Application of apatinib in the preparation of antiviral drugs

Abstract

This invention provides the application of apatinib in the preparation of antiviral drugs, specifically involving the application of the anticancer drug apatinib in inhibiting the replication of Hantanensis virus (HTNV) and Chikungunya virus (CHIKV). Apatinib can significantly inhibit HTNV replication and reduce viral titers at the cellular level. Experiments have shown that after treatment with apatinib, the expression levels of viral structural proteins, the nucleic acid levels of HTNV, and the viral titers in the cell culture supernatant were significantly reduced in HTNV-infected A549 cells, while the drug exhibited low cytotoxicity. Furthermore, apatinib can also inhibit CHIKV replication. This invention provides a novel anti-HTNV candidate drug and offers insights for the rapid development of antiviral drugs.

Description

Technical Field

[0001] This invention relates to the field of antiviral drugs, specifically to the application of the original anticancer drug apatinib in the preparation of antiviral drugs. Background Technology

[0002] Hantaanvirus (HTNV) is a major pathogen causing severe hemorrhagic fever with renal syndrome (HFRS), belonging to the order Bunyaviridae, family Hantaviridae, and genus Hantavirus. The virus was first successfully isolated in 1976 by Korean scientist Lee Ho-wang from the lung tissue of striped field mice in the Hantaan River basin, an endemic area of ​​South Korea. HFRS caused by Hantaanvirus has a rapid onset, severe symptoms, high mortality rate, and lacks specific and effective treatments.

[0003] Chikungunya virus (CHIKV) is the pathogen that causes chikungunya fever, an acute infectious disease. The virus was isolated from the blood of chikungunya fever patients discovered in Xishuangbanna, Yunnan Province in 1987. Chikungunya virus is primarily transmitted through the bite of infected Aedes mosquitoes.

[0004] Current antiviral drug research primarily targets the virus's own structural proteins. While this can achieve highly specific therapeutic effects, it is time-consuming, costly, technically challenging, and prone to leading to drug resistance. Ribavirin is currently used as an antiviral drug in clinical treatment, but clinical data indicates that its viral inhibition effect when used alone is not ideal.

[0005] As obligate parasitic microorganisms, viruses rely entirely on host cells for biosynthesis. Various protein kinases in host cells participate in viral replication or cellular antiviral responses. Targeting these protein kinases as drug targets may yield better antiviral effects, and drugs targeting these targets may have the ability to combat multiple viruses.

[0006] Currently, there are many anticancer drugs that have entered clinical use, targeting protein kinases to inhibit tumor cell growth and angiogenesis. From these anticancer drugs, drugs for antiviral therapy can be screened. Since these drugs have undergone clinical trials and have clear side effects, they can be quickly used to treat emerging and re-emerging viral infectious diseases. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide the application of apatinib in the preparation of antiviral drugs, specifically relating to the application of the anticancer drug apatinib in inhibiting the replication of Hantan virus and CHIKV.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows.

[0009] The application of apatinib in the preparation of antiviral drugs. The viruses in question mainly include Hantan virus and Chikungunya virus.

[0010] Furthermore, the structural formula of apatinib is shown in formula (I):

[0011]

[0012] Furthermore, the application of apatinib in the preparation of anti-Hantan virus drugs.

[0013] Furthermore, the application of apatinib in the preparation of drugs that inhibit Hantan virus replication.

[0014] Furthermore, the use of apatinib in the preparation of anti-Chikungunya virus drugs.

[0015] Furthermore, the use of apatinib in the preparation of drugs that inhibit the replication of Chikungunya virus.

[0016] The beneficial effects of this invention are:

[0017] This invention demonstrates that the anticancer drug apatinib can effectively inhibit the replication of Hantan virus (HTNV), reduce viral titers, and exhibit low cytotoxicity, while also possessing anti-chikungunya virus (CHIKV) activity. This invention provides a novel candidate for anti-HTNV drugs and offers new insights for the development of antiviral drugs. Attached Figure Description

[0018] Figure 1 These are the Western blot results of apatinib inhibiting the production of HTNV nucleocapsid protein at different concentrations.

[0019] Figure 2 This is the result of apatinib reducing the titer of HTNV-infected cell supernatant.

[0020] Figure 3 This is the result of RT-PCR detection of HTNV genomic RNA levels inhibited by apatinib.

[0021] Figure 4 This is the result of apatinib inhibiting the growth of A549 cells.

[0022] Figure 5 This is the result of the test on the efficiency of apatinib in inhibiting the replication of chikungunya virus. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0026] This invention relates to the use of apatinib in the preparation of antiviral drugs, the structural formula of which is shown in formula (I) below:

[0027]

[0028] This invention explores novel applications of anticancer drugs based on their mechanisms of action, aiming to identify potential antiviral uses and verifying their antiviral efficacy through virological methods. It utilizes established anticancer drugs for antiviral therapy, providing a rapid response strategy for treating emerging and re-emerging viral infectious diseases.

[0029] This invention relates to the application of apatinib in the preparation of anti-Hantan virus drugs, specifically the application of apatinib in the preparation of drugs that inhibit Hantan virus replication and proliferation. In in vitro experiments, this drug has demonstrated its ability to effectively inhibit the replication and proliferation of Hantan virus.

[0030] This invention also relates to the use of apatinib in the preparation of anti-Chikungunya virus (CHIKV) drugs. Specifically, it relates to the use of apatinib in the preparation of drugs that inhibit Chikungunya virus replication. This drug has shown in in vitro experiments that it can effectively inhibit Chikungunya virus replication.

[0031] The following is an analysis and explanation of the specific experimental procedures and related data regarding the efficacy of apatinib as an anti-Hantan virus and anti-Chikungunya virus (CHIKV) drug.

[0032] Example 1

[0033] Candidate drugs with anti-HTNV (Hantan virus) effects were screened from the anticancer drug library and their antiviral effects were verified.

[0034] (I) Screening

[0035] A549 cells were digested and passaged into 96-well cell culture plates. When the cell density reached 60%, HTNV was infected at an MOI of 1 (Multiple of Infection = 1). The cells were incubated at 37°C for 2 hours, then the medium was replaced with each drug at a working concentration of 10 μM, and the cells were cultured in a CO2 cell culture incubator at 37°C for 48 hours. After fixing the cells with 4% paraformaldehyde, HTNV nucleocapsid proteins were detected by immunofluorescence to assess HTNV replication. Apatinib was selected as a preliminary drug candidate, showing an inhibitory effect of 83.2% against HTNV.

[0036] (II) Verification

[0037] A549 cells were digested and passaged into 6-well cell culture plates. When the cell density reached 60%, HTNV was infected at an MOI of 1. Two hours post-infection, apatinib was added to the cell culture supernatant at different concentrations, mixed thoroughly, and then incubated in a CO2 cell culture incubator at 37°C for 48 hours. Cells were lysed using RIPA lysis buffer, and total protein was extracted. Quantification was performed using the BCA method, followed by Western blot analysis to determine the amount of HTNV nucleocapsid protein (NP). 10 μg of protein was loaded for each sample. (See attached table). Figure 1 .

[0038] Figure 1 These are Western blot results showing the inhibition of HTNV nucleocapsid protein by apatinib at different concentrations. Figure 1 As shown, apatinib at concentrations above 0.1 μM can significantly reduce the amount of HTNV nucleocapsid protein in cells.

[0039] Example 2

[0040] Identification of the ability of apatinib to inhibit HTNV replication at the cellular level.

[0041] (I) Titer Determination

[0042] A549 cells were digested and passaged into 6-well cell culture plates. When the cell density reached 60%, HTNV was infected at an MOI of 1. Two hours post-infection, the medium was replaced with 10 μM apatinib, and the cells were cultured in a CO2 cell incubator at 37°C for 48 hours. VeroE6 cells were also digested and passaged into a new 96-well plate. After adhesion, the culture supernatant from the 6-well plates was added to freshly seeded 12-well plates, with four replicates per experimental group. The 12-well plates were incubated at 37°C for 2 hours, then replaced with 3% methylcellulose covering medium (w / v) and cultured for another 7 days. VeroE6 cells were fixed with 4% paraformaldehyde and HTNV nucleocapsid protein was detected. HRP-labeled antibody was used as the secondary antibody, and AEC substrate was used as the chromogenic solution. Positive cells appeared as visible brown spots. The number of spots per well was analyzed using ImageJ image analysis software, and the viral titer was determined by the number of spots. Figure 2 .

[0043] Figure 2 This is the result of apatinib reducing the titer of HTNV-infected cell supernatant. Apatinib treatment indicates the apatinib treatment group; DMSO indicates the DMSO treatment group; Control indicates the blank treatment group. Figure 2 As shown, apatinib can significantly reduce the viral titer in the supernatant.

[0044] (II) RT-PCR detection

[0045] A549 cells were digested and passaged into 6-well cell culture plates. When the cell density reached 60%, HTNV was infected at an MOI of 1. Two hours post-infection, the medium was replaced with 10 μM apatinib, and the cells were cultured in a CO2 incubator at 37°C for 24 hours. Cells in the wells were digested with 0.25% trypsin-EDTA, washed twice with PBS, and the liquid was completely discarded. Total RNA was extracted from the cells. 2000 ng of total RNA was reverse transcribed into cDNA. Nucleic acid quantification was performed using SYBR Green as the fluorescent dye, targeting the HTNVS fragment, and using GAPDH as an internal control. Figure 3 .

[0046] Figure 3 This is the result of RT-PCR detection of HTNV genomic RNA levels inhibited by apatinib. Apatinibtreatment indicates the apatinib treatment group; DMSO indicates the DMSO treatment group; Control indicates the blank treatment group; Logcopynumber(Ssegment) indicates the Log copy number (S segment). Figure 3 As shown, apatinib can reduce intracellular HTNVRNA levels.

[0047] (III) Cytotoxicity assay

[0048] A549 cells were digested, passaged, and seeded into 96-well cell culture plates. When the cell density reached 60%, apatinib at concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM was added, and the cells were cultured for 24 h or 48 h. Cell viability was assessed using the CCK8 reagent. After incubation at 37°C for 2 h, absorbance at 450 nm was measured. The median toxic dose (LD50) of the drug was calculated based on the absorbance. (See [reference needed]). Figure 4 .

[0049] Figure 4 This is the result of an assay for the inhibition of A549 cell growth by apatinib. "Cell growth inhibition" indicates the cell growth inhibition rate; "Log2 concentration" indicates the Log2 concentration. Figure 4 As shown, apatinib did not significantly inhibit cell growth at 24h and 48h, with half-maximal inhibitory concentrations (IC50) reaching 203.94 μM (24h) and 42.93 μM (48h).

[0050] (iv) Testing of antiviral efficacy against other viruses

[0051] VeroE6 cells were passaged and seeded into 96-well cell culture plates. Once the cell density reached 80%, the cells were infected with a fluorescently tagged CHIKV replicon. Two hours after infection, 10 μM apatinib was added, and the cells were cultured for another 24 hours before observing cell fluorescence. Figure 5 .

[0052] Figure 5 This is the result of apatinib's inhibition of chikungunya virus replication. Log concentration represents the Log concentration; CHIKV inhibition represents the CHIKV inhibition rate. Figure 5 As shown, apatinib can inhibit the replication ability of CHIKV replicons, and its half-maximal inhibitory concentration (IC50) against CHIKV can be as low as 0.69 μM.

[0053] As can be seen from the above, this invention confirms that the anticancer drug apatinib can effectively inhibit HTNV replication and reduce viral titer in in vitro experiments, while also having anti-CHIKV effects. It can be used as a candidate for novel antiviral drugs and provides ideas for the development of drugs against hemorrhagic fever with renal syndrome.

[0054] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of apatinib in the preparation of antiviral drugs, characterized in that, The application of apatinib in the preparation of anti-Chikungunya virus drugs.

2. The use of apatinib in the preparation of antiviral drugs according to claim 1, characterized in that, The structural formula of apatinib is shown in formula (I): 。 3. The use of apatinib in the preparation of antiviral drugs according to claim 1, characterized in that, The application of apatinib in the preparation of drugs that inhibit the replication of Chikungunya virus.

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