Application of AXL kinase inhibitor as medicine for resisting coxsackie virus B

Abstract

The invention relates to an application of an AXL kinase inhibitor as a medicine for resisting coxsackie virus B. The invention finds that the AXL kinase inhibitor has an extremely significant inhibition effect on mRNA, viral protein and plaque formation of the coxsackie B virus, can reduce the infection ability of the coxsackie B virus, and inhibits the infection of the coxsackie B virus on cells, so that the AXL kinase inhibitor has good anti-coxsackie B virus activity. A further research shows that the AXL kinase inhibitor shows anti-coxsackie B virus activity by inhibiting the activation of an AXL / AKT / ERK signal channel. The good coxsackie B virus resisting activity of the AXL kinase inhibitor provides an effective scheme for treating coxsackie B virus infection and diseases caused by the coxsackie B virus infection.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to the use of an AXL kinase inhibitor as a drug against Coxsackie B virus. Background Art

[0002] Coxsackievirus B (CVB) belongs to the Picornaviridae family and includes six serotypes: CVB1, CVB2, CVB3, CVB4, CVB5, and CVB6. CVB was first isolated from the feces of two paralyzed children in 1948 and was officially named Coxsackievirus in 1963. Coxsackievirus B infection can easily induce human heart or muscle tissue diseases, such as myocarditis. In addition, it is reported that Coxsackievirus B infection may also cause other inflammatory diseases, such as hand, foot and mouth disease, aseptic meningitis, pleurisy, panuveitis, acute pancreatitis, and fulminant meningitis hepatitis. Although the incidence of human CVB infection has increased significantly in recent years and has broken out irregularly around the world, and it is also one of the main pathogenic factors of clinical viral myocarditis, the current research on antiviral drugs for CVB is very limited.

[0003] For CVB infection, there is still a lack of specific antiviral drugs in clinical practice, and there is also a lack of vaccines to prevent CVB infection, which makes clinical treatment, especially the treatment of severe cases, in a very passive state. The strategy of repurposing old drugs is a very promising drug development strategy. Studies have shown that the antifungal drug itraconazole has the activity of inhibiting enterovirus RNA replication, but the effective inhibitory concentration for CVBs is higher than the therapeutic dose. Azithromycin has been found to reduce the replication and release of rhinovirus by stimulating the antiviral response mediated by the IFN pathway. It can inhibit CVB3 in acute infection of human epithelial cells and pancreatic cells, but cannot inhibit other CVB serotypes. Therefore, drugs with good therapeutic effects against Coxsackie B virus are needed. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the problem that the existing anti-Coxsackie B virus drugs are still relatively scarce or have poor effects, and to provide the use of AXL kinase inhibitors as / in the preparation of anti-Coxsackie B virus drugs.

[0005] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0006] Use of AXL kinase inhibitors as anti-Coxsackie B virus drugs.

[0007] Existing studies have found that AXL kinase inhibitors have anti-tumor activity. For example, there have been reports that AXL kinase inhibitors have been used in limited clinical treatment of acute myeloid leukemia.

[0008] The inventors of the present invention have found through research that AXL kinase inhibitors have extremely significant inhibitory effects on the mRNA expression, viral protein expression and plaque formation of Coxsackie B virus, can reduce the infectivity of Coxsackie B virus, and inhibit the infection of Coxsackie B virus to cells, thus having good anti-Coxsackie B virus activity. Further studies have shown that AXL kinase inhibitors exhibit anti-Coxsackie B virus activity by inhibiting the activation of the AXL / AKT / ERK signaling pathway. The good anti-Coxsackie B virus activity of AXL kinase inhibitors provides an effective solution for the treatment of Coxsackie B virus infection and the diseases caused by it.

[0009] Preferably, the AXL kinase inhibitor is at least one of R428, TP-0903 or SGI-7079.

[0010] Among them, R428 is Bemcentinib, with a CAS number of 1037624-75-1. TP-0903 is Dubermatinib, with a CAS number of 1341200-45-0. The CAS number of SGI-7079 is 1239875-86-5.

[0011] More preferably, the AXL kinase inhibitor is R428. Compared with other AXL kinase inhibitors, R428 exhibits significant anti-Coxsackie B virus activity at a lower dose, that is, R428 has better anti-Coxsackie B virus activity.

[0012] Preferably, the anti-Coxsackie B virus serotype is at least one of CVB1, CVB2, CVB3, CVB4, CVB5 or CVB6.

[0013] Preferably, the drug is a drug for preventing and / or treating hand, foot and mouth disease, myocarditis, aseptic meningitis, pleurisy, panuveitis, acute pancreatitis or fulminant meningitis hepatitis induced by Coxsackie B virus.

[0014] Preferably, the drug is a drug for resisting Coxsackie B virus infection mediated by the AXL-AKT-ERK signaling pathway.

[0015] Preferably, the drug is a drug that inhibits the expression of mRNA of Coxsackie B virus.

[0016] More preferably, the mRNA is VP2 mRNA.

[0017] Preferably, the drug is a drug that inhibits protein expression of Coxsackie B virus.

[0018] More preferably, the protein is VP1 protein.

[0019] Preferably, the medicament comprises pharmaceutically acceptable excipients.

[0020] More preferably, the pharmaceutically acceptable excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH adjuster.

[0021] More preferably, the pharmaceutically acceptable excipient is an excipient.

[0022] Preferably, the drug is in the form of injection, capsule, tablet, pill or granule.

[0023] Preferably, the content of the AXL kinase inhibitor in the drug is 1.5 nM to 3200 nM.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention found that AXL kinase inhibitors have extremely significant inhibitory effects on the mRNA, viral proteins and plaque formation of Coxsackie B virus, can reduce the infectivity of Coxsackie B virus, and inhibit the infection of Coxsackie B virus to cells, thereby having good anti-Coxsackie B virus activity. Further studies have shown that AXL kinase inhibitors exhibit anti-Coxsackie B virus activity by inhibiting the activation of the AXL / AKT / ERK signaling pathway. The good anti-Coxsackie B virus activity of AXL kinase inhibitors provides an effective solution for the treatment of Coxsackie B virus infection and the diseases caused by it. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the experimental results of R428 in Example 1 inhibiting the cytopathic effect of CVB1 virus.

[0027] Figure 2 A is an inhibition curve diagram of R428 in Example 1 inhibiting the cytopathic effect of CVB1 virus; Figure 2 B is a statistical graph showing that R428 of Example 1 inhibits the expression of CVB1 virus mRNA; Figure 2 C is a graph showing the experimental results of the effect of R428 in Example 1 on inhibiting the expression of CVB1 viral protein VP1; Figure 2 D is a statistical diagram of the expression effect of R428 in Example 1 on the inhibitory protein VP1.

[0028] Figure 3 A is a diagram showing the experimental results of Western blotting analysis of the R428 action signal pathway in Example 1; Figure 3 B is a statistical diagram of changes in the AXL phosphorylated protein p-AXL in Example 1; Figure 3 C is a statistical diagram of changes in AKT phosphorylation protein p-AKT in Example 1; Figure 3D is the statistical diagram of the changes in ERK phosphorylation protein p-ERK in Example 1.

[0029] Figure 4 A is an inhibition curve diagram of R428 in Example 1 inhibiting the cytopathic effect of CVB3 virus; Figure 4 B is a graph showing the experimental results of R428 in Example 1 for inhibiting viral plaque formation activity.

[0030] Figure 5 A is an inhibition curve diagram of TP-0903 in Example 1 inhibiting the cytopathic effect of CVB1 virus; Figure 5 B is the inhibition curve diagram of TP-0903 in Example 1 inhibiting the cytopathic effect of CVB3 virus.

[0031] Figure 6 A is an inhibition curve diagram of the inhibition of the cytopathic effect of CVB1 virus by SGI-7079 in Example 1; Figure 6 B is the inhibition curve diagram of SGI-7079 in Example 1 in inhibiting the cytopathic effect of CVB3 virus. DETAILED DESCRIPTION

[0032] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0033] Example 1

[0034] This embodiment provides the use of R428 as a drug against Coxsackie B virus. R428 is Bemcentinib, CAS 1037624-75-1, and its structure is as follows:

[0035]

[0036] 1.1 Antiviral activity of R428 against CVB1 virus

[0037] 1.1.1 Inhibition of CVB1 virus cytopathic effect

[0038] A normal cell control group, a virus infection control group, and a drug administration group were set up. Vero cells in the drug administration group were infected with CVB1 virus at a virus dose of 0.04 MOI, and different concentrations of R428 solution (1.56nM, 3.12nM, 6.25nM, 12.5nM, and 25nM) were added at the same time. After culturing in an incubator at 37°C and 5% CO2 for 3 days, the cell pathological state was observed under a microscope. The results are as follows: Figure 1 As shown. Figure 1It can be seen that compared with the normal cell control group (Cell control), the cells in the CVB1 infected control group (Virus control) became diseased, shrunken and died; while in the low-dose administration group (6.25nM), most of the cells grew normally, and only a few cells became diseased; the cells in the high-dose administration group (25nM) were normal, and almost no cells were infected, indicating that R428 can significantly inhibit the infection of Coxsackie B virus to cells.

[0039] After observation under a microscope, the cells in each group were added with the cell viability detection reagent alamarblue, and the cells were incubated at 37°C and 5% CO2 for 4 h. The fluorescence value of each well was measured at 530 nm excitation light and 590 nm emission wavelength, and the inhibition rate and half inhibitory concentration (IC) were calculated using Graphpadprime. 50 ), and finally the IC value of the cytopathic effect of R428 on CVB1 was obtained by three independent repeated experiments. 50 was 8.80±3.37nM. Figure 2 As shown in A, this indicates that R428 significantly inhibits the infection of Coxsackie B virus to cells and has good anti-Coxsackie B virus activity.

[0040] 1.1.2 Inhibition of viral mRNA expression

[0041] A virus infection control group, a drug administration group, and a positive control group were set up. The Vero cells in the drug administration group were infected with CVB1 virus at a virus dose of 0.2MOI, and different concentrations of R428 solution (12.5nM and 25nM) were added at the same time. The Vero cells in the positive control group were infected with CVB1 virus at a virus dose of 0.2MOI, and the drug used at the same time was doxepin hydrochloride (Doxepin) of known activity. After culturing in a 37°C, 5% CO2 incubator for 24 hours, the cells were harvested, total RNA was extracted, and then RT-qPCR was performed to detect the expression of the viral VP2 gene. The results are shown in Figure 2 As shown in B, Figure 2 B shows that R428 almost completely inhibits the expression of VP2 mRNA of CVB1 virus at a concentration of 25 nM (p<0.00001). At a lower concentration, it shows an inhibitory effect on the mRNA of CVB1 virus comparable to that of doxetine hydrochloride at a higher concentration, indicating that R428 significantly inhibits the replication of CVB1 virus.

[0042] 1.1.3 Inhibition of the expression of viral protein VP1

[0043] A virus control group, a drug administration group, and a positive control group were set up. The Vero cells in the drug administration group were infected with CVB1 virus at a virus dose of 0.2MOI, and R428 drug solution (25nM) was added at the same time. The Vero cells in the positive control group were infected with CVB1 virus at a virus dose of 0.2MOI, and the drug used at the same time was doxepin hydrochloride (Doxepin) of known activity. After culturing in a 37°C, 5% CO2 incubator for 24 hours, the cells were collected, lysed by adding RIPA lysis buffer, and then western blotting analysis was performed to detect the expression of viral protein VP1. The results are shown in Figure 2. Figure 2 C and Figure 2 As shown in D, Figure 2 C and Figure 2 D shows that R428 significantly inhibited the expression of viral VP1 protein at a concentration of 25nM (p<0.00001), indicating that R428 significantly inhibited the expression of CVB1 viral protein.

[0044] 1.1.4 Experiment on the mechanism of action of R428 against CVB virus

[0045] A virus control group, a drug administration group, and a positive control group were set up. The Vero cells in the drug administration group were infected with CVB1 virus at a virus dose of 0.2MOI, and R428 drug solution (25nM) was added at the same time. The Vero cells in the positive control group were infected with CVB1 virus at a virus dose of 0.2MOI, and the drug used at the same time was doxepin hydrochloride (Doxepin) of known activity. After culturing in a 37°C, 5% CO2 incubator for 24 hours, protein samples were collected to detect the expression of AXL and its downstream signaling pathways AKT, ERK proteins and their phosphorylated proteins to determine the drug action mechanism. The results are shown in Figure 3 As shown. Figure 3 It can be seen that CVB1 infection can activate the AXL / AKT / ERK signaling pathway, and R428 treatment can inhibit the activation of this signaling pathway, thereby exerting an antiviral effect. This indicates that the AXL / AKT / ERK signaling pathway is involved in viral infection, and AXL kinase inhibitors can be used as drugs to resist Coxsackie B virus infection mediated by the AXL / AKT / ERK signaling pathway.

[0046] 1.2 Antiviral activity of R428 against CVB3 virus

[0047] 1.2.1 Inhibition of CVB3 virus cytopathic effect

[0048] A normal cell control group, a virus infection control group, and a drug administration group were set up. Vero cells in the drug administration group were infected with CVB3 virus at a virus dose of 0.04 MOI, and different concentrations of R428 solution (1.56nM, 3.12nM, 6.25nM, 12.5nM, and 25nM) were added at the same time. After culturing in a 37°C 5% CO2 incubator for 3 days, the cell viability detection reagent alamarblue was added. After reacting for 4 hours, the fluorescence value of each well was measured at 530nm excitation light and 590nm emission wavelength, and the inhibition rate and half inhibitory concentration (IC) were calculated using Graphpad prime. 50 ), and finally the IC value of the cytopathic effect of R428 on CVB3 was obtained by three independent repetitions of the experiment. 50 was 13.18±3.05nM. Figure 4 As shown in A, this indicates that R428 can significantly inhibit the infection of Coxsackie B virus to cells.

[0049] 1.2.2 Inhibition of CVB3 plaque formation effect

[0050] A normal cell control group, a virus infection control group, and a drug administration group were set up. Vero cells in the drug administration group were infected with CVB3 virus at a virus amount of 30-60 PFU / well. After 2 hours in a 37°C, 5% CO2 incubator, the culture medium was replaced with a covering medium containing a gradient concentration of R428 solution (1.56nM, 12.5nM, and 25nM) and 2% FBS and 1% methylcellulose. After continued culture for 72 hours, 10% formaldehyde fixation and 1% crystal violet staining were used, and then the number of plaques and the inhibition rate of plaque formation were calculated. The results are shown in the figure. Figure 4 As shown in B. Figure 4 B shows that R428 inhibits the plaque formation of CVB3 at a concentration of 25 nM. The IC value of R428 inhibiting the plaque formation can be calculated. 50 The value was 10.69±4.41nM, which further indicated that R428 could inhibit the ability of Coxsackie B virus to infect cells.

[0051] Example 2

[0052] This embodiment provides the use of TP-0903 as a drug against Coxsackie B virus. TP-0903 is Dubermatinib, CAS 1341200-45-01, and its structure is as follows:

[0053]

[0054] 2.1 Antiviral Activity of TP-0903 against CVB1 Virus

[0055] 2.1.1 Inhibition of CVB1 virus cytopathic effect

[0056] A normal cell control group, a virus infection control group, and a drug administration group were set up. Vero cells in the drug administration group were infected with CVB1 virus at a virus dose of 0.04 MOI, and different concentrations of TP-0903 solution (6.25nM, 12.5nM, 25nM, 50nM, 100nM, and 200nM) were added at the same time. After culturing in a 37°C, 5% CO2 incubator for 3 days, the cell pathological state was observed under a microscope, and the cell viability detection reagent alamarblue was added. After reacting for 4 hours, the fluorescence value of each well was measured at 530nm excitation light and 590nm emission wavelength, and the inhibition rate and half inhibitory concentration (IC) were calculated using Graphpad prime. 50 ), and finally the IC value of TP-0903 on CVB1 was obtained by three independent repetitions of the experiment. 50 was 132.30±69.40nM. Figure 5 As shown in A, this indicates that TP-0903 can significantly inhibit the infection of Coxsackie B virus to cells.

[0057] 2.2 Antiviral Activity of TP-0903 against CVB3 Virus

[0058] 2.2.1 Inhibition of CVB3 virus cytopathic effect

[0059] A normal cell control group, a virus infection control group, and a drug administration group were set up. Vero cells in the drug administration group were infected with CVB3 virus at a virus dose of 0.04 MOI, and different concentrations of TP-0903 solution (6.25nM, 12.5nM, 25nM, 50nM, 100nM, and 200nM) were added at the same time. After culturing in a 37°C, 5% CO2 incubator for 3 days, the cell viability detection reagent alamarblue was added. After 4 hours of reaction, the fluorescence value of each well was measured at 530nm excitation light and 590nm emission wavelength, and the inhibition rate and half inhibitory concentration (IC) were calculated using Graphpad prime. 50 ), and finally the IC value of TP-0903 on CVB3 was obtained by three independent repeated experiments. 50 is 89.20±19.37nM. Figure 5 As shown in B, this indicates that TP-0903 can significantly inhibit the infection of Coxsackie B virus to cells.

[0060] Example 3

[0061] This embodiment provides the use of SGI-7079 as a drug against Coxsackie B virus. The CAS number of SGI-7079 is 1239875-86-5, and its structure is as follows:

[0062] 2.1 Antiviral activity of SGI-7079 against CVB1

[0063] 2.1.1 Inhibition of CVB1 virus cytopathic effect:

[0064] A normal cell control group, a virus infection control group, and a drug administration group were set up. Vero cells in the drug administration group were infected with CVB1 virus at a virus dose of 0.04 MOI, and different concentrations of SGI-7079 solution (195nM, 391nM, 781nM, 1562.5nM, and 3125nM) were added at the same time. After culturing in a 37°C 5% CO2 incubator for 3 days, the cell pathological state was observed under a microscope, and the cell viability detection reagent alamarblue was added. After 4 hours of reaction, the fluorescence value of each well was measured at 530nm excitation light and 590nm emission wavelength, and the inhibition rate and half inhibitory concentration (IC) were calculated using Graphpad prime. 50 ), and finally the cytopathic effect IC of SGI-7079 on CVB1 was obtained by three independent repeated experiments. 50 was 2.20±0.82μM. Figure 6 As shown in A, this indicates that SGI-7079 can significantly inhibit the infection of Coxsackie B virus to cells.

[0065] 2.2 Antiviral Activity of SGI-7079 against CVB3 Virus

[0066] 2.2.1 Inhibition of CVB3 virus cytopathic effect:

[0067] A normal cell control group, a virus infection control group, and a drug administration group were set up. Vero cells in the drug administration group were infected with CVB3 virus at a virus dose of 0.04 MOI, and different concentrations of SGI-7079 solution (195nM, 391nM, 781nM, 1562.5nM, and 3125nM) were added at the same time. After culturing in a 37°C, 5% CO2 incubator for 3 days, the cell viability detection reagent alamarblue was added. After reacting for 4 hours, the fluorescence value of each well was measured at 530nm excitation light and 590nm emission wavelength, and the inhibition rate and half inhibitory concentration (IC) were calculated using Graphpad prime. 50 ), and finally the cytopathic effect IC of SGI-7079 on CVB3 was obtained by three independent repetitions of the experiment. 50 was 1.05±0.25μM. Figure 6 As shown in B, this indicates that SGI-7079 can significantly inhibit the infection of Coxsackie B virus to cells.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Use of AXL kinase inhibitors as drugs against Coxsackie B virus.

2. The use according to claim 1, It is characterized in that The AXL kinase inhibitor is at least one of R428, TP-0903 or SGI-7079.

3. The use according to claim 1, It is characterized in that The anti-Coxsackie B virus serotype is at least one of CVB1, CVB2, CVB3, CVB4, CVB5 or CVB6.

4. The use according to claim 1, It is characterized in that The medicine is used for preventing and / or treating hand, foot and mouth disease, myocarditis, aseptic meningitis, pleurisy, panuveitis, acute pancreatitis or fulminant meningitis hepatitis induced by Coxsackie B virus.

5. The use according to claim 1, It is characterized in that The drug is a drug for resisting Coxsackie B virus infection mediated by the AXL-AKT-ERK signaling pathway.

6. The use according to claim 1, It is characterized in that The drug is a drug for inhibiting the mRNA expression of Coxsackie B virus.

7. The use according to claim 1, It is characterized in that The drug is a drug for inhibiting protein expression of Coxsackie B virus.

8. The use according to claim 1, It is characterized in that The drug includes pharmaceutically acceptable excipients.

9. The use according to claim 8, It is characterized in that The pharmaceutically acceptable excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH adjuster.

10. The use according to claim 1, It is characterized in that The dosage form of the medicine is injection, capsule, tablet, pill or granule.

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