Application of compound Azelnidipine in the preparation of antiviral drugs

Abstract

This invention discloses the application of the compound Azelnidipine in the preparation of antiviral drugs, relating to the field of biomedical technology. This invention successfully explores the novel application value of the compound Azelnidipine and its pharmaceutically acceptable salts in the prevention and control of African swine fever. Azelnidipine possesses excellent ability to inhibit the replication of African swine fever virus, exerting intervention effects at different stages of viral infection and exhibiting good concentration-dependent action. It also possesses high biocompatibility, causing no significant damage to host cells, thus balancing antiviral efficacy and biocompatibility. Furthermore, this compound can not only be used to prepare various dosage forms of drugs for the prevention and / or treatment of African swine fever virus infection, but can also be extended to serve as an adjuvant for African swine fever vaccines, possessing very broad prospects for industrial application and market value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the compound Azelnidipine in the preparation of antiviral drugs. Background Technology

[0002] African swine fever virus (ASFV) is the sole member of the classical swine fever virus family and is a pathogen that causes a highly destructive and contagious disease in domestic pigs. Clinical symptoms in infected pigs include high fever, cyanosis, bleeding, diarrhea, and even acute death. To date, there is no effective vaccine to prevent African swine fever.

[0003] ASFV can be classified into highly virulent, moderately virulent, and low-virulence strains based on virulence. Genotype II ASFV (China2018 / 1) is a highly virulent strain with a mortality rate as high as 100%; genotype I (SD / DY-I / 21) is a low-virulence strain (10... 6 (TCID50 challenge, no pigs died within 28 days). A recombinant virus of genotype I / II (referred to as "recombinant virus") emerged in December 2021. This strain is highly virulent, with a mortality rate of up to 100%. The emergence of the recombinant virus suggests that ASFV strain mutations are more complex. Currently, there are no effective commercially available drugs for the prevention and treatment of African swine fever.

[0004] Based on the study of the C717R mechanism of action, this invention aims to screen compounds targeting this protein to achieve effective inhibition of ASFV. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the compound Azelnidipine in the preparation of antiviral drugs, thereby addressing the problems existing in the prior art. This invention has found that Azelnidipine has the effect of inhibiting ASFV replication and can be used to prepare drugs or adjuvants for antiviral infections, showing broad application prospects.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of the compound Azelnidipine or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, wherein the virus is African swine fever virus;

[0008] The structural formula of the compound Azelnidipine is:

[0009] .

[0010] Furthermore, the antiviral drug is a drug for the prevention and / or treatment of viral infections.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the excipients are at least one of fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, and colorants.

[0013] Furthermore, the dosage form of the drug is powder for injection, tablets, granules, capsules, pills, or suspension.

[0014] The present invention also provides a drug for combating African swine fever virus, the active ingredient of which includes the compound Azelnidipine or a pharmaceutically acceptable salt thereof;

[0015] The structural formula of the compound Azelnidipine is:

[0016] .

[0017] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0018] Furthermore, the excipients are at least one of fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, and colorants.

[0019] Furthermore, the dosage form of the drug is powder for injection, tablets, granules, capsules, pills, or suspension.

[0020] This invention also provides the use of the compound Azelnidipine or a pharmaceutically acceptable salt thereof in the preparation of vaccine adjuvants for African swine fever virus, wherein the compound Azelnidipine has the following structural formula:

[0021] .

[0022] The present invention discloses the following technical effects:

[0023] This invention successfully uncovers the novel application value of the compound Azelnidipine and its pharmaceutically acceptable salts in the prevention and control of African swine fever (ASF), providing key technical support for solving the current challenges in ASF prevention and control. This invention precisely targets the C717R protein of ASF virus as its core target. Through professional molecular docking and affinity screening, it was determined that Azelnidipine can efficiently bind to this target, demonstrating a clear mechanism of action and strong targeting, overcoming the technical limitations of traditional prevention and control methods that lack a clear target. In terms of antiviral performance, Azelnidipine exhibits excellent ability to inhibit ASF virus replication, exerting intervention effects at different stages of viral infection and showing good concentration-dependent action. It also possesses high biocompatibility, causing no significant damage to host cells, thus balancing antiviral efficacy and biocompatibility. Furthermore, this compound can not only be used to prepare various dosage forms of drugs for the prevention and / or treatment of African swine fever virus infection, but also as an adjuvant for African swine fever vaccines, forming a synergistic prevention and treatment system. This effectively fills the current industry gap of having no effective commercial drugs and dedicated adjuvants for African swine fever, providing a brand-new technical solution for disease prevention and control in the pig farming industry, and has a very broad prospect for industrial application and market value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Figure 1 shows the cell viability test results of PAM cells after treatment with the compound Azelnidipine.

[0026] Figure 2 Fluorescence microscopy observation of the effect of 30 μM azelnidipine on ASFV replication;

[0027] Figure 3 The figure shows the qPCR detection results for testing the effect of 30 μM Azelnidipine on ASFV replication;

[0028] Figure 4 Fluorescence microscopy observations to test the effect of different concentrations of azelnidipine on ASFV replication;

[0029] Figure 5 To test the effect of different concentrations of azelnidipine on ASFV replication, HAD 50Test result image;

[0030] Figure 6 The image shows the results of Western blot analysis to test the effect of different concentrations of azelnidipine on ASFV replication.

[0031] Figure 7 Fluorescence microscopy images showing the effect of Azelnidipine treatment at different time points on ASFV replication; ASFV-G-WT+DMSO serves as the solvent control group.

[0032] Figure 8 To test the effect of Azelnidipine treatment at different time points on ASFV replication, HAD 50 Detection results image; ASFV-G-WT+DMSO is the solvent control group;

[0033] Figure 9 The image shows the Western blot results for testing the effect of Azelnidipine treatment at different time points on ASFV replication; ASFV-G-WT+DMSO serves as the solvent control group. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] The Azelnidipine used in the following examples was purchased from Target Molecule Corp. (TargetMol), USA.

[0040] The alveolar macrophages (PAMs) used in the following examples were isolated from the lung tissue of 3-month-old pigs;

[0041] The ASFV-G-WT strain is a recombinant ASFV virus obtained by adding the green fluorescent protein (GFP) gene to the ASFV CN / GS / 2018 strain through homologous recombination technology. The GFP labeling process does not affect the normal biological function and infectivity of the ASFV CN / GS / 2018 strain.

[0042] ASFV CN / GS2018 is a wild-type African swine fever genotype II strain, provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. ASFV strains obtained by the public through other means can also be used to implement this invention.

[0043] Preparation of Azelnidipine-containing medium: Azelnidipine was diluted with complete medium and added to 96-well plates to make final concentrations of 10 μM, 20 μM and 30 μM, with 3 replicate wells for each gradient.

[0044] Example 1 Compound Screening

[0045] This invention utilizes MOE software to analyze the C717R pattern and identify the inhibitor-binding active site. Molecular docking was performed on compounds from a library containing 23,619 small molecules. Finally, through affinity analysis and other methods, the small molecule compound that binds to C717R—Azelnidipine—was selected. The molecular formula of Azelnidipine is C... 33 H 34 N4O6, structural formula:

[0046] .

[0047] Example 2: Effect of compound Azelnidipine on PAM cell activity

[0048] PAM cells were seeded in 96-well plates with a cell suspension of 50 μL / well. 30 μM azelnidipine was added to each well, for a total volume of 100 μL / well. Three replicates were set up. After culturing for 24 h, medium containing 10% CCK8 was added, and the plates were incubated at 37°C for 1 h. OD values ​​were then measured using a microplate reader. A control group (Mock) was also set up, using blank solvent instead of the azelnidipine solution.

[0049] Cell viability test results as follows Figure 1 As shown in the figure, the results indicate that PAM cell viability is greater than 95% when the concentration of Azelnidipine is 30 μM.

[0050] Example 3: Effect of compound Azelnidipine on ASFV replication

[0051] 1. PAM cells were treated with azelnidipine (30 μM) and then inoculated with ASFV-G-WT at MOI=1. A positive control group (ASFV-G-WT group) without azelnidipine treatment was also established. GFP fluorescence was observed under a fluorescence microscope 24 hours after infection. The results are as follows: Figure 2 As shown, the compound significantly inhibited ASFV replication compared to the positive control group.

[0052] 2. PAM cells were treated with azelnidipine (30 μM) and then inoculated with ASFV-G-WT at MOI=1. A positive control group (ASFV-G-WT group) without azelnidipine treatment and a blank control group (Mock group) that received neither azelnidipine treatment nor ASFV-G-WT inoculation were also established. Twenty-four hours after infection, the cells were repeatedly frozen and thawed at -80°C, and the ASFV copy number was determined by RT-qPCR. The experimental results are as follows: Figure 3 As shown, the compound significantly inhibited ASFV replication compared to the positive control group.

[0053] 3. PAM cells were treated with different concentrations of the compound azelnidipine (0, 10, 20, 30 μM) and then inoculated with ASFV-G-WT at an MOI of 1. A blank control group (named Mock) was also established, which neither received azelnidipine treatment nor received ASFV-G-WT. The following tests were performed 24 hours after infection:

[0054] (1) Observe GFP fluorescence using a fluorescence microscope. The observation results are as follows: Figure 4 As shown, compared with the 0 μM treatment group, the Azelnidipine concentration at 10 μM significantly inhibited ASFV replication.

[0055] (2) After repeated freeze-thaw cycles at -80℃, the half-maximal hemagglutination dose (HAD) was measured. 50 Assess ASFV replication levels. Experimental results are as follows: Figure 5 As shown, compared with the 0 μM treatment group, different concentrations of Azelnidipine significantly inhibited ASFV replication.

[0056] (3) After cell lysis, the ASFVP72 protein level was measured by Western blotting. The experimental results are as follows: Figure 6 As shown, compared with the 0 μM treatment group, different concentrations of Azelnidipine significantly inhibited ASFV replication.

[0057] 4. PAM cells were treated with the compound azelnidipine (30 μM) at different time points (-4, -2, 0, 2, 4 h) and then infected with ASFV-G-WT at MOI=1. A solvent control group (treated at 0 h) was also set up. The following assays were performed 24 hours after infection:

[0058] (1) Observe GFP fluorescence using a fluorescence microscope. The observation results are as follows: Figure 7 As shown, compared with the control group, the compound Azelnidipine also significantly inhibited ASFV replication 4 hours after exposure.

[0059] (2) After repeated freeze-thaw cycles at -80℃, HAD was used. 50 Assess ASFV replication levels. Experimental results are as follows: Figure 8 As shown, compared with the control group, the compound can also significantly inhibit ASFV replication 4 hours after exposure.

[0060] (3) After cell lysis, the ASFVP72 protein level was measured by Western blotting. The experimental results are as follows: Figure 9 As shown, compared with the control group, the compound Azelnidipine significantly inhibited the replication of ASFV.

[0061] The above results indicate that azelnidipine inhibits the expression of the ASFV structural protein P72, reduces the ASFV titer, and inhibits ASFV replication; moreover, the inhibitory effect on ASFV replication is stronger with increasing azelnidipine content, showing a dose-dependent relationship; and the inhibitory effect on ASFV remains strong with changes in exposure time.

[0062] In summary, the embodiments of this invention, using host cells PAMs cells as an example, have demonstrated that Azelnidipine can inhibit ASFV replication in host cells PAMs cells. This indicates that the compound Azelnidipine screened in this invention can inhibit ASF virus replication and can be used to prepare drugs or adjuvants against ASFV virus infection, showing broad application prospects.

[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of the compound Azelnidipine or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, characterized in that, The virus in question is the African swine fever virus; The structural formula of the compound Azelnidipine is: 。 2. The application according to claim 1, characterized in that, The antiviral drug is a drug for the prevention and / or treatment of viral infections.

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

4. The application according to claim 3, characterized in that, The excipients are at least one of fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, and colorants.

5. The application according to claim 3, characterized in that, The dosage form of the drug is powder for injection, tablets, granules, capsules, pills, or suspension.

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