Application of compound Tolimidone

Abstract

The invention discloses application of a compound Tolimidone, and belongs to the technical field of biological medicines. The invention provides an application of a compound Tolimidone in preparation of a medicament for preventing and treating influenza virus, and discloses the effect of human tyrosine kinase LYN on phosphorylation of influenza A virus NP protein, so that the effect of an LYN agonist Tolimidone on inhibiting influenza A virus is clear; the Tolimidone can reduce the replication effect of the influenza A virus in different cells and inhibit the replication of different subtype influenza viruses, so that the Tolimidone has great significance and application value in clinical treatment and prevention of influenza A virus infection. The invention also provides an application of the compound Tolimidone in an influenza virus kit. The invention also provides an application of the compound Tolimidone in health care products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of the compound Tolimidone. Background Art

[0002] The NP protein of influenza A virus is the most highly expressed viral protein in influenza virus infected cells. It plays a central role in the influenza virus replication process and is an ideal drug design target. In the previous study, the present invention found that the tyrosine kinase LYN in the human body can affect the function of NP protein by tyrosine phosphorylation modification of NP protein, restore the function of the host immune system, and resist viral replication. Based on this study, upregulating the kinase activity of LYN and promoting NP phosphorylation can become an effective therapeutic method for inhibiting influenza A virus. For this reason, the use of the compound Tolimidone in anti-influenza virus infection is proposed. Summary of the invention

[0003] The purpose of the present invention is to provide the use of the compound Tolimidone. The compound can target host factors, act as an agonist of host factors, inhibit the replication of influenza viruses, have broad-spectrum antiviral activity, and play an important role in the prevention and treatment of influenza viruses. Compared with existing influenza therapeutic drugs, the compound Tolimidone has a different anti-influenza virus mechanism of action, is a beneficial supplement to existing influenza antiviral drugs, and provides a new option for anti-influenza virus therapeutic drugs.

[0004] Tolimidone, CAS number: 41964-07-2; molecular formula is C 11 H 10 N2O2; also known as: MLR-1023. The chemical structure of the compound Tolimidone is shown below: The specific technical solutions are as follows: In a first aspect, the present invention provides use of the compound Tolimidone in the preparation of a medicament for preventing and treating influenza virus.

[0005] As a further improvement of the present invention, the virus is influenza A virus.

[0006] As a further improvement of the present invention, it includes one or more of preparing antiviral products, preparing virus-inhibiting products, and preparing viral infection prevention products.

[0007] As a further improvement of the present invention, the medicine includes compounds or pharmaceutically acceptable salts of compounds used in preparing antiviral products, preparing virus-inhibiting products or preparing products for preventing viral infection, and pharmaceutically acceptable adjuvants.

[0008] As a further improvement of the present invention, the pharmaceutically acceptable adjuvant includes one or more of pharmaceutically acceptable carriers, excipients, and diluents.

[0009] As a further improvement of the present invention, the dosage form of the drug includes at least one of tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral solutions, injections, and powders.

[0010] As a further improvement of the present invention, the concentration of Tolimidone is not less than 20 μM, preferably 20 μM - 80 μM.

[0011] As a further improvement of the present invention, the virus-inhibiting product is used to inhibit the replication of influenza A virus.

[0012] In a second aspect, the present invention provides the use of the compound Tolimidone in an influenza virus kit, which is a reagent comprising Tolimidone and / or its analogs and / or its derivatives, and a kit comprising Tolimidone and / or its analogs and / or its derivatives in combination with other reagents to form a complete set.

[0013] In a third aspect, the present invention provides the use of the compound Tolimidone in health care products, which are foods having preventive or health-care effects and comprising Tolimidone and / or its analogs and / or its derivatives.

[0014] The beneficial effects of the present invention are as follows: the application of the small molecule compound Tolimidone in antiviral infection reveals the effect of human tyrosine kinase LYN through phosphorylation of influenza A virus NP protein, thereby clarifying that the LYN agonist Tolimidone has the effect of inhibiting influenza A virus; Tolimidone can reduce the replication of influenza A virus in different cells and inhibit the replication of different subtypes of influenza virus, which has great significance and application value for the clinical treatment and prevention of influenza A virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result diagram of Tolimidone promoting NP protein phosphorylation; Figure 2 This is a graph showing the results of a cytotoxicity study of Tolimidone; Figure 3 This is a graph showing the results of Tolimidone inhibiting the replication of influenza A virus at the cellular level; Figure 4 This is a graph showing the results of Tolimidone inhibiting the replication of influenza A virus in mice. DETAILED DESCRIPTION

[0016] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0017] The LYN protein plasmid was constructed by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0018] Cell culture: A549 cells are derived from human lung cancer epithelial cells; U2OS cells are derived from human osteosarcoma cells; cells were cultured in a 5% CO2 incubator (37°C) using DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody.

[0019] Virus source: IAV-PR8 strain was stored in Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0020] Tolimidone (MLR-1023) was dissolved in dimethyl sulfoxide (DMSO; Innochem, D3855) at a concentration of 10 mM and stored at -80°C until use. When used, it was diluted to different concentrations with DMEM for CCK-8 cytotoxicity detection experiments; when used, it was diluted to different concentrations with opti-MEM (Gibco, 31985-070) for antiviral drug intervention experiments.

[0021] Statistical analysis: For comparison of multiple groups, one-way ANOVA with Dunnett's post hoc test was used (GraphPad Prism, USA). Data are expressed as mean ± SEM.

[0022] Embodiment 1, Tolimidone promotes NP protein phosphorylation.

[0023] Normal HEK293T cells were counted and seeded in 6-well plates (2*10 6 100 μM Tolimidone was added to the culture medium at the same time.

[0024] After 24 hours of transfection, the protein samples were lysed and immunoprecipitated with Flag antibody to detect the phosphorylation of NP protein. The results showed that LYN promoted NP protein tyrosine phosphorylation, and Tolimidone further promoted NP protein tyrosine phosphorylation. Figure 1 shown.

[0025] Embodiment 2, Cytotoxicity studies of tolimidone.

[0026] A549 cells were collected at 3×105 Cells were seeded at a density of 100 μL per well in a 96-well plate and cultured overnight at 37°C. Then, cells were treated with different concentrations of Tolimidone (0-80 μM) at 37°C for 24 hours. Cell activity was detected by Cell Counting Kit-8 (CCK-8; Bio-Tech, C0037) according to the product instructions. In brief, CCK-8 (10%) reagent diluted with DMEM was added to the cell culture plate at a volume of 70 μL per well and incubated with the cells in a 37°C incubator for 2 hours, and then the optical density (OD value) was measured at a wavelength of 450 nm.

[0027] In order to monitor the effect of different concentrations of Tolimidone on cell viability, we evaluated the effect of different concentrations of Tolimidone on cell viability. The cell viability assay showed that Tolimidone did not show any cytotoxicity in A5490 cells at a dose below 80 μM. Figure 2 shown.

[0028] Embodiment 3, Tolimidone inhibits influenza A virus infection.

[0029] Normal A549 cells and U2OS cells were counted and seeded in 12-well plates (5*10 5 At the same time, different concentrations of compound Tolimidone (20 μM, 40 μM, 80 μM) were added into the cell culture medium.

[0030] When the cell density reached 90%, IAV-PR8 strain (0.01 MOI) was inoculated, and the cell supernatant was collected at 24 h and 48 h after inoculation.

[0031] EID 50 The determination steps are as follows: prepare 9-11 day old SPF chicken embryos. Inoculate the above cell supernatant and make a 10-fold dilution to 10 -8 , select 10 -2 -10 -8 , inoculate 9-11 day old SPF chicken embryos, three chicken embryos per dilution, 0.1 mL / embryo. After 48 hours of incubation, all chicken embryos were taken out for hemagglutination test. If there was hemagglutination, it was considered positive for infection. Then, the EID was calculated according to the hemagglutination titer formula. 50 That is the virus titer.

[0032] The results of virus titration showed that Tolimidone concentrations greater than 20 µM significantly inhibited the virus titer in A549 cells 24 h and 48 h after influenza virus infection. Figure 3 As shown in A and B. 20-80 µM Tolimidone can also inhibit the viral titer of U2OS cells after influenza virus infection, such as Figure 3 As shown in C and D. This indicates that the compound Tolimidone can inhibit the replication of influenza A virus when the concentration is greater than 20 µM.

[0033] Embodiment 4, Detection of the inhibitory effect of the compound Tolimidone on influenza A virus infection in a mouse model.

[0034] In order to further identify the effect of the compound Tolimidone in inhibiting influenza A virus in vivo, the C57BL / 6N mouse model was selected for testing.

[0035] 25 5-week-old C57BL / 6N female mice were purchased and randomly divided into 5 groups, with 5 mice in each group. The mice were intraperitoneally injected with the drug (the drug solvent was 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, and the drug was the compound Tolimidone). The dosages were 12.5 mg / kg, 25 mg / kg, 50 mg / kg and a blank control group, respectively. The drug was administered for seven consecutive days before the challenge and for seven consecutive days after the challenge, for a total of 14 days. Influenza A virus challenge dose: 10 4 EID 50 / 50 µl. The body weight and survival rate of mice were measured and recorded daily during the challenge period.

[0036] Through the statistics of mouse weight and survival rate, we found that the weight loss of mice at a dose of 25 mg / kg was significantly lower than that of mice at a dose of 25 mg / kg. Figure 4 A) and survival rate ( Figure 4 B) significantly higher than the other groups.

[0037] The above experimental results show that the compound Tolimidone can inhibit the replication of influenza A virus in a mouse model, thereby protecting mice without causing any observable side effects on the mice.

Claims

1. Application of the compound Tolimidone in the preparation of drugs for preventing and treating influenza virus.

2. Use of the compound Tolimidone according to claim 1 in the preparation of a drug for preventing and treating influenza virus, characterized in that: The virus is influenza A virus.

3. Use of the compound Tolimidone according to claim 2 in the preparation of a drug for preventing and treating influenza virus, characterized in that: It includes one or more of the preparation of antiviral products, preparation of virus inhibition products, and preparation of products for preventing viral infection.

4. Use of the compound Tolimidone according to claim 3 in the preparation of a drug for preventing and treating influenza virus, characterized in that: The medicine comprises the compound Tolimidone and / or its analogs and / or its derivatives and / or its pharmaceutically acceptable salts and / or pharmaceutically acceptable adjuvants.

5. Use of the compound Tolimidone according to claim 4 in the preparation of a drug for preventing and treating influenza virus, characterized in that: The pharmaceutically acceptable adjuvant includes one or more of pharmaceutically acceptable carriers, excipients, and diluents.

6. Use of the compound Tolimidone according to claim 4 in the preparation of a drug for preventing and treating influenza virus, characterized in that: The dosage form of the drug includes at least one of tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral solutions, injections, and powders.

7. Use of the compound Tolimidone according to claim 6 in the preparation of a drug for preventing and treating influenza virus, characterized in that: The concentration of tolimidone is not less than 20 μM, preferably 20 μM-80 μM.

8. Use of the compound Tolimidone according to claim 3 in the preparation of a drug for preventing and treating influenza virus, characterized in that: The virus-inhibiting product is used to inhibit the replication of influenza A virus.

9. Use of the compound Tolimidone in an influenza virus test kit, characterized in that: The kit is a reagent containing Tolimidone and / or its analogs and / or its derivatives, and a kit containing Tolimidone and / or its analogs and / or its derivatives in combination with other reagents to form a complete set.

10. Use of the compound Tolimidone in health care products, characterized in that: The health care product is a food having preventive or health-care effects and comprising Tolimidone and / or its analogs and / or its derivatives.

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