Application of compound Barsertib or pharmaceutically acceptable salt thereof in preparation of medicine for resisting African swine fever virus

Abstract

The invention discloses an application of a compound Barsertib or a pharmaceutically acceptable salt thereof in preparation of an anti-African swine fever virus drug, and belongs to the technical field of antiviral drugs. The molecular formula of the compound Barsertib provided by the invention is C26H31FN7O6P, and the structural formula of the compound Barsertib is shown in the description. According to the invention, C717R is taken as a target spot, molecular docking is carried out on a compound library containing 23619 small molecules, and finally, through affinity analysis, structural diversity analysis and visual binding mode analysis, the small molecule compound Barsertib combined with C717R is screened out. The cytotoxicity of the small molecule compound Barsertib is detected through a CCK-8 reagent; the virus replication inhibition capability of the small molecule compound Barsertib is detected by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction), green fluorescence observation and Western blot experiments, and the result shows that the small molecule compound Barsertib can obviously inhibit the ASFV replication.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug technology, and in particular to the use of the compound Barasertib or a pharmaceutically acceptable salt thereof in the preparation of drugs against African swine fever virus. Background Technology

[0002] African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs. It is considered one of the most serious infectious diseases, with virulent strains causing a mortality rate as high as 100% in domestic pigs. Currently, there is no commercially available vaccine for the prevention and control of ASF.

[0003] ASFV, the only member of the Asfarviridae family, is a large enveloped double-stranded DNA virus and the only DNA virus transmitted by arthropods, specifically the genus *Ornithodoros*. ASFV particles are 200 nm in diameter and icosahedral in shape. In 2019, Chinese scientists reconstructed the overall capsid structure of ASFV using high-resolution three-dimensional electron microscopy. The virus has a five-layered structure, consisting of a nucleoid (composed of viral genomic DNA and proteins), a core shell, an inner envelope, a capsid, and an external envelope, from the inside out. The ASFV genome ranges from 170 to 193 kb and encodes 150-200 proteins, most of whose functions remain unknown. These proteins not only perform functions related to viral entry, virion assembly, and expulsion but also regulate the host's innate antiviral immune response.

[0004] The large and complex genome of ASFV makes the identification of effective antigens difficult. Furthermore, live attenuated vaccines pose a risk of environmental spread, hindering vaccine development. Therefore, small molecule drugs have emerged as a potential alternative for the prevention and treatment of African swine fever. Drugs approved by the U.S. Food and Drug Administration (FDA) have undergone extensive studies in terms of safety, pharmacokinetics, and targets, providing a readily available compound library for the discovery of novel antiviral drugs. Screening this FDA-approved drug library would be a rapid and effective method for obtaining drugs against ASFV infection. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the compound barasertib or a pharmaceutically acceptable salt thereof in the preparation of drugs against African swine fever virus (ASFV), thereby addressing the problems existing in the prior art. Given that C717R can induce strong pyroptosis and cytokine release, this invention screened a library of small molecules targeting C717R to find ASFV replication inhibitors. Among the screened molecules, barasertib exhibited potent inhibition of ASFV replication with extremely low cytotoxicity. As a highly selective Aurora B inhibitor, this molecule specifically acts on specific residues of C717R and can be used to prepare drugs or adjuvants against ASFV infection for inhibiting ASFV replication.

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

[0007] In a first aspect, the present invention provides the use of the compound Barasertib or a pharmaceutically acceptable salt thereof in the preparation of an anti-African swine fever virus drug, said compound Barasertib having the molecular formula C 26 H 31 FN7O6P, the structural formula is shown in formula (I):

[0008]

[0009] Formula (I).

[0010] Preferably, the African swine fever virus is genotype II African swine fever virus.

[0011] Preferably, the dosage form of the drug is selected from any one or more of tablets, sprays, granules, capsules, oral liquids, injections, or suspensions.

[0012] Preferably, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0013] Preferably, the excipients include any one or more of the following: excipients, diluents, preservatives, colorants, flavoring agents, wetting agents, suspending agents, stabilizers, isotonic agents, and emulsifiers.

[0014] Preferably, the compound Barasertib or a pharmaceutically acceptable salt thereof is used to inhibit the activity of African swine fever virus.

[0015] Preferably, the compound Barasertib or a pharmaceutically acceptable salt thereof is used to inhibit the expression of the ASFV structural protein P72.

[0016] The present invention discloses the following technical effects:

[0017] This invention discovers a small molecule compound, Barasertib, targeting the C717R protein of African swine fever virus, with the molecular formula C... 26 H 31 FN7O6P, this small molecule compound can inhibit the replication of ASFV, indicating that the compound Barasertib has the effect of inhibiting ASFV replication. It can be used to prepare drugs or adjuvants against African swine fever virus infection and to inhibit the replication of African swine fever virus. Attached Figure Description

[0018] 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.

[0019] Figure 1 The results are from a cytotoxicity assay.

[0020] Figure 2 After the compound was added, the green fluorescence of ASFV-G-WT was observed.

[0021] Figure 3 The results of ASFV replication assay;

[0022] Figure 4 ASFV-G-WT green fluorescence was observed after treating PAMs cells with different concentrations of compounds.

[0023] Figure 5 After treating PAMs cells with different concentrations of compounds, HAD 50 Measurement results;

[0024] Figure 6 Protein levels of ASFV structural protein P72 were detected after treating PAMs cells with different concentrations of compounds.

[0025] Figure 7 ASFV-G-WT green fluorescence was observed after treating PAMs cells with compounds at different time points.

[0026] Figure 8 After treating PAMs cells with compounds at different time points, HAD 50 Measurement results;

[0027] Figure 9 Protein levels of the ASFV structural protein P72 were detected after treating PAMs cells with compounds at different time points. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The ASFV-G-WT strain used in this embodiment of the invention is the type II African swine fever virus strain ASFV CN / GS 2018. This type II African swine fever virus strain ASFV CN / GS 2018 was deposited at the China Center for Type Culture Collection on December 21, 2020, with accession number CCTCC NO:V202096, and the deposit address is Wuhan University, Wuhan, China.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the experimental materials used in the following examples, unless otherwise specified, were all purchased from conventional biochemical reagent companies. Barasertib was purchased from Tausch. ASFV CN / GS 2018 is a wild-type African swine fever genotype II strain, deposited at the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Besides the strain used in this invention, wild-type ASFV strains obtained by the public through other means can also be used to implement this invention.

[0035] Preparation of Barasertib-containing medium: Barasertib 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.

[0036] Example 1: Screening of the compound Barasertib

[0037] Small molecule compounds targeting C717R were screened. Using AlphaFold software to analyze the C717R pattern, inhibitor-binding active sites 1 and 2, covering two amino acids, were identified on the C717R surface. Furthermore, compounds were screened using surface plasmon resonance (SPR) technology. Ultimately, the natural product barasertib was selected, which selectively and efficiently targets C717R.

[0038] Example 2: Effect of compound Barasertib on PAM cell activity

[0039] PAM cells were seeded in 96-well plates at a cell suspension of 50 μL / well, and simultaneously inoculated with 30 μM barasertib at a total volume of 100 μL / well. Three replicate wells were set up. After culturing for 24 h, medium containing 10% CCK8 was added, and after incubation for 1 h, the OD value was measured using a microplate reader. A blank PAM cell culture group without the compound was used as a control group (Mock group).

[0040] Cell viability test results as follows Figure 1 As shown, at a Barasertib concentration of 30 μM, PAM cell viability was greater than 70%.

[0041] Example 3: Effect of compound Barasertib on ASFV replication

[0042] 1. PAMs cells were treated with a compound (30 μM) and then infected with ASFV-G-WT cells at MOI=1. GFP fluorescence was observed under a fluorescence microscope 24 hours after infection. An equal amount of DMSO was added as a positive control.

[0043] Observation results as follows Figure 2As shown, compared with the control group, the compound significantly inhibited the replication of ASFV.

[0044] 2. PAM cells were treated with a compound (30 μM) and then infected with ASFV-G-WT at MOI=1. 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. Simultaneously, an equal amount of DMSO was added as a positive control, and uninoculated blank PAM cells were used as a negative control (Mock group).

[0045] Experimental results are as follows Figure 3 As shown, compared with the positive control group, the compound significantly inhibited ASFV replication.

[0046] 3. PAM cells were treated with different concentrations of the compound (0, 10, 20, 30 μM) and then infected with ASFV-G-WT at MOI=1. Uninoculated blank PAM cells were used as a negative control group (Mock group). The following tests were performed 24 hours later:

[0047] (1) Observe GFP fluorescence using a fluorescence microscope.

[0048] Observation results as follows Figure 4 As shown, compared with the control group, 10 μM of the compound can significantly inhibit ASFV replication;

[0049] (2) After repeated freeze-thaw cycles at -80℃, HAD was used. 50 Assess ASFV replication levels.

[0050] Experimental results are as follows Figure 5 As shown, compared with the control group, the compound significantly inhibited the replication of ASFV;

[0051] (3) After cell lysis, the ASFV P72 protein level was measured by Western blot.

[0052] Experimental results are as follows Figure 6 As shown, compared with the control group, the compound significantly inhibited the replication of ASFV.

[0053] 4. PAMs cells were treated with a compound (30 μM) at different infection time points, including 4 h before infection (-4 h), 2 h before infection (-2 h), simultaneous infection (0 h), 2 h after infection (2 h), 4 h after infection (4 h), and 8 h after infection (8 h). ASFV-G-WT cells were infected with an MOI of 1. An equal amount of DMSO was added as a positive control. The following tests were performed 24 hours after infection:

[0054] (1) Observe GFP fluorescence using a fluorescence microscope.

[0055] Observation results as follows Figure 7 As shown, compared with the control group, the compound can also significantly inhibit ASFV replication 8 hours after exposure;

[0056] (2) After repeated freeze-thaw cycles at -80℃, HAD was used. 50 Assess ASFV replication levels.

[0057] Experimental results are as follows Figure 8 As shown, compared with the control group, administration of the compound 8 hours after exposure to the virus also significantly inhibited the replication of ASFV;

[0058] (3) After cell lysis, the ASFV P72 protein level was measured by Western blot.

[0059] Experimental results are as follows Figure 9 As shown, compared with the control group, the compound significantly inhibited the replication of ASFV.

[0060] The above results indicate that barasertib 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 barasertib content, showing a dose-dependent relationship; and the inhibitory effect on ASFV remains strong with changes in exposure time.

[0061] In summary, the embodiments of this invention, using host cells PAMs cells as an example, have demonstrated that Barasertib can inhibit ASFV replication in host cells PAMs cells, indicating that the Barasertib described in this invention can inhibit ASF virus replication and can be used to prepare drugs or adjuvants against ASF virus infection; it can also be used to prepare antiviral drugs and has broad application prospects.

[0062] 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 Barasertib or a pharmaceutically acceptable salt thereof in the preparation of an anti-African swine fever virus drug, characterized in that, The molecular formula of the compound Barasertib is C 26 H 31 FN7O6P, the structural formula is shown in formula (I): Formula (I).

2. The application according to claim 1, characterized in that, The African swine fever virus mentioned is genotype II African swine fever virus.

3. The application according to claim 1, characterized in that, The dosage form of the drug is selected from any one or more of tablets, sprays, granules, capsules, oral liquids, injections, or suspensions.

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

5. The application according to claim 4, characterized in that, The excipients include any one or more of the following: excipients, diluents, preservatives, colorants, flavoring agents, wetting agents, suspending agents, stabilizers, isotonic agents, and emulsifiers.

6. The application according to claim 1, characterized in that, The compound Barasertib or a pharmaceutically acceptable salt thereof is used to inhibit the activity of African swine fever virus.

7. The application according to claim 1, characterized in that, The compound Barasertib or a pharmaceutically acceptable salt thereof is used to inhibit the expression of the ASFV structural protein P72.

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