New application of compound C75 in preventing or treating African swine fever
Compound C75 addresses the lack of effective drugs for African swine fever by inhibiting the transcription and expression of structural proteins of ASFV, thus achieving effective inhibition and treatment of the African swine fever virus.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LANZHOU VETERINARY RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES(LANZHOU BRANCH CENTER OF CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENTER)
- Filing Date
- 2026-02-13
- Publication Date
- 2026-04-24
AI Technical Summary
Currently, there is a lack of effective commercial vaccines or treatments to combat African swine fever. Existing small molecule compounds have not shown significant inhibitory effects on ASFV, and different viruses have different regulatory effects on fatty acid synthase.
Compound C75, as a fatty acid synthase inhibitor, can inhibit the transcription and expression of ASFV structural proteins and block the production of African swine fever virus particles, and can be used to prepare drugs for the prevention or treatment of African swine fever.
Compound C75 significantly reduces the replication level of African swine fever virus, inhibits the expression of viral structural proteins, and blocks viral infection, providing an effective prevention and treatment strategy.
Smart Images
Figure CN121910718A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a novel use of compound C75 for the prevention or treatment of African swine fever. Background Technology
[0002] African swine fever (ASF) is a highly contagious and virulent disease caused by the African swine fever virus (ASFV). Currently, there are no commercially available vaccines or treatments in China, making the development of new antiviral drugs crucial for treating ASF. Although various active anti-ASFV drugs have been reported, they have not yet been put into practical production and application.
[0003] ASFV is a large cytoplasmic DNA virus that primarily targets monocytes and macrophages, encoding a variety of structural and non-structural proteins. p30 is an early ASFV protein, typically produced 2-4 hours after infection. p72 is an important ASFV capsid protein that protects viral nucleic acid from damage and also participates in the infection process, being produced in the later stages of infection. As an important ASFV antigenic protein, it possesses good immunogenicity and is a major component of the ASFV icosahedron.
[0004] Studies have found that small molecule inhibitors that are effective against most viruses may not be effective against ASFV. For example, AVN 944 can significantly inhibit the replication of monkeypox virus and Zika virus (ZIKV), but has no significant effect on ASFV replication, indicating that small molecule compounds that are applicable to other viruses may not necessarily be able to inhibit ASFV.
[0005] C75 is a small-molecule biological agent and an inhibitor of fatty acid synthase (FASN), which can significantly inhibit fatty acid synthesis. Studies have shown that different viruses regulate fatty acid synthase differently. SARS-CoV-2 and dengue virus (DENV) upregulate the expression of fatty acid synthase FASN, while Newcastle disease virus (NDV) downregulates its expression during infection. Therefore, the inhibitory effect of compound C75 on different viruses also varies. This invention unexpectedly discovered that compound C75 can inhibit the transcription and expression of ASFV structural proteins, inhibit the production of African swine fever virus particles, and has an inhibitory effect on 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. Summary of the Invention
[0006] To address the aforementioned technical problems, this invention has discovered that compound C75 can inhibit the replication of ASFV (African Swine Fever Virus) and can be used to prepare drugs against African swine fever virus infection. Specifically, it includes the following:
[0007] In a first aspect, the present invention provides the use of compound C75 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention of African swine fever, wherein the structural formula of compound C75 is shown in formula (I) below:
[0008]
[0009] Equation (Ⅰ).
[0010] Preferably, compound C75 inhibits the replication of African swine fever virus.
[0011] Preferably, compound C75 inhibits the expression of African swine fever virus p72 protein.
[0012] Preferably, compound C75 inhibits the expression of African swine fever virus p30 protein.
[0013] Preferably, compound C75 inhibits the production of infectious viral particles of African swine fever virus.
[0014] Preferably, the compound C75 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable excipient to form any dosage form.
[0015] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.
[0016] Secondly, the present invention provides the use of compound C75 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever, wherein the structural formula of compound C75 is shown in formula (I) below:
[0017]
[0018] Equation (Ⅰ).
[0019] Preferably, compound C75 inhibits the replication of African swine fever virus.
[0020] Preferably, compound C75 inhibits the expression of African swine fever virus p72 protein.
[0021] Preferably, compound C75 inhibits the expression of African swine fever virus p30 protein.
[0022] Preferably, compound C75 inhibits the production of infectious viral particles of African swine fever virus.
[0023] Preferably, the compound C75 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable excipient to form any dosage form.
[0024] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.
[0025] The beneficial effects of this invention are: This invention unexpectedly discovered that compound C75 can reduce the replication level of African swine fever virus, inhibit the expression of African swine fever virus structural proteins, inhibit the production of African swine fever virus particles, and has the effect of inhibiting African swine fever virus infection; it can be used to prepare drugs or adjuvants against African swine fever virus infection, for inhibiting the replication of African swine fever virus. Attached Figure Description
[0026] Figure 1 The figure shows the experimental results of the inhibitory effect of compound C75 on ASFV-GFP.
[0027] Figure 2 Compound C75 inhibits HAD associated with ASFV CN / GS / 2018. 50 Experimental results diagram.
[0028] Figure 3 Figure showing the results of an experiment in which compound C75 inhibited p72 transcription of ASFV CN / GS / 2018.
[0029] Figure 4 Figure showing the experimental results of compound C75 inhibiting the expression of p72 and p30 proteins of ASFV CN / GS / 2018.
[0030] Figure 5 The figure shows the experimental results of the effect of compound C75 on the cell viability of PAM cells. Detailed Implementation
[0031] 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.
[0032] The experiments described in the following examples obtained biosafety clearance and African swine fever laboratory activity clearance:
[0033] The structural formula of compound C75 described in the following examples is shown in formula (Ⅰ). The compound was purchased from Selleck Chemicals (Selleck, S9819).
[0034]
[0035] Equation (Ⅰ).
[0036] The experimental cells and virus sources described in the following examples are:
[0037] Primary porcine alveolar macrophages (PAMs) were obtained from piglets around 5 weeks of age. After aseptic collection of PAM cells, red blood cells were removed with erythrocyte lysis buffer (purchased from Biosharp). After low-speed centrifugation, the supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 complete medium (purchased from Gibco) containing 10% FBS (purchased from Gibco) and cultured in a 37°C, 5% CO2 incubator.
[0038] The ASFV strain is CN / GS / 2018. The ASFV CN / GS / 2018 isolate was obtained from the National African Swine Fever Regional Laboratory (Lanzhou), belongs to genotype II, and has a viral titer of 5 × 10⁻⁶. 7 TCID50 / mL, representing the 4th generation seed virus after PAM cell propagation. ASFV-GFP is a fluorescent ASFV virus containing GFP prepared in our laboratory.
[0039] Unless otherwise specified, all other reagents used in the examples are common commercially available reagents; unless otherwise specified, all operations described in the examples are procedures known in the art. Unless otherwise specified, all cells and reagents mentioned in the following examples are commercially available.
[0040] Example 1: Preliminary screening of the effect of compound C75 on African swine fever virus.
[0041] Porcine alveolar macrophages (PAM) were cultured in 12-well plates using RPMI 1640 + 10% FBS medium, 2 × 10⁶ cells / well. 6 Cells were treated with ASFV fluorescent toxin (MOI = 0.1) mixed with C75 (25 μM) dissolved in DMSO (<1%) in a microplate ( / well). The control group was treated with ASFV fluorescent toxin (MOI = 0.1) mixed with DMSO (<1%). Cells were then cultured at 37°C and 5% CO2 for 48 h, and fluorescence changes were observed under a microscope.
[0042] The results are as follows Figure 1 As shown, compared with the DMSO control group, the ASFV fluorescence in the compound C75 treatment group was significantly reduced, indicating that compound C75 can inhibit ASFV replication.
[0043] Example 2: Compound C75's HAD against African swine fever virus 50 Impact
[0044] Porcine alveolar macrophages (PAMs) were cultured in 12-well plates using RPMI 1640 + 10% FBS medium, 2 × 10⁻⁶ cells / well. 6Cells in the experimental group were treated with ASFV CN / GS / 2018 (MOI = 0.1) mixed with C75 (25 μM) dissolved in DMSO (<1%), while cells in the infection control group were treated with ASFV CN / GS / 2018 (MOI = 0.1) mixed with DMSO (<1%). Cell plates were then incubated at 37°C and 5% CO2 for 48 h. After three freeze-thaw cycles at -80°C, the cells were used as samples and serially diluted 10-fold with serum-free RPMI 1640 in six dilutions, with each dilution replicated in eight wells. These samples were then seeded into PAM cells coated with red blood cells for culture. The cells were incubated at 37°C and 5% CO2 for 5 days, and red blood cell adsorption in each well was observed daily to calculate HAD. 50 .
[0045] The results are as follows Figure 2 The results showed that, compared with the infection control group, the HAD in the compound C75-treated group was significantly lower. 50 The significant reduction indicates that compound C75 can inhibit the production of infectious viral particles.
[0046] Example 3: Effect of compound C75 on p72 transcription level of African swine fever virus
[0047] Porcine alveolar macrophages (PAM) were cultured in 12-well plates using RPMI 1640 + 10% FBS medium, 2 × 10⁶ cells / well. 6 Cells were treated with different concentrations of C75 (6.25, 12.5, 25 μM) dissolved in DMSO (<1%) using ASFV CN / GS / 2018 (MOI = 0.1) in the experimental group, while the control group was treated with ASFV CN / GS / 2018 (MOI = 0.1) mixed with DMSO (<1%). Cell cultures were then incubated at 37°C and 5% CO2 for 48 h, followed by centrifugation and discarding of the supernatant. Total RNA was extracted using the Trizol method, and cDNA was synthesized using the RT Primer Mix kit. The differences in p72 gene transcription levels were detected by RT-qPCR.
[0048] The total volume of the RT-qPCR reaction system was 10 μL, including 0.4 μL of forward and reverse primers, 2 μL of cDNA, 5 μL of TBGreen™ Premix Ex Taq (TaKaRa), and sterile deionized water was added to bring the volume to 10 μL. The reaction conditions were: 95 ℃ for 2 min; 95 ℃ for 10 s, 60 ℃ for 34 s, for 40 cycles.
[0049] The primer sequences for amplifying p72 are as follows: upstream primer: 5'-TGCGATGATGATTACCTT-3'; downstream primer: 5'-ATTCTCTTGCTCTGGATAC-3'.
[0050] Experimental results are as follows Figure 3 As shown, compound C75 can inhibit the RNA expression level of p72 in the African swine fever virus gene, and the higher the concentration of C75, the stronger the inhibitory effect. When the dose of compound C75 is 12.5 μM, the inhibition rate of p72 RNA expression level is higher than 50%.
[0051] Example 4: Effect of compound C75 on the protein levels of p72 and p30 of African swine fever virus.
[0052] Porcine alveolar macrophages (PAMs) were cultured in 12-well plates using RPMI 1640 + 10% FBS medium, 2 × 10⁻⁶ cells / well. 6 Cells in the experimental group were treated with different concentrations of C75 (6.25 μM, 12.5 μM, 25 μM) dissolved in DMSO (<1%) using ASFV CN / GS / 2018 (MOI = 0.1). Cells in the control group were treated with the same ASFV CN / GS / 2018 (MOI = 0.1) mixed with DMSO (<1%). Cell cultures were then incubated at 37°C and 5% CO2 for 48 h. Cell cultures were collected, centrifuged, and the supernatant was discarded. Total protein was extracted, and the expression differences of p30 and p72 proteins were detected using Western blotting.
[0053] Experimental results are as follows Figure 4 As shown, the protein levels of p72 and p30 in infected cells treated with C75 were significantly reduced, and the reduction effect became more pronounced with increasing C75 concentration. These results indicate that compound C75 can significantly inhibit the expression of p72 and p30 proteins in African swine fever virus.
[0054] Example 5: CCK-8 assay to detect the effect of compound C75 on the cell viability of PAM cells.
[0055] Resuscitate PAM cells at a rate of 1×10 5Cells were seeded into 96-well cell culture plates and cultured at 37°C with 5% CO2 for 6 hours. Cells were then divided into control and experimental groups. Control group cells were supplemented with 0.1% (v / v) dimethyl sulfoxide (DMSO), while experimental group cells were supplemented with RPMI 1640 medium containing 6.25, 12.5, 25, and 50 μM compound C75, respectively. A blank control group containing the same dose of DMSO as the control group was also included. Cells were incubated for 48 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, the cell plate was gently tapped to mix the reagent, and the cells were incubated at 37°C for another 1 hour. After incubation, the absorbance at 450 nm was measured using a microplate reader. The cell viability at the corresponding compound concentration was calculated using the formula: [(experimental group absorbance - blank group absorbance) / (control group absorbance - blank group absorbance)] × 100% = cell viability percentage.
[0056] The results are as follows Figure 5 As shown, compound C75 has no significant effect on cell activity at concentrations of 25 μM and below, and has good safety.
[0057] In summary, the C75 of this invention can inhibit the replication of African swine fever virus and inhibit the transcription of the p72 gene and the expression of p72 and p30 proteins of African swine fever virus in a dose-dependent manner. It can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever.
Claims
1. The use of compound C75 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention of African swine fever, wherein the structural formula of compound C75 is shown in formula (Ⅰ): Equation (Ⅰ).
2. The use of compound C75 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever, wherein the structural formula of compound C75 is shown in formula (Ⅰ): Equation (Ⅰ).
3. The application as described in claim 1 or 2, characterized in that, The compound C75 inhibits the replication of African swine fever virus.
4. The application as described in claim 3, characterized in that, The compound C75 inhibits the expression of the African swine fever virus p72 protein.
5. The application as described in claim 3, characterized in that, The compound C75 inhibits the expression of the African swine fever virus p30 protein.
6. The application as described in claim 3, characterized in that, The compound C75 inhibits the production of infectious viral particles of African swine fever virus.
7. The application as described in claim 1 or 2, characterized in that, The compound C75 or a pharmaceutically acceptable salt thereof may be formulated into any dosage form by adding pharmaceutically acceptable excipients.
8. The application as described in claim 7, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.