Application of CQ0343 compound in medicine for treating PRRS (porcine reproductive and respiratory syndrome)

Abstract

The invention discloses an application of a CQ0343 compound in a medicine for treating PRRS (porcine reproductive and respiratory syndrome), and cell experiments prove that the compound CQ0343 can be effectively combined with a CD163 receptor so as to block an infection pathway of PRRSV (porcine reproductive and respiratory syndrome virus) and effectively inhibit an infection process of the PRRSV; meanwhile, qPCR (quantitative polymerase chain reaction) verifies that the CQ0343 compound can effectively inhibit the replication of PRRSV (porcine reproductive and respiratory syndrome virus), and the CQ0343 compound shows a good PRRSV resisting effect by integrating the results.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and in particular relates to application of a CQ0343 compound in a drug for treating PRRS. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) that seriously harms the pig industry. PRRSV belongs to the order Nidovirales and the family Arteriviridae, and is a single-stranded positive-sense enveloped RNA virus. PRRSV infection can cause serious reproductive disorders in sows, respiratory symptoms in pigs of all ages, and high mortality in suckling piglets. Since it was first reported in 1987, PRRSV has spread rapidly, causing huge economic losses to the pig industry worldwide. It can be said that PRRS has become one of the main reproductive and respiratory diseases in the global pig industry.

[0003] The GP2a and GP4 proteins encoded by PRRSV specifically bind to the SRCR5 domain of the CD163 molecule on the surface of porcine alveolar macrophages (PAM), and are subsequently mediated by internalization into PAM cells to complete the viral infection process. Factors, designing drugs to target the CD163 SRCR5 domain, competitively binding, theoretically can effectively block PRRSV infection and play a role in treating PRRSV infection. Among them, there are also articles confirming this effect [1]. This application uses computer-assisted virtual screening technology and the reported porcine CD163 molecular structure to screen small molecule compounds of the CD163 SRCR5 domain from a library of millions of compounds.

[0004] 1.Zhu J,He X,Bernard D,Shen J,Su Y,WolekA,Issacs B,MishraN,Tian 31;97(5):e0005423.doi:10.1128 / jvi.00054-23.Epub 2023May 3.PMID:37133376;PMCID:PMC10231194. Summary of the invention

[0005] Technical issues solved:

[0006] In view of the deficiencies of the prior art, the present application solves the current problems of the rapid spread of PRRSV, which has caused huge economic losses to the pig industry worldwide, and provides an application of a CQ0343 compound in a drug for treating PRRS.

[0007] Technical solution:

[0008] To achieve the above objectives, this application is implemented through the following technical solutions:

[0009] A use of a CQ0343 compound in a PRRS treatment drug, wherein the CQ0343 compound has a molecular formula of O=C(O)C=3N(CC=1C=CC(=CC=1)OC)C2=CC(C)=CC=C2C=3CNC / C4=C / C=C( / C=C4 / OC)

[0010] OC.

[0011] Furthermore, the CQ0343 compound has the structural formula:

[0012] Furthermore, the CQ0343 compound is easily soluble in water and DMSO.

[0013] Furthermore, the PRRS treatment drug is in an injectable or non-injectable form.

[0014] Furthermore, the PRRS treatment drug is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

[0015] Furthermore, the drug for treating PRRS includes the CQ0343 compound and pharmaceutically acceptable excipients.

[0016] Furthermore, the auxiliary materials include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

[0017] Beneficial effects:

[0018] The present application provides a use of a CQ0343 compound in a drug for treating PRRS, which has the following beneficial effects compared with the prior art:

[0019] 1. The present application screened out compound number CQ0343, which can bind to the CD163 SRCR5 site and inhibit PRRSV infection. Subsequently, the anti-PRRSV effect of the compound was verified by Western Blot, immunofluorescence and qPCR, confirming its effectiveness;

[0020] 2. The compound No. CQ0343 screened in the present application showed an inhibitory effect on the proliferation of PRRSV, and it effectively inhibited the infection of PRRSV immediately at a low dose of 1 μM, confirming the high efficiency of the drug.

[0021] 3. The compound No. CQ0343 screened in this application had no significant effect on cell proliferation, confirming its safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a cytotoxicity graph of the CCK-8 detection compound of the present application;

[0023] Figure 2 The CQ0343 compound of the present application significantly inhibits the proliferation of PRRSV, wherein AC is a Western blot test of the effect of different drug treatments on the proliferation of PRRSV, D is a Western blot test of the effect of different concentrations of drug #0343 on the proliferation of different strains of PRRSV, and E is an immunofluorescence test of the expression of PRRSV Nsp2 protein;

[0024] Figure 3 A is a chemiluminescence scanning band diagram in Example 1 of the present application, and B is a qPCR detection PRRSV titer diagram in Example 1 of the present application. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0026] Compound CQ0343 was purchased from Shanghai Taosu.

[0027] Embodiment 1:

[0028] Application of CQ0343 compound in PRRS treatment, CQ0343 compound molecular formula O=C(O)C=3N(CC=1C=CC(=CC=1)OC)C2=CC(C)=CC=C2C=3CNC / C4=C / C=C( / C=C4 / OC)

[0029] OC, structural formula:

[0030] The Surflex molecular docking module of the Sybyl-X2.1 drug design platform was used for high-throughput virtual screening. The Chemdiv small molecule database (with a library capacity of 1.6 million compounds) was selected, and the compounds in Chemdiv were screened in the first round of the "drug-like five rules" using the "CompoudFiltering" module. The ligand-binding pocket (LBP) in the crystal structure of the SRCR5 domain of pCD163 was then selected as the active cavity for molecular docking virtual screening, and some parameters were modified to speed up the second round of virtual screening. Specifically, the "Max conformations per Fragment"

[0031] Change the default value of 20 to 10, and change the "Max number of rotatable bonds per molecule" from the default value of 100 to 50. Cancel the default "Per-DockMinimization" and "Post-DockMinimization" options. Change the "Maximum number of poses per ligand" from the default value of 20 to 3, that is, only retain the top 3 molecular conformations of each ligand molecule to speed up the docking speed. Screen out small molecule compounds with molecular score values ​​in the top 1%. Finally, based on the results of the second round of screening, restore the Surflex molecular docking parameters to the default values. Select the top 500 target compounds for manual screening and structural clustering, and finally confirm 100 target compounds for in vitro activity testing.

[0032] CCK-8 assay for drug toxicity:

[0033] Porcine alveolar macrophages (PAM) were cultured in 1640 medium containing 10% fetal bovine serum and penicillin-streptomycin. When the cell confluence reached more than 95%, trypsin was used for digestion for 5 minutes, and the supernatant was removed after centrifugation at 800rpm. Then the cells were counted, and 2000 cells were inoculated in each well of a 96-well cell culture plate. When the cells grew to a confluence of 40-50%, different concentrations of CQ0343 compounds (2.5μM, 5μM, 10μM) were added, and the cells were cultured for 24 and 48 hours, respectively. Subsequently, 10 microliters of CCK-8 solution was added to each well, and a control well with no cells and only cell culture medium was set up. After continuing to culture at 37°C for 2 hours, the OD450 absorbance was measured, and the experimental group was compared with the DMSO group to statistically calculate the drug cytotoxicity, and 33 drugs that had no significant inhibitory effect on cell proliferation were screened out. The results are shown as follows Figure 1 shown.

[0034] CQ0343 significantly inhibits the proliferation of PRRSV

[0035] PAM cells were passaged normally and inoculated with 6×10 5 cells / well to a 6-well plate. When the cells proliferate to 40-50%, pretreat the cells with 5 μM of different drugs for 1 hour. After 1 hour, aspirate and discard the supernatant, inoculate 0.5 MOI of WUH3 strain PRRSV virus solution, continue incubation at 37°C for 1 hour, and replace with fresh culture medium. 36 hours after virus infection, remove the supernatant, wash the cells twice with pre-cooled PBS, add 120 μL RAPA lysis buffer to each well, place on ice for 5 minutes, scrape the cells with a cell scraper and continue to lyse on ice for 20 minutes, invert and mix every 5 minutes. After the lysis is completed, centrifuge at 1000 rpm for 5 minutes, take 80 μL of supernatant, add 20 μL of loading buffer, boil in boiling water for 5 minutes, and store in a -20°C refrigerator for later use.

[0036] Prepare 10% SDS denaturing gel, preheat the sample to be loaded in a 95℃ metal bath in advance, load 30μL of sample into each well, first adjust to 80V constant voltage electrophoresis, and when the protein molecular weight marker is stratified, adjust the voltage to 120V and continue electrophoresis until bromophenol blue is electrophoresed to the bottom of the glass plate, stop the electrophoresis. Prepare the transfer solution in advance and precool it in a 4℃ refrigerator for use. Pour the precooled transfer solution into a container 5 minutes before transfer, soak the sponge, filter paper and NC membrane. After sufficient soaking, follow the "sandwich" principle, adjust to 100V constant voltage, and transfer for 100 minutes. After the transfer, use 5% skim milk powder to block at room temperature for 30 minutes. After blocking, incubate the primary antibody of PRRSVN protein and β-actin at 4℃ overnight. The next day, wash the membrane 3 times with PBST for 5 minutes each time, then incubate the secondary antibody at room temperature for 2 hours. After washing 3 times with PBST, use a chemiluminescence instrument to scan the bands. The results are as follows Figure 2 Middle A, Figure 2 Medium B and Figure 2 As shown in C, it was found that drug CQ0343 was superior to other drugs in significantly inhibiting the expression of PRRSV N protein.

[0037] To further verify the anti-PRRSV effect of CQ0343, the present application tested the inhibitory effects on WUH3 and FJ1402 strains at different concentrations. 56-well plates were inoculated with 10 cells / well. When the cells proliferated to 40-50%, the cells were treated with 0, 0.25, 0.5, and 1 μM CQ0343 drugs for 1 hour. WUH3 and FJ1402 viruses were inoculated at 0.5 MOI, respectively, and the complete culture medium was replaced after 1 hour of virus adsorption. The cells continued to be cultured, and the cells were lysed with RAPA lysis buffer at 24, 36, and 48 hours, and protein samples were collected (the method is the same as above). The protein samples were transferred to a PVDF membrane after SDS denaturing gel electrophoresis, blocked with skimmed milk powder, and incubated with antibodies to N protein and β-actin. After incubation with secondary antibodies, the color was developed. The results showed that the drug CQ0343 significantly inhibited the proliferation of different PRRSV strains at different time points, and the expression of N protein was significantly inhibited, such as Figure 2 As shown in D.

[0038] Furthermore, the present application uses immunofluorescence to detect the expression of Nsp2. A 24-well plate was placed with cells and 1.5×10 5 PAM cells were treated with DMSO and 0.5μM and 1μM CQ0343 for 1 hour, and then inoculated with 0.5MOI of WUH3 or FJ1402 PRRSV. After 1 hour of cell inoculation, fresh culture medium was replaced and cultured for 36 hours. The culture medium was aspirated, PBS was gently added to wash twice, 1ml of 4% paraformaldehyde was added to restore to room temperature, and fixed at room temperature for 30 minutes. The paraformaldehyde was then aspirated and washed twice with PBS. 0.2% Triton X-100 was added to break the membrane for 5 minutes, and 2% BSA was added to completely cover the slide, and blocked at room temperature for 1 hour. A light-proof humid box was used as an incubation box, and the PRRSV Nsp2 primary antibody was incubated at 4°C overnight. The slides incubated with the primary antibody were returned to the well plate, sufficient PBS was added, and the shaker was slowly shaken at room temperature for 10 minutes, and the washing was repeated 3 times. Then, the cells were incubated with red fluorescent labeled secondary antibody for 1 hour, and finally, DAPI was diluted with PBS at a ratio of 1:1000 to stain the cell nucleus. After staining, images were collected using a laser confocal microscope and subsequent image analysis was performed. The results are shown in Figure 2 As shown in E, 1 μM CQ0343 can significantly inhibit the proliferation of PRRSV, and the inhibitory effect on PRRSV is even stronger when the concentration reaches 5 μM.

[0039] CQ0343 binds to CD163 to block PRRSV adsorption and inhibit PRRSV replication

[0040] The binding of CQ0343 to CD163 molecules was detected by thermal stability experiment. PAM cells were subcultured and spread on 10cm dishes. When the cells grew to 90%, RAPA protein lysate was added to prepare protein samples, and 5μM compound CQ0343 was added. A control group with DMSO was set up, and then the protein samples were placed in 45℃, 49℃, 53℃, 57℃, 61℃, 65℃, 69℃ and 73℃ water baths, respectively. After 30 minutes, SDS denatured gel was run, and 30μL was loaded into each well. After the gel run, 100V constant voltage was passed and the membrane was transferred for 100min. After the transfer, 5% skim milk powder was used to block at room temperature for 30 minutes, and then the CD163 protein primary antibody was incubated at 4℃ overnight. The next day, the membrane was washed 3 times with PBST for 5 minutes each time, and then the secondary antibody was incubated at room temperature for 2 hours. After washing 3 times with PBST, the bands were scanned using a chemiluminometer. The results are as follows Figure 3 As shown in Figure A, it was found that after adding the CQ0343 compound, the degradation of CD163 was significantly reduced, confirming that CQ0343 can bind to CD163.

[0041] Furthermore, the present application verifies through different treatment methods which link of adsorption, internalization, replication and release during PRRSV infection the compound CQ0343 has an inhibitory effect on.

[0042] Adsorption experiment: inoculation 1.5×10 5 PAM cells were added to 24-well cell culture plates. When the cells grew to 40-60%, DMSO and CQ0343 (5 μM) were used to treat the cells for 1 hour, and then 0.5 MOI of PRRSV was inoculated and adsorbed to the surface of PAM cells at 4°C for 2 hours. The supernatant was discarded. After washing the cells with ice PBS, total RNA was extracted and reverse transcribed into cDNA. The virus titer was detected by absolute qPCR.

[0043] Internalization assay: inoculate 1.5×10 5 PAM cells were added to 24-well cell culture plates. When the cells grew to 40-60%, 0.5 MOI of PRRSV was inoculated and adsorbed to the surface of PAM cells at 4°C for 2 hours, and then the cells were washed twice with PBS. The cells were treated with DMSO and CQ0343 (5 μM) for 3 hours respectively, and finally the total cell RNA was extracted and reverse transcribed into cDNA. The virus titer was detected by absolute qPCR.

[0044] Replicate experiment: inoculate 1.5×10 5PAM cells were added to a 24-well cell culture plate. When the cells grew to 40-60%, 0.5 MOI of WUH3 and FJ11402 PRRSV was inoculated and adsorbed to the surface of PAM cells at 4°C for 2 hours. After washing twice with PBS, complete medium was replaced and cultured at 37°C for 6 hours. Then, the cells were incubated with DMSO and CQ0343 (5uM) for 1, 2, 3, and 4 hours, respectively. After washing the cells with pre-cooled PBS, total RNA was extracted and reversely transcribed into cDNA. The virus titer was detected by absolute qPCR.

[0045] Release experiment: inoculate 1.5×10 5 PAM cells were added to a 24-well cell culture plate. When the cells grew to 40-60%, 0.5 MOI of WUH3 and FJ11402 PRRSV were inoculated and adsorbed to the surface of PAM cells at 4°C for 2 hours. After washing twice with PBS, complete medium was replaced and cultured at 37°C for 18 hours. The cells were then incubated with DMSO and CQ0343 (5uM) for 15, 30, and 45 minutes, respectively. After washing the cells with pre-cooled PBS, total RNA was extracted and reverse transcribed into cDNA. The virus titer was detected by absolute qPCR.

[0046] The specific experimental method is as follows (qPCR detection of PRRSV titer), and the results are as follows Figure 3 As shown in B: CQ0343 compound can significantly inhibit the adsorption and proliferation of PRRSV.

[0047] qPCR detection of PRRSV titer

[0048] Add 1ml 4℃ pre-cooled Trizol to the PAM cells in the 24-well plate, mix with a pipette, let stand at room temperature for 5 minutes, add 0.2ml chloroform, vortex and mix, let stand at room temperature for 3 minutes, and centrifuge at 12000g for 15 minutes. Prepare a new 1.5ml nuclease-free EP tube, carefully aspirate the supernatant from the previous step, add 0.5ml isopropanol to it, mix by inversion, and let stand at room temperature for 10 minutes. Then centrifuge at 4℃ centrifuge at 12000g for 10 minutes. Discard the supernatant, and a small amount of white precipitate can be seen at the bottom of the tube. Invert the centrifuge tube on a clean filter paper to dry, add pre-cooled 75% ethanol prepared in advance with nuclease-free water, oscillate and mix, and then centrifuge at 4℃ low-temperature centrifuge at 7500g for 5 minutes. Discard the supernatant again, invert on a clean filter paper to dry, and wait for the ethanol to evaporate completely. Add an appropriate volume of nuclease-free water to dissolve the RNA, and measure the RNA concentration using an ultra-microsample spectrophotometer. Then perform qPCR as follows:

[0049] (1) Removal of genomic DNA

[0050]

[0051]

[0052] Mix well, spin briefly, and place in PCR machine at 42°C for 2 min.

[0053] (2) Reverse transcription system

[0054] Element Dosage 5×HiSciptIIqRTSuperMixII 4ul Reaction solution of step 1 16ul Total 20ul

[0055] Mix well and spin briefly before placing in PCR machine, 50℃, 15min, 85℃, 5sec

[0056] (3) RT-PCR reaction system

[0057]

[0058] The sample was added using an eight-tube strip equipped with an RT-qPCR instrument. After the sample was added, the amplification reaction was performed on the RT-PCR instrument. The amplification primers were as follows: PRRSV-F such as SEQ ID NO.1: GCAATTGTGTCTGTCGTC, and PRRSV-R such as SEQ ID NO.2: CTTATCCTCCCTGAATCTGAC;

[0059] The amplification procedure is as follows:

[0060]

[0061] In summary, this study, through early computer-assisted virtual screening and later experimental verification, confirmed that the compound can bind to porcine CD163, thereby blocking the adsorption and infection process of PRRSV. At the same time, CQ0343 entering the cell can also significantly inhibit the replication of PRRSV.

[0062] Finally, it should be noted that the above implementation modes are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in this field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A use of a CQ0343 compound in a drug for treating PRRS, characterized in that: The molecular formula of the CQ0343 compound is O=C(O)C=3N(CC=1C=CC(=CC=1)OC)C2=CC(C)=CC=C2C=3CNC / C4=C / C=C( / C=C4 / OC)OC.

2. The use of the CQ0343 compound according to claim 1 in a drug for treating PRRS, characterized in that: The structural formula of the CQ0343 compound is:

3. The use of the CQ0343 compound according to claim 1 in a drug for treating PRRS, characterized in that: The CQ0343 compound is freely soluble in water and DMSO.

4. The use of the CQ0343 compound according to claim 1 in a drug for treating PRRS, characterized in that: The PRRS treatment medicine is in the form of an injection or non-injection dosage form.

5. The use of the CQ0343 compound according to claim 1 in a drug for treating PRRS, characterized in that: The PRRS treatment medicine is in the form of injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

6. The use of the CQ0343 compound according to claim 1 in a drug for treating PRRS, characterized in that: The PRRS treating drug comprises a CQ0343 compound and pharmaceutically acceptable adjuvants.

7. The use of the CQ0343 compound according to claim 6 in a drug for treating PRRS, characterized in that: The auxiliary materials include one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

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