New use of the compound tafenoquine for the prevention or treatment of african swine fever

Abstract

The application belongs to the technical field of African swine fever treatment, and particularly relates to a new use of a compound, i.e., tafenoquine, for preventing or treating African swine fever. It is accidentally found that the compound tafenoquine has no antiviral activity on Japanese encephalitis virus, but can reduce the replication level of African swine fever virus, inhibit the expression of structural proteins of the African swine fever virus, has the effect of inhibiting the infection of the African swine fever virus, can be used for preparing a medicine or an adjuvant against the infection of the African swine fever virus, and is used for inhibiting the replication of the African swine fever virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel use of the compound tafenoxanone for the prevention or treatment of African swine fever. Background Technology

[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by the African swine fever virus (ASFV). It causes fever and widespread hemorrhage in both domestic and wild pigs, with a mortality rate as high as 100%. Currently, there are no commercially available vaccines or effective treatments; prevention relies solely on strict quarantine measures. Once an infected herd is identified, culling is mandatory. Therefore, developing effective anti-ASF drugs is a crucial direction for the current control of this disease.

[0003] ASFV is the only known DNA-borne virus, belonging to the African swine fevervirus family and the African swine fevervirus genus. ASFV is an enveloped icosahedral virus with five structures from the outside in: outer membrane, protein capsid, inner membrane, core capsid, and nucleoid. Its genome is 170-190 kb in length and encodes 150-200 viral proteins. The primary host cell for this virus is the monocyte-macrophage.

[0004] The p30 protein, encoded by the viral gene CP204L, is located on the viral inner membrane and is an early protein in the viral replication phase, assisting in viral internalization and entry into the cell. The p72 protein, encoded by the B646L gene, is located on the viral protein capsid and is an important protein that makes up the protein coat. It is a late protein in the viral replication phase. Both have good immunogenicity and can serve as ideal detection targets.

[0005] Tafenoquine (TFQ) is an 8-aminoquinoline antimalarial drug with broad-spectrum activity against a variety of protozoan parasites. In 2018, the U.S. FDA approved the drug for the treatment of Plasmodium vivax, but its target is still unclear.

[0006] Furthermore, there is limited research on the drug's effects on viruses, with studies limited to Severe Acute Respiratory Syndrome Coronavirus II. This invention unexpectedly discovered that TFQ has no effect on Japanese encephalitis virus replication, but it can reduce ASFV replication levels and inhibit the expression of viral proteins p72 and p30, thus inhibiting African swine fever virus infection. It can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever, showing great application potential. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a novel use of tafenoxanone for the prevention or treatment of African swine fever, specifically including the following:

[0008] In a first aspect, the present invention provides the use of the compound tafenoxane or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention of African swine fever virus infection, wherein the compound tafenoxane has the structural formula shown in formula (Ⅰ) below:

[0009]

[0010] Preferably, the compound tafenoxane or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

[0011] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0012] Secondly, the present invention provides the use of the compound tafenoxane or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating African swine fever virus infection, wherein the compound tafenoxane has the structural formula shown in formula (Ⅰ) below:

[0013]

[0014] Preferably, the compound tafenoxane or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

[0015] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0016] Thirdly, the present invention provides the use of the compound tafenoxane or a pharmaceutically acceptable salt thereof in the preparation of adjuvants for African swine fever virus vaccines, wherein the structural formula of the compound tafenoxane is shown in formula (Ⅰ) below:

[0017]

[0018] Preferably, the compound tafenoxane or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

[0019] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0020] The beneficial effects of this invention are: This invention unexpectedly discovered that the compound tafenoxane has no effect on the replication of Japanese encephalitis virus, but can reduce the replication level of African swine fever virus and inhibit the expression of African swine fever virus structural proteins, thus having the effect of inhibiting African swine fever virus infection. 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

[0021] Figure 1 Results of Western blotting analysis of the expression levels of ASFV structural proteins p72 and p30;

[0022] Figure 2 RT-qPCR results of transcriptional levels of ASFV B646L and CP204L genes;

[0023] Figure 3 Viral titer HAD 50 Test results;

[0024] Figure 4 Results of toxicity assays on 293T cells at different concentrations of tafenoxanol;

[0025] Figure 5 Results of the effects of different concentrations of tafenoxanone on the activity of PAM cells;

[0026] Figure 6 Results of the effect of tafenoxanone on the protein levels of Japanese encephalitis virus (JEV);

[0027] Figure 7 Results of the effect of tafenoxanone on the transcriptional level of Japanese encephalitis virus (JEV). Detailed Implementation

[0028] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further described below in conjunction with specific embodiments. However, the scope of protection of this invention is not limited to the embodiments described below.

[0029] The experiments described in the following examples obtained biosafety clearance and African swine fever laboratory activity clearance:

[0030] In accordance with the requirements for a Biosafety Level 3 (BSL-3) laboratory and related biosafety for African swine fever, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, through a hierarchical reporting process involving the Lanzhou Veterinary Research Institute's Biosafety Committee, Laboratory Animal Ethics Committee, the Chinese Academy of Agricultural Sciences' Biosafety Committee, the Lanzhou Veterinary Research Institute's Laboratory Animal Ethics Committee, and the Lanzhou Veterinary Research Institute's Biosafety Committee, obtained permission from the Ministry of Agriculture to conduct research on highly pathogenic ASFV pathogens and related animals. This permit has been registered with the Ministry of Agriculture and Rural Affairs and meets the national biosafety level requirements.

[0031] Unless otherwise specified, all reagents described in the following examples are commercially available.

[0032] The African swine fever virus is the genotype II African swine fever virus strain ASFV CN / GS / 2018, which was isolated by our team and preserved in the Foot-and-Mouth Disease Epidemiology Team Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0033] Example 1: Effect of tafenoxanone on ASFV

[0034] 1. Effects of tafenoxanone on ASFV protein levels

[0035] PAM cells were seeded in 12-well plates. After adhesion, tafenoxane at concentrations of 2, 4, 6, and 8 μM was added to the PAM cells for 6 h. A positive control group (DMSO) and a negative control group (Mock) were also included. Subsequently, virus solution with an MOI of 1 was added, and the cells were cultured at 37°C and 5% CO2 for 24 h. Cells and supernatant were then scraped for Western blotting (WB) to detect the expression of proteins p30 and p72. First, cells were scraped from the wells, centrifuged at 1000×g for 5 min, and the supernatant was discarded. The cells were washed twice with PBS, and RIPA lysis buffer was added. The cells were then lysed on ice and shaken for 3 h. After lysis, the cells were centrifuged at 12000×g for 15 min at 4°C. The supernatant was collected, and protein concentration was determined using the BCA method. Loading buffer was added, and the cells were denatured at 100°C for 15 min. Protein electrophoresis was then performed at a constant voltage of 100V. After electrophoresis, the proteins were transferred in situ onto a nitrocellulose membrane at a constant current of 250 mA for 2 h. After transfer, the nitrocellulose membrane was blocked at room temperature for 1 hour with 5% skim milk powder prepared with TBST. After blocking, it was washed three times with TBST for 5 minutes each time. After washing, it was incubated overnight at 4°C with p72, p30, and the internal control β-actin primary antibody. Subsequently, it was washed three times with TBST for 10 minutes each time. Secondary antibody was added and incubated at room temperature for 1 hour, followed by washing three times with TBST for 10 minutes each time. Finally, it was visualized using ECL developing solution.

[0036] The results are as follows Figure 1 As shown, compared with the DMSO control group, tafenoxane treatment was able to inhibit the expression levels of ASFV structural proteins p72 and p30 in a dose-dependent manner, and could significantly inhibit ASFV replication.

[0037] 2. Effects of tafenoxanone on ASFV transcriptional levels

[0038] PAM cells were seeded in 12-well plates. After adhesion, tafenoxane at concentrations of 2, 4, 6, and 8 μM was added to the PAM cells for 6 h. A negative control group (DMSO) and a blank control group (Mock) were also included. Subsequently, ASFV virus solution with an MOI of 1 was inoculated. After culturing at 37°C and 5% CO2 for 24 h, cells and supernatant were scraped for RT-qPCR experiments to detect the transcriptional levels of ASFV B646L and CP204L genes. First, cells were scraped from the wells, centrifuged at 1000×g for 5 min, the supernatant was discarded, and the cells were washed twice with PBS. RNA was extracted from the cell samples using the Trizol method, and the RNA concentration was measured. PrimeScript was used to analyze the RNA. TM Reverse transcription was performed using the RT reagent Kit with gDNA ERASER kit (Takara). Following the kit instructions, 2 μL of 5×gDNA Eraser Buffer, 1 μL of gDNA Eraser, and 7 μL of RNA were added to a 10 μL system and mixed thoroughly. The mixture was then incubated at 42°C for 2 min to remove residual DNA. The DNA-free RNA was then added to a 20 μL reaction system and reverse transcribed to obtain cDNA, which served as the template. Primer sequences designed based on the ASFV CP204L gene sequence (GenBank: MK333184.1), ASFVB646L gene sequence (GenBank: MK333180.1), and porcine GAPDH gene sequence (GenBank: NM001206359.1) from the NCBI database are shown in Table 1.

[0039] Table 1 Primers

[0040]

[0041] Using SYBR Green qPCR (Servicebio), 40 cycles of amplification were performed under the following reaction program: 95℃ for 30s, 95℃ for 15s, and 60℃ for 30s. The results were then analyzed.

[0042] The results are as follows Figure 2 As shown, compared with the DMSO control group, tafenoxane treatment can inhibit the transcriptional levels of ASFVB646L and CP204L genes in a dose-dependent manner and can significantly inhibit ASFV replication.

[0043] 3. Effect of tafenoxanone on ASFV viral titer

[0044] PAM cells were seeded in 96-well plates. After adhesion, the PAM cells were treated with 2, 4, 6, and 8 μM tafenoxane for 6 h. A negative control group (DMSO) was set up. Then, the cells were seeded with serially diluted virus solution (10×). -1 -10 -9 After stabilization, 2% porcine erythrocytes were added, and pericellular erythrocyte adsorption (HAD) was observed around infected cells within 4-7 days post-infection. The 50% HAD was calculated using the Reed-Muench method. 50 This is used to display the virus replication level.

[0045] The results are as follows Figure 3 As shown, tafenoxane treatment reduced viral titers compared to the DMSO control group, indicating that the compound tafenoxane can significantly inhibit ASFV replication.

[0046] Example 2 Security

[0047] PAM cells and 293T cells were seeded into 96-well plates and cultured for 12 hours until adherence. Then, a specified concentration of tafenoxane was added, and the cells were incubated for 24 hours. Afterward, 10 μL of CCK-8 was added to each well, and the cells were incubated for 2-4 hours. The cells were then analyzed using a microplate reader at OD500. 450 Cell viability is calculated by reading values ​​at specific wavelengths.

[0048] Safety results for 293T cells are as follows Figure 5 As shown, compared to the DMSO group, within the concentration range that has effective inhibitory activity against African swine fever, tafenoxane did not show a significant difference in the survival rate of PAM cells and 293T cells, indicating good safety.

[0049] Safety results for PAM cells, such as Figure 4 As shown, compared with the DMSO group, treatment with tafenoxane at concentrations less than 10 μM did not significantly affect the survival rate of PAM cells, indicating good safety.

[0050] Example 3: Effect of tafenoxanone on Japanese encephalitis virus

[0051] 1. Effects of tafenoxanone on protein levels of Japanese encephalitis virus (JEV)

[0052] 293T cells were seeded in 12-well plates and allowed to adhere for 12 hours. Then, 8 μM tafenoxane was added to the cells for 6 hours. A positive control group (DMSO) and a negative control group (Mock) were also included. The cells were then inoculated with virus solution at an MOI of 1 and cultured at 37°C with 5% CO2 for 24 hours. Afterward, cells were scraped for Western blotting (WB) to detect the expression of the NS3 protein. First, cells were scraped from the wells, centrifuged at 1000×g for 5 minutes, and the supernatant was discarded. The cells were washed twice with PBS, and lysed with RIPA lysis buffer on ice for 3 hours. After lysis, the cells were centrifuged at 12000×g for 15 minutes at 4°C. The supernatant was collected, and the protein concentration was determined using the BCA method. Loading buffer was added, and the cells were denatured at 100°C for 15 minutes. Then, protein electrophoresis was performed at a constant voltage of 100V. After electrophoresis, the protein was transferred in situ onto a nitrocellulose membrane at a constant current of 250 mA for 2 hours. After transfer, the nitrocellulose membrane was blocked at room temperature for 1 hour with 5% skim milk powder prepared with TBST. After blocking, it was washed three times with TBST for 5 minutes each time. After washing, it was incubated overnight at 4°C with NS3 and internal control GAPDH primary antibody. Then, it was washed three times with TBST for 10 minutes each time. Secondary antibody was added and incubated at room temperature for 1 hour, followed by washing three times with TBST for 10 minutes each time. Finally, it was visualized using ECL developing solution.

[0053] The results are as follows Figure 6 As shown, compared with the DMSO control group, tafenoxane treatment could not reduce the expression level of the non-structural protein NS3 of JEV, nor could it inhibit JEV replication.

[0054] 2. Effects of tafenoxanone on JEV transcription levels

[0055] 293T cells were seeded in 12-well plates and, after 12 h of adhesion, were treated with 8 μM tafenoxane for 6 h. A positive control group (DMSO) and a negative control group (Mock) were also included. Subsequently, the cells were inoculated with virus solution at an MOI of 1 and cultured at 37°C with 5% CO2 for 24 h. Cells and supernatant were then scraped for RT-qPCR to detect gene transcription levels. First, cells were scraped from the wells, centrifuged at 1000×g for 5 min, the supernatant was discarded, and the cells were washed twice with PBS. RNA was extracted from the cell samples using the Trizol method, and the RNA concentration was measured. PrimeScript was used to analyze the RNA. TMReverse transcription was performed using the RT reagent kit with gDNAERASER (Takara). Following the kit instructions, 2 μL of 5x gDNAEraser Buffer, 1 μL of gDNA Eraser, and 7 μL of RNA were added to a 10 μL system and mixed thoroughly. The mixture was then incubated at 42°C for 2 min to remove residual DNA. The DNA-free RNA was then added to a 20 μL reaction system for reverse transcription to obtain cDNA, which served as the template.

[0056] Using SYBR Green qPCR (Servicebio), 40 cycles of amplification were performed under the following reaction program: 95℃ for 30s, 95℃ for 15s, and 60℃ for 30s. The results were then analyzed.

[0057] The primer sequences used for qPCR are shown in Table 2 below:

[0058] Table 2 Primers used in qPCR

[0059]

[0060] The results are as follows Figure 7 As shown, compared with the DMSO control group, tafenoxane treatment could not downregulate the transcription level of the non-structural protein NS3 gene of JEV, nor could it inhibit JEV replication.

[0061] In summary, the tafenoxane described in this invention can dose-dependently inhibit the transcription of ASFV B646L and CP204L genes and the expression levels of their encoded structural proteins p72 and p30, significantly inhibiting ASFV replication, but it has no inhibitory activity against Japanese encephalitis virus. It can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever.

[0062] The present invention has been illustrated with the above embodiments to demonstrate its detailed features and methods. However, the present invention is not limited to the above-described detailed features and methods, meaning that the present invention does not necessarily depend on the above-described detailed features and methods for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the components used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection and disclosure scope of the present invention.

Claims

1. The use of the compound tafenoxane or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention of African swine fever virus infection, wherein the structural formula of the compound tafenoxane is shown in formula (Ⅰ): Equation (Ⅰ).

2. The application as described in claim 1, characterized in that, The compound tafenoxane or a pharmaceutically acceptable salt thereof, when combined with pharmaceutically acceptable excipients, is formulated into any pharmaceutically acceptable dosage form.

3. The application as described in claim 2, characterized in that, The dosage form includes any one of the following: tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

4. The use of the compound tafenoxane or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating African swine fever virus infection, wherein the structural formula of the compound tafenoxane is shown in formula (Ⅰ) below: Equation (Ⅰ).

5. The application as described in claim 4, characterized in that, The compound tafenoxane or a pharmaceutically acceptable salt thereof, when combined with pharmaceutically acceptable excipients, is formulated into any pharmaceutically acceptable dosage form.

6. The application as described in claim 5, characterized in that, The dosage form includes any one of the following: tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

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