New uses of inhibitors, inhibitor screening models

Abstract

The present application belongs to the field of biotechnology, and particularly relates to different characteristics of a first inhibitor, a second inhibitor and a third inhibitor in coping with human influenza virus replication and avian influenza virus replication. The present application finds that the inhibitors can have the functions of inhibiting or promoting human influenza and / or avian influenza through research, and the efficacy of the inhibitors can be judged by researching the trend of the functions and the tissue half quantity of infection, thereby providing strong support for subsequent development of drugs reducing drug resistance, experiments and development of human / avian influenza drugs. Meanwhile, the present application also provides a model obtained from the screening result of the present application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a new use of an inhibitor and an inhibitor screening model. Background Technology

[0002] Influenza viruses are among the most prominent human respiratory pathogens. Currently, according to the World Health Organization, influenza viruses cause approximately 290,000 to 650,000 deaths globally each year. H1N1 and H3N2 subtypes of influenza viruses are the main seasonally circulating strains. Furthermore, multiple subtypes of avian influenza viruses (i.e., H9N2, H5N1, and H7N9) are widespread in my country, causing enormous economic losses and posing a significant threat to public health. They also continuously spread across host species, infecting mammals and humans. H5, H7, and H9 subtypes can cause sporadic infections in humans, and the highly pathogenic H5N1 avian influenza (HPAI) virus can rapidly develop into severe viral pneumonia with a high mortality rate (60%). Moreover, due to the lack of immunity to H5, H7, and H9 viruses in humans, the emergence of pandemic viruses adapted to humans could be devastating. To date, hundreds of people have died from the H5N1 virus, and there have been multiple cases of human infection with the highly pathogenic H7N9 avian influenza and young children infected with the low pathogenic H9N2 avian influenza virus. However, existing anti-influenza drugs are prone to inducing viral resistance or causing teratogenic side effects. Neuraminidase inhibitors (zanamivir and oseltamivir) are currently the most widely used anti-influenza drugs, but clinical studies have shown that influenza viruses have acquired resistance to these drugs through mutation. Newly marketed anti-influenza drugs, such as RNA polymerase inhibitors (favipiravir and baloxavir), have also caused drug-resistant mutations in influenza viruses within a short period. Therefore, there is an urgent need to develop novel broad-spectrum anti-influenza drugs that can effectively prevent viral drug resistance mutations and deliver medication precisely to the lungs.

[0003] The main objective of this case is to identify substances from existing reagents that can promote or inhibit the replication of human / avian influenza. Summary of the Invention

[0004] The purpose of this invention is to provide a variety of inhibitors that traditionally exhibit inhibitory effects on tumor cells or other cells. This invention, through research, has discovered that these inhibitors can inhibit or promote the growth of human influenza and / or avian influenza. The efficacy of these inhibitors can be determined by studying functional trends and tissue half-infection levels, providing strong support for the subsequent development of drugs to reduce drug resistance, experimental studies, and the development of drugs for human / avian influenza.

[0005] The technical solution of this invention is as follows:

[0006] The first inhibitor is intended for use in manufacturing agents that inhibit the replication of human influenza virus and avian influenza virus.

[0007] The first inhibitor is one or a combination of Orteronel, KU-0063794, WYE-354, GSK429286A, Pimobendan, AST-1306, SIB 1757, Ibrutinib (PCI-32765), WAY-600, Nepicastat (SYN-117)HCl, CP-91149, Vistusertib (AZD2014), AMG-333, GSK2656157, VE-822, Savolidinib (AZD6094, HMPL-504), BDA-366, BMS-345541, Dynasore, BI-78D3, Nec-1s (7-Cl-O-Nec1), Atuveciclib (BAY-1143572), and LDN-193189 2HCl.

[0008] A search revealed the existing uses of the aforementioned inhibitors, as follows:

[0009] Orteronel: WO2023107861A1, as a reagent for novel hormone therapy for cancer;

[0010] KU-0063794: ZA202304456A, as a mTOR inhibitor for cancer treatment;

[0011] WYE-354: TW202308624A is intended for the treatment of HPV infection or for the prevention or treatment of SARS-CoV-2 infection; although the document includes influenza A virus, it does not indicate that it has an inhibitory effect on human influenza or avian influenza.

[0012] GSK429286A: CN115433715A, a ROCK pathway inhibitor in culture medium for inducing iPSC differentiation to obtain macrophages;

[0013] Pimobendan: WO2017036284A1, a phosphodiesterase inhibitor in pharmaceutical compositions for the treatment of tumors;

[0014] AST-1306: AU2023200428A1, an EGFR TKI reagent used to treat cancer;

[0015] SIB 1757:WO2023279193A2, an mGluR5 blocker for use in combination with phosphodiesterase inhibitors that alleviate symptoms of Fragile X syndrome;

[0016] Ibrutinib (PCI-32765): JP2023052878A, is used as a Src family tyrosine kinase inhibitor;

[0017] WAY-600: WO2023102379A1, as a mTOR inhibitor for cancer treatment;

[0018] Nepicastat (SYN-117) HCl: EP3490554B1, used as an inhibitor for cancer treatment;

[0019] CP-91149: WO2021174195A2, is used as a glycogen phosphorylase inhibitor in the treatment of cancer with thyroid drugs;

[0020] Vistusertib (AZD2014): HK40080890A, used as a reagent in the AR+ breast cancer treatment method;

[0021] AMG-333: No patent information is available to know its specific uses, but according to supplier information, MG-333 is an effective highly selective TRPM8 antagonist.

[0022] GSK2656157: CN114601835A, Application of PERK inhibitors in drugs for preventing and treating mitochondrial damage;

[0023] VE-822: US20230248728A1, an ATR inhibitor used in cancer treatment;

[0024] Savolitinib (AZD6094, HMPL-504): WO2023031781A1, a combination of drugs containing a tead inhibitor and its use in the treatment of cancer;

[0025] BDA-366: WO2023131305A1, as a BCL-2 inhibitor for cancer treatment;

[0026] BMS-345541:WO2023131576A1, as an IKK2 / IKKP inhibitor in cancer treatment;

[0027] Dynasore: US20210196732A1, as a preparation to delay brain aging;

[0028] BI-78D3: US20230001193A1, as a JNK inhibitor used to increase the sensitivity of cancer cells to alternating electric fields;

[0029] Nec-1s(7-Cl-O-Nec1): CN113069546A, its application as an inhibitor of RIPK1 targeting macrophages;

[0030] Atuveciclib (BAY-1143572): MX2020003270A, a CDK9 inhibitor used to treat cancer;

[0031] LDN-193189 2HCl: CN105617377A discloses its ability to inhibit the upregulation of matrix proteins related to bacterial adhesion on the surface of lung cells caused by influenza virus infection, inhibit the adhesion of respiratory pathogens to lung cells caused by influenza virus infection, inhibit the settlement and / or infection of respiratory pathogens in the lungs caused by influenza virus infection, and prevent and / or treat bacterial pneumonia secondary to influenza virus. It does not indicate that it can inhibit or promote the replication of influenza virus.

[0032] Preferably, the first inhibitor is one or a combination of VE-822, BDA-366, Nepicastat (SYN-117)HCl, and GSK2656157.

[0033] Meanwhile, the present invention also provides the use of a second inhibitor as a reagent for manufacturing a substance that promotes the replication of human influenza virus and avian influenza virus.

[0034] The second inhibitor is one or a combination of Dabrafenib Mesylate, HJC0350, JNK Inhibitor IX, SAR125844, GSK1324726A (I-BET726), SP2509, GSK2292767, and VPS34-IN1.

[0035] A search revealed the existing uses of the aforementioned inhibitors, as follows:

[0036] Dabrafenib Mesylate: WO2023111829A1, used as an anti-tumor API;

[0037] HJC0350: No patent information is available to know its specific use. According to supplier information, HJC0350 is a potent and selective epac2 inhibitor.

[0038] JNK Inhibitor IX: CN116200330A, used to inhibit the reduction of MAPK8 gene expression;

[0039] SAR125844: HK40073845A, an RTK inhibitor for targeted cancer therapy;

[0040] GSK1324726A (I-BET726): No patent information is available to know its specific use. According to supplier information, GSK1324726A (I-BET726) is an abet protein inhibitor.

[0041] SP2509: CN116445408A, as an LSD1 inhibitor in promoting iPSC differentiation into HSC and maintaining HSC stemness;

[0042] GSK2292767:WO2023039578A1, as a PI3K6 inhibitor for cancer treatment;

[0043] VPS34-IN1: US20220193056A1, as an EZH inhibitor for cancer treatment;

[0044] Preferably, the second inhibitor is one or more combinations of HJC0350, GSK2292767 and VPS34-IN1.

[0045] Meanwhile, the present invention also provides the use of a third inhibitor as a reagent for manufacturing a reagent that promotes or inhibits the replication of human influenza virus and has no effect on the replication of avian influenza virus, or the use of a reagent for manufacturing a reagent that promotes or inhibits the replication of avian influenza virus and has no effect on the replication of human influenza virus.

[0046] The third inhibitor is one or a combination of JNJ-1661010, Bedaquiline, CPI-360, and EPZ005687.

[0047] A search revealed the existing uses of the aforementioned inhibitors, as follows:

[0048] JNJ-1661010: US20190060220A1, for the treatment of accelerating the healing of surface damage;

[0049] Bedaquiline: MX2023004635A, used to prevent and / or reduce the likelihood of tumor metastasis and recurrence in patients;

[0050] CPI-360: WO2023143608A1, a histone methyltransferase inhibitor used to treat cancer;

[0051] EPZ005687: AU2023201580A1, an LSDI inhibitor used to treat cancer;

[0052] Preferably, the third inhibitor is any one of Bedaquiline, CPI-360, and EPZ005687.

[0053] In the above-mentioned uses, the human influenza virus is the H1N1 subtype human influenza virus; the avian influenza virus is one or more of the H9N2 subtype avian influenza virus, H5N1 subtype avian influenza virus, and H7N9 subtype avian influenza virus.

[0054] In the above-described uses, the virus replicates in a culture medium or in the body of an animal, including a human.

[0055] In addition, this invention also discloses an inhibitor screening model, which is constructed by the following method:

[0056] Step 1: Culture cells that can be infected with human influenza virus and avian influenza virus in a culture medium;

[0057] Step 2: Infect the cells in the culture medium obtained in Step 1 with human influenza virus and avian influenza virus, respectively;

[0058] Step 3: Remove the virus infection solution from the culture medium in Step 2, add DMEM culture medium containing TPCK-trypsin and 1% penicillin, and add a small molecule inhibitor to the culture medium. The infection group without small molecule inhibitor treatment but with DMSO added to a final concentration of 0.1% is used as the negative control group. At the same time, amantadine treatment group and oseltamivir treatment group are set up. Then, the culture is carried out in a 37℃ CO2 incubator.

[0059] Step 4: Determine the TCID of the virus in the supernatant of each sample from Step 3. 50 value;

[0060] Step 5: Based on the TCID of the sample from Step 4 50 Values ​​determine the effect of inhibitors.

[0061] In the above inhibitor screening model, the human influenza virus is the H1N1 subtype human influenza virus; the avian influenza virus is one or more of the H9N2 subtype avian influenza virus, H5N1 subtype avian influenza virus, and H7N9 subtype avian influenza virus.

[0062] The beneficial effects of this invention are as follows:

[0063] 1. This invention specifically screens different drugs and studies their inhibitory effects on different subtypes of influenza viruses, making it possible to repurpose existing drugs; it can also provide a reagent basis for laboratory research on the relevant properties of viruses.

[0064] 2. In addition to developing drugs targeting human influenza H1N1, the research results of this invention further enable the development of broad-spectrum antiviral drugs targeting multiple subtypes of human and avian influenza viruses, as well as drugs specifically targeting avian influenza viruses. This will help prevent the cross-species transmission of avian influenza and is of great significance for early warning of emerging viruses. This invention not only provides a rich reserve of drugs for combating various subtypes of influenza viruses and those originating from different hosts, but also provides a theoretical basis for in-depth research into the mechanisms by which influenza viruses adapt to their hosts.

[0065] 3. Based on the current problems and research status of antiviral clinical treatment, this invention uses the method described above to infect mammalian cells with different subtypes of human and avian influenza viruses, and screens 339 small molecule inhibitors that specifically target mammalian cell signal transduction and metabolic pathways. This invention not only establishes a method for screening inhibitors against different subtypes of influenza viruses, but also lays the foundation for further in-depth research on the correlation between the screened inhibitor targets and influenza viruses. Attached Figure Description

[0066] Figure 1 This is a schematic diagram of the well plate culture in Example 1;

[0067] Figure 2A BDA-366 is a TCID inhibitor against the H1N1 influenza virus. 50 Line graph;

[0068] Figure 2B BDA-366 is a TCID inhibitor against the H5N1 influenza virus. 50 Line graph;

[0069] Figure 2C BDA-366 is a TCID inhibitor against the H7N9 influenza virus. 50 Line graph;

[0070] Figure 2D BDA-366 is a TCID inhibitor against the H9N2 influenza virus. 50 Line graph;

[0071] Figure 2E It is the inhibitor VE-822 against the TCID of H1N1 influenza virus. 50 Line graph;

[0072] Figure 2F It is the inhibitor VE-822 against the TCID of H5N1 influenza virus. 50 Line graph;

[0073] Figure 2G It is the inhibitor VE-822 against the TCID of H7N9 influenza virus. 50 Line graph;

[0074] Figure 2H It is the inhibitor VE-822 against the TCID of H9N2 influenza virus. 50 Line graph;

[0075] Figure 2I It is the inhibitor Nepicastat (SYN-117) HCl against the TCID of H1N1 influenza virus. 50 Line graph;

[0076] Figure 2J It is the inhibitor Nepicastat (SYN-117) HCl against the TCID of H5N1 influenza virus. 50 Line graph;

[0077] Figure 2K It is the inhibitor Nepicastat (SYN-117) HCl against the TCID of H7N9 influenza virus. 50 Line graph;

[0078] Figure 2L The inhibitor Nepicastat (SYN-117) HCl is a TCID inhibitor against the H9N2 influenza virus. 50 Line graph;

[0079] Figure 2M It is the inhibitor GSK2656157, a TCID inhibitor against the H1N1 influenza virus. 50 Line graph;

[0080] Figure 2N It is the inhibitor GSK2656157, a TCID inhibitor against the H5N1 influenza virus. 50 Line graph;

[0081] Figure 2O It is the inhibitor GSK2656157, a TCID inhibitor against the H7N9 influenza virus. 50 Line graph;

[0082] Figure 2P It is the inhibitor GSK2656157, a TCID inhibitor against the H9N2 influenza virus. 50 Line graph;

[0083] Figure 3A It is the inhibitor VPS34-IN1 against the TCID of H1N1 influenza virus. 50 Line graph;

[0084] Figure 3B It is the inhibitor VPS34-IN1 against the TCID of H5N1 influenza virus. 50 Line graph;

[0085] Figure 3CIt is the inhibitor VPS34-IN1 against the TCID of H7N9 influenza virus. 50 Line graph;

[0086] Figure 3D It is the inhibitor VPS34-IN1 against the TCID of H9N2 influenza virus. 50 Line graph;

[0087] Figure 3E It is the inhibitor GSK2292767, a TCID inhibitor against the H1N1 influenza virus. 50 Line graph;

[0088] Figure 3F It is the inhibitor GSK2292767, a TCID inhibitor against the H5N1 influenza virus. 50 Line graph;

[0089] Figure 3G It is the inhibitor GSK2292767, a TCID inhibitor against the H7N9 influenza virus. 50 Line graph;

[0090] Figure 3H It is the inhibitor GSK2292767, a TCID inhibitor against the H9N2 influenza virus. 50 Line graph;

[0091] Figure 3I HJC0350 is a TCID inhibitor against the H1N1 influenza virus. 50 Line graph;

[0092] Figure 3J HJC0350 is a TCID inhibitor against the H5N1 influenza virus. 50 Line graph;

[0093] Figure 3K HJC0350 is a TCID inhibitor against the H7N9 influenza virus. 50 Line graph;

[0094] Figure 3L HJC0350 is a TCID inhibitor against the H9N2 influenza virus. 50 Line graph;

[0095] Figure 4A It is the inhibitor Bedaquiline against the TCID of H1N1 influenza virus. 50 Line graph;

[0096] Figure 4B It is the inhibitor Bedaquiline against the TCID of H9N2 influenza virus. 50 Line graph;

[0097] Figure 4CIt is the inhibitor EPZ005687, a TCID inhibitor against the H1N1 influenza virus. 50 Line graph;

[0098] Figure 4D It is the inhibitor EPZ005687, a TCID inhibitor against the H9N2 influenza virus. 50 Line graph;

[0099] Figure 4E CPI-360 is a TCID inhibitor against the H1N1 influenza virus. 50 Line graph;

[0100] Figure 4F CPI-360 is a TCID inhibitor against the H9N2 influenza virus. 50 Line graph;

[0101] Figures 2A to 4F TCID involved 50 In the curve graph, the vertical axis is Virus titer (TCID). 50 / ml xlog10), which means viral titer (unit: TCID50 / ml x log10); the horizontal axis is hpi (hours postinfection), which means hours after infection. Detailed Implementation

[0102] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this does not constitute any limitation on the present invention.

[0103] Example 1

[0104] I. A549 cells were infected with H1N1 subtype human influenza virus and H9N2 subtype avian influenza virus, and then treated with 339 small molecule inhibitors respectively; the 339 small molecule inhibitors are shown in Tables 1-4.

[0105] (1) A549 cells were passaged in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% carbon dioxide (CO2) incubator. When the cells reached the 3rd to 5th generation, A549 cells were seeded into the wells of a 96-well plate 24 hours before infection and the 96-well plate was placed in a 37°C CO2 incubator for culture.

[0106] (2) When the cells grew to a density of approximately 80%-90%, A549 cells were infected with H1N1 subtype human influenza virus and H9N2 subtype avian influenza virus at an MOI of 0.1. After virus adsorption for 1 hour, the virus infection solution was aspirated, and the cells were gently washed three times with PBS. DMEM medium containing TPCK-trypsin and 1% penicillin antibiotics was added, along with a small molecule inhibitor at a final concentration of 10 μM. The infection group without the small molecule inhibitor but with DMSO at a final concentration of 0.1% served as the negative control group. Simultaneously, amantadine and oseltamivir treatment groups were also established. The cells were then cultured in a 37℃ CO2 incubator. Figure 1 The diagram shown is a schematic diagram of small molecule inhibitor screening.

[0107] (3) Cells were removed 24 hours after infection, and cell supernatant was collected. The sample was temporarily stored at -80°C.

[0108] (4) After all cell supernatant samples treated with different inhibitors have been collected, the viral titer of all samples was determined, and the TCID of each sample was calculated according to the Reed-Muench method. 50 value.

[0109] Table 1 List of Inhibitors

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] Table 2 List of Inhibitors

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] Table 3 List of Inhibitors

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Table 4 List of Inhibitors

[0129]

[0130]

[0131]

[0132] The results can be referenced. Figures 2A to 2P , Figures 3A to 3L , Figures 4A to 4F ;

[0133] Among them, the inhibitors that inhibit the replication of human and avian influenza are Orteronel, KU-0063794, WYE-354, GSK429286A, Pimobendan, AST-1306, SIB 1757, Ibrutinib (PCI-32765), WAY-600, Nepicastat (SYN-117)HCl, CP-91149, Vistusertib (AZD2014), AMG-333, GSK2656157, VE-822, Savolidinib (AZD6094, HMPL-504), BDA-366, BMS-345541, Dynasore, BI-78D3, Nec-1s (7-Cl-O-Nec1), Atuveciclib (BAY-1143572), and LDN-193189. The 24 small molecule inhibitors of 2HCl that showed significant inhibitory effects on avian and human influenza were identified. Inhibitors that promoted the replication of both human and avian influenza included Dabrafenib Mesylate, HJC0350, JNK Inhibitor IX, SAR125844, GSK1324726A (I-BET726), SP2509, GSK2292767, and VPS34-IN1. Inhibitors with different effects on the replication of human and avian influenza included JNJ-1661010, Bedaquiline, CPI-360, and EPZ005687.

[0134] II. TCID of all 339 small molecule inhibitors screened 50 Measurement

[0135] (1) MDCK cells (Madison canine kidney cell line) were passaged in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% carbon dioxide (CO2) incubator. When the cells reached the 3rd to 5th generation, MDCK cells were seeded into the wells of a 96-well plate 24 hours before infection and the 96-well plate was placed in a 37°C CO2 incubator for culture.

[0136] (2) When the cell density is 70% to 80%, the serum-containing DMEM medium is aspirated and the cells are washed three times with PBS buffer.

[0137] (3) Remove the cell supernatant sample from the -80℃ freezer 30 minutes in advance and place it on ice to thaw naturally. Prepare eight 1.5mL sterile centrifuge tubes, and add 450μL of DMEM containing TPCK-trypsin (final concentration of 2μg / mL) and 1% penicillin antibody to each tube. Add 50μL of the virus stock solution to the first tube and vortex to mix. Aspirate 50μL of the mixture and add it to the second tube. Serial dilute the mixture tenfold until the eighth tube is reached. Label the tubes and place them on ice temporarily.

[0138] (4) Samples of each dilution were seeded into 96-well plates at a dose of 100 μL / well, with three replicates for each dilution. After incubation for approximately 36-48 hours, the hemagglutination of each well was determined and detected by indirect immunofluorescence (IFA).

[0139] IFA testing method steps:

[0140] 1. Washing: Aspirate the cell culture medium, wash the cells once with PBS buffer, and discard the washing solution.

[0141] 2. Fixation: Prepare fixative solution according to the ratio of ethanol:acetone = 3:2. Add 50 μL of pre-cooled fixative solution to each cell well and incubate at room temperature for 15 min.

[0142] 3. Washing: Discard the fixative and wash the cells three times with PBS buffer.

[0143] 4. Primary antibody incubation: Dilute the influenza virus NP protein monoclonal antibody 5000 times with PBS, add 30 μL to each well, and incubate at 37°C for 1 h (or at 4°C overnight).

[0144] 5. Washing: Remove the primary antibody from the wells and wash the cells with PBS buffer. Repeat the washing process three times.

[0145] 6. Secondary antibody incubation: Dilute FITC-labeled goat anti-mouse IgG 400 times with PBS, add 30 μL to each well, and incubate at 37°C in the dark for 1 h.

[0146] 7. Washing: Remove the secondary antibody from the wells and wash the cells with PBST (0.5 mL Tween-20 added to 1000 mL PBS). After each addition of washing buffer, wash on a shaker for 3 min and then discard the washing buffer. Repeat the washing process 3 times.

[0147] 8. Results Observation: Cells emitting green fluorescence under a fluorescence microscope were considered virus-infected positive cells. TCID was calculated using the Reed-Muench method. 50 .

[0148] Preliminary screening yielded three classes of inhibitors, namely inhibitors that inhibit human and avian influenza replication: Orteronel, KU-0063794, WYE-354, GSK429286A, Pimobendan, AST-1306, SIB1757, Ibrutinib (PCI-32765), WAY-600, Nepicastat (SYN-117)HCl, CP-91149, Vistusertib (AZD2014), AMG-333, GSK2656157, VE-822, Savolidinib (AZD6094, HMPL-504), BDA-366, BMS-345541, Dynasore, BI-78D3, Nec-1s (7-Cl-O-Nec1), Atuveciclib (BAY-1143572), and LDN-193189. Of the 24 small molecule inhibitors that showed significant inhibitory effects on avian and human influenza, 8 inhibitors that promoted the replication of both human and avian influenza were identified: Dabrafenib Mesylate, HJC0350, JNK InhibitorIX, SAR125844, GSK1324726A (I-BET726), SP2509, GSK2292767, and VPS34-IN1. Inhibitors with different effects on the replication of human and avian influenza were also obtained: JNJ-1661010, Bedaquiline, CPI-360, and EPZ005687.

[0149] III. Continue to validate the viral growth kinetics of the screened small molecule inhibitors using H1N1, H5N1, H7N9, and H9N2 influenza subtypes.

[0150] (1) A549 cells were passaged in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibody at 37°C in a 5% carbon dioxide (CO2) incubator. When the cells reached the 3rd to 5th generation, A549 cells were seeded into the wells of a 6-well plate 24 hours before infection. The 96-well plate was then placed in a 37°C CO2 incubator for culture.

[0151] (2) When the cells grew to a density of about 80%-90%, A549 cells were infected with H1N1, H5N1, H7N9 and H9N2 subtype influenza viruses at an MOI of 0.1. After the virus was adsorbed for 1 hour, the virus infection solution was aspirated, the plate was gently washed three times with PBS, DMEM medium containing TPCK-trypsin and 1% double antibiotics was added, and a small molecule inhibitor with a final concentration of 10 μM was added to the medium. The cells were then cultured in a CO2 incubator at 37℃.

[0152] (3) Collect 100 μL of cell supernatant at 12h, 24h, 36h and 48h after infection and label each tube; store the cell supernatant in a -80℃ freezer for later use.

[0153] (4) After the samples at each time point are collected, the viral titer of all samples is determined uniformly, and the TCID of each sample is calculated according to the Reed-Muench method. 50 value.

[0154] (5) One day in advance, seed MDCK cells in a 96-well plate. When the cell density is 70% to 80%, discard the serum-containing DMEM medium and wash three times with PBS buffer.

[0155] (6) Remove the frozen virus solution or tissue organs from the -80℃ freezer 30 minutes in advance and place them on ice to thaw naturally. Prepare eight 1.5mL sterile centrifuge tubes, and add 450μL of DMEM containing TPCK-trypsin (final concentration of 2μg / mL) and 1% penicillin antibody to each tube. Add 50μL of the original virus solution to the first tube and vortex to mix. Aspirate 50μL of the mixture and add it to the second tube. Serially dilute the mixture tenfold until the eighth tube is reached. Label the tubes and place them on ice temporarily.

[0156] (7) Virus of various dilutions was inoculated into 96-well plates at a dose of 100 μL / well, with three replicates for each dilution. After incubation for approximately 36-48 hours, hemagglutination was measured in each well, and indirect immunofluorescence (IFA) detection was performed. IFA detection was performed according to the above procedure, and multi-step growth curves of different influenza subtypes on A549 cells under inhibitor treatment were plotted based on the results. The virus growth curves after influenza virus infection and subsequent inhibitor treatment are shown in Figure C.

[0157] Further analysis of viral growth kinetics revealed that VE-822, BDA-366, Nepicastat (SYN-117)HCl, and GSK2656157 significantly inhibited the replication of multiple influenza virus subtypes in mammalian cells, providing more options for combating various influenza viruses. Conversely, HJC0350, GSK2292767, and VPS34-IN1 significantly promoted the growth and replication of multiple influenza virus subtypes in mammalian cells, suggesting that these inhibitors target and regulate influenza virus replication in host cells, providing a theoretical basis for further research into the mechanisms of influenza virus adaptation to the host. Furthermore, Bedaquiline and CPI-360 specifically promoted the replication of H9 subtype avian influenza virus in mammalian cells, but had no effect on H1 subtype human influenza. EPZ005687 specifically inhibited the replication of H9 subtype avian influenza virus, but had no effect on H1 subtype human influenza, indicating that their targets specifically regulate the replication of avian influenza virus in mammalian cells, potentially facilitating cross-host transmission of avian influenza virus. To provide a rich stockpile of drugs to combat various subtypes of influenza viruses and to provide drugs that target different subtypes from different host sources.

[0158] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of the first inhibitor as a reagent for manufacturing an inhibitor of human influenza virus replication and an inhibitor of avian influenza virus replication; The first inhibitor is one or a combination of VE-822, BDA-366, Nepicastat (SYN-117) HCl, and GSK2656157.

2. The use according to claim 1, characterized in that the human influenza virus is an H1N1 subtype human influenza virus; and the avian influenza virus is one or more of the H9N2 subtype avian influenza virus, H5N1 subtype avian influenza virus, and H7N9 subtype avian influenza virus.

3. The use according to claim 1 or 2, characterized in that the virus replicates in a culture medium or in the body of an animal, including a human.

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