Application of Indisulam in preparation of antiviral drugs

Abstract

The invention provides application of Indisulam in preparation of antiviral drugs, and relates to the technical field of medicines. The invention discloses that Indisulam can be applied to preparation of antiviral drugs for the first time, and molecular glue Indisulam can influence splicing of M genes of influenza viruses by targeting RBM39 protein so as to inhibit replication of the influenza viruses. A new functional component is provided for antiviral drugs, especially anti-influenza A virus drugs.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to the use of Indisulam in the preparation of antiviral drugs. Background Art

[0002] Influenza A virus (IAV) is a respiratory virus that causes zoonotic diseases. It is highly contagious and can easily trigger seasonal epidemics or even global pandemics, posing a serious threat to public health. Based on their mechanism of action, anti-influenza drugs primarily include ion channel inhibitors such as amantadine, neuraminidase inhibitors such as oseltamivir, and influenza virus acidic polymer (PA) inhibitors such as mabasolvir. Due to the continuous mutation and recombination of the influenza virus genome, resistance to antiviral drugs targeting viral components is becoming increasingly serious. For example, amantadine is no longer used to treat influenza, while resistance to oseltamivir and mabasolvir is becoming increasingly prominent. Therefore, there is an urgent need to develop novel, host-targeted anti-influenza drugs.

[0003] Pre-mRNA alternative splicing is an essential component of the influenza virus lifecycle and relies on the involvement of multiple host factors. Therefore, host factors involved in influenza virus mRNA alternative splicing are attractive targets for the development of novel antiviral drugs. The RNA-binding motif 39 (RBM39) protein contains a conserved RNA recognition motif (RRM) that binds to RNA molecules to select splice sites and recruit spliceosomal components for mRNA splicing. Indisulam and dCeMM1 are molecular glue degraders that bind to the ubiquitin-proteasome system, promoting RBM39 degradation by binding to the DCAF15-CRL4 E3 ubiquitin ligase. Indisulam is a potential anti-tumor drug for acute myeloid leukemia, malignant melanoma, and colorectal cancer and is currently in Phase II clinical trials. The role of RBM39 in influenza virus mRNA splicing and the potential of molecular glue degraders targeting RBM39 for influenza virus treatment remain unknown and require urgent elucidation. Summary of the Invention

[0004] To solve the above problems, the present invention first discovered that Indisulam can be used to prepare antiviral drugs and has an inhibitory effect on influenza A virus.

[0005] In one aspect, the present application provides the use of Indisulam in the preparation of antiviral drugs.

[0006] Indisulam (E 7070) is a carbonic anhydrase inhibitor with prior art documentation of anticancer activity. Its CAS# is 165668-41-7.

[0007] Furthermore, the virus includes one or more of influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

[0008] Furthermore, the virus is influenza A virus.

[0009] The present application has demonstrated for the first time through experiments that Indisulam has an EC50 of 0.1018 μM, a CC50 of 187.1 μM, and a selectivity index of 1852.47 against influenza A virus, and has a good anti-influenza effect, especially against influenza A virus.

[0010] Furthermore, the Indisulam inhibits the virus by regulating the level of RBM39.

[0011] The regulation of RBM39 level is to downregulate the RBM39 level. Downregulation of RBM39 level leads to splicing errors of influenza A virus mRNA, specifically affecting the splicing of the M gene, thereby achieving an antiviral effect.

[0012] On the other hand, the present application also provides an antiviral drug, which includes Indisulam.

[0013] Those skilled in the art can select the concentration of Indisulam according to actual conditions, and no specific limitation is made here.

[0014] In a preferred embodiment, the concentration of Indisulam used may be 0.0625-4 μM.

[0015] Furthermore, the virus includes one or more of influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

[0016] Furthermore, the virus is influenza A virus.

[0017] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0018] The present application may also add excipients, which may be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0019] The drug of the present application can be prepared by a general method, wherein one or more diluents or carriers can be added. Those skilled in the art can select the carrier and specific dosage form of the drug according to their needs.

[0020] Furthermore, the dosage form of the drug can be pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions and / or suspensions.

[0021] The present invention has the following beneficial effects: This application demonstrates for the first time that RBM39 can serve as a splicing hub, becoming a new target for "host-targeted antiviral drugs" and achieving anti-influenza virus effects by degrading RBM39.

[0022] This application also discloses for the first time that indisulam can be used in the preparation of antiviral drugs. The molecular glue indisulam can inhibit influenza virus replication by targeting the RBM39 protein and affecting the splicing of the influenza virus M gene. This provides a new active ingredient for antiviral drugs, especially those against influenza A virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 This is a statistical graph of RBM39 expression levels in Example 1; Figure 2 Western blot detection of the inhibition of influenza virus replication by knocking down RBM39 protein; Figure 3 Western blot detection of the inhibition of influenza virus replication by degradation of RBM39; Figure 4 This is a statistical graph of the viral growth inhibition titer of dCeMM1; Figure 5 This is a statistical chart comparing the relative values ​​of the inhibition titers of influenza virus growth by Indisulam and dCeMM1; Figure 6 Western blot analysis comparing the inhibition of influenza virus splicing by Indisulam and dCeMM1; Figure 7 This is the EC50 statistical graph of Indisulam; Figure 8 This is the CC50 statistical chart of Indisulam; Figure 9 This is an image of IAV mRNA splicing in RBM39 knockdown cell lines; Figure 10 Statistical graph showing that Indisulam inhibits IAV mRNA splicing; Figure 11 This is an image of IAV mRNA splicing under RBM39 degradation. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, in the following embodiments, all reagents or instruments used without manufacturer indication are conventional products that can be purchased commercially. If specific conditions are not indicated in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0027] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were commercial products, and the specific operations were performed according to the kit instructions. Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art, and can be specifically performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0028] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0029] In the following examples, unless otherwise specified, % represents vt%, i.e., volume percentage.

[0030] The culture medium involved in the following examples is as follows: The cell culture medium was DMEM containing 10% FBS. RBM39 is an RNA-binding protein that binds to pre-mRNA by recognizing specific RNA motifs, recruiting spliceosomal proteins, and playing an important role in pre-mRNA maturation. The M2 protein of the influenza virus is an indispensable ion channel protein for the influenza virus, and plays a vital role in the invasion of host cells by the influenza virus and the maturation and release of progeny virus particles. The M2 protein is translated from the M2 mRNA produced by the splicing of M mRNA. The influenza virus lacks a splicing machinery and relies on host factors to complete its own mRNA splicing. This application verified for the first time that RBM39 can serve as a splicing hub and may become a new target for "host-targeted antiviral drugs", achieving an antiviral effect by degrading RBM39.

[0031] Example 1 Construction and verification of RBM39 knockdown cell line 1.1 sgRNA design and expression vector construction (1) The NCBI number of RBM39 mRNA is NC_000020.11. The guide RNA (sgRNA) sequence targeting RBM39 was designed as follows: sgRNA1: 5'-GTGCCATCCGAGGAAAGATT-3' (SEQ ID No. 1); sgRNA2: 5'-CTCAAGAAGTCGAGATCGAA-3' (SEQ ID No. 2).

[0032] (2) The sgRNA primers synthesized by the above-mentioned company were phosphorylated. The reaction system is shown in Table 1.

[0033] Table 1

[0034] The mixed reaction mixture was placed in a PCR instrument and annealed to obtain double-stranded sgRNA. The annealing program was as follows: 37°C for 30 minutes; 95°C for 5 minutes; 95°C-25°C at -5°C / s; and 25°C for 1 minute. After the program was completed, the annealed double strands were removed and the primers were diluted 1:1000 with water.

[0035] (3) Use BsmB I cut the pLentiCRISPR V2 vector and react at 55°C for 4 h.

[0036] (4) The target fragment (about 15,000 bp in size) from step (3) was recovered by nucleic acid gel electrophoresis. The vector was ligated with the diluted double-stranded sgRNA obtained in step (2) using T4 ligase. The reaction system is shown in Table 2.

[0037] Table 2

[0038] (5) The ligation product obtained in step (4) was transformed into STBL3 competent cells, coated with LB plates containing ampicillin and cultured at 37°C. A single colony was picked and sent for sequencing verification. The plasmid pLentiCRISPR V2 was obtained.

[0039] 1.2 Construction of RBM39 knockdown cell lines 16-24 h before transfection, 293T cells were plated at 2×10 6 The cells were plated at a concentration of 1.6 × 10 cells / dish in a 6 cm dish. Transfection was performed after the cell density reached 70%-80%. The transfection ratio was pLentiCRISPR V2:psPAX2:pMD2.G = 4:3:1, plasmid 5.4 μg, PEI 13.5 μL. 48 h after transfection, the cell supernatant was collected and filtered through a 0.22 μM sterile filter. A 12-well plate with A549 cells plated 18 h in advance at a density of 1.6 × 10 5 / mL, aspirate the residual culture medium in the wells, wash twice with PBS, add 500 μL of supernatant, and keep a group of WT cells without supernatant as a control. After 48-72 h of infection, replace the culture medium with puromycin (final concentration of 1 μg / mL) and continue to culture until all cells in the WT group die to obtain RBM39-depletion cells.

[0040] 1.3 Detection of RBM39 expression in RBM39-depleted cells using qRT-PCR (1) Design qRT-PCR primers (see Table 3 for specific primer sequences) to detect RBM39 gene expression.

[0041] Table 3

[0042] (2) Inoculate control (WT) A549 cells and RBM39-depletion cells into 12-well plates and culture in an incubator for 15-20 hours. After the cells have grown confluently, discard the culture medium in the corresponding wells, wash twice with PBS, and add 500 μL of Trizol.

[0043] (3) Extract RNA and reverse transcribe the extracted RNA into cDNA using a reverse transcription kit.

[0044] (4) Prepare the qRT-PCR reaction solution. The system is shown in Table 4. The primers used are RBM39-F / R in Table 3.

[0045] Table 4

[0046] (5) Add the reaction solution from step (4) to a qRT-PCR-specific well plate (three replicate wells per group), cover the well plate with a dedicated membrane, centrifuge the well plate to the bottom of the well plate, place it in a dedicated qRT-PCR instrument, and run it according to the instrument's default program.

[0047] (6) The data were processed and plotted using GraphPadPrism 10.1.2. The results are shown in the figure below. Figure 1 As shown in the figure, sgAAVS1 refers to the control group, with RBM39 expression at 100%; sgRBM39_1 refers to the group knocking down RBM39 using sgRNA1, with RBM39 expression at 60%; and sgRBM39_2 refers to the group knocking down RBM39 using sgRNA2, with RBM39 expression at 25%. This demonstrates that transduction of sgRNA1 and sgRNA2 reduces RBM39 expression, generating an RBM39 knockdown cell line.

[0048] Example 2 Evaluation of viral proliferation in RBM39 knockout cell lines The RBM39 knockdown cell line (RBM39-depletion cells) constructed in Example 1 (using the RBM39 knockdown group using sgRNA2 as an example) was used to test its viral proliferation. The specific testing method is as follows: Control (WT) A549 cells and RBM39-depletion cells constructed in Example 1 were seeded into 12-well plates and cultured in an incubator for 20 hours. WSN virus was then infected at an MOI of 0.01. 500 μL of infection medium containing the desired virus was added to each well and allowed to adsorb at room temperature for 1 hour. The infection medium containing the original virus was aspirated, the cells were washed twice with PBS, and 1 mL of virus-free virus maintenance medium (DMEM containing 0.5% fetal bovine serum and 0.5 μg / mL TPCK-trypsin) was added. At 9, 24, and 48 hours after infection, the medium in the corresponding wells was discarded. After washing twice with PBS, the cells were lysed by adding 70 μL of RIPA lysis buffer, incubated on ice for 30 minutes, and then centrifuged at 12,000 rpm for 15 minutes. 65 μL of the lysate was added to 13 μL of protein loading buffer and boiled at 98°C for 8 minutes. The cells were cooled to room temperature, briefly centrifuged, and subjected to SDS-PAGE electrophoresis. After electrophoresis, the protein samples in the gel were transferred to a nitrocellulose membrane at a current of 280 mA for 90 minutes. Blocking was performed with 5% skim milk powder for 1 hour. Primary antibodies RBM39 (67420-1-Ig, Proteintech), β-actin (66009-1-Ig, Proteintech); M1 (GTX636675, GeneTex), NA (GTX125974, GeneTex), and NP (GTX125989, GeneTex) were diluted 1:1000 and incubated at room temperature for 2 hours. The membranes were then washed with TBST (four times, 5 minutes each). Fluorescent secondary antibodies Rb800 (926-32213, LI-COR) and Mu680 (926-68073, LI-COR) were diluted 1:10000 and incubated at room temperature for 30 minutes. The membranes were then washed with TBST (four times, 5 minutes each). The membranes were then developed using a dual-color infrared laser imaging system. The results are shown in Figure 2. Figure 2 As shown in Figure 3, influenza virus replication was significantly attenuated in the RBM39-depleted cell line. This result demonstrates that reducing RBM39 expression can inhibit influenza virus replication.

[0049] Example 3 Detection of the Anti-IAV Effect of Degrading RBM39 3.1 Western Blot Detection of the Anti-IAV Effect of Indisualm on RBM39 Degradation Approximately 15-20 hours after A549 cells were seeded into 12-well plates, different drug concentrations (0, 0.1, 0.3, and 1 μM) of Indisulam were added directly to the cell culture medium (DMSO was used as the control group). After gentle shaking, the cells were incubated in a 37°C, 5% CO2 incubator for 1 hour and then infected with WSN virus. The drug-pretreated A549 cells were infected at an MOI of 0.001. One hour after infection, the medium was replaced with the corresponding dose of DMSO or Indisulam in the virus maintenance medium. 48 hours after viral infection, the cell culture supernatant was collected and used to determine viral titer using a plaque assay. Simultaneously, the cells were lysed with RIPA buffer, and protein samples were collected for Western blot analysis of influenza virus replication. Subsequent steps were the same as in Example 2.

[0050] The results are as follows Figure 3 As shown, with the increase of the dose of Indisulam, the influenza virus replication was significantly weakened.

[0051] 3.2 Plaque assay to detect the anti-IAV effect of dCeMM1 on RBM39 degradation (1) About 15-20 hours after A549 cells were seeded into 12-well plates, different drug concentrations (0, 0.625, 0.125, 0.5 μM) of dCeMM1 (DMSO was used as the control group) were added directly to the cell culture medium. After gentle shaking, the cells were cultured in a 37°C, 5% CO2 incubator for 1 hour and then infected with WSN virus. The drug-pretreated A549 cells were infected at an MOI of 0.001. After 1 hour of infection, the medium was replaced with the virus maintenance medium containing the corresponding dose of dCeMM1 / DMSO. 72 hours after virus infection, the cell culture supernatant was collected and used to detect the virus titer by plaque assay.

[0052] (2) Prepare MDCK cells one day in advance and plate them in 12-well plates (approximately 1.2 × 10 cells per well). 5 The cell density is about 70%-80%. -3 -10 -5Prepare a serial dilution of the virus (triplicate wells per group). Prepare 450 μL of virus solution for each dilution. Use a vortex mixer to ensure uniform mixing of the virus during the dilution process. Aspirate the MDCK cell culture medium and wash the cells twice with PBS. Add 400 mL of the virus dilution solution to each well as needed for the experiment, mix gently, and incubate in an incubator for 2 hours. Melt 2% low-melting-point agarose (LMT) in a microwave oven and keep warm in a 37°C water bath. Dilute 2% LMT 1:1 with preheated virus maintenance medium (2× DMEM containing 1% BSA and 0.5 μg / mL TPCK-trypsin) to a final concentration of 1%. After virus incubation, wash once with PBS to remove unadsorbed virus particles. Add approximately 1.2 mL of the diluted 1% agar solution to each well. Incubate at room temperature for 30 minutes. Once the agar has completely solidified, invert the well and incubate in an incubator. Observe the results after 72 hours. Fix with 4% tissue fixative at room temperature for 10 min, remove the agar, stain with crystal violet for 5 min, rinse with tap water, observe the results, and calculate the virus titer in the cell supernatant.

[0053] The calculation formula is:

[0054] The obtained data were processed using GraphPadPrism 10.1.2 software, and the results were as follows. Figure 4 As shown in Figure 3, the titer of influenza virus in A549 cells treated with dCeMM1 was significantly lower than that in control cells (treated with DMSO).

[0055] Comparison of anti-IAV effects between dCeMM1 and Indisualm According to Example 3 3.1, protein samples were collected for Western Blot and the supernatant virus was collected to determine the virus titer. Figure 5 and Figure 6 As shown, Figure 5 The results showed that at the same concentration (0.125 μM), Indisulam had a stronger inhibitory effect on influenza virus than dCeMM1. Figure 6 The results showed that Indisulam could inhibit the expression of influenza virus M2 protein at a lower concentration than dCeMM1, indicating that Indisulam has a stronger anti-influenza virus effect.

[0056] Example 4 Evaluation of the potential of Indisulam in anti-IAV applications 4.1 Plaque assay to detect the anti-IAV effect of degraded RBM39 The supernatant was collected and the virus titer was determined according to the experiment in Example 3 3.1. The concentrations of Indisulam were 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 μM, respectively. The specific operation process was the same as 3.2 (2). The results showed that the titer of influenza virus in A549 cells with Indisulam was significantly lower than that in control cells (with DMSO). The obtained data were processed using GraphPadPrism 10.1.2 software, as shown in Figure 3. Figure 7 As shown, the EC50 of Indisulam against IAV is 0.1018 μM.

[0057] 4.2 Detection of Indisulam Cytotoxicity Using CCK-8 A549 cells were seeded in 96-well plates (10,000 cells / well) and pre-cultured in an incubator for 15-20 hours. 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 μM drugs were added to the 96-well plates (three replicates per group). The plates were cultured in an incubator for 24 hours. Then, 10 μL of CCK-8 solution was added to each well. The plates were incubated in an incubator for 1 hour. The absorbance at 450 nm in each well was measured using a microplate reader (SpectraMaxS). The data were processed using GraphPadPrism 10.1.2 software, as shown in Figure 5. Figure 8 The CC50 of Indisulam is shown to be 187.1 μM.

[0058] The calculation formula involved above is:

[0059] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: absorbance of control wells (containing cells, culture medium, CCK-8 solution, but no drug); Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but no cells or drug).

[0060] 4.3 Calculation of the Selectivity Index to Evaluate the Potential of Indisulam in Anti-IAV Applications According to the selection index calculation formula, the drug selection index of Indisulam for influenza virus is 1852.47.

[0061] The formula for calculating the selection index is:

[0062] Favipiravir has an EC50 of 341 nM and a CC50 of 100 μM against influenza virus. Its selectivity index is 293. This indicates that compared to conventional antiviral drugs, Indisulam has a superior antiviral effect against influenza virus. Furthermore, this example demonstrates that this method is non-toxic to cells and highly safe.

[0063] Example 5 Detection of IAV mRNA splicing in RBM39 knockdown cell lines (1) Design semi-qRT-PCR primers (see Table 5 for the specific primer sequences) to detect the expression and splicing of influenza virus M / NS genes.

[0064] Table 5

[0065] (2) The constructed control (WT) A549 cells and RBM39-depletion cells (the group with RBM39 knockdown using sgRNA1 is sgRBM39#1, the group with RBM39 knockdown using sgRNA2 is sgRBM39#2, and sgControl is a blank control) were seeded into 12-well plates and cultured in an incubator for 15-20 hours. WSNs were infected at an MOI of 1. After 1 hour of infection, the virus maintenance medium was replaced with the virus maintenance medium containing the corresponding dose of Indisulam. After 5 hours of infection, the culture medium was discarded, the cells were washed twice with PBS, and 500 μL of Trizol was added; (3) RNA was extracted using the Trizol method, and the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (without adding Radom in the second step of reverse transcription).

[0066] (4) Perform PCR on the cDNA reverse transcribed in the previous step (primers used are rtM-F1, rtM-F2, rtM-R, rtNS-F1, rtNS-F2, and rtNS-R in Table 1). The system is shown in Table 6.

[0067] Table 6

[0068] (5) Place the mixed solution from step (4) in a PCR instrument. The M gene amplification program is shown in Table 7, and the NS gene amplification program is shown in Table 8.

[0069] Table 7

[0070] Table 8

[0071] (6) Prepare a 2% agarose gel and take 15 μL of PCR product for nucleic acid electrophoresis at 80V for 90 min. After the electrophoresis is completed, develop it on the ChemiDoc imaging system. The results are as follows Figure 9 As shown, RBM39 mainly inhibits the virus by affecting the splicing of the influenza virus M gene.

[0072] Example 6 Detection of IAV mRNA splicing under RBM39 degradation 6.1 qRT-PCR detection of M / NS gene expression and splicing (1) Design qRT-PCR primers (see Table 9 for specific primer sequences) to detect influenza virus M / NS gene expression and splicing.

[0073] Table 9

[0074] (2) A549 cells were seeded into 12-well plates and cultured in an incubator for 15-20 h. 1 h before infection, 1 μM Indisulam (DMSO for the control group) was added to the wells to infect WSNs at an MOI of 1. 1 h after infection, the virus maintenance medium was replaced with the appropriate dose of DMSO or Indisulam. At 4 h, 6 h, and 8 h after infection, the medium in the corresponding wells was discarded, the wells were washed twice with PBS, and 500 μL Trizol was added.

[0075] (3) Perform RT-qPCR according to step 1.3 of Example 1, using primers qM1-F, qM1-R, qM2-F, qM2-R, qNS1-F, qNS1-R, qNS2-F, and qNS2-R.

[0076] (4) After the data were processed, they were plotted using GraphPadPrism 10.1.2. The results are shown in the figure below. Figure 10 As shown, the results demonstrated that Indisulam inhibits influenza virus replication mainly by affecting the splicing of the influenza virus M gene.

[0077] 6.2 Detection of M / NS gene expression and splicing by semi-qRT-PCR (1) The primer sequences are the same as those in Example 5 (1). (2) A549 cells were seeded into 12-well plates and cultured in an incubator for 15-20 h. DMSO or (1 μM) Indisulam was added to the wells 1 h before infection, and the A549 cells were infected at an MOI of 1. One h after infection, the virus maintenance medium was replaced with the corresponding dose of DMSO or Indisulam. 8 h after infection, the medium was discarded, the cells were washed twice with PBS, and 500 μL of Trizol was added.

[0078] (3) Perform RT-PCR according to Example 5 (3)-(5).

[0079] (4) Prepare a 2% nucleic acid gel and take 15 μL of PCR product for nucleic acid electrophoresis at 80V for 90 min. After the electrophoresis is completed, the nucleic acid gel is developed on the ChemiDoc imaging system. The results are as follows Figure 11 As shown, the results prove that Indisulam inhibits influenza virus replication mainly by affecting the splicing of the influenza virus M gene.

[0080] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. Use of Indisulam in the preparation of antiviral drugs.

2. The use according to claim 1, characterized in that The virus includes one or more of influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

3. The use according to claim 2, characterized in that The virus is influenza A virus.

4. An antiviral drug, characterized in that: Such drugs include Indisulam.

5. The antiviral drug according to claim 4, characterized in that The virus includes one or more of influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

6. The antiviral drug according to claim 5, characterized in that The virus is influenza A virus.

7. The antiviral drug according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The antiviral drug according to claim 7, characterized in that The dosage form of the drug can be pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions and / or suspensions.

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