Application of Dasabuvir in preparation of orthopoxvirus inhibitor

Abstract

The invention relates to the field of biological medicine, in particular to application of Dasabuvir in preparation of orthopoxvirus inhibitors, the Dasabuvir is applied to a F13L membrane protein specific binding regulating agent and is specifically bound with palmitoylation sites on F13L membrane protein, formation of orthopoxviruses can be inhibited, the invention further provides application of the Dasabuvir in preparation of the orthopoxvirus inhibitors, the Dasabuvir is an FDA medicine, and the Dasabuvir can be applied to preparation of the orthopoxvirus inhibitors. The safety of the Dasabuvir is widely authenticated, and the Dasabuvir is successfully applied to clinical treatment, so that the Dasabuvir can be used as an effective orthopoxvirus inhibitor, the use safety can be ensured, and a new direction can be provided for inhibition of orthopoxviruses.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of Dasabuvir in the preparation of orthopoxvirus inhibitors. Background Art

[0002] Poxviridae are the largest and most complex viruses in nature. They are a large class of DNA viruses that are usually transmitted through direct contact or respiratory secretions. They can infect humans and animals, often causing skin lesions. Some of the deadly orthopoxviruses still pose a threat to human health. The orthopoxvirus genus includes a variety of viruses, including but not limited to smallpox virus, monkeypox virus, vaccinia virus, cowpox virus, mousepox virus and camelpox virus.

[0003] Poxvirus is a double-stranded DNA virus that replicates in the cytoplasm of host cells. Its replication cycle includes multiple stages, such as immature virions, mature virions in cells, etc. Poxviruses spread in various ways, including discharge through microvilli on the cell surface, acquisition of envelope by budding to form large vacuoles, etc.

[0004] In recent years, migrasomes, as a newly discovered organelle, are considered to be closely related to cell migration. Migrasomes can participate in the transmission of signal molecules related to poxvirus infection, thereby affecting the replication and spread of the virus. At the same time, poxviruses may use the release mechanism of migrasomes to promote the migration of virus particles into host cells during infection. Virus particles may be encapsulated in migrasomes and released into the extracellular matrix by interacting with integrins or other markers in migrasomes. Subsequently, these migrasomes containing virus particles can be taken up by surrounding host cells, thereby promoting the spread of the virus.

[0005] Monkeypox virus has attracted widespread attention due to its global spread. Its evolutionary branches and transmission characteristics have become a hot topic of research. This invention aims to develop small molecule inhibitors against orthopoxviruses through the FDA-approved drug Dasabuvir, which can provide specific treatment for orthopoxviruses represented by monkeypox virus, thereby reducing the potential threat of orthopoxviruses to human health. Through this innovative approach, we hope to contribute to global public health security and protect people from these deadly viruses. Summary of the invention

[0006] The object of the present invention is to provide the use of Dasabuvir in the preparation of orthopoxvirus inhibitors;

[0007] Another object of the present invention is to provide an application in a F13L membrane protein specific binding regulator.

[0008] The technical solution adopted to achieve the purpose of the present invention is:

[0009] F13 is a key membrane protein of the orthopoxvirus genus and is closely related to the formation of poxvirus enveloped virus particles (EEV). It is highly conserved in orthopoxviruses such as smallpox, monkeypox, vaccinia, cowpox, mousepox and camelpox, with an amino acid sequence similarity of over 90%. The amino acid sequence similarity of F13 between vaccinia virus and monkeypox virus is as high as 99.5%.

[0010] Therefore, the present application provides an application of Dasabuvir in a F13L membrane protein specific binding regulator, and the specific experimental steps are as follows:

[0011] (1) Acquisition of F13 protein structure: Alphafold2 was used to simulate the F13L proteins of monkeypox virus, vaccinia virus and variola virus in the orthopoxvirus genus; the F13L of monkeypox virus (pLDDT = 92.062) was based on the simulated structure of the F13L protein of the MPXV_USA_2022_MA001 virus strain by Zheng L. et al. (Liangzhen Zheng, Jintao Meng and Mingzhi Lin et al. Structure prediction of the entire proteome of monkeypox variants. Acta Materia Medica. 2022. Vol. 1(2): 260-264); the F13L of vaccinia virus (pLDDT = 91.9) and variola virus (pLDDT = 92.0) were based on the simulated structure of the F13L protein of the MPXV_USA_2022_MA001 virus strain by Zheng L. et al. (Liangzhen Zheng, Jintao Meng and Mingzhi Lin et al. Structure prediction of the entire proteome of monkeypox variants. Acta Materia Medica. 2022. Vol. 1(2): 260-264), respectively. The amino acid sequence of the virus strain F13L of Human / India / Ind3 / 1967 (Http: / / www.Uniprot.org) was then directly simulated from scratch using AlphaFold v2.0, and the simulated F13L protein structure with the highest pLDDT value was used for subsequent research.

[0012] (2) Virtual docking: The 3D structures of the F13L proteins of monkeypox virus, vaccinia virus and smallpox virus were imported respectively using the software MOE2019. They were first preprocessed using “QuickPrep”, and then the “SiteFinder” command was used to search for amino acid sites where small molecules might dock. Pocket sites including the palmitoylation active site were found, and then the “Dummies” command was used to construct virtual sites for small molecule docking.

[0013] Select candidate small molecule compounds from the EMBL-EBI small molecule compound database CHEMBL (https: / / www.ebi.ac.uk / chembl / ), download the SD format file and import it into MOE2019, and then use the "Dock" command to simulate docking at the Dummies virtual site. According to the comparison of docking results, the small molecule compounds are ranked, and the small molecule compound with the best docking result is selected for synthesis for subsequent virus infection experiments.

[0014] After screening, the small molecule compounds obtained are Dasabuvir, Dasabuvir (C 26 H 27 The structural formula of N3O5S is:

[0015]

[0016] Through the above screening, Dasabuvir had the best docking results with the palmitoylated F13 protein receptor molecules on monkeypox virus, vaccinia virus and smallpox virus in the orthopoxvirus genus;

[0017] (3) Verification of infection experiment: Vaccinia virus was used as the experimental object, and different concentration gradients of Dasabuvir were set to perform virus plaque assay and virus titer assay to obtain IC50 value.

[0018] Furthermore, the present application provides a use of Dasabuvir in the preparation of an orthopoxvirus inhibitor;

[0019] Furthermore, according to the final results of the infection experiment, the IC50 of Dasabuvir in inhibiting the spread of orthopoxvirus was 7.623 μmol / L;

[0020] Furthermore, when the IC50 of Dasabuvir is 8.051 μmol / L or 12.51 μmol / L, the ability of orthopoxvirus production in cells can be inhibited, thereby inhibiting the infectivity of the virus.

[0021] Furthermore, when the IC50 of Dasabuvir is 8.051 μmol / L, it can inhibit the ability of orthopoxvirus production in Huh7.5.1, thereby inhibiting the infectivity of the virus;

[0022] Furthermore, when the IC50 of Dasabuvir is 12.51 μmol / L, it can inhibit the production of orthopoxvirus in HeLa cells, thereby inhibiting the infectivity of the virus;

[0023] Furthermore, the orthopoxvirus is at least one of smallpox virus, monkeypox virus, and vaccinia virus.

[0024] It can be seen that the virus inhibition experiment confirmed that Dasabuvir can specifically bind to the palmitoylated active site of the orthopoxvirus viral protein F13, thereby inhibiting the spread of orthopoxviruses and the formation of enveloped viruses (EEV), and can effectively inhibit orthopoxviruses represented by VacciniaWR. Therefore, small molecule compounds targeting F13 are the first choice for the development of poxvirus-specific drugs, and have good drug development prospects and potential.

[0025] Therefore, the present invention also claims to protect the use of the small molecule Dasabuvir as a specific binding inhibitor of the F13 protein, which will not be limited to the inhibitor of the aforementioned orthopoxvirus virus, but will further extend to the protection of the poxvirus family containing the highly conserved F13 protein, and even other virus species.

[0026] Furthermore, the inhibitor forms mentioned in the present application include but are not limited to: capsules, tablets, pastes, injections, sprays, and aerosols.

[0027] Furthermore, the inhibitor is in the form of a liquid preparation.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. Dasabuvir is an FDA drug, and its safety has been widely certified and has been successfully used in clinical treatment. The application of Dasabuvir in F13L membrane protein specific binding regulator can not only ensure the safety of use, but also provide a new strategy for the inhibition of orthopoxviruses represented by monkeypox virus. Further experimental studies have shown that Dasabuvir can be used as an effective orthopoxvirus inhibitor. In laboratory studies, it has shown a significant inhibitory effect on orthopoxviruses, which provides important evidence for the potential application of Dasabuvir as an orthopoxvirus inhibitor and provides a new direction for future antiviral treatment.

[0030] 2. Dasabuvir has multifaceted inhibitory effects on orthopoxviruses. Dasabuvir can inhibit the migrasome induced by orthopoxviruses, especially when the IC50 of Dasabuvir is 7.623μmol / L, it can inhibit the spread of orthopoxviruses. Dasabuvir not only has an inhibitory effect on the spread of orthopoxviruses, but also has a good inhibitory effect on the production of viral EEV. When the IC50 of Dasabuvir is 8.051μmol / L or 12.51μmol / L, it can significantly inhibit the production of viral EEV, thereby inhibiting the formation of orthopoxviruses in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Virtual docking results of small molecule inhibitor Dasabuvir and F13L protein of monkeypox virus;

[0032] Figure 2 Virtual docking results of small molecule inhibitor Dasabuvir and vaccinia virus F13L protein;

[0033] Figure 3 Virtual docking results of small molecule inhibitor Dasabuvir and F13L protein of smallpox virus;

[0034] Figure 4 Effect of Dasabuvir on migrasome production by poxvirus transmission pathway in Huh7.5.1 cells;

[0035] Figure 5 The number of migrasomes in Huh7.5.1 cells after being affected by Dasabuvir;

[0036] Figure 6 Effects of Dasabuvir on migrasome production by poxvirus transmission pathways in HeLa cells;

[0037] Figure 7 The number of migrasomes in HeLa cells after exposure to Dasabuvir;

[0038] Figure 8 The toxic effect of Dasabuvir on BSC-1 cells and the spread of poxvirus;

[0039] Fig. 9 The inhibitory effect of Dasabuvir on poxvirus EEV in Huh7.5.1 cells;

[0040] Fig.10 The toxic effect of Dasabuvir on Huh7.5.1 cells;

[0041] Fig.11 The effect of Dasabuvir on poxvirus IMV in Huh7.5.1 cells;

[0042] Fig.12 The inhibitory effect of Dasabuvir on poxvirus EEV in HeLa cells;

[0043] Fig.13 The toxic effect of Dasabuvir on HeLa cells;

[0044] Fig.14 The effect of Dasabuvir on poxvirus IMV in HeLa cells;

[0045] Fig.15 The effect of Dasabuvir on the production of actin tail of poxvirus in Huh7.5.1 cells;

[0046] Fig.16 Quantify the effect of Dasabuvir on poxvirus actin tail production in Huh7.5.1 cells;

[0047] Fig.17 The effect of Dasabuvir on the production of actin tail of poxvirus in HeLa cells;

[0048] Fig.18 Quantitative statistics of the effect of Dasabuvir on poxvirus actin tail production in HeLa cells. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0051] All data were processed using GraphPad Prism 10.1.2 statistical software, and the experimental data were expressed as mean ± standard error. One-way analysis of variance was used to compare data between groups. P < 0.05 was considered statistically significant.

[0052] Example 1

[0053] HeLa and BSC-1 (African green monkey kidney) cells (purchased from American Type Culture Collection, ATCC) were cultured in DMEM medium containing 1% penicillin / streptomycin (P / S) and 10% fetal bovine serum (FBS) in an incubator at 37°C and 5% CO2.

[0054] The VacciniaWR virus strain was used as the experimental strain, and the virus was amplified in HeLa cells. After discarding the supernatant, the cells were repeatedly sonicated at low temperature for 3 times (15s / time) and then packaged and stored at -80°C. HeLa and BSC-1 cells were cultured in DMEM containing 10% fetal bovine serum, 100U / mL penicillin, and 100U / mL streptomycin at 37°C in a 5% CO2 incubator and passaged every 3 to 4 days.

[0055] Example 2

[0056] Acquisition of F13L protein structure: The F13L of monkeypox virus (pLDDT=92.062) adopts the simulated structure of the F13L protein of the MPXV_USA_2022_MA001 virus strain of Zheng L. et al.; the F13L of vaccinia virus (pLDDT=91.9) and smallpox virus (pLDDT=92.0) adopts the amino acid sequence of F13L of Vaccinia virus (strain Western Reserve) and Variola virus (isolate Human / India / Ind3 / 1967) virus strain respectively (Http: / / www.Uniprot.org), and then AlphaFold v2.0 is used to simulate directly from scratch, and the simulated F13L protein structure with the highest pLDDT value is used for subsequent research.

[0057] Virtual docking: The 3D structures of F13L proteins of monkeypox virus, vaccinia virus and smallpox virus were imported into MOE2019 software. First, they were preprocessed using "QuickPrep", and then the "SiteFinder" command was used to search for amino acid sites where small molecules could be docked. Pocket sites including phosphodiesterase active amino acid sites were found, and then the "Dummies" command was used to construct virtual sites for small molecule docking. Candidate small molecule compounds were selected from the EMBL-EBI small molecule compound database CHEMBL (https: / / www.ebi.ac.uk / chembl / ), and the SD format files were downloaded and imported into MOE2019, and then the "Dock" command was used to simulate docking at the Dummies virtual site.

[0058] According to the comparison of docking results, the small molecule compounds were ranked, and the small molecule compounds with the best docking results were selected for synthesis and used in subsequent virus infection experiments.

[0059] The docking results are as follows Figure 1 , 2 , 3, Figure 1 , 2 , 3 represent the structural formula respectively

[0060] The docking data are shown in Table 1:

[0061] Table 1 Docking data

[0062]

[0063] It can be seen from the figure that Dasabuvir can bind to the corresponding amino acid in the palmitoylation active site of F13L. Therefore, we have reason to predict that Dasabuvir can inhibit orthopoxviruses represented by monkeypox virus, vaccinia virus and smallpox virus.

[0064] Example 3 Effect of Dasabuvir on Poxvirus-Induced Migrasome Formation

[0065] The slides were spread in six-well plates. After 24 hours of culture, Huh7.5.1 / HeLa cells reached a density of 50%, and then infected with VACVA4-YFP at an MOI of about 5. Two hours later, the medium was changed and Dasabuvir was added. After 30 hours, the cells were collected and fixed with 4% PFA and stained with WGA (wheat germ agglutinin staining). The working solution concentration was 80nM and observed through an Olympus 100x oil immersion lens.

[0066] The experimental results are as follows Figure 4 The results show that the migrasomes induced by VACV infection are reduced under the action of Dasabuvir. Figure 5 The figure shows the integration of migrasome quantitative statistical results through the software Graphpad Prism. Figure 5 It can be seen that the number of migrasomes decreased significantly under the action of Dasabuvir.

[0067] like Figure 6 Shown is the effect of Dasabuvir on migrasome production by poxvirus transmission pathways in HeLa cells, combined with Figure 7 The number of migrasomes was counted after HeLa was affected by Dasabuvir. The results showed that the migrasomes induced after HeLa was infected with VACV would be reduced under the action of Dasabuvir.

[0068] Example 4 Dasabuvir inhibits viral spread and cytotoxicity

[0069] After 48 hours of culture, when the BSC-1 / HeLa / Huh7.5.1 cells were basically grown into a monolayer, trypsin was added for digestion and transferred to a 96-well sterile cell culture plate, 100 μL per well, and cultured in a cell culture incubator for 18 to 24 hours to allow the cells to grow into a monolayer for use in the toxicity experiment of Dasabuvir on BSC-1 cells.

[0070] The Dasabuvir stock solution was diluted with serum-free DMEM culture medium to prepare five concentration gradients (1, 5, 10, 15, 20 μmol / L). Then, different concentrations of drugs were added to the cell culture wells from which the supernatant was discarded, 100 μL per well, and each concentration was repeated 3 wells. At the same time, cell control wells were set up. No drug was added to the control wells, only culture medium was added. Then, 100 μL of cell culture medium was added to each well, and the wells were placed in a cell culture incubator at 37°C. After 48 hours of culture, 5 μL of CCK-8 (proteintech detection reagent) was added to each well. The incubation was continued for 2 hours and then the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader, and the results were recorded.

[0071] The cell survival rate was calculated as follows: cell survival rate (%) = OD value of the experimental well / OD value of the control well × 100%. The maximum non-toxic concentration range of the inhibitor to the cells was found out through the cell survival rate.

[0072] The experimental results are as follows Figure 8 As shown, compared with the control, Dasabuvir below 7 μmol / L had almost no cytotoxicity to BSC-1 / HeLa / Huh7.5.1 cells, and the CC50 value was 28.26 μmol / L. Combined with the observation under the microscope, it can be seen that BSC-1 cells have no lesions, which shows that Dasabuvir has good safety.

[0073] BSC-1 cells were cultured and plated in six-well plates. The culture medium was replaced with DMEM containing 2% fetal bovine serum. VACVWR was diluted and infected for 2 hours and then replaced with complete culture medium (10% fetal bovine serum + 1% anti-streptomycin penicillin). Different concentration gradients of Dasabuvir (1, 5, 10, 15, 20 μmol / L) were added. After 50 hours, the culture medium was discarded and the cells were stained with 1% crystal violet. The plaque size was counted using imageJ.

[0074] The virus plaque assay is a biological method used to detect and measure the ability of viruses to reproduce and spread. The virus's spread is evaluated by observing the size and number of plaques. The experimental results are as follows: Figure 8As shown, the results showed that Dasabuvir had a significant inhibitory effect on the size of viral plaques. When the half-inhibitory concentration was calculated to be 7.623 μmol / L, it had a strong inhibitory ability on the spread of vaccinia virus. When the CC50 value was 28.26 uM, Dasabuvir had almost no cytotoxicity to cells, and observation under a microscope showed that no lesions occurred in the cells, indicating that Dasabuvir had good safety.

[0075] Example 5 Experiment on the toxicity of Dasabuvir to Huh7.5.1 cells and its inhibitory effect on poxvirus

[0076] When Huh7.5.1 cells were cultured to a density of 80%, they were infected with VACVWR at an MOI of about 1. After 2 hours, the medium was changed and Dasabuvir was added. The cells were cultured in a CO2 incubator for 24 hours, and the EEV in the supernatant was collected for a virus titer experiment. The supernatant was diluted in gradients and infected with BSC-1 cells. After 2 hours, the culture medium was changed to complete medium, and crystal violet staining was performed after 50 hours. 0.5 mL of 0.1% crystal violet solution was added to each well, incubated at room temperature for 5 minutes, and the virus PFU was calculated based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / inoculated virus amount.

[0077] After infecting with VACV WR at an MOI of about 3 for 2 hours, change the medium and add Dasabuvir, incubate in a CO2 incubator for 24 hours, collect IMVs in the cells, and perform a virus titer experiment. After gradient dilution of the cells, infect BSC-1 cells, change to complete medium for 2 hours, and perform crystal violet staining after 50 hours. Add 0.5mL of 0.1% crystal violet solution to each well, incubate at room temperature for 5 minutes, and finally calculate the virus PFU based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / inoculated virus amount.

[0078] The results are as follows Fig. 9 As shown in Figure 2, after adding Dasabuvir, when the IC50 value was 8.051 μmol / L, Dasabuvir could selectively inhibit the production of viral EEV in Huh7.5.1 cells, but did not affect the production of IMV. Fig.11 This suggests that Dasabuvir disrupts a critical step in the viral life cycle, thereby leading to a reduction in EEV formation.

[0079] like Fig.10 As shown, the CC50 of Dasabuvir can be calculated to be 38.95 μmol / L through cytotoxicity experiments.

[0080] Example 6 Experiment on the toxicity of Dasabuvir to HeLa cells and its inhibitory effect on poxvirus

[0081] When HeLa cells were cultured to a density of 80%, they were infected with VACV WR at an MOI of about 1. After 2 hours, the medium was changed and Dasabuvir was added. The cells were cultured in a CO2 incubator for 24 hours, and the EEV in the supernatant was collected for a virus titer experiment. The supernatant was diluted in gradients and infected with BSC-1 cells. After 2 hours, the culture medium was changed to complete medium, and crystal violet staining was performed after 50 hours. 0.5 mL of 0.1% crystal violet solution was added to each well, incubated at room temperature for 5 minutes, and the virus PFU was calculated based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / amount of virus inoculated.

[0082] After infecting with VACVWR with an MOI of about 3 for 2 hours, change the medium and add Dasabuvir, incubate in a CO2 incubator for 24 hours, collect IMVs in the cells, and perform a virus titer experiment. After gradient dilution of the cells, infect BSC-1 cells, change to complete medium for 2 hours, and perform crystal violet staining after 50 hours. Add 0.5mL of 0.1% crystal violet solution to each well, incubate at room temperature for 5 minutes, and finally calculate the virus PFU based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / inoculated virus amount.

[0083] The results are as follows Fig.12 As shown in Figure 2, in HeLa cells, when IC50 = 12.51 μmol / L, Dasabuvir also observed a parallel selective inhibition of EEV, such as Fig.14 As shown in the results, Dasabuvir did not affect the generation of IMV, that is, it had little effect on viral replication, which indicates that Dasabuvir seems to disrupt a key step in the viral life cycle, resulting in reduced EEV formation. Fig.13 As shown, the CC50 of Dasabuvir was 31.34 μmol / L through cytotoxicity experiment.

[0084] Example 7 Inhibitory Effect of Dasabuvir on the Production of Actin Tail by Poxvirus

[0085] Considering that EEV production is closely linked to actin tail dynamics necessary for viral egress, the effect of Dasabuvir on actin tail length was further evaluated.

[0086] Huh7.5.1 and HeLa cells were infected with VACV A4-YFP (a VACV expressing YFP tagged to the A4 protein) at an MOI of about 5. After 2 hours, the medium was changed and 15 μmol / L Dasabuvir was added. After 24 hours, the cells were collected and fixed with 4% PFA, blocked with 5% goat serum for 10 minutes, and stained with phalloidin (working solution concentration was 80 nM). The cells were observed using an Olympus 100X oil immersion lens. The length of the actin tail was counted and quantified using ImageJ.

[0087] The experimental results are as follows Fig.15 , 16 The effect of Dasabuvir on the production of actin tail of poxvirus in Huh7.5.1 cells is shown, and the data were quantified and statistically analyzed using ImageJ and Graphpad Prism, as shown in Fig.17 , 18 As shown, the same method was used to operate on HeLa cells, and it was concluded that 15μmol / L Dasabuvir treatment can lead to a significant decrease in the length of the actin tail of Huh7.5.1 / HeLa, further proving that Dasabuvir disrupts the formation of EEV and subsequently releases VACV from infected cells.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Dasabuvir as a F13L membrane protein specific binding regulator.

2. The use according to claim 1, characterized in that: The application is achieved by specific binding of Dasabuvir to the palmitoylation active site of F13L membrane protein.

3. Application of Dasabuvir in the preparation of orthopoxvirus inhibitors.

4. The use according to claim 3, characterized in that: The IC50 of the Dasabuvir used in the invention for inhibiting the proliferation of orthopoxvirus is 7.623 μmol / L.

5. The use according to claim 3, characterized in that: The IC50 of the Dasabuvir used in the invention is 8.051 μmol / L or 12.51 μmol / L when inhibiting the formation of orthopoxvirus in cells.

6. The use according to claim 3, characterized in that: The orthopoxvirus includes, but is not limited to, smallpox virus, monkeypox virus, vaccinia virus, cowpox virus, mousepox virus, and camelpox virus.

7. The use according to claim 6, characterized in that: The orthopoxvirus is at least one of smallpox virus, monkeypox virus, and vaccinia virus.

8. Use of an orthopoxvirus inhibitor in orthopoxvirus treatment according to claim 7, characterized in that: The orthopoxvirus is monkeypox virus.

9. The use according to any one of claims 3 to 8, characterized in that: The inhibitor forms include, but are not limited to, capsules, tablets, pastes, injections, sprays, and aerosols.

10. The use according to claim 9, characterized in that: The inhibitor is in the form of a liquid preparation.

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