Use of a compound of formula CQ0343 in the manufacture of a medicament for treating PRRS

Abstract

The application discloses application of a CQ0343 compound in a PRRS medicine. Through cell experiments, it is proved that the compound CQ0343 can be effectively combined with a CD163 receptor, and then the infection path of PRRSV is blocked, and the infection process of PRRSV is effectively inhibited. Meanwhile, through qPCR verification, the compound CQ0343 can effectively inhibit the replication of PRRSV. According to the above results, the compound CQ0343 has good anti-PRRSV effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of agriculture, and particularly relates to application of a CQ0343 compound in a PRRS medicine. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) that seriously endangers the pig industry. PRRSV belongs to the arterivirus family of the order Nidovirales and is a single-stranded, enveloped RNA virus. PRRSV infection can cause severe reproductive disorders in sows, respiratory symptoms in pigs of all ages, high mortality in nursing piglets, and other symptoms. Since it was first reported in 1987, PRRSV has spread rapidly and caused significant economic losses to the pig industry worldwide. PRRS has become one of the major reproductive and respiratory diseases in the global pig industry.

[0003] GP2a and GP4 proteins encoded by PRRSV specifically bind to the SRCR5 domain of porcine alveolar macrophage (PAM) surface CD163 molecules, and are then mediated to endocytose into PAM cells, completing the infection process of the virus. Factors that target the CD163 SRCR5 domain and competitively bind to it can effectively block PRRSV infection and play a role in treating PRRSV infection in theory. Some articles have confirmed this effect [1]. This application uses computer-aided virtual screening technology and the reported structure of pig CD163 molecules to screen small molecule compounds of CD163 SRCR5 domain from millions of compound libraries.

[0004] 1. Zhu J, He X, Bernard D, Shen J, Su Y, Wolek A, Issacs B, Mishra N, Tian X, Garmendia A, Tang Y. Identification of New Compounds against PRRSV Infection by Directly Targeting CD163. J Virol. 2023 May 31; 97(5): e0005423. doi: 10.1128 / jvi.00054-23. Epub 2023 May 3. PMID: 37133376; PMCID: PMC10231194. SUMMARY

[0005] Technical problems solved:

[0006] In response to the deficiencies of the existing technology, 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 a use of the 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 CQ0343 compound is used in a remedy for PRRS, 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 structural formula of the CQ0343 compound is:

[0012] Furthermore, the CQ0343 compound is readily 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 remedy for PRRS, which has the following beneficial effects compared to the prior art:

[0019] 1. This application screened compound number CQ0343, which binds to the CD163 SRCR5 site and inhibits PRRSV infection. Subsequently, Western Blot, immunofluorescence, and qPCR were used to verify the compound's anti-PRRSV effect, confirming its effectiveness.

[0020] 2. The compound numbered CQ0343 screened in this application showed an inhibitory effect on PRRSV proliferation, and it effectively inhibited PRRSV infection immediately at a low dose of 1 μM, confirming the high efficiency of the drug.

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

[0022] Figure 1 This is a graph showing the cytotoxicity of the CCK-8 assay compounds used in this application;

[0023] Figure 2 The CQ0343 compound of the present application significantly inhibits the proliferation of PRRSV, wherein AC is a Western blot test showing the effect of different drug treatments on PRRSV proliferation, D is a Western blot test showing the effect of different concentrations of drug #0343 on the proliferation of different strains of PRRSV, and E is an immunofluorescence test showing 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 will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0026] Compound CQ0343 was purchased from Shanghai Taosu.

[0027] Example 1:

[0028] Application of CQ0343 compound in treating PRRS, wherein the molecular formula of 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)

[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 capacity of 1.6 million compounds) was selected, and the compounds in Chemdiv were screened using the "CompoudFiltering" module for the first round of "drug-like five rules". The ligand-binding site (LBP) in the crystal structure of the SRCR5 domain of pCD163 was then selected as the active cavity for molecular docking virtual screening. Some parameters were modified to speed up the second round of virtual screening. Specifically, the "Max conformations per Fragment" was set to

[0031] Reduce the default value of 20 to 10, and the "Maximum number of rotatable bonds per molecule" from the default value of 100 to 50. Disable the default "Per-Dock Minimization" and "Post-Dock Minimization" options. Reduce the "Maximum number of poses per ligand" from the default value of 20 to 3, which means that only the top three molecular conformations of each ligand molecule are retained, speeding up docking. Select small molecules with molecular scores 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 ultimately 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 95% or more, trypsin was used for digestion for 5 minutes, and the supernatant was removed after centrifugation at 800 rpm. The cells were then counted, and 2,000 cells were inoculated into a 96-well cell culture plate per well. When the cells grew to 40-50% confluence, different concentrations of CQ0343 compounds (2.5 μM, 5 μM, 10 μM) 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 further culture at 37°C for 2 hours, the OD450 absorbance was measured. 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 below. Figure 1 shown.

[0034] CQ0343 significantly inhibits PRRSV proliferation

[0035] PAM cells were normally passaged and inoculated with 6x10 5 When the cells proliferated to 40-50%, the cells were pretreated with 5 μM of different drugs for 1 hour, respectively. After 1 hour, the supernatant was aspirated, and 0.5 MOI of WUH3 strain PRRSV virus solution was inoculated, and the incubation was continued at 37°C for 1 hour, and then fresh culture medium was replaced. After 36 hours of virus infection, the supernatant was removed, and the cells were washed twice with pre-cooled PBS, 120 μL of RAPA lysis solution was added to each well, and the cells were lysed on ice for 5 minutes. After the cells were scraped, the cells were lysed on ice for 20 minutes, and the mixture was inverted every 5 minutes. After lysis, the cells were centrifuged at 1000 rpm for 5 minutes, and 80 μL of supernatant was taken, 20 μL of loading buffer was added, and the mixture was boiled in boiling water for 5 minutes, and then stored in a refrigerator at -20°C for standby.

[0036] A 10% SDS denaturing gel was prepared, and the samples to be loaded were preheated in a 95°C metal bath. 30 μL was loaded into each well, and electrophoresis was performed at a constant voltage of 80 V. When the protein molecular weight marker was layered, the voltage was adjusted to 120 V, and the electrophoresis was continued. When the bromophenol blue electrophoresis reached the bottom of the glass plate, the electrophoresis was stopped. The transfer solution was prepared in advance and pre-cooled in a refrigerator at 4°C for standby. 5 minutes before transfer, the pre-cooled transfer solution was poured into the container, and the sponge, filter paper and NC membrane were soaked. After sufficient soaking, the "sandwich" principle was followed, and the constant voltage was adjusted to 100 V, and the transfer was performed for 100 minutes. After the transfer was completed, 5% skimmed milk powder was used for room temperature blocking for 30 minutes. After blocking, the primary antibodies for PRRSV N protein and β-actin were incubated at 4°C overnight. The next day, the membrane was washed with PBST for 3 times, each for 5 minutes, and then the secondary antibody was incubated at room temperature for 2 hours. After washing with PBST for 3 times, the chemiluminescence instrument was used to scan the bands, and the results are shown in Figure 2 Fig. A, Figure 2 Fig. B and Figure 2 Fig. C, it is found that the drug CQ0343 is better than other drugs in significantly inhibiting the expression of PRRSV N protein.

[0037] To further verify the anti-PRRSV effect of CQ0343 drug, the application detects the inhibition of WUH3 and FJ1402 strains at different concentrations. PAM cells were passaged, and 6x10 5Cells / well were inoculated into 6-well plates. 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 with 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 skim milk powder, and incubated with antibodies to N protein and β-actin. After incubation with secondary antibodies, the cells were colored. 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. Cells were placed on 24-well plates and 1.5×10 5 PAM cells were treated with DMSO and 0.5 μM and 1 μM CQ0343 for 1 hour, respectively. PAM cells were then inoculated with 0.5 MOI of PRRSV (WUH3 or FJ1402). One hour after inoculation, fresh medium was replaced and cultured for 36 hours. The medium was aspirated, and the cells were gently washed twice with PBS. 1 ml of 4% paraformaldehyde, which had been brought to room temperature, was added and fixed at room temperature for 30 minutes. The paraformaldehyde was then aspirated, and the cells were washed twice with PBS. The membrane was permeabilized with 0.2% Triton X-100 for 5 minutes, and 2% BSA was added to completely cover the slides, and the cells were blocked at room temperature for 1 hour. Incubate the slides with the primary antibody against PRRSV Nsp2 overnight at 4°C in a humidified, light-proof chamber. After the primary antibody incubation, the slides were returned to the plate, and sufficient PBS was added. The slides were then washed for 10 minutes at room temperature with gentle shaking. This was repeated three 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 nuclei. After staining, images were collected using a laser confocal microscope and subsequent image analysis was performed. The results are shown in Figure 2. Figure 2 As shown in Figure 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, blocks PRRSV adsorption, and inhibits PRRSV replication

[0040] The binding of CQ0343 to CD163 molecules was detected by thermal stability experiment. PAM cells were passaged and plated on 10 cm dishes. When the cells grew to 90%, RAPA protein lysate was added to prepare protein samples, and 5 μM compound CQ0343 was added. A DMSO control group was set up, and the protein samples were placed in 45 ° C, 49 ° C, 53 ° C, 57 ° C, 61 ° C, 65 ° C, 69 ° C and 73 ° C water baths, respectively. After 30 minutes, SDS denaturing gel was run, 30 μL was loaded into each well, and after running the gel, 100 V constant voltage was passed and the membrane was transferred for 100 minutes. 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 ° C overnight. The next day, the membrane was washed 3 times with PBST for 5 minutes each time, and then incubated with secondary antibody at room temperature for 2 hours. After washing 3 times with PBST, the bands were scanned using a chemiluminescence instrument. The results are as follows Figure 3 As shown in Figure A, it was found that the degradation of CD163 was significantly reduced after the addition of CQ0343 compound, 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 plated onto 24-well cell culture plates. After cell growth reached 40-60%, the cells were treated with DMSO and CQ0343 (5 μM) for 1 hour, respectively. PRRSV was then inoculated at a 0.5 MOI and allowed to adsorb to the PAM cell surface for 2 hours at 4°C. The supernatant was discarded. After washing the cells with ice-cold PBS, total RNA was extracted and reverse transcribed into cDNA. Viral titers were determined by absolute qPCR.

[0043] Internalization experiment: inoculate 1.5×10 5 PAM cells were plated in 24-well cell culture plates. After the cells grew to 40-60% viability, 0.5 MOI of PRRSV was inoculated and allowed to adsorb to the PAM cell surface for 2 hours at 4°C. The cells were then washed twice with PBS. The cells were treated with DMSO and CQ0343 (5 μM) for 3 hours, respectively. Total RNA was extracted and reverse transcribed into cDNA. Viral titers were determined by absolute qPCR.

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

[0045] Release experiment: inoculate 1.5×10 5 PAM cells were plated in 24-well cell culture plates. When the cells grew to 40-60%, 0.5 MOI of WUH3 and FJ11402 PRRSV was inoculated and adsorbed to the PAM cell surface 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 1 ml of 4°C pre-chilled Trizol to the PAM cells in a 24-well plate. Mix thoroughly by pipetting. Let stand at room temperature for 5 minutes. Add 0.2 ml of chloroform, vortex to mix thoroughly, let stand at room temperature for 3 minutes, and centrifuge at 12,000 g for 15 minutes. Prepare a new 1.5 ml nuclease-free EP tube. Carefully aspirate the supernatant from the previous step. Add 0.5 ml of isopropanol to the tube, mix thoroughly by inversion, and let stand at room temperature for 10 minutes. Centrifuge at 12,000 g for 10 minutes at 4°C. Discard the supernatant. A small amount of white precipitate may be visible at the bottom of the tube. Invert the tube onto clean filter paper to dry. Add pre-chilled 75% ethanol prepared in nuclease-free water and vortex to mix thoroughly. Centrifuge at 7,500 g for 5 minutes at 4°C. Discard the supernatant again and invert onto clean filter paper to dry until the ethanol evaporates. Add an appropriate volume of nuclease-free water to dissolve the RNA and measure the RNA concentration using an ultra-microsample spectrophotometer. qPCR is then performed as follows:

[0049] (1) Removal of genomic DNA

[0050]

[0051]

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

[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, then place 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 subsequent experimental verification, confirmed that the compound can bind to porcine CD163, thereby blocking the adsorption and infection process of PRRSV. Furthermore, CQ0343, once inside the cell, can significantly inhibit PRRSV replication.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and are not limiting. 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 the preparation of a medicament for treating PRRS, characterized in that: The structural formula of the CQ0343 compound is: 。 2. The use of the CQ0343 compound according to claim 1 in the preparation of a medicament for treating PRRS, characterized in that: The PRRS treatment medicine is in the form of an injection or non-injection dosage form.

3. The use of the CQ0343 compound according to claim 1 in the preparation of a medicament 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.

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

5. The use of the CQ0343 compound according to claim 4 in the preparation of a medicament for treating PRRS, characterized in that: The auxiliary material is selected from one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

Images