Use of compound azd1390 in the manufacture of a medicament for the treatment of an infection with an african swine fever virus
Patent Information
- Application Number
- CN202611459629.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-20
- Publication Date
- 2026-10-30
AI Technical Summary
关于AZD1390在抗病毒领域，尤其是在抗非洲猪瘟病毒感染方面的应用，尚无任何报道
[0031]（1）本发明意外发现，在非洲猪瘟病毒感染宿主细胞后给予小分子化合物AZD1390，能够显著抑制病毒的复制，有效降低宿主细胞中非洲猪瘟病毒的滴度，且该抑制作用呈现明显的剂量依赖关系；即，本发明首次发现了化合物AZD1390具有显著的抗非洲猪瘟病毒活性，能够有效抑制ASFV在宿主细胞中的复制，拓宽了AZD1390的应用领域。
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Figure CN122998150A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a compound AZD1390 in the preparation of drugs against African swine fever virus infection. Background Technology
[0002] African swine fever (ASF) is an acute, highly contagious, and fatal infectious disease caused by the African swine fever virus (ASFV). It can infect both domestic and wild pigs, with an acute mortality rate of up to 100%. ASFV is currently the only known large double-stranded DNA virus that can be transmitted via insect vectors. Infection can cause a series of clinical symptoms, including high fever, hemorrhage, poor blood clotting, and splenomegaly.
[0003] Currently, there are no safe and effective commercial vaccines available for the prevention and control of African swine fever (ASF), nor are there any approved antiviral drugs for the treatment of ASFV infection. Therefore, implementing strict biosafety measures and developing antiviral agents capable of inhibiting ASFV replication are key aspects of current ASF control efforts. In recent years, researchers have conducted extensive screening work on ASFV inhibitors, discovering several small molecule compounds with anti-ASFV activity. However, overall, the available candidate molecules for anti-ASFV drugs remain very limited, and there is an urgent need to develop more novel and highly effective anti-ASFV drugs.
[0004] AZD1390 is an orally active, highly selective inhibitor of ATM (ataxia-telangiectasia mutant protein) kinase that crosses the blood-brain barrier. Its IC50 inhibitory activity at the cellular level inhibits ATM kinase activity. 50 With a concentration of 0.78 nM, AZD1390 exhibits excellent selectivity (>10,000-fold) for other members of the PIKK kinase family and a broader kinase spectrum. AZD1390 blocks the ATM-dependent DNA damage response pathway, inhibiting ATM autophosphorylation and the phosphorylation of its downstream targets Chk2 and Rad50. Currently, AZD1390 is primarily being investigated as a radiosensitizer for use in cancer therapy. There are no reports on the application of AZD1390 in antiviral fields, particularly in combating African swine fever virus infection. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention unexpectedly discovered that compound AZD1390 has a significant inhibitory effect on the replication of African swine fever virus, providing a new pharmaceutical raw material for African swine fever virus infection and offering a new prevention and treatment strategy for combating African swine fever virus infection. Specifically, it includes the following:
[0006] In a first aspect, the present invention provides the use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, wherein the structural formula of compound AZD1390 is shown in formula (I) below:
[0007]
[0008] Equation (Ⅰ).
[0009] Preferably, the virus is African swine fever virus.
[0010] Preferably, the African swine fever virus includes, but is not limited to, genotype II African swine fever virus.
[0011] Secondly, the present invention provides the use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of adjuvants for African swine fever virus vaccines, wherein the structural formula of compound AZD1390 is shown in formula (I) below:
[0012]
[0013] Equation (Ⅰ).
[0014] Preferably, the African swine fever virus includes, but is not limited to, genotype II African swine fever virus.
[0015] Thirdly, the present invention provides the use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing African swine fever virus infection, wherein the structural formula of compound AZD1390 is shown in formula (Ⅰ) below:
[0016]
[0017] Equation (Ⅰ).
[0018] Preferably, the African swine fever virus includes, but is not limited to, genotype II African swine fever virus.
[0019] Fourthly, the present invention provides the use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever virus infection, wherein the structural formula of compound AZD1390 is shown in formula (I) below:
[0020]
[0021] Equation (Ⅰ).
[0022] Preferably, the African swine fever virus includes, but is not limited to, genotype II African swine fever virus.
[0023] Fifthly, the present invention provides a pharmaceutical composition for the prevention or treatment of African swine fever, wherein the active ingredient of the pharmaceutical composition comprises compound AZD1390 or a pharmaceutically acceptable salt thereof, the structural formula of compound AZD1390 being shown in formula (I) below:
[0024]
[0025] Equation (Ⅰ).
[0026] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.
[0027] Preferably, the pharmaceutical composition can be formulated into any pharmaceutically acceptable dosage form.
[0028] Preferably, the dosage form includes powder for injection, capsules, tablets, and suspensions.
[0029] Preferably, the African swine fever virus includes, but is not limited to, genotype II African swine fever virus.
[0030] The beneficial effects of this invention are:
[0031] (1) The present invention unexpectedly discovered that administering the small molecule compound AZD1390 after African swine fever virus infection of host cells can significantly inhibit viral replication and effectively reduce the titer of African swine fever virus in host cells, and the inhibitory effect shows a clear dose-dependent relationship; that is, the present invention is the first to discover that the compound AZD1390 has significant anti-African swine fever virus activity and can effectively inhibit ASFV replication in host cells, thus broadening the application field of AZD1390.
[0032] (2) The compound AZD1390 can significantly inhibit the formation of African swine fever virus factories, thereby blocking the assembly and maturation process of the virus and exerting antiviral effects from the virus assembly stage. The mechanism of action is clear.
[0033] (3) This invention provides new drug candidate molecules for the clinical treatment of African swine fever virus, which is of great significance for the prevention and control of African swine fever epidemic. Attached Figure Description
[0034] Figure 1 The effect of different concentrations of the small molecule compound AZD1390 on the cytotoxicity of BMDM cells was analyzed.
[0035] Figure 2 This is a schematic diagram showing the red fluorescence intensity of the control group and the treatment group with different concentrations of DMSO under the condition of infection with the recombinant ASFV-RFP-P54 virus at the local ASFV virus factory.
[0036] Figure 3This is a schematic diagram showing the intensity of green fluorescence observed 2 hours after ASFV-II-GFP infection of BMDM cells and 24 hours after treatment with 0.5 μmol concentration of AZD1390.
[0037] Figure 4 This is a schematic diagram showing the detection of ASFV P72 mRNA 2 hours after BMDM cells were infected with ASFV-II-GFP and treated with 0.5 μmol of AZD1390.
[0038] Figure 5 Two hours after ASFV-II-GFP infection of BMDM cells, the cells were treated with 0.5 μmol of AZD1390, and the whole virus was collected for TCID testing. 50 Schematic diagram of experimental results. Detailed Implementation
[0039] In the following embodiments, the chemical name of the small molecule compound AZD1390 (also referred to as T5175 in the following examples or figures) is: 7-fluoro-1-isopropyl-3-methyl-8-(6-(3-(piperidin-1-yl)propoxy)pyridin-3-yl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one. The molecular formula of AZD1390 is C 27 H 32 FN5O2, with a molecular weight of 477.57, was dissolved in sterile DMSO (dimethyl sulfoxide, purchased from Solarbio) to prepare a 10 mmol / L stock solution. After aliquoting, it was stored at -20°C and diluted to the required concentration with DMEM medium before experiments. Its chemical structural formula is as follows: .
[0040] In the following implementation, the cells were: porcine primary bone marrow-derived macrophages (BMDM), isolated from healthy pigs, cultured in DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, purchased from Gibco), 100 μg / mL penicillin and 100 μg / mL streptomycin, and cultured in a 37°C, 5% CO2 incubator. Cells in good condition were selected for subsequent experiments.
[0041] In the following implementation, the virus strains are: fluorescently labeled African swine fever virus strain ASFV-II-GFP, preserved in the laboratory, which carries the green fluorescent protein gene (prepared by those skilled in the art using conventional techniques), and whose viral replication can be observed through green fluorescent signals after cell infection; and recombinant virus ASFV-RFP-P54, which locates the viral factory, expressing a fusion protein of red fluorescent protein and P54 protein, and can be used to label the viral factory for observing its formation (prepared by those skilled in the art using conventional techniques); the virus titer is determined and then aliquoted and stored at -80℃. The African swine fever virus strain ASFV-II is ASFV CN / GS / 2018.
[0042] In the following implementations, the reagents are: CCK-8 cell proliferation-toxicity assay kit; ASFV P72 gene-specific primers.
[0043] In the following implementations, the instruments and equipment include: fluorescence microscope, real-time quantitative PCR instrument, CO2 incubator, ELISA reader, biosafety cabinet, centrifuge, etc.
[0044] Example 1: Safety screening of AZD1390 in BMDM cells
[0045] BMDM cells were loaded at 5 × 10 3 Cells were seeded at a density of 100 μL / well in 96-well cell culture plates, with 100 μL of DMEM complete medium added to each well. The plates were incubated at 37°C with 5% CO2 for 24 h until cells adhered stably. The original medium was discarded, and fresh DMEM complete medium containing different concentrations of AZD1390 was added to each well. The concentration gradients were 0 μmol / L, 0.0005 μmol / L, 0.005 μmol / L, 0.05 μmol / L, 0.5 μmol / L, and 5 μmol / L, with three replicates for each concentration. The 0 μmol / L group (containing only an equal amount of DMSO) served as a blank control. After culturing for another 24 h, 10 μL of CCK-8 reagent was added to each well, gently mixed, and incubated for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.
[0046] The results are as follows Figure 1 As shown, when the concentration of AZD1390 was 0.0005 μmol / L, 0.005 μmol / L, 0.05 μmol / L, 0.5 μmol / L and 5 μmol / L, the survival rate of BMDM cells remained above 80% and there was no obvious cytotoxicity.
[0047] Example 2: Inhibitory effect of AZD1390 on ASFV viral factory formation
[0048] The impact of AZD1390 on the formation of ASFV viral factories was directly determined by observing red fluorescently marked viral factories using a fluorescence microscope. The steps are as follows:
[0049] BMDM cells were loaded at 2×10 5 Cells were seeded at a density of cells / well in 6-well cell culture plates and cultured for 24 h. The experiment was divided into treatment groups and control groups: the treatment groups were added with DMEM complete medium containing 0.05 μmol / L, 0.5 μmol / L, and 5 μmol / L LAZD1390, respectively; the control group was added with DMEM complete medium containing an equal volume of DMSO. After 2 h of pretreatment, the treatment solution was discarded, the cells were washed twice with PBS buffer, and ASFV-RFP-P54 virus solution was added for infection. After 1 h of incubation, the unadsorbed virus solution was discarded, and fresh DMEM complete medium containing the corresponding concentration of LAZD1390 or an equal volume of DMSO was added. After 24 h of further culture, the cells were washed with PBS, and the red fluorescence signal was observed and photographed using a fluorescence microscope. The number and fluorescence intensity of the red fluorescent spots (i.e., virus factories) were counted.
[0050] The results are as follows Figure 2 As shown, a large number of bright red fluorescent spots were visible in the control group cells, indicating that ASFV virus factories were formed in large numbers; while the number of red fluorescent spots in the AZD1390 treatment group was significantly reduced, the fluorescence intensity was significantly reduced, and the inhibitory effect was more obvious as the drug concentration increased.
[0051] The above results indicate that AZD1390 can dose-dependently inhibit the formation of ASFV viral factories, thereby blocking the assembly and maturation process of the virus.
[0052] Example 3: Inhibitory effect of AZD1390 on ASFV replication fluorescence expression
[0053] The effect of AZD1390 on the overall replication level of ASFV was observed using green fluorescence. The specific steps are as follows:
[0054] BMDM cells were loaded at 2×10 5Cells were seeded at a density of [number] cells / well in 6-well cell culture plates (pre-placed with sterile coverslips) and cultured for 24 h until cell confluence reached 70%–80%. The culture medium was discarded, and the cells were washed twice with PBS. BMDM cells were then infected with ASFV-II-GFP virus solution. After incubation for 1 h, the unadsorbed virus solution was discarded, and the cells were washed twice with PBS. Two h post-infection, the cells were divided into two groups: the drug-treated group received DMEM complete medium containing 0.5 μmol / L AZD1390; the control group received DMEM complete medium containing an equal volume of DMSO. Cells were cultured for another 24 h. After washing with PBS, the green fluorescence signal was observed and captured using a fluorescence microscope, and the proportion and intensity of green fluorescent positive cells were recorded.
[0055] The results are as follows Figure 3 As shown, a large number of strong green fluorescent signals were observed in the control group cells, indicating massive viral replication; while the number of green fluorescent positive cells was significantly reduced and the fluorescence intensity was significantly weakened in the AZD1390 treatment group. This demonstrates that administration of AZD1390 after viral infection can significantly inhibit ASFV replication in BMDM cells.
[0056] Example 4: qPCR detection of viral P72 gene transcription level
[0057] BMDM cells were seeded into 6-well plates and infected with ASFV-II-GFP according to the method in Example 3. Two hours after infection, the cells were treated with medium containing 5 μmol / L AZD1390 or an equal volume of DMSO. After 24 hours of treatment, the cells were subjected to three freeze-thaw cycles, and the whole virus was collected. The cells were then inactivated by high-temperature inactivation for 5 minutes and centrifuged. The supernatant was used as a template for absolute quantification.
[0058] The results are as follows Figure 4 As shown, compared with the DMSO control group, the transcription level of the ASFV P72 gene was significantly reduced in the AZD1390 treatment group, demonstrating that AZD1390 effectively inhibits ASFV replication at the transcriptional level.
[0059] Example 5 TCID 50 Experimental detection of viral titer
[0060] Virus collection: BMDM cells were seeded into 6-well plates and infected with ASFV-II-GFP according to the method in Example 3. Two hours after infection, the cells were treated with medium containing 0.5 μmol / L AZD1390 or an equal volume of DMSO. After 24 hours of treatment, the cell culture supernatant and cell lysate were collected, and the cells were subjected to three freeze-thaw cycles. The supernatant was collected by centrifugation and used for virus titer determination.
[0061] TCID 50Assay: The collected virus solution was serially diluted 10-fold and seeded into BMDM cells cultured in 96-well plates, with 8 replicates per dilution. The plates were incubated at 37°C in a 5% CO2 incubator for 5–7 days. Cytopathic effect (CPE) was observed daily, and the number of positive wells was recorded. The viral titer (TCID) was calculated using the Reed-Muench method. 50 / mL).
[0062] The results are as follows Figure 5 As shown, the viral titer in the DMSO control group remained at a high level, while the viral titer in the AZD1390 treatment group decreased significantly, by 1 to 2 orders of magnitude.
[0063] This invention demonstrates at the infectious virus particle level that administration of AZD1390 after viral infection can effectively reduce the titer of African swine fever virus in host cells and inhibit the release of progeny viruses.
[0064] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.
[0065] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. The use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of antiviral drugs, wherein the structural formula of compound AZD1390 is shown in formula (Ⅰ) below: Equation (Ⅰ).
2. The application as described in claim 1, characterized in that, The virus in question is the African swine fever virus.
3. The use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of adjuvants for African swine fever virus vaccines, wherein the structural formula of compound AZD1390 is shown in formula (Ⅰ): Equation (Ⅰ).
4. The use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention of African swine fever virus infection, wherein the structural formula of compound AZD1390 is shown in formula (Ⅰ) below: Equation (Ⅰ).
5. The use of compound AZD1390 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever virus infection, wherein the structural formula of compound AZD1390 is shown in formula (Ⅰ): Equation (Ⅰ).
6. The application according to any one of claims 2-5, characterized in that, The African swine fever virus mentioned is genotype II African swine fever virus.
7. A pharmaceutical composition for the prevention or treatment of African swine fever, characterized in that, The active ingredient of the pharmaceutical composition includes compound AZD1390 or a pharmaceutically acceptable salt thereof. The structural formula of compound AZD1390 is shown in formula (Ⅰ) below: Equation (Ⅰ).
8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.
9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition can be formulated into any pharmaceutically acceptable dosage form.
10. The pharmaceutical composition according to claim 9, characterized in that, The dosage forms include powder for injection, capsules, tablets, and suspensions.