Application of lycorine in preparation of medicine for preventing or treating poxvirus infection related diseases

Abstract

The invention relates to the medical field and the biotechnical field, in particular to application of lycorine in preparation of a medicine for preventing or treating poxvirus infection related diseases. Through screening of lycorine compounds and antiviral experiments, it is found for the first time that lycorine can limit in-vitro amplification of orthopoxvirus representative member vaccinia virus. In addition, lycorine can also significantly inhibit in-vitro amplification of various other pox viruses such as vaccinia virus, mucinoma virus and the like.

Description

Technical Field

[0001] The invention relates to the fields of medicine and biotechnology, and in particular to application of lycorine in preparing medicines for preventing or treating diseases related to poxvirus infection. Background Art

[0002] Monkeypox is a zoonotic disease. Monkeypox and its variants have been distributed sporadically in many countries and regions.

[0003] Monkeypox virus is a member of the poxvirus family and belongs to the genus Orthopoxvirus of the subfamily Chordovirinae, which also includes variola virus (VARV, the pathogen of smallpox), cowpox virus (VACV, the basis of smallpox vaccine), cowpox virus (CPXV, circulated in rodents in northern Europe) and mousepox virus (ECTV, an infectious pathogen of mice). Their antigenic characteristics are similar, and there is immune cross-protection between monkeypox virus and smallpox virus and cowpox virus. Poxvirus is a large double-stranded DNA virus, marked by the monkeypox epidemic caused by human monkeypox virus in 2022. In terms of participation in DNA replication, the virus is about 98% identical to VACV at the sequence level, mainly replicated and assembled in perinuclear virus factories, and viral DNA synthesis is independent of the host cell nucleus.

[0004] Human monkeypox virus is mainly transmitted through sexual contact between men with characteristic skin lesions, fever and myalgia in the oral and anogenital areas, followed by a centrifugal secondary rash. There are currently no specific anti-monkeypox virus drugs for monkeypox patients. The available antiviral drugs are mainly Tecovirimat and Brincidofovir, the former mainly blocking viral transmission by interfering with virion assembly, while the latter targets viral DNA replication as a chain terminator. To date, there are still few highly specific anti-pox virus therapeutics, and the existing drugs lack sufficient definition of their effectiveness in large-scale clinical use, and long-term use carries the risk of viral immune escape. MPXV and VACV have been shown to have similar gene expression profiles, and VACV is the most well-studied orthopoxvirus and was originally used to eradicate smallpox through vaccination. There is currently no vaccine specifically designed to prevent monkeypox virus infection. Due to the existence of cross-immunity, the smallpox vaccine (based on vaccinia virus) is recommended in the current monkeypox epidemic, but the vaccine supply is limited and is only provided to high-risk populations. Therefore, it is crucial to develop new substances that target host factors or virus-host interactions to resist orthopoxvirus infection.

[0005] Lycorine is a natural alkaloid extracted from the plant family Chrysanthemiaceae and has been shown to be an orally active SCAP inhibitor. ADRB2It is also a melanoma angiogenesis inhibitor and has research potential for prostate cancer and metabolic diseases, but whether lycorine is related to poxvirus inhibition remains to be studied. Summary of the invention

[0006] At present, the main prevention and treatment measures for monkeypox virus are still vaccination and the use of broad-spectrum antiviral drugs, and there is still a lack of effective targeted therapeutic preparations in the clinic. The present invention aims to screen antiviral drugs with significant effects by evaluating the effects of β2 adrenergic receptor agonist-related drugs against CPXV, and determine their potential antiviral effects and molecular mechanisms, in order to find a safe and effective therapeutic drug for anti-pox virus infection, and provide a theoretical basis and technical direction for preventing related complications caused by pox virus infection.

[0007] The purpose of the present invention is to improve the singleness and limitations of the current clinical poxvirus drug preparation treatment, and to provide a β2 adrenergic receptor agonist compound that has an inhibitory effect on the replication of multiple poxvirus family members such as cowpox virus (CPXV), vaccinia virus (VACV), and myxoma virus (MYXV). This type of drug is expected to become a potential drug for treating poxvirus-related infections by promoting the binding interaction between neurotransmitters and their receptors, providing a potential drug direction to be developed for preventing the occurrence and spread of poxvirus infections.

[0008] In a first aspect, the present invention provides a use of lycorine in the preparation of a medicament for preventing or treating diseases related to poxvirus infection.

[0009] As a preferred embodiment of the present invention, the poxvirus is an orthopoxvirus or a rabbitpoxvirus.

[0010] Further preferably, the poxvirus is vaccinia virus (VACV), cowpox virus (CPXV) or myxoma virus (MYXV) of the genus Leporopoxvirus.

[0011] As a preferred embodiment of the present invention, the drug is used to inhibit poxvirus replication and amplification and / or poxvirus toxicity.

[0012] In a second aspect, the present invention provides a drug for inhibiting poxvirus infection, wherein the drug contains lycorine as the only effective active ingredient.

[0013] As a preferred embodiment of the present invention, the drug is prepared by compounding the lycorine with a pharmaceutically acceptable excipient or carrier.

[0014] The drug is prepared into an oral preparation or an injection preparation according to a pharmaceutically acceptable method. The oral preparation includes capsules, tablets, granules or liquid preparations, and the injection preparation includes liquid for injection or powder for injection. During the specific preparation, those skilled in the art can choose to add appropriate excipients according to the selected dosage form, such as wetting agents, flavoring agents, antioxidants, suspending agents, thickeners, viscosity enhancers, pH regulators, osmotic pressure regulators, colorants, capsids, coating materials, etc.

[0015] The poxviruses described in the present invention include vaccinia virus (VACV), cowpox virus (CPXV), and myxoma virus (MYXV) of the genus Leporipoxvirus, but are not limited to the above viruses. In fact, lycorine has a certain therapeutic effect on various poxvirus infections and is suitable for inhibiting the replication, proliferation and / or toxicity of various poxviruses.

[0016] As a preferred embodiment of the present invention, in the application of the drug in the treatment of CPXV infection, the concentration of lycorine for cell treatment is 2.5 to 5 μM / mL. More preferably, it is 2.5 μM / mL. The cells are host cells susceptible to CPXV infection, preferably Vero cells. The effect is preferably an inhibitory effect on poxvirus in vitro. Lycorine can limit the in vitro proliferation ability of vaccinia virus (CPXV) to the greatest extent within the concentration range of 2.5 μM / mL.

[0017] Furthermore, the present invention adopts the scheme of adding drugs after virus infection, and detects CPXV virus 4h, 8h, and 12h after adding drugs. CPXV001, CPXV003, CPXV004, CPXV005, CPXV007, CPXV009, CPXV010, CPXV012 The RNA levels of related genes were measured to further evaluate the effect of lycorine in inhibiting viral replication in vitro, and it was clarified that lycorine prevented CPXV from entering cells.

[0018] Preferably, in the application of the drug for treating VACV infection, the concentration of lycorine for cell treatment is 2.5-5 μM / mL, more preferably 2.5 μM / mL, and the cells are VACV-infected host cells, preferably B-SC-1 cells. Lycorine can significantly inhibit the proliferation of VACV in vitro within a concentration range of 2.5 μM / mL.

[0019] In another preferred embodiment of the present invention, the inhibitory effect of lycorine on VACV was verified, and the results showed the antiviral effect of lycorine on VACV in the pre-dosing, synchronous dosing and post-infection dosing modes, indicating that lycorine has a consistent inhibitory effect on multiple poxviruses.

[0020] Furthermore, the present invention adopts a scheme of adding drugs after virus infection, and by measuring the expression levels of the virus's own proteins D8L, L1R, and E3L expressed by VACV virus replication without drug treatment and at 4h, 8h, 12h, and 24h of drug treatment, the inhibitory effect of lycorine on viral replication is further evaluated, and the process of lycorine preventing VACV-related poxviruses from replicating and assembling viral proteins is clarified.

[0021] In another preferred embodiment of the present invention, the antiviral effect of lycorine on MYXV was verified, indicating that lycorine has an antiviral effect on MYXV in a post-infection administration mode, indicating that lycorine has a wide range of inhibitory effects on a variety of poxviruses.

[0022] In a preferred embodiment of the present invention, lycorine as a β2 adrenergic receptor agonist has an inhibitory effect on poxviruses such as CPXV, VACV, MYXV, etc. The screening method of lycorine as a β2 adrenergic receptor agonist is to evaluate it through antiviral experiments.

[0023] Furthermore, the present invention utilizes the interaction between the molecular structure of the lycorine compound and the molecular conformation of ADRB2 to perform a molecular docking experiment.

[0024] The research results of the present invention have the following benefits: Through screening of lycorine compounds and antiviral experiments, the present invention discovered for the first time that lycorine can maximally limit the in vitro amplification of vaccinia virus (CPXV), a representative member of the orthopoxvirus genus, within a concentration range of 2.5 to 5 μM / mL. In addition, lycorine can also significantly inhibit the in vitro amplification of a variety of other poxviruses, such as vaccinia virus (VACV) and myxoma virus (MYXV). The use of lycorine drugs to inhibit this process is expected to become a therapeutic strategy for combating poxvirus infection-related diseases, providing a potential drug research direction for subsequent anti-poxvirus-related drug development, and a promising path for the treatment and intervention of future monkeypox epidemics and other potential poxvirus-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the crystal violet staining result of Vero cells infected with CPXV treated with different concentrations of lycorine; Figure 2 is the effect of lycorine at different concentrations on B-SC-1 cells infected with VACV; Figure 3 It is the inhibitory effect of adding drugs at different time points on poxvirus; Figure 4 is the effect of lycorine on the expression level of viral mRNA in CPXV-infected Vero cells; Figure 5is the effect of lycorine on the expression level of viral genes after VACV infection of B-SC-1 cells; Figure 6 is the effect of lycorine on the expression level of viral proteins in B-SC-1 cells infected with VACV at different treatment times; Figure 7 It is lycorine that inhibits viral infection in in vivo experiments; Figure 8 The inhibition of Lycorine on the myxoma virus of the genus Leporinovirus; the first row is the GFP fluorescence image, and the second row is all the cells under bright field; Fig. 9 is the frequency of interaction between lycorine and ADRB2 protein structure; Fig.10 It is the molecular docking result of the interaction between lycorine and ADRB2 protein structure; Fig.11 A is the binding conformation of lycorine and ADRB2 protein structure, and B is the enlarged view of the red frame in A. DETAILED DESCRIPTION

[0026] The present invention is described more clearly and completely through the following examples, but the examples described are only part of the embodiments of the present invention, not all of them. The embodiments are to help understand the present invention and should not be used to limit the scope of protection of the present invention.

[0027] Sources of materials used in the examples: Viruses and cells: CPXV-GFP (Brighton Red strain) (NCBI: 10243) strain, which is a strain that has been disclosed in the prior art and the public can learn about it through literature resource sharing; VACV-GFP (Western Reserve strain) (NCBI: 10254) and MYXV-GFP (Lausanne strain) (NCBI: 31530) strains, which are publicly available strains in the prior art and can be learned by the public through literature resource sharing; CPXV (Brighton Red strain) was propagated in CV-1 cells. A derivative of CPXV (vCpx-gfp) was generated by inserting an enhanced green fluorescent protein (EGFP) cassette between the open reading frames CPXV-107 and CPXV-108 of the CPXV genome. The transfer plasmid was transfected into CPXV-infected CV-1 cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. The resulting EGFP-tagged CPXV virus (CPXV-gfp) produced fluorescent green development. The EGFP cassette in the viral gene of VACV-GFP (Western Reserve strain) (NCBI: 10254) was driven by a previously described synthetic VACV early / late promoter (vv sE / L). The MYXV-GFP (Lausanne strain) (NCBI: 31530) strain was also labeled with EGFP using the above method to obtain green fluorescent virions for subsequent experimental observation. Vero cells were purchased from ATCC (NA: Cat# CRL-1586); BSC-1 was purchased from ATCC (Cat# CCL-26).

[0028] Reagents and antibodies: Antibodies VACWR117 Antibody, L1R Antibody, A27L Antibody, E3L Antibody, and D8LAntibody were purchased from Huamei Biotechnology for preparation in this experiment, HRP-labeled goat anti-mouse IgG was purchased from Cell Signaling, fetal bovine serum was purchased from Gibco, and BCA protein quantification kit was purchased from Thermo Fisher Scientific. Other reagents not mentioned in the present invention are all commercially available products.

[0029] Example 1 Lycorine inhibits poxvirus infection in a concentration-dependent manner This experiment further evaluated the effects of different concentrations of lycorine on Vero cells infected with CPXV. The specific steps are as follows:

[0030] Vero cells were inoculated with CPXV-GFP at MOI = 1, and 10, 5, 2.5, and 1.25 uM / mL of lycorine were added 2 hours after infection. After 48 hours, the cells were fixed with 4% PFA solution for 30 minutes and stained with crystal violet. The cells were rinsed with slow water flow, and the number of virus holes was counted by naked eyes. The results were plotted using Graphpad Prism (version 8.0.2, Graphpad Software Diego, CA, USA).

[0031] The results show that ( Figure 1 ), the inhibitory effect of lycorine on CPXV is concentration-dependent.

[0032] The same method was used to further evaluate the effects of lycorine at different concentrations on B-SC-1 cells infected with VACV. Figure 2 As shown, the inhibitory effect of lycorine on VACV is concentration-dependent.

[0033] Example 2 Evaluation of the inhibitory effect of adding drugs at different time points on poxvirus This experiment further evaluated the effect of adding lycorine at different times on Vero infection with CPXV, using three methods and nine time points: (0) Method of infecting the virus in advance by adding drugs: Treat Vero cells with lycorine 1h / 2h / 4h in advance, then inoculate CPXV and incubate for 2h. Discard the original virus solution, wash with PBS, add cell maintenance solution, and after 48h, fix the Vero cells with 4% PFA for 30min, stain with crystal violet, and observe the number of virus holes with the naked eye to evaluate the antiviral effect of the prevention mode.

[0034] (1) Simultaneous drug addition during virus infection: Add a mixture of CPXV and lycorine (at a concentration of 2.5 μM) to Vero cells at the same time, and measure the virus inhibition effect as described above after 48 hours.

[0035] (2) Method of drug addition after virus infection: First, CPXV was inoculated into Vero cells and treated for 1h / 2h / 4h / 8h / 12h, respectively. The virus infection solution was discarded, the cells were washed with PBS, and lycorine (2.5μM) was added. After 48h of treatment, the virus inhibition effect was determined to evaluate the therapeutic effect of lycorine in the early, middle and late stages of virus infection.

[0036] The results are as follows Figure 3 As shown. Figure 3 It can be seen that lycorine mainly plays an inhibitory role after viral infection.

[0037] Example 3 Effect of Lycorine Treatment on the Expression of Viral mRNA in CPXV-Infected Vero Cells The present invention further adopts the scheme of adding drugs after virus infection as in Example 2, and by measuring the CPXV virus encoding gene without and after drug addition CPXV006, CPXV008, CPXV011, CPXV013, CPXV016, CPXV017 The mRNA level of lycorine was further evaluated to inhibit CPXV at the mRNA level. Figure 4 As shown, CPXV006, CPXV008, CPXV011, CPXV013, CPXV016, CPXV017 The expression of is related to virus adsorption and invasion. Therefore, this experiment clarified that lycorine enters host cells by blocking CPXV adsorption.

[0038] In addition, the present invention also evaluates the effect of VACV virus infection on the expression level of VACV virus genes at different times after lycorine treatment in B-SC-1 cells. The specific technical scheme is the same as above, and the mRNA levels of E3L (gene expressed in the early stage of infection), D13L (gene expressed in the middle stage of infection), and A3 (gene expressed in the late stage of infection) of VACV virus infection without drug addition and 4h, 8h, 12h, and 24h after drug addition are measured. The results show that ( Figure 5 ), Lycorine inhibits the expression of genes throughout the VACV virus infection process, and plays a blocking role in the virus adsorption process.

[0039] Example 4 Evaluation of the effect of lycorine on viral protein expression levels at different treatment times after VACV infection of B-SC-1 cells The present invention further adopts the scheme of adding drugs after virus infection as in Example 2 above, and further evaluates the effect of lycorine on inhibiting viruses by measuring the expression of D8L, L1R, and E3L of VACV virus's own proteins at 2h, 4h, 8h, 12h, and 24h after drug addition, and clarifies that lycorine prevents the process of VACV-related poxvirus replication and assembly of viral proteins. Figure 6 shown.

[0040] Example 5 Validation of Lycorine for the Treatment of Virus-Infected Mice in In Vivo Experiments The above implementation cases have proved that Lycorine can inhibit the replication and proliferation of poxvirus in vitro. The present invention further conducted an in vivo animal experiment, and the experimental mouse strain was C57BL / 6 background mice. The operation was as follows:

[0041] C57BL / 6 male mice of the same age were selected and randomly divided into two groups, with 10 mice in each group. The operation was as follows: the control group: only CPXV virus 1*10 7 / treatment group: intraperitoneal injection of CPXV virus 1*10 7 / mouse + 10mg / kg lycorine. After 3.5 days of normal water and food feeding, the mice were killed, and the liver, fat, and spleen were collected using DMEM containing 2.5% fetal bovine serum as the storage medium. The tissues were crushed and ground using a freezing grinder, and the virus-infected tissues were diluted in multiples. The virus titers of different organs were determined by counting the number of plaques on Vero cells to determine the inhibitory effect of lycorine on viruses in vivo.

[0042] The results are as follows Figure 7 As shown, Lycorine also has the effect of inhibiting poxvirus infection in vivo.

[0043] Example 6 The effect of lycorine (2.5 μM / ml) on myxoma virus (MYXV) of the genus Lepoxvirus in the post-infection dosing mode was observed after 48 hours, and the GFP fluorescence carried by the virus was also observed, which also had an antiviral effect and significantly inhibited the virus proliferation in the host cells. Figure 8 shown.

[0044] Example 7 Verification of the molecular interaction between lycorine and β2-adrenergic receptor (ADRB2) The present invention further adopts a molecular docking experiment to analyze the molecular structure of the lycorine drug and the conformational change of the ADRB2 molecule.

[0045] like Figures 9-11 Lycorine binds to Phe193 of the ADRB2_MOUSE structure to form an Arene-H interaction, and also forms a hydrogen bond interaction with Asp133 of the ADRB2_MOUSE protein.

[0046] The above results indicate that lycorine targeting ADRB2 significantly inhibits the in vitro infection of poxvirus family members such as CPXV.

[0047] It should be understood that although the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it should be understood by those skilled in the art that various changes in form and details may be made therein and any combination of various embodiments may be made without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. Use of lycorine in the preparation of a drug for preventing or treating diseases related to poxvirus infection.

2. The use according to claim 1, characterized in that: The poxvirus is an orthopoxvirus or a rabbitpoxvirus.

3. The use according to claim 2, characterized in that: The poxvirus is a myxoma virus of the genus vaccinia virus, cowpox virus or rabbitpox virus.

4. The use according to claim 1, characterized in that: The drug is used for inhibiting the replication and expansion of poxvirus.

5. The use according to claim 1, characterized in that: The drug is used to inhibit poxvirus toxicity.

6. The use according to claim 1, characterized in that: The lycorine is used for preparing a beta 2 adrenergic receptor agonist.

7. A drug for inhibiting poxvirus infection, characterized in that: The drug contains the lycorine described in claim 1 as the only effective active ingredient.

8. The drug according to claim 7, characterized in that The content of lycorine in the medicine is 2.5-5 μM / mL.

9. The drug according to claim 7, characterized in that The medicine is prepared by compounding the lycorine with pharmaceutically acceptable auxiliary materials.

10. The drug according to claim 7, characterized in that The medicine is an oral preparation or an injection preparation.

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