Application of compound EG-011 and pharmaceutical composition thereof in resisting influenza A virus

Abstract

The invention discloses an application of a compound EG-011 and a pharmaceutical composition thereof in resisting influenza A virus. It is found for the first time that EG-011 treated cells can increase the density of actin branches so as to prevent IAV from being transported from plasma membranes to cell nucleuses, and proliferation of IAV in host cells is effectively inhibited. In-vivo experiments prove that the EG-011 can significantly inhibit the proliferation of IAV in the body of a mouse and improve the survival rate of the mouse. The EG-011 has a clear action mechanism, has a good anti-influenza effect, can be used as a lead compound of a novel anti-influenza virus drug, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of a compound EG-011 with anti-influenza A virus activity and its pharmaceutical composition in the prevention or treatment of diseases related to influenza virus infection. Background Art

[0002] Influenza A virus (IAV) is a zoonotic virus with strong infectivity and fast transmission speed, which can cause seasonal influenza epidemics or even global influenza pandemics, posing a serious threat to the livestock and poultry breeding industry and human health. IAV has a high degree of variability, which may lead to the failure of drugs and vaccines developed against its protein structure as the virus mutates, bringing huge challenges to prevention and control work. Currently, the main anti-influenza drugs clinically used include neuraminidase inhibitors (such as oseltamivir, zanamivir) and polymerase inhibitors (such as favipiravir, baloxavir), etc. Although these drugs can relieve the symptoms of influenza virus infection to a certain extent, their action targets mainly focus on viral proteins. With the increase in the frequency of use, more and more virus strains show drug resistance, resulting in a decline in drug efficacy. In recent years, antiviral strategies targeting host factors have received extensive attention. Relevant studies have shown that after influenza virus infects host cells, it must rely on the actin cytoskeleton system in the cell to complete the transport from the plasma membrane to the nucleus. Therefore, intervening in the dynamic structure of actin is considered a potential and effective antiviral means. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide the use of compound EG-011 in the prevention or treatment of diseases related to influenza A virus infection.

[0004] The technical solution of the present invention is: the use of EG-011 or its pharmaceutically acceptable derivatives, salts, esters or prodrugs in the preparation of a drug for preventing or treating influenza A virus infection, and the structural formula of the said EG-011 is as follows:

[0005]

[0006] EG-011 is a small molecule compound, which can significantly inhibit the replication of IAV in host cells. This compound shows good antiviral activity in both cell experiments and animal experiments, and has low toxicity and good biosafety.

[0007] EG-011 inhibits the transport of influenza virus from the plasma membrane to the nucleus by inducing the branched polymerization of actin and increasing the actin branch density, and significantly reduces the replication efficiency of the virus in host cells.

[0008] EG-011 does not directly target viral proteins, but acts on the actin cytoskeleton system within host cells. Therefore, it is not likely to induce viral drug-resistant mutations and has potential broad-spectrum antiviral advantages, being applicable to different subtypes of influenza A virus.

[0009] EG-011 has significant inhibitory effects on a variety of influenza A virus subtypes, including but not limited to H1N1, H3N2, H5N1, H7N9, and H9N2, etc., demonstrating broad-spectrum antiviral activity.

[0010] EG-011 can be used as a single drug or in combination with other drugs having anti-influenza activity to form a synergistic drug composition, so as to improve the therapeutic effect and reduce the risk of drug resistance.

[0011] Preferably, the synergistic drugs include but not limited to oseltamivir, zanamivir, peramivir, favipiravir, and their pharmaceutically acceptable salts or derivatives.

[0012] Furthermore, the drug composition contains EG-011 or its pharmaceutically acceptable derivatives, salts, esters, or prodrugs as active ingredients.

[0013] Preferably, the drug composition can be prepared into various dosage forms suitable for clinical use, including but not limited to liquid injections, powder aerosols, oral suspensions, tablets, capsules, or nebulized inhalants.

[0014] Preferably, EG-011 is applicable to various administration routes, including oral, intramuscular, intravenous, and nebulized inhalation, etc., having flexible clinical administration routes.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The present invention discovers for the first time that EG-011 has anti-influenza virus activity, especially against influenza A virus. EG-011 can inhibit the proliferation of the virus by inducing the branched polymerization of actin to inhibit the transport of IAV from the plasma membrane to the nucleus.

[0017] (2) In the IAV-infected mouse model, EG-011 shows good in vivo antiviral effects, can improve the survival rate of mice, reduce the viral titer in lung tissues, and alleviate lung injury.

[0018] (3) The mechanism of action of EG-011 is different from that of traditional anti-influenza drugs targeting viral proteins. It exerts its effect by targeting the actin cytoskeleton system of host cells. Since it does not directly act on viral proteins, EG-011 significantly reduces the risk of drug resistance, providing a new strategy and theoretical basis for the research and development of anti-influenza virus drugs. Brief Description of the Drawings

[0019] Figure 1 : EG-011 treatment increases the density of actin branches; (A) Indirect immunofluorescence assay was used to detect the effect of EG-011 treatment on F-actin; (B) Statistical analysis of the length, number of branches, and number of nodes of protein filaments after EG-011 treatment.

[0020] Figure 2 : EG-011 treatment inhibits the transport of IAV from the plasma membrane to the nucleus.

[0021] Figure 3 : EG-011 treatment inhibits the proliferation of IAV in cells, *P<0.05, ***P<0.001, mean±SD(n=3).

[0022] Figure 4 : Flow chart of the experiment on EG-011 treatment and IAV-infected mice.

[0023] Figure 5 : EG-011 treatment alleviates the weight loss of mice caused by IAV infection, *P<0.05, **P<0.01, ***P<0.001, mean±SEM(n=10).

[0024] Figure 6 : EG-011 treatment improves the survival rate of IAV-infected mice, ***P<0.001, mean±SD(n=10).

[0025] Figure 7 : EG-011 treatment reduces the virus titer in the lungs of IAV-infected mice. **P<0.01, ***P<0.001, mean±SD(n=3). Specific implementation manners

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from commercial channels unless otherwise specified.

[0027] Example 1:

[0028] EG-011 treatment increases the density of actin branches

[0029] (1) Seed PK-15 cells (porcine kidney cells) in confocal dishes (Biosharp, BS-20-GJM) and culture them in a 37 °C cell incubator. When the cell confluence rate reaches 30%-40%, wash the cells once with serum-free DMEM (Biochannel, BC-M-005), dilute EG-011 (Selleck, E1332) to 50 nM with serum-free DMEM, add the diluted EG-011 to the cells, and set up an untreated control group. Incubate in a 37 °C cell incubator for 1.5 h.

[0030] (2) After incubation, wash the cells 5 times with PBS (Biosharp, BL302A), and then fix them with 4% paraformaldehyde (Biosharp, BL539A) for 10 min.

[0031] (3) Discard the paraformaldehyde solution, wash 5 times with PBS, add 0.2% Triton-X 100 (Solarbio, T8200) (dissolved in PBS), and permeabilize for 10 min.

[0032] (4) Discard the waste liquid, wash 5 times with PBS, add 1% BSA (BioFroxx, 4240GR500) (dissolved in PBS), and block at room temperature for 1 h.

[0033] (5) Discard the waste liquid, stain F-actin with the TraKine TM F-actin staining kit (Abbkine, KTC4008), and incubate at room temperature for 1 h.

[0034] (6) After incubation, wash the cells 5 times with PBS.

[0035] (7) Observe under a laser confocal microscope according to the operating requirements.

[0036] (8) Perform statistical analysis on the obtained images using ImageJ v1.8.0.

[0037] The results are as Figure 1 shown in A and B below. Treatment with EG-011 significantly increased the length, number of branches, and number of nodes of actin filaments.

[0038] Example 2:

[0039] EG-011 treatment inhibits the transport of IAV from the plasma membrane to the nucleus

[0040] (1) Seed PK-15 cells in confocal dishes and culture them in a 37 °C cell incubator. When the cell confluence rate reaches 30%-40%, wash the cells once with serum-free DMEM. Dilute EG-011 to 50 nM with serum-free DMEM, add the diluted EG-011 to the cells, and set up an untreated control group. Incubate at 37 °C in a cell incubator for 1 h;

[0041] (2) Wash the cells twice with serum-free DMEM. Infect the cells with A / swine / Hubei / 221 / 2016 (HuB / H1N1) at an MOI of 50, and add 50 nM EG-011. Incubate the virus with the cells on ice for 1 h;

[0042] (3) After incubating for 1 h, wash the cells twice with serum-free DMEM, and add 50 nM EG-011 diluted with serum-free DMEM. Incubate at 37 °C in a cell incubator for 50 min;

[0043] (4) Stain F-actin according to the operation steps in (3)-(6) of Example 1;

[0044] (5) Discard the waste liquid, add the corresponding primary antibody: rabbit polyclonal anti-IAV NP (GeneTex, GTX125989), and incubate at room temperature for 2 h;

[0045] (6) Discard the waste liquid, wash 5 times with PBS, add the corresponding secondary antibody: Cy3-labeled goat anti-rabbit (ABclonal, AS007), and incubate at room temperature for 1 h;

[0046] (7) Discard the waste liquid, wash 5 times with PBS, add DAPI (Beyotime, C1002), and incubate at room temperature for 10 min;

[0047] (8) Discard the waste liquid, wash 5 times with PBS, and observe under a laser confocal microscope according to the operation requirements.

[0048] (9) Perform statistical analysis on the obtained images using ImageJ v1.8.0.

[0049] The results are as Figure 2 shown. Treatment with EG-011 inhibited the transport of IAV from the plasma membrane to the nucleus.

[0050] Example 3:

[0051] EG-011 treatment inhibits the proliferation of IAV in cells

[0052] (1) Seed PK-15 cells in a 12-well plate and culture them in a cell incubator at 37°C. When the cell confluence rate reaches 90%-100%, wash the cells once with serum-free DMEM, dilute EG-011 to 50 nM with serum-free DMEM, add the diluted EG-011 to the cells, and set up an untreated control group. Incubate for 1 h in a cell incubator at 37°C;

[0053] (2) Wash the cells twice with serum-free DMEM, inoculate the cells with A / swine / Hubei / 221 / 2016 (HuB / H1N1) at 0.01 MOI, and add 50 nM EG-011. Incubate for 1 h in a cell incubator at 37°C;

[0054] (3) Wash the cells twice with serum-free DMEM, add 50 nM EG-011 diluted with serum-free DMEM, and incubate for 12 h in a cell incubator at 37°C;

[0055] (4) After 12 h, collect the virus supernatant and store it at -80°C;

[0056] (5) TCID 50 Determine the virus titer by experiment:

[0057] a. Seed MDCK cells in a 96-well plate and culture them in a cell incubator at 37°C;

[0058] b. When the cell confluence rate reaches 90%-100%, perform a 10-fold serial dilution of the virus supernatant collected in (4) with serum-free DMEM;

[0059] c. Wash the cells in the 96-well plate twice with serum-free DMEM, inoculate the diluted virus in b. into the 96-well plate, and culture for 1 h in a cell incubator at 37°C;

[0060] d. Discard the virus solution in the 96-well plate, wash the cells twice with serum-free DMEM, add DMEM culture medium containing 0.3 μg / mL TPCK, and culture for 72 h in a cell incubator at 37°C;

[0061] e. After 72 h, transfer the cell supernatant in the 96-well plate to a U-shaped hemagglutination plate, and determine whether there is virus in the well through a hemagglutination experiment;

[0062] f. Calculate the TCID of the virus using the Reed-Muench method 50 .

[0063] The results are as Figure 3 shown, and EG-011 treatment inhibits the proliferation of IAV in cells.

[0064] Example 4:

[0065] Intraperitoneal injection of EG-011 can reduce the mortality rate of mice infected with IAV

[0066] (1) According to the experimental flow chart shown in Figure 4 , prepare 54 6-8-week-old female BALB / c SPF mice, and randomly divide them into 3 groups (group A, group B, and group C). Group A is the PBS negative control group, and group B and group C are intraperitoneally injected with EG-011 (10% DMSO, 40% PEG300, 5% Tween-80, 45% normal saline, 10 mg / kg EG-011) and the control solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% normal saline), respectively. The intraperitoneal injection time is 1 day before influenza virus infection and on days 0 to 4 of infection;

[0067] (2) One day after intraperitoneal injection of EG-011 in mice, the mice were anesthetized by inhalation of ether. On day 0, the mice in group A were instilled with 50 μL of PBS into the nasal cavity as a mock infection control, and the mice in group B and group C were instilled with 30 PFU of HuN / H1N1 (50 μL), respectively. The body weight changes of the mice were recorded daily within two weeks after infection, and their survival status was observed;

[0068] (3) On days 3 and 5 after infection, 3 mice in each group were randomly sacrificed and dissected, and the lung tissues were extracted and homogenized in 0.8 mL of PBS containing 1% penicillin-streptomycin. The homogenate was centrifuged at 12,000 r / min at 4 °C for 20 min, and the supernatant was collected. The virus titer in the lungs was measured according to the operation steps in (5) of Example 3.

[0069] The results are as shown in Figure 5 、 6 、7. Treatment with EG-011 can alleviate the weight loss of mice caused by IAV infection, improve the survival rate of mice infected with IAV, and reduce the virus titer in the lungs of mice infected with IAV.

Claims

1. Use of EG-011 or its pharmaceutically acceptable derivatives, salts, esters or prodrugs in the preparation of a drug for preventing or treating influenza A virus infection, wherein the structural formula of the EG-011 is as follows:

2. The application according to claim 1, wherein The dosage form of the drug is any one of liquid injection, powder aerosol, oral suspension, tablet or capsule.

3. The application according to claim 1, characterized in that The drug further comprises oseltamivir, zanamivir, peramivir, favipiravir or its pharmaceutically acceptable salt or derivative.

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