Use of isohumulonol in the preparation of a drug for inhibiting sindbis virus replication

Abstract

The application provides application of isohomoflavucrol in preparation of a medicine for inhibiting replication of Senecavirus, and belongs to the technical field of medicines. 50 Through in vitro cytotoxicity determination, fluorescence observation, Western blot detection and TCID virus titer detection verification, it is found that isohomoflavucrol has no obvious toxicity to BHK-21, PK-15 and HEK293-T cells in the concentration range of 0-60 muM, and can inhibit replication of SVV classical strains and new epidemic strains in a concentration-dependent and time-dependent manner, significantly reduce the expression amount of virus VP3 protein and virus titer, and the half effective concentration is as low as 11.78 muM-20.53 muM. The application provides a safe, efficient and widely applicable anti-SVV drug candidate which can be prepared into injection, tablets and various dosage forms, and has important clinical application value and market prospect for prevention and control of SVV infection.

Description

Technical Field

[0001] This invention relates to novel uses of known compounds, belonging to the field of pharmaceutical technology, specifically to the use of isoflavones in the preparation of drugs that inhibit Seneca virus replication. Background Technology

[0002] Seneca Valley Virus (SVV) is a non-enveloped, single-stranded, positive-sense RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Its genome is approximately 7.3 kb in length and contains one open reading frame (ORF). The encoded polyprotein, after cleavage by proteases, forms four structural proteins (VP1, VP2, VP3, and VP4) and seven non-structural proteins. Among these, the VP3 protein, as a core component of the viral capsid, directly reflects viral replication activity and is a key indicator for evaluating the inhibitory effect of antiviral drugs.

[0003] Senecavirus (SVV) primarily infects pig herds, with piglets and finishing pigs being the most susceptible groups. Clinical symptoms mainly include fever, blisters and ulcers around the mouth, nose, and hooves, and lameness. In severe cases, it can cause diarrhea, dehydration, and death in piglets, and also lead to reproductive disorders in sows, resulting in significant economic losses to the global pig industry. In recent years, with the continuous emergence of SVV variants, especially the outbreaks of new circulating strains in 2025, its transmissibility and pathogenicity have significantly increased compared to the classic strain. Existing biosecurity control measures are no longer sufficient to effectively contain the spread of Senecavirus.

[0004] Currently, there are no commercially available or effective treatments for SVV globally, making the development of safe and effective anti-SVV drugs an urgent need for swine disease prevention and control. Isoxanthohumol is a natural flavonoid compound widely found in natural products such as hops and beer. Its CAS number is 521-48-2, and its molecular formula is C5. 21 H 22 O5, molecular weight 354.40. Isoflavone possesses biological activities such as antitumor, anti-inflammatory, antioxidant, and adipogenesis inhibition. Isoflavone can induce tumor cell apoptosis, autophagy, and migration. Isoflavone can be used in research on tumors, metabolic diseases, and inflammatory diseases. However, to date, there are no reports on its use in inhibiting Seneca virus replication, and its anti-SVV activity and mechanism of action remain unknown. Summary of the Invention

[0005] The purpose of this invention is to overcome the lack of anti-SVV drugs in the prior art, and to provide a new use for isoflavones, specifically its application in the preparation of drugs that inhibit Seneca virus replication, thus providing a safe and efficient drug candidate for the prevention and control of SVV infection.

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

[0007] The application of isoflavol in the preparation of drugs that inhibit Seneca virus replication.

[0008] Among them, Seneca virus includes, but is not limited to, the classic SVV CHhb17 strain and the newly circulating NM2025-08 strain.

[0009] As described above, the effective concentration of isoflavone for inhibiting Seneca virus replication is 1 μM to 60 μM.

[0010] As described above, the concentrations of isoflavone in drugs that inhibit Seneca virus replication are 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, and 40 μM.

[0011] As described above, optionally, the dosage form of the drug is a tablet, suspension, dry suspension, injection, or powder.

[0012] Optionally, the drug is a single active ingredient drug, including isoflavone as the active ingredient and a pharmaceutically acceptable carrier. Further, the carrier can be a liquid or solid carrier, wherein liquid carriers include, but are not limited to, physiological saline and phosphate buffer; solid carriers include, but are not limited to, one or more of lactose, starch, microcrystalline cellulose, and mannitol, etc., and are prepared into dosage forms such as tablets, suspensions, dry suspensions, injections, and powders using conventional pharmaceutical processes, particularly suitable for the drug administration needs of pig herds in different farming scenarios.

[0013] In this invention, isoflavol is used in the preparation of drugs that inhibit Seneca virus replication, and also in the preparation of anti-Seneca virus drugs.

[0014] This invention also provides the use of isoflavone in the preparation of medicaments for the treatment or prevention of Seneca virus infection.

[0015] A drug for inhibiting Seneca virus replication, the active ingredient of which comprises isoflavone, said drug having inhibitory effects on both the classical CHhb17 strain and the emerging NM2025-08 strain of Seneca virus.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention provides a novel use of isoflavone for inhibiting Seneca virus replication. This invention utilizes in vitro cell experiments (cytotoxicity assay, fluorescence observation, Western blot detection, TCID). 50 Virus titer detection, EC 50 The anti-SVV activity and safety of isoflavone were systematically verified by calculation. The experimental results showed that:

[0018] (1) Isoflavone has low toxicity to cells and good safety: CCK-8 assay showed that after treating BHK-21, PK-15 and HEK293-T cells with isoflavone in the concentration range of 0-60 μM for 24 h, the cell survival rate remained above 85%, indicating that it has no obvious toxicity to host cells within the experimental concentration range.

[0019] (2) Significantly inhibits SVV replication: Fluorescence observation experiments showed that 20 μM isoflavone could reduce the fluorescence intensity of rSVV-GFP virus in BHK-21 cells by more than 60%; Western blot detection confirmed that it had a concentration-dependent and time-dependent inhibitory effect on the expression of SVV VP3 protein, and the expression of VP3 was significantly inhibited after treatment with 20 μM concentration for 24 h.

[0020] (3) Wide range of applications: TCID 50 The test results showed that isoflavone had a significant inhibitory effect on both the classic SVV CHhb17 strain and the newly circulating NM2025-08 strain. At concentrations of 20 μM and above, it could reduce the viral titer of both strains by 2-3 log values, and it could exert a stable inhibitory effect in BHK-21, PK-15 and HEK293-T cell types.

[0021] (4) Highly effective anti-SVV activity: EC50 curve fitting results showed that isoflavone inhibited SVV replication by 11.78 μM in BHK-21 cells and by 11.78 μM in HEK293-T cells. 50 The concentration was 20.53 μM, indicating that it has highly efficient anti-SVV activity. Attached Figure Description

[0022] Figure 1 The graph shows the toxicity assay of isoflavone on BHK-21, PK-15, and HEK293-T cells, demonstrating the drug's safety.

[0023] Figure 2 The image shows the fluorescence of rSVV-GFP in BHK-21 cells after treatment with different concentrations of isoflavone, visually demonstrating the inhibitory effect of the drug on SVV replication.

[0024] Figure 3 Western blot images of VP3 protein of SVV CHhb17 strain in three cell lines after treatment with different concentrations of isoflavone, demonstrating the concentration-dependent inhibitory effect of the drug on the expression of SVV structural proteins.

[0025] Figure 4Western blot images of VP3 protein of SVV CHhb17 strain in three cell lines after isoflavone treatment at different time points demonstrate the time-dependent inhibitory effect of the drug on the expression of SVV structural proteins.

[0026] Figure 5 TCID values ​​of SVV CHhb17 and NM2025-08 strains in three cell lines after treatment with different concentrations of isoflavone. 50 The test results demonstrate the inhibitory effect of the drug on different prevalent strains of SVV;

[0027] Figure 6 The TCID50 values ​​of SVV CHhb17 strain titers in three cell lines treated with isoflavone at different time points demonstrate the sustained inhibitory effect of the drug on SVV proliferation.

[0028] Figure 7 The EC50 curves show the inhibition of SVV CHhb17 virus replication by isoflavone in BHK-21 and HEK293-T cells, demonstrating the high efficacy of the drug against SVV. Detailed Implementation

[0029] This invention investigates the regulatory effect of isoflavones on SVV replication in vitro and finds that isoflavones can be used to prepare drugs that inhibit Seneca virus replication.

[0030] The active ingredient involved in this invention is isoxanthohumol, also known as isoxanthohumol; CAS number: 521-48-2; molecular formula: C 21 H 22 O5; Molecular weight: 354.40; Chemical structure formula as follows:

[0031] Extensive experimental studies have revealed that isoflavones exert their antiviral effects by directly targeting viral components or host cell pathways, such as inhibiting viral adsorption / replication, regulating immunity, or combating oxidative stress.

[0032] Unless otherwise specified, the technical means employed in the embodiments of this invention are conventional means well known to those skilled in the art; the experimental methods and conditions involved can be specifically operated by referring to relevant experimental manuals, publicly available literature, or product instructions provided by reagent manufacturers. Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available products, and their purity is analytical grade or higher; the biological carriers, cell lines, and experimental animals involved can all be obtained through commercial channels. Unless otherwise expressly defined, the meanings of all technical and scientific terms used in this invention are consistent with the meanings commonly understood by those skilled in the art. Modifications or substitutions made to this invention without departing from the spirit and substance of this invention are all within the scope of protection of this invention.

[0033] The following cells were used in the examples: BHK-21 (hamster kidney cells), PK-15 (pig kidney cells), and HEK293-T (human embryonic kidney cells) were all preserved by the High-Tech Laboratory of Animal Biological Agents, Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. Cell culture conditions: DMEM medium containing 10% fetal bovine serum (Wuhan Saiweier Biotechnology Co., Ltd., catalog number G8003-100ML), 100U / mL penicillin, and 100μg / mL streptomycin was used for static culture in a 37℃, 5% CO2 incubator, with a passage period of 36-48h.

[0034] Virus strains: SVV CHhb17 strain (classic circulating strain): isolated and preserved from clinically ill pig samples by the Beijing Key Laboratory of Animal Husbandry and Veterinary Disease Prevention and Control Technology, Beijing Academy of Agricultural and Forestry Sciences, GenBank accession number: MG983756.1; SVV NM2025-08 strain (new circulating strain in 2025): isolated from vesicular fossa samples from clinically ill pigs in a large-scale pig farm in 2025, GenBank accession number: PX254463.1; recombinant virus rSVV-GFP: donated by Professor Liu Fuxiao of Qingdao Agricultural University, which can stably express GFP protein and is used for the visualization of SVV virus replication.

[0035] Drugs and reagents: Isoxanthohumol: purchased from MedChemExpress (MCE) (catalog number HY-N2584A), solvent: dimethyl sulfoxide (DMSO), purity ≥98%; Anti-SVV VP3 rabbit polyclonal antibody: provided by the Beijing Key Laboratory of Animal Disease Prevention and Control Technology, Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, titer 1:1000; β-actin mouse monoclonal antibody: purchased from Wuhan Saiwei Biotechnology Co., Ltd., catalog number: GB15001-100, titer 1:1000; HRP-labeled goat anti-rabbit secondary antibody: purchased from Beijing Lamborghide Trading Co., Ltd., catalog number S0101, titer 1:5000; Western blot chemiluminescence substrate kit: purchased from Beijing Lamborghide Trading Co., Ltd., catalog number E1070; TransDetect Cell Counting Kit (CCK) Cell proliferation and cytotoxicity assay kit: purchased from Beijing TransGen Biotech Co., Ltd., catalog number FC101-02; other reagents: DMEM medium, fetal bovine serum, RIPA protein lysis buffer, protein marker, SDS-PAGE gel preparation kit, etc., all purchased from Wuhan Saive Biotechnology Co., Ltd.

[0036] Example 1: Effects of isoflavones on cell viability

[0037] BHK-21, PK-15, and HEK293-T cells were seeded into 96-well plates and cultured for 24 hours. When the cell confluence reached 90%, a series of different concentrations of isoflavones were added: 0 μM (i.e., an equal volume of DMSO), 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 60 μM. After 24 hours of culture, the toxicity of isoflavones to the cells was detected using a CCK-8 assay. Blank wells (containing only cell culture medium) and control wells (containing both cells and culture medium) were also included. 10 μL of CCK-8 solution was added to each well of the cell culture plate, and the plates were incubated for 1–4 hours. The absorbance at 450 nm was then measured using a Bio-Rad iMark microplate reader, and cell viability was calculated. The calculation formulas are: Cell viability = [(As-Ab) / (Ac-Ab)] × 100%; Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%. As: Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drug); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drug).

[0038] Experimental results are as follows Figure 1 As shown in the figure, the horizontal axis represents DMSO, which is dimethyl sulfoxide, with a final concentration of 60 μM. The survival rate of the three cell types remained above 85% in each drug concentration group, with no significant difference compared to the control group. This confirms that isoflavone has no obvious toxicity to host cells within the experimental concentration range and has good safety.

[0039] Example 2: Effect of different concentrations of isoflavone on rSVV-GFP replication

[0040] BHK-21 cells and HEK293-T cells were seeded in 24-well plates and cultured for 24 hours until adherence. Then, isoflavones were added to the cells at final concentrations of 0 μM, 1 μM, 10 μM, and 20 μM, respectively, to infect them with rSVV-GFP (MOI=1). After 12 hours of further culture, the cells were observed and photographed using a fluorescence microscope. Simultaneously, DAPI (4',6-diamidinyl-2-phenylindole) was used to infect the cells. DAPI is a blue fluorescent nucleic acid dye that specifically binds to double-stranded DNA in the cell nucleus and does not interact with cytoplasmic components; it is used to indicate cell number. The results are shown below. Figure 2 The study includes four sets of fluorescence microscopy images: fluorescence images of BHK-21 cells and HEK293-T cells in the control group (0 μM, DMSO only), the 1 μM drug treatment group, the 10 μM drug treatment group, and the 20 μmol / L drug treatment group. The green fluorescence in the images represents the green fluorescent protein expressed by the rSVV-GFP virus. The intensity of the green fluorescence of rSVV-eGFP is used to indicate the viral replication level. The blue fluorescent dots represent the total number of cells in the sample. By combining the images above and below, the proportion of green fluorescence in the blue fluorescence can be used to determine whether the recombinant virus is affected by drug treatment.

[0041] The experimental results showed that a large amount of strong green fluorescence was visible in the control group, indicating that the virus was replicating in large quantities; the fluorescence intensity of the 10μM group was slightly reduced; the fluorescence area and brightness of the 20μM group were reduced by more than 60%, and the fluorescence intensity was significantly reduced, confirming that the 20μM concentration of isoflavone can effectively inhibit the replication of SVV in BHK-21 cells.

[0042] Example 3: Inhibitory effect of different concentrations of isoflavone on viral VP3 protein expression.

[0043] BHK-21, PK-15, and HEK293-T cells were seeded into 12-well plates, and isoflavones were added to these cells at final concentrations of 0 μM, 10 μM, 20 μM, and 40 μM, respectively. The cells were then infected with the SVV CHhb17 strain (MOI=1). Four drug concentration groups (0 μM (control group), 10 μM, 20 μM, and 40 μM) were also established for the BHK-21, PK-15, and HEK293-T cells, respectively. After 12 h of culture, total protein was extracted, and the samples were subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked, and incubated with primary antibodies (anti-VP3 monoclonal antibody and anti-β-actin monoclonal antibody) and secondary antibodies. Chemiluminescence staining was then performed to detect the expression of viral VP3 protein and the intracellular reference protein β-actin. Results Figure 3 As shown, the left figure shows the effect of isoflavone treatment on the expression of viral protein VP3 in BHK-21 cells. The bands from left to right represent: blank group without virus and drug, control group containing virus and only DMSO solvent, virus and 10 μM kaempferol, virus and 20 μM isoflavone, and virus and 40 μM isoflavone. The middle figure shows the effect of kaempferol treatment on PK-15 cells. The first figure shows the effect of kaempferol treatment on the expression of the viral protein VP3. The bands, from left to right, represent: a blank group without virus or drug; a control group containing virus and only DMSO solvent; a group containing virus and 10 μM isoflavone; a group containing virus and 20 μM isoflavone; and a group containing virus and 40 μM isoflavone. The second figure shows the effect of kaempferol treatment on the expression of the viral protein VP3 in HEk-293T cells. The bands, from left to right, represent: a blank group without virus or drug; a control group containing virus and only DMSO solvent; a group containing virus and 10 μM isoflavone; a group containing virus and 20 μM isoflavone; and a group containing virus and 40 μM isoflavone. The upper part shows the VP3 protein Western blot bands, and the lower part shows the corresponding time point internal control protein β-actin Western blot bands.

[0044] The experimental results showed that in the three cell types, the gray value of the VP3 protein band gradually decreased with increasing drug concentration. The VP3 expression level in the 20μM group was only 20%-30% of that in the control group, indicating that isoflavone has a concentration-dependent inhibition effect on SVV VP3 protein expression.

[0045] Example 4: Inhibitory effect of isoflavone treatment on viral VP3 protein expression at different time points

[0046] BHK-21, PK-15, and HEK293-T cells were seeded into 12-well plates and cultured for 24 hours. After adherence, isoflavones at a final concentration of 20 μM were added to the cells, and the cells were infected with SVV CHhb17 strain (MOI=1). Cells were collected at 6, 9, and 12 hours, and total protein was extracted and analyzed by Western blot.

[0047] The results are as follows Figure 4 As shown, the upper part is the Western blot protein band diagram of VP3 protein, and the lower part is the Western blot protein band diagram of the internal reference protein β-actin at the corresponding time point.

[0048] The experimental results showed that the expression level of VP3 protein in the control group (-) gradually increased over time, reaching a peak at 12h; the expression level of VP3 protein in the drug-treated group (+) increased slowly, with an inhibition rate of over 70% at 12h, indicating that isoflavone's inhibition of SVV replication is time-dependent and the inhibitory effect increases with the extension of treatment time.

[0049] Example 5: Effects of different concentrations of isoflavone on the toxicity of SVV strains CHhb17 and NM2025-08.

[0050] BHK-21, PK-15, and HEK293-T cells were seeded into 96-well plates and cultured for 24 hours until adherence. Then, isoflavones at final concentrations of 0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 40 μM were added to each cell type to infect the SVVCHhb17 and NM2025-08 strains (MOI=1). After 12 hours of culture, cells and culture supernatants were collected and subjected to three freeze-thaw cycles at -80°C. The supernatant was serially diluted 10-fold to obtain the virus for detection. The virus solution was serially diluted 10-fold in DMEM medium containing 2% fetal bovine serum, with dilution gradients from 10⁻¹ to 10⁻¹. 8 Discard the original culture medium in the 96-well plate, add 100 μL of diluted virus solution to each well, and set up 4 replicate wells per group. Incubate at 37℃ and 5% CO2 for 48 h, observe and record the cytopathic effect (CPE) in each well, and define positive cytopathic effect as the appearance of shrunken cells, cell shedding, and lysis.

[0051] The results are as follows Figure 5 As shown in the experimental results, the viral titers of both strains decreased significantly with increasing drug concentration. The titers of the 20 μM and above concentration groups were about 2 log values ​​lower than those of the control group. Moreover, the inhibitory effect on the newly circulating NM2025-08 strain was not significantly different from that on the classic CHhb17 strain, confirming that isoflavone has an inhibitory effect on different circulating SVV strains.

[0052] Example 6: Inhibitory effect of isoflavone treatment on viral titers at different time points

[0053] BHK-21, PK-15, and HEK293-T cells were seeded into 96-well plates and cultured for 24 hours until adherence. Then, isoflavones at a final concentration of 20 μM were added to each cell type, followed by infection with SVV CHhb17 strain (MOI=1). After 12 hours of culture, cells and culture supernatant were collected and subjected to three freeze-thaw cycles at -80°C. The supernatant was serially diluted 10-fold to obtain the virus for detection. The virus solution was then serially diluted 10-fold in DMEM medium containing 2% fetal bovine serum, with dilution gradients from 10⁻¹ to 10⁻¹. 8 Discard the original culture medium in the 96-well plate, add diluted virus solution to each well, and set up 4 replicate wells per group. Incubate at 37℃ and 5% CO2 for 48 hours, observe and record the cytopathic effect (CPE) in each well, and define positive cytopathic effect as the appearance of cell rounding, shedding, or lysis. At the same time, add an equal volume of DMSO to the above three cell types as a control group and perform the same experiment.

[0054] The results are as follows Figure 6 As shown in the figure, *** indicates significant differences (P < 0.01). The experimental results showed that the viral titer in the control group (DMSO) increased rapidly over time, reaching a peak at 12 h; the increase in viral titer in the isoflavone treatment group was significantly inhibited. Isoflavone treatment significantly inhibited SVV replication at 6 h, 9 h, and 12 h post-infection. At 6 h and 9 h, viral titers in BHK-21 cells, PK-15 cells, and HEK-293T cells decreased by 1.5-fold, and at 12 h, they decreased by 2-fold, 1.5-fold, and 1.5-fold, respectively. The significant decrease in titer at 12 h confirms that isoflavone can continuously inhibit SVV proliferation and prevent the increase in viral titer.

[0055] Example 7: EC50 curve of isoflavone inhibiting the replication of SVV CHhb17 strain

[0056] BHK-21 cells and HEK293-T cells were seeded in 96-well plates, pretreated with serially concentrated drugs for 1 h, and then infected with SVV CHhb17 strain (MOI=1). After 12 h of culture, changes in viral infection were detected and the inhibition rate was calculated. Nonlinear regression analysis was performed using GraphPadPrism software to fit the ECG. 50 The curve was calculated and the half-maximal effective concentration was determined.

[0057] Plot the fitted curve with the logarithm of drug concentration (logμM) as the x-axis, with a concentration range of 1-40μM; and the viral replication inhibition rate (%) as the y-axis. Figure 7The figure contains two curves, which represent the EC50 fitting curves of isoflavone inhibition of SVV replication in BHK-21 cells and HEK293-T cells, respectively.

[0058] The experimental results showed that the EC50 of isoflavone in BHK-21 cells was 11.78 μM, and the EC50 in HEK293-T cells was 20.53 μM, indicating that it has highly efficient anti-SVV activity in both cell types.

[0059] In summary, isoflavones can significantly inhibit SVV replication and exhibit anti-SVV activity. Isoflavones can be used to prepare drugs that inhibit SVV replication, particularly suitable for the prevention and control of Seneca virus infection.

Claims

1. Application of isoflavol in the preparation of drugs that inhibit Seneca virus replication.

2. The application according to claim 1, characterized in that, The effective concentration of isoflavone in the drug for inhibiting Seneca virus replication is 1 μM to 60 μM.

3. The application according to claim 1, characterized in that, The dosage forms of the drug are tablets, suspensions, dry suspensions, injections, and powders.

4. The application according to claim 1, characterized in that, The drug is a single active ingredient drug, which includes isoflavone as the active ingredient and a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The carrier can be a liquid carrier or a solid carrier, wherein the liquid carrier includes, but is not limited to, physiological saline and phosphate buffer; the solid carrier includes, but is not limited to, one or more of lactose, starch, microcrystalline cellulose, and mannitol, and is prepared into tablets, suspensions, dry suspensions, injections, and powders by conventional pharmaceutical processes.

6. The use of isoflavone in the preparation of drugs for the treatment or prevention of Seneca virus infection.

7. A drug for inhibiting Seneca virus replication, characterized in that, The active ingredient of the drug includes isoflavone, and the drug has inhibitory effects on both the classical strain CHhb17 and the newly circulating strain NM2025-08 of Seneca virus.

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