Compound Zafirlukas and obtaining method and application thereof

Abstract

The invention relates to the technical field of new application of a compound Zafirlukas, in particular to a compound Zafirlukas and an obtaining method and application thereof.According to the compound Zafirlukas and the obtaining method and application thereof, a high-throughput screening technology is applied, 113 types of compound libraries which possibly have the inhibition effect on PEDV are screened, the compound Zafirlukas with the good antiviral effect is obtained, and test results show that the Zafirlukas has the good inhibition effect on PEDV and PDCoV, and the compound Zafirlukas can be used for preparing the compound Zafirlukas with the good antiviral effect on PEDV and PDCoV. The compound has broad-spectrum anti-virus capability on coronavirus, Zafirlukas has more than 90% of inhibition effect on PEDV (porcine epidemic diarrhea virus) after being used within 7 hours after virus infection, and in addition, the compound also has inhibition effect on common pathogenic bacteria of animals, such as escherichia coli, bacillus subtilis and shigella freundii, so that the compound Zafirlukas has good anti-virus and antibacterial effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of new uses of compound Zafirlukast, in particular to compound Zafirlukast, its obtaining method and application. BACKGROUND

[0002] Zafirlukast is also known as Zafirlukast in Chinese, with chemical formula of C 31 H 33 N3O6S, and a molecular weight of 575.68. The prior art found that cysteinyl leukotrienes (CysLTR) is a lipid metabolite of arachidonic acid produced by 5-lipoxygenase catalysis. In the process of 5-lipoxygenase catalysis, the intermediate product leukotriene A4 (LTA4) can produce LTC4, LTD4, LTE4 through two metabolic pathways. LTC4, LTD4, LTE4 all have cysteine at the sixth position, and cysteinyl leukotrienes are named accordingly. Zafirlukast can inhibit inflammation mediated by CysLTs, so it is widely used in asthma and allergic rhinitis. In addition, Zafirlukast has also been reported to be a new type of TI inhibitor, which has an anti-thrombosis effect and does not cause bleeding. In the aspect of anti-virus, the prior art shows that Zafirlukast can participate in the immune blockage of the TNF signaling pathway and its downstream signaling pathway (such as MAPK and ERK1 / 2) of two viruses of flavivirus genus, Zika virus (ZIKV) and dengue virus (DENV), and also offsets the changes of key genes involved in cell lipid metabolism induced by ZIKV, which makes Zafirlukast show potential for the development of therapeutic drugs for ZIKV and DENV. However, there is no research on the related aspects of compound Zafirlukast in inhibiting diarrhea virus and bacteria.

[0003] Porcine epidemic diarrhea (PED) is a zoonotic disease caused primarily by porcine epidemic diarrhea virus (PEDV). Currently, the PED situation is complex, and clinically, it is difficult to distinguish between single infections or mixed infections caused by TGEV, PoRV, PDCoV, SADS-CoV, or PEDV. Therefore, prevention and control efforts prioritize accurate detection and timely isolation. Vaccines are also a key tool for PED prevention and control, but the rapid mutation of viruses presents significant challenges in vaccine development and updating. Furthermore, in addition to viral infections, certain bacteria, such as Escherichia coli and Staphylococcus aureus, can also contaminate the pig intestine and cause diarrhea. Therefore, to effectively prevent and control PED, drug research is necessary to develop compounds that effectively inhibit PED-related viruses and control a broad spectrum of bacteria, including Escherichia coli and Staphylococcus aureus. [Summary of the invention]

[0004] In view of the above, it is necessary to develop relevant drugs for porcine epidemic diarrhea, which can effectively inhibit the growth of porcine epidemic diarrhea-related viruses, and at the same time effectively prevent and control a wider spectrum of related compounds such as Escherichia coli and Staphylococcus aureus.

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

[0006] The use of the compound Zafirlukas in the preparation of anti-coronavirus drugs, the structural formula of the compound Zafirlukas 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 Zafirlukas against porcine epidemic diarrhea virus is 0.74 μM, and the selectivity index is 15.5.

[0010] Furthermore, the half-maximal inhibitory concentration of Zafirlukas against Porcine deltacoronavirus is 0.39 μM.

[0011] Furthermore, the Zafirlukas inhibits 3CL of PEDV pro .

[0012] A method for screening the compound Zafirlukas, 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 with a compound concentration of 10 μM; collecting samples 12 hours later and measuring TCID 50 .

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

[0014]

[0015] Furthermore, the bacteria inhibited by the antibacterial drug are Escherichia coli, Bacillus subtilis 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 an inhibitory effect on PEDV, and obtains Zafirlukas, a compound with good antiviral effect. The test results show that Zafirlukas has a good inhibitory effect on PEDV and PDCoV, indicating that the compound Zafirlukas has a broad-spectrum antiviral ability against coronaviruses. After research, the half-maximal inhibitory concentration (IC50) of the compound against PEDV is 50 ) was 0.74 μM, the selectivity index (SI) was 15.5, and the IC 50The inhibitory effect of Zafirlukas on PEDV was 0.39 μM. Delayed dosing experiments showed that the inhibitory effect of Zafirlukas on PEDV gradually decreased with delayed dosing. However, when administered within 7 hours of infection, Zafirlukas exhibited over 80% inhibition of PEDV. Furthermore, the present application investigated the compound's inhibitory effects on PEDV adsorption, invasion, and replication, finding that Zafirlukas'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 that the compound Zafirlukas exhibits excellent antiviral and antibacterial properties.

Brief Description of the Drawings

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

[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 1 shows the inhibitory effect of different concentrations of the compound Zafirlukas on PEDV; Figure A shows the inhibitory effect of different concentrations of Zafirlukas on the ability of PEDV to infect cells; Figure B shows the effect of different concentrations of Zafirlukas on the expression ability of PEDVN protein.

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

[0024] Figure 7 The effect of compound Zafirlukas on the adsorption stage of PEDV.

[0025] Figure 8 The effect of compound Zafirlukas on the invasion stage of PEDV.

[0026] Figure 9The effect of compound Zafirlukas on the replication stage of PEDV; 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 PEDVM protein at 12 h and 24 h after virus incubation, where the left graph is for 12 h and the right graph is for 24 h.

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

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

[0030] Figure 13 Compound Zafirlukas and PEDV3CL pro Molecular docking diagram of Zafirlukas and PEDV3CL; A is the molecular docking diagram of Zafirlukas and PEDV3CL pro Molecular docking 2D diagram of Zafirlukas and PEDV 3CL; Figure B shows the molecular docking of Zafirlukas 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 detection reagent (CCK-8) was purchased from Abbkine; protease inhibitor tablets (A32965) were purchased from Thermo Fisher; WB protein-free blocking buffer (1% in 1× PBST) and sensitive ECL luminescent solution were both 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. The compound library was purchased from Med Chemexpress Biotechnology, Inc., USA.

[0038] 4. Main reagent formula:

[0039] Complete medium: 50 mL FBS, 10 mL streptomycin-azidin mixture, 5 mL glutamine, 450 mL DMEM mixed and placed at 4°C for standby.

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

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

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

[0043] Transfer membrane buffer: Tris base 2.9 g, glycine 1.45 g, constant volume to 400 mL with double distilled water, add 100 mL anhydrous methanol before use, mix thoroughly and store at low temperature.

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

[0045] PBST: 1× PBS buffer 500 mL, 250 μL Tween-20 mixed.

[0046] Protein lysate: 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 method

[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%, the cells were washed three times with PBS, the virus solution to be tested was diluted 10-fold with virus maintenance solution to make 10 -1 -10 -6 gradients, 8 replicate wells were made for each concentration gradient, and a negative control with only virus maintenance solution was made for each 96-well plate. 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 were produced, 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 at 1.0 x 105cells / mL to 96-well plates in 100 μL per well, and after the cells grew to 80%, the cells were washed with PBS for three times, and the compounds were diluted with virus maintenance solution, 6 replicates for each concentration, and set the blank control wells with virus maintenance solution without cells. After 24 h, the cells were washed with PBS for three times, and the in vitro cytotoxicity of the compounds was evaluated according to the procedure of the Cell Counting Kit-8 (CCK-8), because the highly water-soluble tetrazolium salt WST-8 in CCK-8 can be reduced to water-soluble orange formazan by dehydrogenase in cells under the action of an electron mediator, and the number of living cells can be indirectly determined by measuring the absorbance at 450 nm with a microplate reader. The cytotoxicity was calculated according to the following formula: Cytotoxicity (%) = {(absorbance of experimental wells) - (absorbance of blank wells)} / { (absorbance of control wells) - (absorbance of blank wells)} x 100%.

[0053] 6. Western Blot experimental method: 10% SDS-PAGE protein gel was prepared according to the instructions of the Sivier protein gel kit, and the electrophoresis device was prepared. The protein gel was assembled into the electrophoresis tank, and the electrophoresis liquid was added. The comb on the protein gel was gently pulled out by hand, and the marker and protein sample were added to the well after the power was started. The concentrated gel was electrophoresed at 80V, and the separation gel was electrophoresed at 120V. When the bromophenol blue in the sample reached the bottom of the protein gel, the power was turned off. After electrophoresis, the PVDF membrane of appropriate size was cut and soaked with anhydrous methanol to activate the PVDF membrane. A layer of sponge was placed on one side of the black membrane transfer clamp, covered with two layers of filter paper soaked with transfer solution, and then the cut protein gel was placed on the filter paper, and the PVDF membrane was covered on the gel, followed by filter paper, sponge, forming a "sandwich" structure. The "sandwich" was placed in the membrane transfer tank and the power was turned on. The constant current transfer was 200mA for 2h. After the transfer was completed, the rapid blocking solution was blocked for 30min. After blocking, the PBST was washed on the shaker for three times, 10min each time. After washing, the primary antibody was added and incubated overnight on the shaker at 4°C. After incubation of the primary antibody, the PBST was washed for three times, 10min each time, and the secondary antibody was added and incubated for 1h. After incubation of the secondary antibody, the PBST was washed for three times, 10min each time, and then the PVDF membrane was added to the developing solution and exposed to the exposure machine. The data was saved.

[0054] 7. Half maximal inhibitory concentration determination: 2.0 x 10 5Vero CCL 81 cells at a concentration of 1 mL / well were plated into 12-well plates. When the cells reached 80% of the bottom, the plates were washed three times with PBS buffer. The plates were then inoculated with PEDV at a 0.1 MOI and incubated in a cell culture incubator for 2 hours. After 2 hours, the virus solution was discarded and replaced with virus maintenance solution. While adding maintenance solution, the drug was added to the maintenance solution in a geometric or arithmetic progression. After 12 hours, the 12-well plates were frozen and thawed three times at -80°C. The virus solution was collected into a centrifuge tube and centrifuged at 8000 rpm for 10 minutes. The supernatant was collected and the virus titer was measured. The nonlinear fitting log(inhibitor) vs. normalized response - Variableslope module in GraphPad Prism was used to plot the virus titer at different concentrations.

[0055] 8. Compound screening method: Through literature review, this study sorted out 113 compounds that may have inhibitory effects on PEDV. 5 Vero CCL 81 cells with a concentration of 1 cell / mL were plated into 12-well plates, with 500 μL of cell suspension per well. After the cells grew to 80%, they were washed three times with PBS buffer and a mixture of 0.1 MOI of virus and 10 μM was added. The mixture was diluted with PEDV maintenance medium. The virus and compound mixture was incubated on the cells for 2 hours, the virus solution was discarded, and the same concentration of compound diluted with PEDV maintenance medium was added. After 12 hours of culture, samples were collected and TCID was measured. 50 The toxicity of the samples was calculated using the Reed-Muench method: inhibition rate = {1-(toxicity of the experimental group / toxicity of the control group)} × 100%.

[0056] 9. Result analysis:

[0057] (1) PEDV virus proliferation curve determination: The virus amount at 12h, 24h, 36h and 48h was measured and the growth curve was drawn. The results are as follows Figure 2 As shown in the figure, the virus is in the plateau phase from 12 to 24 hours, and the virus titer curve is stable. After the virus proliferates for more than 24 hours, it begins to show a downward trend. When the virus proliferates for more than 36 hours, the virus titer decreases significantly.

[0058] (2) Determination of the antiviral activity of 113 compounds: In order to screen out compounds that can inhibit PEDV from the 113 compounds in the compound library, a compound concentration of 10 μM was used to treat 0.1 MOIPEDV-infected cells during the screening process, and samples were collected 12 hours later to measure the TCID 50 , the results are as follows Figure 3 Among these 113 compounds, 33 showed an inhibitory effect of more than 95% on PEDV, as shown in Table 1. Figure 3 The compounds above the middle horizontal line are compounds with an inhibition effect of 95% or more. According to the screening results, the proportion of compounds with an inhibition effect of 95% or more is 29.2%, and there are 75 compounds with an inhibition effect of 80%, accounting for 66.37%, which is more than half of the total number.

[0059] Table 1 Compounds with PEDV inhibition rate greater than 95%

[0060]

[0061] (3) Vero CCL 81 cell toxicity assay: In the previous study, we screened a total of 33 compounds that can inhibit PEDV by 95% or more. Based on the visual cell apoptosis during screening and the cost of synthesizing the compounds, we selected NO. 4, NO. 43, NO. 51, NO. 57, and NO. 112 for this study. The cell toxicity of these five compounds on Vero CCL-81 cells was determined.

[0062] (4) Then dilute NO. 4, NO. 43, NO. 51, NO. 57, and NO. 112 by a certain ratio and apply them to Vero CCL81 cells for 24 hours. Then use the CCK-8 kit to determine the cell activity of the compounds. The results are shown in Figure 4 Figure 4 The CC 50 50 of compound NO. 51 is 18.92 μM, the CC 50 50 of compound NO. 57 is 66.61 μM, and the CC50 of compound NO. 112 is 11.47 μM. Based on the cost of synthesis, cell toxicity, and development potential, we finally selected NO. 112 Zafirlukas (hereinafter referred to as ZA) for further study. The chemical formula of the compound is as follows:

[0063]

[0064] (5) Determination of the half-inhibitory concentration of Zafirlukas: To determine the half-inhibitory concentration of Zafirlukas on PEDV, we performed gradient dilution of the compound. The concentration of Zafirlukas was 0.047 μM, 0.19 μM, 0.75 μM, 3 μM, and 12 μM. The compound was added when the Vero CCL81 was infected with PEDV. After two hours of incubation of the cells and virus, the liquid was discarded, and the same concentration of PEDV maintenance solution was added. DMSO was set as the negative control. Each concentration gradient was repeated twice. The samples were collected at 12 hours to measure the TCID 50 ​and Western Blot, the results are as follows Figure 5 Shown: From Figure 5 As can be seen in A, Zafirlukas' IC 50 The concentration was 0.74 μM, from Figure 5 The Western Blot results in B show that Zafirlukast inhibits PEDV in that the expression level of N protein remains almost unchanged before the Zafirlukast concentration reaches 12 μM, and the expression level of N protein decreases after 12 μM.

[0065] (6) Zafirlukas’ Selective Index: The Selective Index (SI) of a drug is an important indicator for evaluating the potential of drug development. 50 and IC 50 The larger the ratio, the stronger the antiviral effect of the drug. The specific results are shown in Table 2:

[0066] Table 2 Zafirlukas's selection index

[0067]

[0068] As can be seen from Table 2, the SI index of Zafirlukas is 15.5.

[0069] Example 2:

[0070] This example studies the mechanism by which Zafirlukas inhibits PEDV replication:

[0071] 1. Delayed drug administration experiment: 2.0×10 5 Vero CCL81 cells with a concentration of 100 cells / mL were plated into 12-well plates, with 1 mL per well. When the cells covered 80% of the bottom, they were washed three times with PBS buffer and inoculated with 0.1 MOI of PEDV. The cells were incubated in a cell culture incubator for 2 hours. After 2 hours, the virus solution was discarded and the virus maintenance solution was added. Zafirlukas was removed from the wells at 0h, 1h, 3h, 5h and 7h. 800 μL of supernatant was transferred to a 1.5 mL centrifuge tube and added to a 10.31 μM concentration (10.31 μM is Zafirlukas CC) 80 After 12 hours, the 12-well plate was placed at -80°C and frozen and thawed three times. The virus solution was collected into a centrifuge tube and centrifuged at 8000 rpm for 10 minutes. The supernatant was collected and the virus titer was measured.

[0072] 2. Effect of compounds on the adsorption stage of PEDV: Using 2.0×10 5Vero CCL81 cells were plated into 12-well plates at a concentration of 1 mL per well. When the cells reached 80% confluency, the cells were washed three times with 4°C pre-chilled PBS buffer. A pre-chilled mixture of 0.1 μM IPEDV and 10.31 μM Zafirlukas was then added to the 12-well 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 buffer. RNA was then extracted to detect PEDV M protein mRNA expression. Primers for PEDV M protein mRNA were F: 5'-GGTTGCTACTGGCGTACAGGTA-3'; R: 5'-GAAGCATTGACTGAACGACCAACA-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 VeroCCL81 cells (100 μg / mL) were plated into 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 10.31 μM Zafirlukas was then added to the treated cells. A DMSO group was also included as a negative control. 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 M protein mRNA expression using RT-qPCR.

[0074] Effect of Compounds on PEDV Replication: 12-well plates of Vero CCL81 cells, grown to 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 the virus maintenance medium was replenished. 10.31 μM Zafirlukas was added to the 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 2×105 LLC-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. Zafirlukas was added in serial dilutions simultaneously with the virus maintenance medium. DMSO was also included 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 decreased with the virus infection time, but the compound had an inhibitory effect of more than 80% at the latest time point, indicating that Zafirlukas had a good inhibitory effect on PEDV within 7 hours.

[0079] (2) Effect of Zafirlukas on the adsorption stage: In this study, 10.31 μM Zafirlukas and 0.1 MOIPEDV were mixed and added to Vero CCL81 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 PEDVM protein mRNA was detected by RT-qPCR. The results are shown in Figure 2. Figure 7 The PEDV genome copy number in the Zafirlukast group was significantly lower than that in the control group, indicating that Zafirlukast inhibited the virus adsorption stage.

[0080] (3) Effect of Zafirlukas on the invasion stage: Vero CCL81 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 10.31 μM Zafirlukas 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 As shown in the figure, during the virus invasion phase, the copy number of PEDV M protein showed no inhibitory effect in the DMSO group and the Zafirlukas group, indicating that Zafirlukas's inhibition of PEDV was not at the invasion phase.

[0081] (4) Effect of Zafirlukas on the replication stage: PEDV-infected cells were treated with 10.31 μM Zafirlukas 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, Zafirlukas showed significant inhibition of PEDV virus titers and mRNA levels during the replication phase, with N protein expression significantly reduced compared to the DMSO group. Therefore, we speculate that the inhibitory effect of Zafirlukas on PEDV is most likely during the replication phase.

[0082] (5) Cytotoxicity assay of Zafirlukas against LLC-PK1: This study investigated the significant inhibitory effect of Zafirlukas against PEDV. PDCoV is a porcine enterovirus that is a coronavirus that seriously harms the pig farming economy. This study selected PDCoV as the experimental subject. The cytotoxicity of the drug against LLC-PK1 was determined, and the results showed that ( Figure 11 ):CC of LLC-PK1 cells treated with Zafirlukas at a concentration of 100 μM 50 At 24.78 μM.

[0083] (6) Inhibition of PDCoV (Porcine deltacoronavirus, PDCoV) by Zafirlukas: To explore the inhibitory effect of Zafirlukas on other coronaviruses, the laboratory isolated PDCoV virus was used as the research object to verify the broad-spectrum antiviral effect of Zafirlukas. The LLC-PK1 cells grown to 80% were washed three times with PBS buffer, then 0.1 MOI of PDCoV was added and incubated at 37°C in a cell incubator for 2h, then the virus solution was discarded and supplemented with virus maintenance solution. While supplementing the virus maintenance solution, Zafirlukas was added at a concentration gradient of 4-fold dilution from 100 μM. After 24h, the virus titer was measured, and the results are shown in Figure 12 : The 50% inhibition concentration of Zafirlukas was 0.39 μM, indicating that the compound had an inhibitory effect on PDCoV.

[0084] (7) Prediction of possible target points by molecular docking: To further explore the inhibitory mechanism of Zafirlukas on PEDV, the study used molecular docking to predict. Because 3CL pro plays an important role in the replication of PEDV, the study used AutoDock to score and evaluate the docking, with a score of 8.1 for Zafirlukas. Then the study used Schrodinger Glide module to simulate molecular docking, and finally used pymol for molecular docking visualization. The results are shown in Figure 13 : The compound has binding activity to the 41st histidine of 3CL pro . Therefore, it can be predicted that the inhibition of Zafirlukas on PEDV is through the inhibition of 3CL pro of PEDV to block the cleavage of polyprotein and thus inhibit viral replication.

[0085] Example 3:

[0086] Previous studies have shown that the compound Zafirlukas has a therapeutic effect on bronchitis, therefore, the research group selected Porcine reproductive and respiratory syndrome virus (PRRSV) and Avian influenza virus (AIV) for antiviral experiments, and determined the antiviral activity according to the method of Example 2. The results showed that the compound Zafirlukas had no inhibitory effect on PRRSV and AIV, indicating that although it is the same or similar disease, the inhibitory effect of the compound on different viruses is not consistent, and the drug has high specificity for viral strains.

[0087] Example 4:

[0088] This example studies the inhibitory effect of compound Zafirlukas on common animal pathogenic bacteria that cause diarrhea, as follows:

[0089] 1. Pathogenic bacteria: Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Shigella flexneri, and Pseudomonas stutzeri were used as test bacteria for antibacterial activity experiments. Among them, Escherichia coli, Staphylococcus aureus, and Pseudomonas stutzeri were cultured at 28°C using MHA medium, and Bacillus subtilis and Shigella flexneri were cultured at 37°C using 2216E medium.

[0090] 2. The method for determining antibacterial activity is as follows: After the bacteria are activated, a bacterial suspension is prepared, and then compound Zafirlukas is gradient-diluted: concentrations of 0.047 μM, 0.19 μM, 0.75 μM, 3 μM, and 12 μM are prepared; an experimental group, a blank control group, and a negative control group are designed for experiments, and three parallel samples are prepared for each compound concentration; the experimental results are observed after 24 h of culture in a 96-well cell culture plate, wherein the reagents added to each group are as follows:

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

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

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

[0094] 3. Determination of minimum bactericidal concentration (MBC): 1 μL of mixed culture was taken from each well of the incubated cell culture plate and added dropwise to MHA or 2216E plates, which were then cultured for 24 h, and the formation of bacterial colonies was observed. The specific test results are shown in Table 3:

[0095] Table 3: Results of activity determination of compound Zafirlukas

[0096]

[0097] As can be seen from Table 3, the results show that the compound Zafirlukas has antibacterial activity against Escherichia coli, Bacillus subtilis and Shigella flexneri, but has no inhibitory effect on Staphylococcus aureus and Pseudomonas stutzeri. The minimum inhibitory concentration for Escherichia coli is 0.047-0.19 μM, and the minimum bactericidal concentration is 0.19-0.75 μM; the minimum inhibitory concentration and minimum bactericidal concentration for Staphylococcus aureus are both 0.75-3 μM; the minimum inhibitory concentration for Shigella flexneri is 0.19-0.75 μM, and the minimum bactericidal concentration is 0.75-3 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 Zafirlukas with an inhibitory effect on PEDV was selected by a high-throughput method. The results showed that Zafirlukas had a half-maximal inhibitory concentration (IC 50 ) was 0.74 μM, with a selectivity index (SI) of 15.5. Delayed dosing experiments showed that Zafirlukas's inhibitory effect on PEDV gradually decreased with delayed dosing. However, when administered within 7 hours of viral infection, Zafirlukas's inhibitory effect on PEDV reached over 90%. This study then investigated the compound's inhibitory effects on PEDV during its adsorption, invasion, and replication phases. Zafirlukas showed no significant inhibitory effect during viral adsorption and invasion, but exhibited a significant inhibitory effect during viral replication, suggesting that Zafirlukas's inhibition of PEDV may occur during the replication phase. Furthermore, the results of this study showed that Zafirlukas had a 50% inhibitory concentration of 0.39 μM against PDCoV. There is no inhibitory effect on PRRSV and AIV, indicating that Zafirlukas has a certain broad-spectrum antiviral potential against coronaviruses. It also has inhibitory effects on common animal pathogens: Escherichia coli, Bacillus subtilis and Shigella flexneri, indicating that the compound Zafirlukas 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 the present invention. Therefore, the scope of the present invention shall be determined by the appended claims.

Claims

1. The use of compound Zafirlukas in the preparation of anti-coronavirus drugs, characterized in that The structural formula of the compound Zafirlukas 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 Zafirlukas has a half-maximal inhibitory concentration of 0.74 μM and a selectivity index of 15.5 against porcine epidemic diarrhea virus.

4. The use according to claim 1, characterized in that The half-maximal inhibitory concentration of Zafirlukas against Porcine deltacoronavirus is 0.39 μM.

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

6. A method for screening the compound Zafirlukas 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 Zafirlukas in the preparation of antibacterial drugs, characterized in that: The structural formula of the compound Zafirlukas is as follows:

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

Images