Use of AXL kinase inhibitors as anti-Coxsackie B virus drugs

Abstract

The present invention relates to the use of AXL kinase inhibitors as anti-coxsackie B virus drugs. The present invention discovered that AXL kinase inhibitors have extremely significant inhibitory effects on coxsackie B virus mRNA, viral proteins, and plaque formation, reducing the infectivity of coxsackie B virus and inhibiting coxsackie B virus infection of cells, thereby exhibiting excellent anti-coxsackie B virus activity. Further studies have shown that AXL kinase inhibitors exhibit anti-coxsackie B virus activity by inhibiting activation of the AXL / AKT / ERK signaling pathway. The excellent 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.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more particularly 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 officially named Coxsackievirus in 1963. Coxsackievirus B infection can easily induce cardiac or muscle diseases, such as myocarditis. Furthermore, CVB infection has been reported to cause other inflammatory diseases, such as hand, foot, and mouth disease, aseptic meningitis, pleuritic pain, panuveitis, acute pancreatitis, and fulminant meningitis hepatitis. Despite a significant increase in the incidence of CVB infection in humans in recent years, with sporadic outbreaks worldwide, and despite being one of the leading causes of clinical viral myocarditis, research on antiviral drugs targeting CVB is currently very limited.

[0003] For CVB infection, there is still a lack of specific antiviral drugs in the clinic, 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 rhinoviruses 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, there is a need for drugs with good therapeutic effects against Coxsackie B virus. Summary of the Invention

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

[0005] The above-mentioned purpose of the present invention is achieved through 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 discovered through research that AXL kinase inhibitors significantly inhibit coxsackie B virus mRNA expression, viral protein expression, and plaque formation, reducing the infectivity of coxsackie B virus and inhibiting its infection of cells, thereby demonstrating excellent anti-coxsackie B virus activity. Further studies have shown that AXL kinase inhibitors exhibit anti-coxsackie B virus activity by inhibiting activation of the AXL / AKT / ERK signaling pathway. The excellent anti-coxsackie B virus activity of AXL kinase inhibitors provides an effective treatment for coxsackie B virus infection and the diseases it causes.

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

[0010] Among them, R428 is bemcentinib, CAS number 1037624-75-1. TP-0903 is dubermatinib, CAS number 1341200-45-0. SGI-7079 is CAS number 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 Coxsackie B virus mRNA.

[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 regulator.

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

[0022] Preferably, the dosage form of the drug is 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 discovered that AXL kinase inhibitors have a highly significant inhibitory effect on coxsackie B virus mRNA, viral proteins, and plaque formation, reducing the infectivity of coxsackie B virus and inhibiting its infection of cells, thereby exhibiting excellent anti-coxsackie B virus activity. Further studies have shown that AXL kinase inhibitors exhibit anti-coxsackie B virus activity by inhibiting activation of the AXL / AKT / ERK signaling pathway. The excellent anti-coxsackie B virus activity of AXL kinase inhibitors provides an effective treatment for coxsackie B virus infection and the diseases it causes. 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 CVB1 virus mRNA expression; Figure 2 C is a graph showing the experimental results of the R428 inhibitory effect on the expression of CVB1 viral protein VP1 in Example 1; Figure 2 D is a statistical diagram of the expression effect of R428 inhibitory protein VP1 in Example 1.

[0028] Figure 3 A is the experimental result of Western blotting analysis of the R428 signaling pathway in Example 1; Figure 3 B is a statistical diagram of the changes in 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 a statistical diagram of 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 2 for inhibiting the cytopathic effect of CVB1 virus; Figure 5 B is the inhibition curve of TP-0903 in Example 2 in inhibiting the cytopathic effect of CVB3 virus.

[0031] Figure 6 A is the inhibition curve of SGI-7079 in Example 3 for inhibiting the cytopathic effect of CVB1 virus; Figure 6 B is the inhibition curve of SGI-7079 in Example 3 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 example provides the use of R428 as a drug against Coxsackie B virus. R428 is Bemcentinib, CAS No. 1037624-75-1, and its structure is as follows: .

[0035] 1.1 Antiviral Activity of R428 against CVB1 Virus

[0036] 1.1.1 Inhibition of CVB1 virus cytopathic effect

[0037] 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 incubation 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 pathological, shrunken and died; while in the low-dose administration group (6.25nM), most cells grew normally, with only a small number of cells becoming pathological; and in the high-dose administration group (25nM), the cells were normal, with almost no cells infected, indicating that R428 can significantly inhibit the infection of Coxsackie B virus to cells.

[0038] After observation under a microscope, cells in each group were added with the cell viability detection reagent alamarblue and incubated at 37°C, 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 (IC50) were calculated using Graphpad Prime. 50 ), and finally the IC of the cytopathic effect of R428 on CVB1 was obtained after three independent repetitions of the experiment. 50 was 8.80±3.37nM, and the results were as follows 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.

[0039] 1.1.2 Inhibitory effect on viral mRNA expression

[0040] 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. 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 CVB1 virus mRNA equivalent to that of a higher concentration of doxetine hydrochloride, indicating that R428 significantly inhibits the replication of CVB1 virus.

[0041] 1.1.3 Inhibitory effect on the expression of viral protein VP1

[0042] A virus control group, a drug administration group, and a positive control group were set up. Vero cells in the drug administration group were infected with CVB1 virus at a viral dose of 0.2 MOI, and R428 drug solution (25nM) was added at the same time. Vero cells in the positive control group were infected with CVB1 virus at a viral dose of 0.2 MOI, 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 with RIPA lysis buffer, and then subjected to western blotting analysis 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.

[0043] 1.1.4 R428 anti-CVV activity mechanism study

[0044] 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 viral 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 viral 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 pathway AKT, ERK proteins and their phosphorylated proteins to determine the drug's mechanism of action. The results are shown in Figure 2. 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.

[0045] 1.2 Antiviral Activity of R428 against CVB3 Virus

[0046] 1.2.1 Inhibition of CVB3 virus cytopathic effect

[0047] 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 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 the cytopathic effect of R428 on CVB3 was obtained by three independent repetitions of the experiment. 50 was 13.18±3.05nM, and the results were as follows Figure 4 As shown in A, this indicates that R428 can significantly inhibit the infection of Coxsackie B virus to cells.

[0048] 1.2.2 Inhibition of CVB3 plaque formation

[0049] 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 30-60 PFU / well. After 2 hours of incubation at 37°C and 5% CO2, the culture medium was replaced with an overlay medium containing gradient concentrations of R428 solution (1.56nM, 12.5nM, and 25nM) and 2% FBS and 1% methylcellulose. After further incubation for 72 hours, the cells were fixed with 10% formaldehyde and stained with 1% crystal violet. The number of plaques and the inhibition rate of plaque formation were then calculated. The results are shown in Figure 2. Figure 4 As shown in B. Figure 4 As shown in B, R428 inhibited the plaque formation of CVB3 at a concentration of 25 nM. The IC value of R428 for inhibiting the plaque formation was calculated to be 50 The value was 10.69±4.41nM, which further indicated that R428 could inhibit the ability of Coxsackie B virus to infect cells.

[0050] Example 2

[0051] This example provides the use of TP-0903 as a drug against Coxsackie B virus. TP-0903 is Dubermatinib, CAS No. 1341200-45-01, and its structure is as follows: .

[0052] 2.1 Antiviral Activity of TP-0903 against CVB1

[0053] 2.1.1 Inhibition of CVB1 virus cytopathic effect

[0054] 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 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 (IC50) were calculated using Graphpad Prime. 50 ), and finally the IC value of TP-0903 on CVB1 cytopathic effect was obtained after three independent repetitions of the experiment. 50 was 132.30±69.40nM, and the results were as follows Figure 5 As shown in A, this indicates that TP-0903 can significantly inhibit the infection of Coxsackie B virus to cells.

[0055] 2.2 Antiviral Activity of TP-0903 against CVB3 Virus

[0056] 2.2.1 Inhibition of CVB3 virus cytopathic effect

[0057] 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 (IC50) were calculated using Graphpad Prime. 50 ), and finally the IC value of TP-0903 on CVB3 cytopathic effect was obtained after three independent repetitions of the experiment. 50 was 89.20±19.37nM, and the results were as follows Figure 5 As shown in Figure B, this indicates that TP-0903 can significantly inhibit the infection of Coxsackie B virus to cells.

[0058] Example 3

[0059] 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: .

[0060] 2.1 Antiviral Activity of SGI-7079 against CVB1 Virus

[0061] 2.1.1 Inhibition of CVB1 virus cytopathic effect:

[0062] 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 cytopathic 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 (IC50) were calculated using Graphpad Prime. 50 ), and finally the cytopathic effect IC of SGI-7079 on CVB1 was obtained by three independent repetitions of the experiment. 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.

[0063] 2.2 Antiviral Activity of SGI-7079 against CVB3 Virus

[0064] 2.2.1 Inhibition of CVB3 virus cytopathic effect:

[0065] 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 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 (IC50) 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, and the results were as follows Figure 6 As shown in B, this indicates that SGI-7079 can significantly inhibit the infection of Coxsackie B virus to cells.

[0066] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of an AXL kinase inhibitor in the preparation of a drug against Coxsackie B virus, characterized in that: The AXL kinase inhibitor is R428; The anti-Coxsackie B virus serotype is at least one of CVB1 and CVB3.

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

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

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

5. The application according to claim 1, characterized in that: The drug is a drug for inhibiting protein expression of Coxsackie B virus.

6. The application according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.

7. The application according to claim 6, 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 regulator.

8. The application according to claim 1, characterized in that: The dosage form of the medicine is injection, capsule, tablet, pill or granule.

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