cell incubator for culture, and the lesions were observed daily. After the lesions caused more than 50% of the cells to fall off, the culture flask was frozen and thawed 3 times at -80°C, the virus solution was collected into a centrifuge tube, centrifuged at 8000 r / min for 10 min, and the supernatant was collected and stored at -80°C.

[0050] 3. Virus propagation curve determination: 2.0 x 10 5 cell / mL of Vero CCL-81 cells were plated into a 12-well plate, 1 mL per well, and when the cells covered 80% of the bottom, they were inoculated with 0.1 MOI of PEDV, incubated in the cell incubator for 2 h, and then the virus solution was discarded and virus maintenance solution was added. Samples were collected at 12 h, 24 h, 36 h, and 48 h and the titer was determined.

[0051] 4. PEDV TCID 50 determination: 1.8 x 10 5 cell / mL of Vero CCL-81 cells were plated into a 96-well plate, 100 μL per well, and after the cells grew to 80%, they were washed three times with PBS, the virus solution to be tested was diluted 10 -1 -10 -6 times with virus maintenance solution, each concentration gradient was done in 8 replicate wells, and each 96-well plate had only virus maintenance solution as a negative control. After the virus solution was added to the cells and incubated in the cell incubator for 1 h, the virus solution was discarded and 100 μL of virus maintenance solution was added per well, and the cell lesions were observed every 24 h. After no new lesions appeared, the TCID 50 of the virus was calculated according to the Reed-Muench method based on the lesion situation of each dilution gradient.

[0052] 5. Cytotoxicity experiment: 1.8 x 10 5Vero CCL-81 cells were plated into 96-well plates at a concentration of 100 μL per well. After reaching 80% cell growth, the cells were washed three times with PBS. Compounds were serially diluted in virus maintenance medium, with six replicates per concentration. A blank control was established with virus maintenance medium alone. After 24 hours, the cells were washed three times with PBS. In vitro cytotoxicity of the compounds was assessed using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. The highly water-soluble tetrazolium salt WST-8 in CCK-8 is reduced by cellular dehydrogenases to form a water-soluble orange formazan under the action of an electron mediator. The absorbance at 450 nm using a microplate reader provides an indirect measure of viable cell count. Cytotoxicity was calculated using the following formula: Cytotoxicity (%) = {(absorbance of experimental well) - (absorbance of blank well)} / {(absorbance of control well) - (absorbance of blank well)} × 100%.

[0053] 6. Western Blot Method: Prepare a 10% SDS-PAGE gel according to the instructions for the Seville protein gel kit. Prepare the electrophoresis apparatus, assemble the gel, and place it in the electrophoresis tank. Add the electrophoresis buffer. Gently remove the comb from the gel by hand, add the marker and protein sample to the wells, and turn on the power. Run the stacking gel at 80V and the separating gel at 120V. Wait until the bromophenol blue in the sample has run to the bottom of the gel, then turn off the power. After the electrophoresis is complete, cut a PVDF membrane to the appropriate size and activate it by soaking it in anhydrous methanol. Place a sponge on one side of a black transfer cassette, covering two layers of filter paper soaked in transfer buffer. Place the cut gel on the filter paper, then place the PVDF membrane on top of the gel. Place the filter paper and then the sponge on top, forming a "sandwich" structure. Place the "sandwich" in the transfer tank, connect the power supply, and transfer the membrane at a constant current of 200mA for 2 hours. After transfer, block the membrane with rapid blocking buffer for 30 minutes. Wash the membrane three times with PBST on a shaker for 10 minutes each. Add the primary antibody and incubate overnight on a shaker at 4°C. After incubation with the primary antibody, wash the membrane three times with PBST for 10 minutes each. Add the secondary antibody and incubate for 1 hour. After incubation with the secondary antibody, wash the membrane three times with PBST for 10 minutes each. Then, add the PVDF membrane to the developer and expose it on an imager to save the data.

[0054] 7. Determination of half-maximal inhibitory concentration: use 2.0×10 5Vero CCL-81 cells were plated at 1.0 x 105cell / mL into 12-well plates, 1 mL per well, and washed three times with PBS buffer when the cells were 80% confluent on the bottom, then inoculated with 0.1 MOI of PEDV and incubated in the cell incubator for 2 h, and then the virus liquid was discarded and replaced with virus maintenance liquid. At the same time as adding the maintenance liquid, the drug was added to the maintenance liquid in an arithmetic or geometric sequence concentration gradient, and after 12 h, the 12-well plates were frozen and thawed three times at -80°C, and the virus liquid was collected into centrifuge tubes, centrifuged at 8000 r / min for 10 min, and the supernatant was collected and titrated, and the virus titers at different concentrations were plotted using the non-linear fitting log(inhibitor) vs. normalized response - Variable slope module in GraphPad Prism.

[0055] 8. Compound screening method: 113 compounds that may have inhibitory effects on PEDV were compiled by literature review. 2.0 x 105 5 Vero CCL-81 cells were plated at 1.0 x 105cell / mL into 12-well plates, 1 mL per well, and washed three times with PBS buffer when the cells were 80% confluent on the bottom, then inoculated with 0.1 MOI of PEDV and incubated in the cell incubator for 2 h, and then the virus liquid was discarded and replaced with virus maintenance liquid. At the same time as adding the maintenance liquid, the drug was added to the maintenance liquid in an arithmetic or geometric sequence concentration gradient, and after 12 h, the 12-well plates were frozen and thawed three times at -80°C, and the virus liquid was collected into centrifuge tubes, centrifuged at 8000 r / min for 10 min, and the supernatant was collected and titrated, and the virus titers at different concentrations were plotted using the non-linear fitting log(inhibitor) vs. normalized response - Variable slope module in GraphPad Prism. 50 The virus titer of the sample was calculated using the Reed-Muench method, and the inhibition rate was calculated as {1-(experimental group titer / control group titer)} x 100%.

[0056] 9. Results analysis:

[0057] (1) PEDV virus proliferation curve determination: The virus amount at 12 h, 24 h, 36 h, and 48 h was determined and a growth curve was plotted, as shown in Figure 2 From the figure, it can be seen that the virus was in a plateau phase at 12-24 h, the virus titer curve was smooth, and the virus proliferation began to decrease after more than 24 h, and the virus titer decreased significantly after more than 36 h.

[0058] (2) Antiviral activity determination of 113 compounds: In order to screen out compounds that can inhibit PEDV from the 113 compounds in the compound library, a concentration of 10 μM of the compound was selected for treatment of cells infected with 0.1 MOI of PEDV, and the TCID 50 was measured at 12 h, and the results are shown in Figure 3 Of the 113 compounds, 33 had an inhibition effect on PEDV of more than 95%, as shown in Table 1. Figure 3 The compounds above the middle horizontal line have an inhibitory effect of more than 95%. From the screening results, it can be seen that the inhibitory effect of more than 95% accounts for 29.2%, and there are 75 compounds with an inhibitory effect of 80%, accounting for 66.37%, which is more than half of the total.

[0059] Table 1 Compounds with an inhibition rate of more than 95% on PEDV

[0060]

[0061] (3) Vero CCL-81 cytotoxicity assay: In the aforementioned study, we screened out 33 compounds that could inhibit PEDV by more than 95%. Based on the visual observation of cell apoptosis during screening and the cost of compound synthesis, this study screened out five compounds, namely, NO.4, NO.43, NO.51, NO.57, and NO.112, and measured the cytotoxicity of these five compounds on Vero CCL-81 cells.

[0062] (4) Then, NO.4, NO.43, NO.51, NO.57, and NO.112 were diluted in multiple ratios and acted on Vero CCL-81 cells for 24 h. The cell activity of the compounds was then determined using a CCK-8 kit. The results were as follows: Figure 4 As shown, Figure 4 Middle: Compounds No. 4 and No. 43 had no significant cytotoxicity at 170 μM and 60 μM, and the CC of compound No. 51 was 50 The CC of 18.92μM and NO.57 50 The CC of 66.61μM and NO.112 50 The result is 11.47 μM. Based on the consideration of synthesis cost, cytotoxicity and development potential, this study finally selected NO.57 Talnetant (hereinafter referred to as TA) for subsequent research. The chemical formula of this compound is as follows:

[0063]

[0064] (5) Determination of the half-maximal inhibitory concentration of Talnetant: In order to determine the half-maximal inhibitory concentration of Talnetant against PEDV, this study performed a gradient dilution of the compound, with Talnetant concentrations of 0.27μM, 1.09μM, 4.37μM, 17.5μM, and 70μM. The compound was added while PEDV infected Vero CCL-81 cells. After two hours of incubation of cells and viruses, the solution was discarded and the PEDV maintenance solution with the same compound concentration was added. DMSO was set as a negative control. Two wells were repeated for each concentration gradient. Samples were collected 12 hours later and TCID was measured. 50and Western Blot, the results are shown in Figure 5 Figure 5 As shown in A, the IC 50 of Talnetant is 2.57 μM, from Figure 5 B, the Western Blot results show that the inhibition of Talnetant on the expression of PEDV N protein is dose-dependent, the higher the concentration of Talnetant, the lower the expression of N protein.

[0065] (6) Selective index of Talnetant: the selective index (SI) of a drug is an important indicator for evaluating the potential of drug development, which is the ratio of CC 50 to IC 50 , the larger the ratio, the stronger the antiviral efficacy of the drug, and the specific results are shown in Table 2:

[0066] Table 2 Selective index of Talnetant

[0067]

[0068] As can be seen from Table 2, the SI index of Talnetant is 25.92.

[0069] Example 2:

[0070] This example studies the mechanism of Talnetant inhibiting PEDV replication:

[0071] 1. Delayed administration experiment: 2.0 x 10 5 cell / mL of Vero CCL-81 cells were plated into a 12-well plate, 1 mL per well, and when the cells were 80% full, they were washed three times with PBS buffer, inoculated with 0.1 MOI of PEDV, and incubated in a cell incubator for 2 h. After 2 h, the virus solution was discarded and the virus maintenance solution was added. 800 μL of supernatant was taken out from the well at 0 h, 1 h, 3 h, 5 h and 7 h, and 38.1 μM concentration (38.1 μM is the CC 80 of Talnetant) of Talnetant was vortexed and mixed. After 12 h, the 12-well plate was frozen and thawed three times at -80℃, and the virus solution was collected into a centrifuge tube. After centrifugation at 8000 r / min for 10 min, the supernatant was collected and the titer was determined.

[0072] 2. Effect of compound on adsorption stage of PEDV: 2.0 x 10 5 ​Vero CCL-81 cells were plated at 1 mL / well in 12-well plates. When the cells reached 80% confluency, the plates were washed three times with pre-chilled PBS at 4°C. A pre-chilled mixture of 0.1 μM IPEDV and 38.1 μM Talnetant was then added to the plates. A DMSO group was also included as a negative control. The plates were incubated at 4°C for 1 hour. After incubation, the viral suspension was discarded and any unbound virus was washed with pre-chilled PBS. RNA was extracted to measure PEDV M protein mRNA expression. Primers for PEDV M protein mRNA were used: F: 5'-GGTTGCTACTGGCGTACAGGTA-3'; R: 5'-GAAGCATTGACTGAACG ACCAACA-3'. A standard curve was constructed using RNA extracted from samples of known toxicity. The standard curve was used to determine the values ​​for the experimental groups. The results were analyzed for significance. P < 0.05 was marked with *, indicating a significant difference; P < 0.01 was marked with **, indicating an extremely significant difference; and P < 0.001 was marked with ***, indicating a very significant difference. Graphs were generated using GraphPad Prism.

[0073] 3. Effect of compounds on the invasion stage of PEDV: One day before the experiment, 2.0×10 5 Vero CCL-81 cells (100 μg / mL) were plated onto 12-well plates. When the cells reached 80% growth, they were washed three times with 4°C pre-chilled PBS buffer. PEDV at a 0.1 MOI was added to the cells and incubated at 4°C for 1 hour. Unbound virions were then washed away with pre-chilled PBS buffer. Virus maintenance solution containing 38.1 μM talnetant was then added to the treated cells. A DMSO group was also included as a negative control, and the cells were incubated in a 37°C cell culture incubator for 2 hours. After incubation, free virions were washed away with PBS buffer, and total RNA was extracted and analyzed for PEDV mRNA expression using RT-qPCR.

[0074] Effect of Compounds on PEDV Replication: 12-well plates of Vero CCL-81 cells, reaching 80% confluence, were washed three times with PBS buffer and inoculated with 0.1 μM PEDV virus. The cells were incubated in a 37°C cell culture incubator for 2 hours. After incubation, the virus solution was discarded and supplemented with virus maintenance medium. 38.1 μM talnetant was added to the supplemented maintenance medium. DMSO was used as a negative control. Four replicates were performed for each group. Protein and virus samples were collected at 12 and 24 hours, and PEDV N protein expression and M protein mRNA expression were analyzed by Western blotting and qPCR, respectively.

[0075] 5. Detection of half-maximal inhibitory concentration of PDCoV: using 2×10 5LLC-PK1 cells were plated in 12-well plates at a concentration of 1 cell / mL. The cells were washed three times with PBS buffer and infected with PDCoV at a 0.1 MOI for 2 hours. The solution was then discarded and replaced with virus maintenance medium. Talnetant was added in serial dilutions simultaneously with the virus maintenance medium, with DMSO as a negative control. After 24 hours, the virus titers were collected and measured. The half-maximal inhibitory concentration (50%) of the compound was calculated using GraphPad Prism based on the titers at each concentration gradient.

[0076] 6. PDCoV titer determination: 2.2×10 5 LLC-PK1 cells were plated in 96-well plates at a concentration of 10 cells / mL. When the cells grew to 80%, the cells were washed three times with PBS buffer. The virus to be tested was diluted with virus maintenance solution at a tenfold ratio of 10:1. -1 Serial dilution to 10 -8 Each concentration gradient was set up with 8 replicate wells, and each 96-well plate was set up with a column of negative control with only maintenance solution added. -8 To 10 -1 Add virus dilution. Observe the CPE of each dilution every day, and finally calculate the TCID of the virus using the Reed-Muench method based on the CPE of each dilution. 50 .

[0077] 7. Experimental results:

[0078] (1) The inhibitory effect of delayed administration on PEDV: Figure 6 As shown in the figure: the inhibitory effect of the compound on PEDV gradually decreases with the virus infection time, but the compound has an inhibitory effect of more than 80% at the latest time point, indicating that Talnetant has a good inhibitory effect on PEDV within 7 hours.

[0079] (2) Effect of Talnetant on the adsorption stage: In this study, 38.1 μM Talnetant and 0.1 MOIPEDV were mixed and added to Vero CCL-81 cells grown to 80% of the monolayer cells and treated at 4°C for 1 hour. The negative control group was added with 1‰ DMSO. After 1 hour, the supernatant was discarded and the cells were washed three times with PBS buffer to wash away the unbound virus particles on the cells. RNA was extracted from the treated cells and the level of PEDV M protein mRNA was detected by RT-qPCR. The results are shown in Figure 2. Figure 7 The PEDV genome copy number in the Talnetant group was not significantly different from that in the DMSO group, indicating that Talnetant had no inhibitory effect on the virus adsorption stage.

[0080] (3) Effect of Talnetant on the invasion stage: Vero CCL-81 cells were infected with 0.1 MOI of PEDV and incubated at 4°C for 1 hour. After incubation, unbound virus particles were washed away. The washed cells were added with virus maintenance solution containing 38.1 μM Talnetant and incubated in a cell culture incubator for 2 hours. The control group was treated with DMSO and an equal amount of virus. The supernatant was discarded and the virus that did not enter the cells was washed away with PBS buffer. Total RNA was extracted and the level of PEDV M protein mRNA was detected by RT-qPCR. The results are as follows: Figure 8 During the virus invasion phase, the number of copies of the PEDV M protein showed no inhibitory effect in the DMSO group or the Talnetant group, proving that Talnetant's inhibition of PEDV was not during the invasion phase.

[0081] (4) Effect of Talnetant on the replication stage: PEDV-infected cells were treated with 38.1 μM Talnetant for 12 h and 24 h, and DMSO was used as a negative control. The virus titer and PEDV N protein expression levels were analyzed, and RNA was extracted. The level of PEDV M protein mRNA was detected by RT-qPCR. The results are as follows: Figure 9 and Figure 10 As shown. Figure 9 and Figure 10 As can be seen, Talnetant showed significant inhibition of PEDV virus titer and mRNA levels during the replication phase, and N protein expression was significantly reduced compared to the DMSO group. Therefore, we speculate that the inhibitory effect of the compound Talnetant on PEDV is most likely during the replication phase.

[0082] (5) Cytotoxicity test of Talnetant on LLC-PK1: This study investigated the significant inhibitory effect of Talnetant on PEDV. PDCoV is a coronavirus that is also a serious threat to the pig farming economy. This experiment selected PDCoV as the experimental object. The cytotoxicity of the drug on LLC-PK1 was measured, and the results showed that ( Figure 11 ):Within the concentration of 100 μM, the activity of LLC-PK1 cells treated with Talnetant remained above 50%.

[0083] (6) Talnetant's inhibitory effect on PDCoV (Porcine deltacoronavirus, PDCoV): In order to explore the inhibitory effect of Talnetant on other coronaviruses, this study used the PDCoV virus isolated in the laboratory as the research object to verify the broad-spectrum antiviral effect of Talnetan. After the LLC-PK1 cells grown to 80% were washed three times with PBS buffer, 0.1MOI of PDCoV was added and incubated in a 37°C cell culture incubator for 2 hours, and then the virus solution was discarded and supplemented with virus maintenance solution. Talnetant was added at the same time as the virus maintenance solution was supplemented. The concentration gradient was 4-fold dilution of Talnetant starting from 100μM. After 24 hours, samples were collected to measure the virus titer. The results are as follows: Figure 12 As shown: Talnetant inhibits 50% at a concentration of 6.4 μM, indicating that the compound has an inhibitory effect on PDCoV.

[0084] (7) Molecular docking prediction of possible targets: In order to further explore the inhibitory mechanism of Talnetant on PEDV, this study chose to use molecular docking to predict. pro This study used AutoDock to evaluate the docking score, and the score of Talnetant was 8.4. The Glide module was used to simulate molecular docking, and pymol was used to visualize the molecular docking. Figure 13 Shown: Compounds for 3CL pro Therefore, it can be predicted that the inhibition of PEDV by Talnetant is through the inhibition of 3CL pro To block the cleavage of polyprotein and thus inhibit viral replication.

[0085] Example 3:

[0086] To further study the antiviral activity of the compound Talnetant, referring to the method of Example 2, the applicant conducted antiviral experiments on different viruses: porcine reproductive and respiratory syndrome virus (PRRSV) and avian influenza virus (AIV). It was found that the compound Talnetant had no inhibitory effect on these two viruses, PRRSV and AIV.

[0087] Example 4:

[0088] This example studies the inhibitory effect of the compound Talnetant on common animal pathogens, as follows:

[0089] 1. Pathogens: Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Shigella flexneri, and Pseudomonas stutzeri were used as test strains for antimicrobial activity experiments. Escherichia coli, Staphylococcus aureus, and Pseudomonas stutzeri were cultured in MHA medium at 28°C, while Bacillus subtilis and Shigella flexneri were cultured in 2216E medium at 37°C.

[0090] 2. The method for determining antibacterial activity is as follows: After activating the bacterial strain, prepare a bacterial suspension, and then dilute the compound Talnetant in a gradient manner: the concentrations are 0.27μM, 1.09μM, 4.37μM, 17.5μM, and 70μM. Design experimental groups, blank control groups, and negative control groups for the experiment, with 3 replicates for each compound concentration. Culture in 96-well cell culture plates for 24 hours and observe the experimental results. The reagents added to each group are as follows:

[0091] Experimental group: 50 μL compound Talnetant solution + 50 μL bacterial suspension;

[0092] Blank control: 50 μL sterile water + 50 μL bacterial suspension;

[0093] Negative control: 50 μL working medium + 50 μL of the lowest concentration of compound Talnetant.

[0094] 3. Determination of Minimum Bactericidal Concentration (MBC): Pipette 1 μL of the mixed culture from each well of the incubated cell culture plate and drop it onto an MHA or 2216E plate. Incubate for 24 hours and observe colony formation. The specific test results are shown in Table 3:

[0095] Table 3 Talnetant activity test results of compounds

[0096]

[0097] As can be seen from Table 3, the results show that the compound Talnetant has antibacterial activity against Escherichia coli, Staphylococcus aureus and Shigella flexneri, but has no inhibitory effect on Bacillus subtilis and Pseudomonas stutzeri. The minimum inhibitory concentrations for Escherichia coli and Staphylococcus aureus are both 0.27-1.09 μM, and the minimum bactericidal concentrations are both 1.09-4.37 μM; the minimum inhibitory concentration for Shigella flexneri is 4.37-17.5 μM, and the minimum bactericidal concentration is 4.37-17.5 μM.

[0098] In summary, this application selected 113 compounds that may have an inhibitory effect on PEDV to form a small compound library. The compound Talnetant with an inhibitory effect on PEDV was selected by a high-throughput method. The results showed that the half-maximal inhibitory concentration (IC 50 ) was 2.57μM, and the selectivity index (SI) was 25.92; the results of the delayed administration experiment showed that the inhibitory effect of Talnetant on PEDV gradually decreased with the delay of administration time, but when the drug was used within 7 hours after infection with the virus, Talnetant had an inhibitory effect on PEDV of more than 80%. This study then explored the inhibitory effect of the compound on the adsorption, invasion, and replication stages of PEDV, and found that Talnetant did not show obvious inhibitory effect in the virus adsorption and invasion stages, but in the virus replication stage, Talnetant, indicating that Talnetant's inhibition on PEDV may occur in the PEDV replication stage. In addition, the results of this study also showed that: Talnetant's IC for PDCoV 50 The concentration of Talnetant was 6.4 μM. It had no inhibitory effect on PRRSV and AIV, indicating that Talnetant has a certain broad-spectrum antiviral potential against coronaviruses. It also has inhibitory effects on common animal pathogens: Escherichia coli, Staphylococcus aureus and Shigella flexneri, indicating that the compound Talnetant of the present application has good antiviral and antibacterial effects.

[0099] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. The use of the compound Talnetant in the preparation of anti-coronavirus drugs, characterized in that: The structural formula of the compound Talnetant is as follows:

2. The use according to claim 1, characterized in that The coronavirus is porcine deltacoronavirus and / or porcine epidemic diarrhea virus.

3. The use according to claim 1, characterized in that The half-maximal inhibition concentration of the talnetant against porcine epidemic diarrhea virus is 2.57 μM, and the selection index is 25.

92.

4. The use according to claim 1, characterized in that The half-maximal inhibitory concentration of the Talnetant against Porcine deltacoronavirus is 6.4 μM.

5. The use according to claim 1, characterized in that The talnetant inhibits the viral 3C-like protease of PEDV.

6. A method for screening the compound Talnetant according to claim 1, characterized in that: The method comprises the following steps: screening 113 compounds in a compound library to identify compounds that have an inhibitory effect on porcine epidemic diarrhea virus; treating cells infected with 0.1 MOI porcine epidemic diarrhea virus with a compound concentration of 10 μM; collecting samples 12 hours later to measure the TCID 50 .

7. Use of the compound Talnetant in the preparation of antibacterial drugs, characterized in that: The structural formula of the compound Talnetant is as follows:

8. The use according to claim 7, characterized in that The bacteria inhibited by the antibacterial drug are Escherichia coli, Staphylococcus aureus and / or Shigella flexneri.

Images