Application of disulfiram in the preparation of orthopoxvirus inhibitors

Abstract

This invention relates to the field of biomedicine, and in particular to the application of disulfiram in the preparation of orthopoxvirus inhibitors. Disulfiram exhibits a significant inhibitory effect on orthopoxvirus by specifically binding to palmitic acid on the MPXV gp045 membrane protein. When the IC50 of disulfiram is 825 nM, it can significantly inhibit the spread of orthopoxvirus. In HeLa and Huh7.5.1 cells, its IC50 is 3022 nM and 7125 nM, respectively, which can significantly inhibit the formation of EEV, thereby preventing the further spread of orthopoxvirus and providing a more comprehensive intervention for anti-orthopoxvirus therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of disulfiram in the preparation of orthopoxvirus inhibitors. Background Technology

[0002] Poxviridae viruses are the largest and most complex viruses in nature. They are a large group 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 more lethal poxviruses still pose a threat to human health. The genus Orthopoxvirus includes a variety of viruses, including but not limited to smallpox virus, monkeypox virus, vaccinia virus, cowpox virus, mousepox virus, and camelpox virus.

[0003] Poxviruses are double-stranded DNA viruses that replicate in the cytoplasm of host cells. Their replication cycle includes multiple stages, such as immature virions and mature virions within the cell. Poxviruses spread in various ways, including through expulsion via cell surface microvilli and by acquiring an envelope through budding and forming large vacuoles.

[0004] Monkeypox virus has garnered widespread attention due to its global spread, with its evolutionary branches and transmission characteristics becoming a hot research topic. The applicant has developed a small-molecule inhibitor against orthopoxvirus through experimental simulation, providing a specific treatment for orthopoxviruses, including monkeypox, thereby reducing their potential threat 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

[0005] The purpose of this invention is to provide the application of disulfiram in the preparation of orthopoxvirus inhibitors.

[0006] Another object of the present invention is to provide an application of a disulfiram-containing vaccinia virus inhibitor.

[0007] The technical solution adopted to achieve the purpose of this invention is:

[0008] Studies of monkeypox virus (MPXV) have revealed that gp045 is a membrane protein encoded by a specific gene. The amino acid sequence of the gp045 membrane protein is shown in SEQ ID NO.1:

[0009] MWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIASFCCNPLSTT

[0010] RGALIFDKLKEVSEKGIKIIVLLDERGKRNLGELQSHSPDINFITVNIDKKNNVGLLLGC

[0011] FWVSDDERCYVGNASFTGGSIHTIKTLGVYSDYPPLATDLRRRFDTFKAFNSAKNSWLNL

[0012] CSAACCLPVSTAYHIKNPIGGVFFTDSPEHLLGYSRDLDTDVVIDKLKSAKTSIDIEHLA

[0013] IVPTTRVDGNSYYWPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALCVQN

[0014] DLSVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQLVSKAKKIFER

[0015] DWVSSHSKSLKI.

[0016] This application provides the use of disulfiram as a specific binding regulator of gp045 membrane protein, and the specific experimental steps are as follows:

[0017] (1) Obtaining the structure of gp045 membrane protein: De novo simulation of gp045 protein of monkeypox virus MPXV_USA_2022_MA001 virus strain was performed using AlphaFold 2; at the same time, the simulated structure of gp045 of vaccinia virus was also simulated using AlphaFold 2.

[0018] (2) Virtual docking: The 3D structure of the gp045 membrane protein of monkeypox virus was imported using the software MOE2019. First, it was preprocessed using "QuickPrep", and then the "SiteFinder" command was used to search for amino acid sites that small molecules may dock with. Pocket sites including palmitoylated active sites were found. Then, the "Dummies" command was used to construct virtual docking sites for small molecules.

[0019] Among the screened small molecules, we discovered molecule C. 10 H 20 N2S4, also known as disulfiram, is what we currently see on the market. 10 H 20 The structural formula of N2S4 is as follows:

[0020]

[0021] Currently, disulfiram is commonly used clinically to treat alcohol dependence. It works by inhibiting the activity of aldehyde dehydrogenase, thereby preventing the further metabolism of acetaldehyde, leading to the accumulation of acetaldehyde in the body and causing unpleasant physiological reactions, thus helping patients avoid drinking alcohol.

[0022] Select disulfiram, download the SD format file and import it into MOE2019, and then use the "Dock" command to perform simulated docking at the Dummies virtual site.

[0023] Through the aforementioned screening, it was found that disulfiram can bind to palmitic acid at C185 and C186 of palmitoylated gp045 membrane protein. Disulfiram is an FDA-approved drug with proven safety. This discovery not only expands the application scope of disulfiram but also provides a new approach for the treatment of monkeypox virus.

[0024] (3) Verification of infection experiment: Using vaccinia virus as the experimental subject, different concentration gradients of disulfiram were set up to conduct virus plaque experiments to obtain IC50. 50 The IC value is obtained through viral titer experiments. 50 value.

[0025] Furthermore, this application provides the use of disulfiram in the preparation of orthopoxvirus inhibitors.

[0026] Currently, besides monkeypox, smallpox virus and vaccinia virus have been confirmed to contain the gp045 protein, a key envelope protein of orthopoxviruses. Therefore, in-depth research on the gp045 protein can not only enhance our understanding of monkeypox virus, but also has the potential to be applied to the research of other viruses in the orthopoxvirus genus.

[0027] Furthermore, based on the final results of the infection experiment, disulfiram's IC50 value was [value missing] when inhibiting the spread of orthopoxvirus. 50 The IC value is 825 nM, when disulfiram's IC 50 At concentrations of 3022 nM and 7125 nM, the ability of orthopoxvirus EEV to be generated was inhibited in HeLa and Huh7.5.1 cells, thereby suppressing viral infectivity.

[0028] Therefore, experiments demonstrating viral inhibition show that disulfiram can specifically bind to the active site of the orthopoxvirus protein gp045, thereby inhibiting the spread of orthopoxvirus and the formation of enveloped viruses (EEVs). It can effectively inhibit orthopoxviruses, represented by monkeypoxvirus. Therefore, small molecule compounds targeting gp045 are the first choice for developing effective drugs against poxviruses and have good prospects and potential for drug development.

[0029] Therefore, this invention also claims protection for the use of the small molecule disulfiram as a specific binding inhibitor of the gp045 protein, not limited to the aforementioned inhibitors of viruses in the Orthopoxvirus genus, but further extending to the scope of protection for the Poxviridae family, which contains the highly conserved gp045 protein, and even other viral species.

[0030] Furthermore, the inhibitor forms mentioned in this application include, but are not limited to: capsules, tablets, pastes, injections, sprays, and aerosols.

[0031] Furthermore, the inhibitor is in the form of a liquid formulation.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. Disulfiram is an FDA-approved drug with a well-established safety profile in clinical practice. Virtual docking studies have shown that disulfiram specifically binds to the gp045 membrane protein and exhibits significant inhibitory activity against orthopoxvirus. Laboratory studies have further confirmed its potential as an orthopoxvirus inhibitor. The application of disulfiram as a specific binding modulator of the MPXV gp045 protein provides a new, safe, and effective strategy for anti-orthopoxvirus treatment, opening new avenues for future antiviral therapy.

[0034] 2. Disulfiram has multiple applications in the preparation of orthopoxvirus inhibitors. Disulfiram can inhibit orthopoxvirus-induced migrasome production, especially when the IC50 of disulfiram is 825 nM, it can significantly inhibit the spread of orthopoxvirus. Disulfiram not only inhibits the spread of orthopoxvirus, but also has a good inhibitory effect on the formation of viral EEVs. In HeLa and Huh7.5.1 cells, its IC50 is 3022 nM and 7125 nM, respectively, which can significantly inhibit the formation of EEVs, thereby preventing the further spread of orthopoxvirus and providing a more comprehensive intervention method for anti-orthopoxvirus therapy.

[0035] 3. This application's research and application of the actin tail of poxviruses reveals its crucial role in poxvirus infection and transmission, making it a potential target for antiviral drug development. By designing and screening drugs that specifically inhibit actin tail function, the viral transmission process can be effectively interfered with. Furthermore, during drug development, evaluating the inhibitory effects of candidate drugs on actin tail function allows for the screening of compounds with antiviral activity, providing experimental evidence for further drug optimization and clinical application, and promoting the development of poxvirus treatment-related drugs. Attached Figure Description

[0036] Figure 1 AlphaFold 2 mimics the membrane protein structure of monkeypox virus gp045;

[0037] Figure 2 AlphaFold 2 mimics the membrane protein structure of vaccinia virus gp045;

[0038] Figure 3 Virtual docking results of the small molecule inhibitor disulfiram with the gp045 membrane protein of monkeypox virus;

[0039] Figure 4 The small molecule inhibitor disulfiram binds to the gp045 membrane protein of monkeypox virus.

[0040] Figure 5 The toxic effects of disulfiram on BSC-1 cells and the spread of poxviruses;

[0041] Figure 6 Inhibitory effect of disulfiram on vaccinia virus EEV in HeLa cells;

[0042] Figure 7 The toxic effects of disulfiram on HeLa cells;

[0043] Figure 8 The effect of disulfiram on vaccinia virus IMV in HeLa cells;

[0044] Figure 9 Inhibitory effect of disulfiram on poxvirus EEV in Huh7.5.1 cells;

[0045] Figure 10 The toxic effects of disulfiram on Huh7.5.1 cells;

[0046] Figure 11 The effect of disulfiram on vaccinia virus IMV in Huh7.5.1 cells;

[0047] Figure 12 The effect of disulfiram on vaccinia virus actin tail production in Huh7.5.1 cells;

[0048] Figure 13 Quantitative analysis of the effect of disulfiram on actin tail production by vaccinia virus in Huh7.5.1 cells;

[0049] Figure 14 The effect of disulfiram on actin tail production by vaccinia virus in HeLa cells;

[0050] Figure 15 Quantitative analysis of the effect of disulfiram on the production of actin tail by vaccinia virus in HeLa cells. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0053] All data were processed using GraphPad Prism 10.1.2 statistical software. Experimental data are expressed as mean ± standard error. One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0054] Example 1

[0055] HeLa, Huh7.5.1 cells (laboratory stock) and BSC-1 (African green monkey kidney) cells (purchased from the American 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.

[0056] Using Vaccinia virus WR strain as the experimental strain, the virus was amplified in HeLa cells. After discarding the supernatant, the cells were repeatedly sonicated at low temperature three times (15 seconds each time) before being aliquoted and stored at -80℃. HeLa and BSC-1 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin in a 37℃, 5% CO2 incubator, and passaged every 3-4 days.

[0057] Example 2

[0058] Obtaining the gp045 protein structure: The simulated structure of the gp045 protein of the monkeypox virus strain MPXV_USA_2022_MA001 was obtained through de novo simulation using AlphaFold2, with palmitoylation modifications added at C13, C185, and C186, and the addition of disulfiram. The simulated gp045 membrane protein structure using AlphaFold2 is shown below. Figure 1 As shown.

[0059] Simultaneously, the simulated structure of gp045 of vaccinia virus was also simulated using AlphaFold 2, as shown in the figure below. Figure 2 As shown.

[0060] Virtual docking: The 3D structure of the monkeypox virus gp045 protein was imported into MOE2019 software. First, preprocessing was performed using "QuickPrep," then the "SiteFinder" command was used to search for potential amino acid sites for small molecule docking. Pocket sites, including palmitoylated active amino acid sites, were identified. Then, the "Dummies" command was used to construct virtual docking sites for the small molecule. Disulfiram was selected, downloaded as an SD file, and imported into MOE2019. Then, the "Dock" command was used to simulate docking at the Dummies virtual sites. The docking results are shown below. Figure 3 As shown, the docking sites are as follows Figure 4 As shown.

[0061] Combination Figure 3 It can be seen that disulfiram can bind to palmitic acid at C185 and C186 of the palmitoylation active site of gp045. Therefore, we have reason to predict that disulfiram can inhibit monkeypox virus, vaccinia virus, and orthopoxviruses represented by smallpox virus.

[0062] Example 3: Disulfiram inhibits virus spread.

[0063] BSC-1 cells were cultured and spread evenly in six-well plates. The culture medium was replaced with DMEM containing 2% fetal bovine serum. After 2 hours of inoculation with diluted VACVWR, the medium was replaced with complete medium (10% fetal bovine serum + 1% anti-streptomycin penicillin). Different concentration gradients of disulfiram (100, 500, 1000, 1500, 2000, 4000 nM) were added. After 50 hours, the culture medium was discarded and stained with 1% crystal violet. The size of the empty spots was counted using ImageJ.

[0064] The viral plaque assay is a biological method used to detect and measure the ability of viruses to multiply and spread. It assesses viral diffusion by observing the size and number of plaques. The experimental results are as follows: Figure 5 As shown, the results indicate that disulfiram significantly inhibits the size of viral plaques. The calculated half-maximal inhibitory concentration (WMC) of 825.1 nM strongly inhibits the spread of vaccinia virus.

[0065] Example 4: Cell toxicity of disulfiram

[0066] When BSC-11 cells were basically confluent after 48 hours of culture, trypsin was added for digestion, and the cells were transferred to 96-well sterile cell culture plates at 100 μL per well. The plates were then incubated in a cell culture incubator for 18–24 hours to allow the cells to grow into a monolayer for use in disulfiram toxicity experiments on BSC-1 cells.

[0067] Validated using the CCK8 kit, disulfiram showed almost no cytotoxicity to BSC-1 cells, and microscopic observation revealed no cytopathic effects, indicating that disulfiram has good safety.

[0068] Disulfiram stock solution was serially diluted with serum-free DMEM medium to prepare five concentration gradients. Then, 100 μL of the drug at different concentrations was added to cell culture wells after the supernatant was discarded, with each concentration repeated in triplicate. A cell control well was also set up, with no drug added but only culture medium. 100 μL of cell culture medium was added to each well, and the wells were placed in a cell culture incubator at 37°C for 48 hours. After incubation, 5 μL of CCK-8 (proteintech assay reagent) was added to each well. The incubation was continued for 2 hours, and then the absorbance of each well was measured at 450 nm using a microplate reader, and the results were recorded.

[0069] The cell survival rate is calculated as follows: Cell survival rate (%) = OD value of experimental well / OD value of control well × 100%. The maximum non-toxic concentration range of the inhibitor on cells is determined by the cell survival rate.

[0070] Experimental results are as follows Figure 5As shown in the cell activity diagram, it does not affect BSC-1.

[0071] Example 5: Experiment on the toxic effects of disulfiram on HeLa cells and its inhibitory effect on poxviruses.

[0072] When HeLa cells reached a density of 80%, they were infected with VCV WR at an MOI of approximately 1. After 2 hours, the medium was changed and disulfiram was added. The cells were then incubated in a CO2 incubator for 24 hours. EEVs in the supernatant were collected for virus titer assays. The supernatant was serially diluted and used to infect BSC-1 cells. After 2 hours, the medium was replaced with 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, and the cells were incubated at room temperature for 5 minutes. Finally, the viral PFU was calculated based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / inoculated virus amount.

[0073] Infect cells with VCV WR at an MOI of approximately 3 for 2 hours, then change the medium and add disulfiram. Incubate in a CO2 incubator for 24 hours, collect IMV from the cells, and perform virus titer experiments. Infect BSC-1 cells with serially diluted cells, replace with complete medium after 2 hours, and perform crystal violet staining after 50 hours. Add 0.5 mL of 0.1% crystal violet solution to each well, incubate at room temperature for 5 minutes, and finally calculate the viral PFU based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / inoculated virus amount.

[0074] The results are as follows Figure 6 As shown, after the addition of disulfiram, when the IC50 value is 3022 nM, disulfiram can selectively inhibit the generation of viral EEV in HeLa cells, but does not affect the generation of IMV. Figure 8 As shown, this indicates that disulfiram disrupts a key step in the viral life cycle, thus leading to a reduction in EEV formation.

[0075] like Figure 7 As shown in the cytotoxicity assay, cell activity was observed, and the cells did not show any effect on HeLa cells.

[0076] Example 6: Experiment on the toxic effects of disulfiram on Huh7.5.1 cells and its inhibitory effect on poxvirus.

[0077] When Huh7.5.1 cells reached a density of 80%, they were infected with VACVWR at an MOI of approximately 1. After 2 hours, the medium was changed and disulfiram was added. The cells were then incubated in a CO2 incubator for 24 hours. EEVs in the supernatant were collected for virus titer assays. The supernatant was serially diluted and used to infect BSC-1 cells. After 2 hours, the medium was replaced with 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, and the cells were incubated at room temperature for 5 minutes. Finally, the viral PFU was calculated based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / inoculated virus amount.

[0078] Infect cells with VCV WR at an MOI of approximately 3 for 2 hours, then change the medium and add disulfiram. Incubate in a CO2 incubator for 24 hours, collect IMV from the cells, and perform virus titer experiments. Infect BSC-1 cells with serially diluted cells, replace with complete medium after 2 hours, and perform crystal violet staining after 50 hours. Add 0.5 mL of 0.1% crystal violet solution to each well, incubate at room temperature for 5 minutes, and finally calculate the viral PFU based on the number of plaques on the cell plate: PFU / mL = average number of plaques × virus dilution / inoculated virus amount.

[0079] The results are as follows Figure 9 As shown, after the addition of disulfiram, when the IC50 value was 7125 nM, disulfiram selectively inhibited the generation of viral EEV in Huh7.5.1 cells, but did not affect the generation of IMV. Figure 11 As shown, this indicates that disulfiram disrupts a key step in the viral life cycle, thus leading to a reduction in EEV formation.

[0080] like Figure 10 As shown, cytotoxicity experiments indicate that disulfiram has low cellular activity and good safety profile.

[0081] Example 7: Inhibitory effect of disulfiram on actin tail production of poxvirus

[0082] Considering that EEV generation is closely related to the actin tail dynamics necessary for viral export, the effect of disulfiram on actin tail length was further evaluated.

[0083] Huh7.5.1 and HeLa cells were infected with VACVA4-YFP (a VACV expressing YFP tagged to the A4 protein) with an MOI of approximately 5. The medium was changed after 2 hours, and 15 μmol / L disulfiram was added. After 24 hours, cells were collected, fixed with 4% PFA, blocked with 5% sheep serum for 10 minutes, stained with pseudocyclopeptide (working solution concentration 80 nM), and observed using an Olympus 100X oil immersion microscope. The length of the actin tail was statistically analyzed and quantified using ImageJ.

[0084] Experimental results are as follows Figure 12 , 13 The image shows the effect of disulfiram on the production of actin tails by poxviruses in HeLa cells, and the data were quantified and statistically analyzed using ImageJ and Graphpad Prism. Figure 14 , 15 As shown, the same method was used to operate on Huh7.5.1 cells, and it was found that treatment with 15 μmol / L disulfiram resulted in a significant reduction in the length of the actin tail in Huh7.5.1 / HeLa, further demonstrating that disulfiram disrupts the formation of EEVs, which then release VCVs from infected cells.

[0085] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of disulfiram for the preparation of an orthopoxvirus inhibitor, characterized in that: The application is achieved by specific binding of disulfiram to palmitic acid sites on gp045 membrane proteins.

2. Use according to claim 1, characterized in that: IC of disulfiram 50 is 825 nM capable of inhibiting orthopoxvirus spread.

3. Use according to claim 1, characterized in that: IC of disulfiram 50 capable of inhibiting orthopoxvirus formation at 3022 nM or 7125 nM.

4. Use according to any one of claims 1 to 3, characterized in that: The orthopoxvirus is at least one of a smallpox virus, a monkeypox virus, and a vaccinia virus.

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