An antiviral ADE drug screening method with TRIM54 ubiquitination inhibition STAT2 as a target

Abstract

The present application relates to a kind of antiviral ADE drug screening method with the target of inhibiting TRIM54 ubiquitination STAT2, and a series of antiviral ADE effect drugs are screened based on the screening system: Baricitinib, Rivaroxaban and the like, new application field is expanded for these drugs, and candidate drug is provided for the blank of current no ADE treatment scheme, and it has important biological significance to the virus prevention and treatment with ADE effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for screening antiviral ADE drugs that targets the inhibition of TRIM54 ubiquitination and degradation of STAT2. Background Technology

[0002] Antibody-dependent enhancement (ADE) refers to the phenomenon where antibodies with weak or no neutralizing capacity, produced after vaccination or viral infection, form complexes with viral particles. These complexes not only fail to block viral infection but actually promote viral infection and increase viral replication by disrupting the interferon signaling pathway. The ADE phenomenon was first described by Halstead et al. in dengue fever infection in 1973. Later, it was found that ADE is widespread in many flavivirus infections, such as Japanese encephalitis virus, Zika virus, and dengue virus. ADE is also a significant challenge in the prevention and control of SARS-CoV-2. Currently, there are no specific drugs available for treating ADE, making it a major obstacle in the prevention and control of viral infections. Therefore, exploring the mechanisms of ADE to discover targets for its inhibition is of great importance for the prevention and control of viral diseases.

[0003] Signal transducers and activators of transcription (STATs) are a unique family of DNA-binding proteins. The STAT family includes seven structurally and functionally related proteins: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. They typically respond to various extracellular cytokine and growth factor signals and are a class of SH2 signaling molecules that bind to phosphorylated tyrosine residues. STATs are nuclear transcription factors, but in resting cells, they are located in the cytoplasm. Among them, STAT2, once activated, translocates to the nucleus and binds to specific DNA, influencing gene transcription and participating in cell growth, differentiation, survival, and apoptosis. Current research indicates that STAT2 gene deletion or overexpression has a significant impact on tumorigenesis and development, and is closely related to tumor angiogenesis, tumor proliferation, and tumor apoptosis.

[0004] The tripartite motif (TRIM) family is named for its three conserved domains. These three domains, from N-terminus to C-terminus, consist of a ring domain, one or two B-box domains, and a coiled-coil domain. Additionally, the family possesses a variable C-terminus. Currently, nearly 70 TRIM protein members have been identified and recognized in the human genome, and the functions of these family members are receiving increasing attention. Studies have shown that TRIM family members participate in many important biological processes, including cell differentiation, proliferation, development, and apoptosis. Most TRIMs possess E3 ubiquitin ligase activity and are involved in various physiological processes, including cell proliferation, DNA repair, signal transduction, and transcription. Recent studies have also revealed a strong association between TRIMs and cancer development.

[0005] However, there are currently no reports on using the interaction between STAT2 and TRIM54 as a target for blocking ADE effects. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for screening antiviral ADE drugs targeting the inhibition of TRIM54 ubiquitination and STAT2 degradation. This method involves fusing TRIM54 and STAT2 with a first fluorescent protein (FP1) and a second fluorescent protein (FP2), respectively, to form FP1-TRIM54 and FP2-STAT2. After cell transfection, the interaction between TRIM54 and STAT2 is evaluated by detecting the intensity of fluorescence resonance transfer (FRET) using flow cytometry or an ELISA reader, thereby screening for compounds that specifically inhibit ADE.

[0007] The first objective of this invention is to provide a method for screening antiviral ADE drugs that target the inhibition of TRIM54 ubiquitination and degradation of STAT2, comprising the following steps:

[0008] S1. Construct a first plasmid and a second plasmid respectively, introduce the first plasmid and the second plasmid into cells, and detect FRET signals; the first plasmid and the second plasmid contain genes encoding TRIM54 and STAT2 respectively, the first plasmid and the second plasmid contain genes encoding a first fluorescent protein and a second fluorescent protein respectively, and the first fluorescent protein and the second fluorescent protein can undergo fluorescence resonance energy transfer.

[0009] S2. Co-incubate the test compound with the cells described in step S1, and screen for drugs that inhibit the ADE effect of the virus based on changes in FRET signal.

[0010] Furthermore, the nucleotide sequence of the gene encoding TRIM54 is shown in SEQ ID NO.1.

[0011] Furthermore, the nucleotide sequence of the gene encoding STAT2 is shown in SEQ ID NO.2.

[0012] Furthermore, test compounds with reduced FRET signals were screened as candidate antiviral ADE-effect drugs.

[0013] Furthermore, in one embodiment of the present invention, the first fluorescent protein is cyan fluorescent protein CFP, and the second fluorescent protein is yellow fluorescent protein YFP.

[0014] Furthermore, during drug screening, cells inoculated with a third plasmid were used as a positive control. The third plasmid contains genes encoding the first fluorescent protein and genes encoding the second fluorescent protein.

[0015] Furthermore, during drug screening, cells inoculated with the fourth and fifth plasmids were used as negative controls. The fourth and fifth plasmids contain genes encoding the first fluorescent protein and the second fluorescent protein, respectively.

[0016] Furthermore, FRET signals were detected by flow cytometry or fluorescence enzyme-linked immunosorbent assay (ELISA).

[0017] Furthermore, in one embodiment of the present invention, the cells are 293T cells.

[0018] This invention provides a novel target for inhibiting viral ADE effects, wherein the target is TRIM54 and STAT2:

[0019] Human monocytes and macrophages primarily express three classes of activated FcγRs: FcγRIa, FcγRIIa, and FcγRIIIa. While there is evidence that FcγRIIIa can participate in mediating dengue virus adverse drug reaction (ADE), more evidence supports that FcγRIIa, rather than FcγRIa and FcγRIIIa, is the key receptor for ADE. For example, studies conducted in Vietnam, Cuba, Pakistan, and Mexico have shown that FcγRIIa polymorphism is closely related to the prognosis of dengue virus infection. Recent evidence from multiple laboratories further indicates that FcγRIIa participates in mediating ADE of dengue, Zika, and Ebola viruses. The form in which antibodies are present may affect the ADE effect; for example, fucose modification of antibodies affects their affinity for FcRs, and dimer antibodies are more likely to induce ADE effects than monomeric antibodies. Furthermore, the ITAM motif in the cytoplasmic region of FcγRIIa is a key domain mediating viral ADE effects, while the ITIM motif in FcγRIIb plays a protective role.

[0020] The applicant's research revealed that immune complexes, acting on human monocytes via FcγRIIa on the cell membrane surface, enhance viral susceptibility. During this process, TRIM54 expression significantly increased, and it inhibited the IFN-I signaling pathway by degrading STAT2, leading to increased viral susceptibility in monocytes and macrophages. Interference with TRIM54 expression significantly reversed the ADE effect, suggesting that the interaction between TRIM54 and STAT2 may be a link in the immune complex-induced enhancement of monocyte viral sensitivity, indicating its potential as a target for blocking the ADE effect. Since TRIM54 is not constitutively expressed, but only in monocytes and macrophages acted upon by immune complexes, specific intervention in the TRIM54 / STAT2 interaction does not have a broad impact on the IFN-I-related signaling pathway, exhibiting high specificity. Therefore, screening for small molecule compounds that target the TRIM54 / STAT2 interaction and inhibit the ADE effect has significant biological implications for the prevention and control of viruses exhibiting ADE.

[0021] A second objective of this invention is to provide the application of a substance that inhibits the interaction between TRIM54 and STAT2 in the preparation of antiviral ADE-effect drugs.

[0022] A third objective of this invention is to provide an application of a substance characterizing the interaction between TRIM54 and STAT2 in the screening of antiviral ADE-effect drugs.

[0023] The fourth objective of this invention is to provide the use of Baricitinib or Rivaroxaban in the preparation of drugs that inhibit the ADE effect of viruses.

[0024] Furthermore, the Baricitinib or Rivaroxaban is used to block the interaction between TRIM54 and STAT2.

[0025] A fifth object of the present invention is to provide a drug that counteracts the ADE effect of viruses, wherein the drug comprises Baricitinib or Rivaroxaban.

[0026] By means of the above-described solution, the present invention has at least the following advantages:

[0027] This invention discloses a novel target for inhibiting viral adverse reaction-related impairment (ADE) and provides an application for treating ADE by targeting STAT2, the substrate of the E3 ubiquitin ligase TRIM54, which is used for ubiquitination degradation. This invention expresses TRIM54 and STAT2 by fusing them with a first fluorescent protein (FP1) and a second fluorescent protein (FP2), respectively, to form FP1-TRIM54 and FP2-STAT2. After transfection into cells, the interaction between TRIM54 and STAT2 is evaluated by detecting the intensity of fluorescence resonance transfer (FRET) using flow cytometry or an enzyme-linked immunosorbent assay (ELISA). This platform allows for large-scale screening to obtain small molecule compounds that specifically inhibit the TRIM54 / STAT2 interaction. Further molecular biology verification of the inhibitory effect of these small molecules on the TRIM54 / STAT2 interaction is conducted, and finally, the inhibitory effect of the compounds on ADE is verified. Ultimately, a series of small molecule compounds targeting the TRIM54 / STAT2 interaction and inhibiting ADE have been screened, which has significant biological implications for the prevention and control of viruses exhibiting ADE.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 The diagram shows the construction of eukaryotic plasmids. a) is a schematic diagram of the vector pCMV-N-CFP; b) is the amplification product of the TRIM54 gene (left), with the results of EcoRI and XhoI digestion of plasmid pCMV-N-CFP-TRIM54; c) is the amplification product of the YFP gene (left), with the results of EcoRI and XhoI digestion of plasmid pCMV-N-CFP-YFP; d) is a schematic diagram of the vector pCMV-C-YFP; e) is the amplification product of the STAT2 gene (left), with the results of BamHI and HindIII digestion of plasmid pCMV-C-YFP-STAT2.

[0031] Figure 2 The expression of CFP-TRIM54 and YFP-STAT2 in 293T cells is shown; where a) is the expression of YFP-STAT2 and CFP-TRIM54 as analyzed by Western blotting; b) is the fluorescence intensity of YFP-STAT2 and CFP-TRIM54 as analyzed by flow cytometry.

[0032] Figure 3For the flow cytometry analysis of the binding efficiency of TRIM54 and STAT2: the binding rate of CFP and YFP, the binding rate of CFP-YFP, and the binding rate of CFP-TRIM54 and YFP-STAT2;

[0033] Figure 4 For the detection and analysis of fluorescence resonance energy transfer intensity by microplate reader: the binding rate of CFP and YFP, the aggregation rate of CFP-YFP, and the binding rate of CFP-TRIM54 and YFP-STAT2;

[0034] Figure 5 These are the initial screening results for small molecule compounds; where a represents the results from the ELISA reader analysis; and b represents the results from the flow cytometry analysis.

[0035] Figure 6 These are the results of secondary screening of small molecule compounds; where a represents the results of enzyme-linked immunosorbent assay (ELISA) analysis, and b represents the results of flow cytometry analysis.

[0036] Figure 7 To demonstrate the inhibitory effect of the compound on the degradation of STAT2 by TRIM54, the plasmid encoding Flag-TRIM54 was transfected into 293T cells. Nine hours later, the compound was added to the experimental group, while the same dilution of DMSO (Med) was added to the control group. After 24 hours, the cells were collected, and Western blotting was performed to detect the protein level of STAT2 in the cell lysate.

[0037] Figure 8 To demonstrate the inhibitory effect of the compound on the interaction between TRIM54 and STAT2, the plasmid encoding Flag-TRIM54 was transfected into 293T cells. After 24 hours, cells were collected, and cell lysates were incubated with Anti-Flag Affinity Gel and the compound (10 μM) at 4°C for 12 hours. The control group was treated with the same dilution of DMSO (Med). The expression level of STAT2 in the immunoprecipitation complex was then detected by Western blotting.

[0038] Figure 9 The analysis focused on the effects of the compounds on ADE. In this context, 'a' represents K562 cells incubated with RPMI, 4G2, DENV2, or IC (DENV2+4G2) for 72 h, followed by flow cytometry analysis; 'b' represents K562 cells treated with DENV2 or IC incubated with the same dilution of DMSO (Med) for 72 h, followed by flow cytometry analysis; and 'c' represents K562 cells treated with IC incubated with the same amount of the compounds (1, 3, 10, 30 μM) for 72 h, followed by flow cytometry analysis.

[0039] Figure 10 This is a schematic diagram of the principle of fluorescence resonance energy transfer; where a represents the co-expression of fluorescent proteins CFP and YFP in 293T cells; b represents the expression of the fusion protein CFP-YFP in 293T cells; and c represents the co-expression of CFP-TRIM54 and YFP-STAT2 in 293T cells. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] The purpose of this invention is to provide a drug screening platform targeting the TRIM54 / STAT2 interaction based on fluorescence resonance energy transfer (FRET) technology. FRET refers to the process where, when two fluorescent chromophores are sufficiently close, the donor molecule absorbs photons of a certain frequency and is excited to a higher electronic energy state. Before this electron returns to its ground state, energy is transferred to a nearby acceptor molecule through dipole interactions. In short, when two fluorescent proteins are close together, the emission light emitted by one fluorescent protein under excitation light can serve as the excitation light for the other fluorescent protein, exciting it to emit light. A schematic diagram of the FRET principle is shown below. Figure 10 In the system of this invention, the selected fluorescent proteins are CFP (ex: 405nm; em: 440 / 50nm) and YFP (ex: 488nm; em: 530 / 30nm). When both fluorescent proteins are present but not close to each other, each fluorescent protein can only be excited by its own excitation light, emitting its own emission light. Figure 10 a). However, when the two fluorescent proteins are brought close enough by an external force, after excitation with the Violet excitation light (405 nm) of CFP, not only is the emission light VL1 (440 / 50 nm) of CFP excited, but the emission light VL2 (620 / 15 nm) of YFP is also excited. Therefore, this invention constructs a fusion protein of CFP and YFP (CFP-YFP). Figure 10 b) As a positive control for detecting FRET signal. Plasmids encoding CFP-TRIM54 and YFP-STAT2 were also constructed, and the interaction efficiency between TRIM54 and STAT2 was evaluated by detecting the fluorescence intensity of intracellular FRET channels. Figure 10 c).

[0042] During the experiment, the compounds to be screened were added to cells co-expressing CFP-TRIM54 and YFP-STAT2 to a final concentration of 10 μM. After treatment with the drugs for 24 h, the results were detected by microplate reader and flow cytometry. The inhibitory effect of the drugs on the interaction between TRIM54 and STAT2 was analyzed by fluorescence resonance energy transfer intensity.

[0043] The compound was added to 293T cells overexpressing TRIM54, and its effect on reversing TRIM54 degradation of STAT2 was analyzed by Western blotting (WB). The compound was added to cell lysates containing TRIM54 and STAT2, and its effect on blocking the TRIM54 / STAT2 interaction was analyzed by co-immunoprecipitation (Co-IP). An ADE model was constructed by infecting K562 cells with an immune complex formed from DENV2 and monoclonal antibody 4G2 (a specific monoclonal antibody against DENV1 E protein). The compound was then applied to this ADE model, and the viral load in K562 cells was detected by flow cytometry to analyze the inhibitory effect of the compound on ADE.

[0044] Example 1: Construction of plasmids encoding CFP-YFP, CFP-TRIM54, and YFP-STAT2

[0045] First, we used human monocyte mRNA and pCMV-C-YFP plasmid as templates to perform PCR amplification of the encoding TRIM54 (…). Figure 1 b left) and YFP ( Figure 1 The DNA fragment of c (left) was used. After digestion with EcoRI and XhoI, the PCR product was inserted into the expression vector pCMV-N-CFP ( Figure 1 a) The plasmids pCMV-N-CFP-TRIM54 (hereinafter referred to as CFP-TRIM54) and pCMV-N-CFP-YFP (hereinafter referred to as CFP-YFP) were obtained. They were identified by enzyme digestion. Figure 1 After sequencing (bc right), the sequence alignment was correct. Next, we used human monocyte mRNA as a template to perform PCR amplification of the DNA fragment encoding STAT2 ( Figure 1 The PCR product was digested with BamHI and HindIII and then inserted into the expression vector pCMV-C-YFP (e left). Figure 1 d) The plasmid pCMV-C-YFP-STAT2 (hereinafter simply referred to as YFP-STAT2) was obtained. It was identified by enzyme digestion. Figure 1 Sequencing was performed after (e right), and the sequence alignment was correct.

[0046] Example 2: Validation of the fusion expression of proteins CFP-TRIM54 and YFP-STAT2

[0047] The two plasmids encoding CFP-TRIM54 and YFP-STAT2 were separately transfected into 293T cells and cultured for 48 h before Western blot and flow cytometry analysis. The theoretical molecular weight of CFP-TRIM54 is 67 kDa, and the theoretical molecular weight of YFP-STAT2 is 121 kDa. Figure 2 As shown in figure a, the Western blot results are consistent with the theoretical values, indicating successful fusion protein expression. Further flow cytometry was used to detect the fluorescence intensity of the fluorescent proteins CFP and YFP of the two fusion-expressed proteins. Figure 2 As shown in b, fluorescence of CFP protein and YFP protein was detected in the VL1 channel and BL1 channel, respectively, and the fluorescence intensity met the requirements for subsequent experiments.

[0048] Example 3: Flow cytometry detection of intracellular TRIM54 and STAT2 interaction

[0049] To determine the intensity of the FRET signal using flow cytometry, we prepared FRET negative and positive controls. Plasmids pCMV-N-CFP (CFP) and pCMV-C-YFP (YFP) were simultaneously transfected into 293T cells as a negative control (CFP+YFP). In this control group, fluorescence was detected in both the CFP channel (ex: 405nm; em: 440 / 50nm) and the YFP channel (ex: 488nm; em: 530 / 30nm). Since CFP and YFP do not interact, the FRET channel (ex: 405nm; em: 512 / 25nm) should have no signal. Plasmid pCMV-N-CFP-YFP (CFP-YFP) was transfected into 293T cells as a positive control. In this control group, fluorescence was detected in both the CFP (ex: 405nm; em: 440 / 50nm) and YFP (ex: 488nm; em: 530 / 30nm) channels. Furthermore, due to the sufficiently close spatial distance between the two fluorescent proteins in CFP-YFP, theoretically, all cells expressing CFP-YFP could generate FRET signals. Flow cytometry analysis results are as follows: Figure 3As shown, after removing dead and adherent cells, CFP and YFP double-positive cells were circled, and the FRET signaling channel gating was adjusted. The negative control group (CFP+YFP) should be 0% (0.07% in the figure), and the positive control group (CFP-YFP) should be 100% (93.16% in the figure). Simultaneously, plasmids pCMV-N-CFP-TRIM54 (CFP-TRIM54) and pCMV-C-YFP-STAT2 (YFP-STAT2) were co-transfected into 293T cells (CFP-TRIM54+YFP-STAT2), and flow cytometry was used for analysis, employing the gating settings for negative (CFP+YFP) / positive control (CFP-YFP). Figure 3 As shown, the proportion of FRET channel-positive cells in the CFP-TRIM54+YFP-STAT2 group was 42.98%, which means that the binding rate of TRIM54 and STAT2 was approximately 42.98%. Using this system, we can quantify the degree of interaction between TRIM54 and STAT2, which will facilitate subsequent quantitative analysis of the efficiency of small molecule drugs in inhibiting the interaction between TRIM54 and STAT2.

[0050] Example 4: Detection of intracellular TRIM54-STAT2 interaction using a fluorescent microplate reader

[0051] Flow cytometry can evaluate the TRIM54 / STAT2 interaction at the single-cell level and can eliminate the influence of differences in transfection efficiency. However, as a drug screening method, its detection efficiency is relatively low. Therefore, we also explored a drug screening method based on the detection of FRET signals using a fluorescence microplate reader.

[0052] We transfected 293T cells with plasmids pCMV-N-CFP (CFP), pCMV-C-YFP (YFP), pCMV-N-CFP, pCMV-C-YFP (CFP+YFP), pCMV-N-CFP-YFP (CFP-YFP), pCMV-N-CFP-TRIM54 (CFP-TRIM54), pCMV-C-YFP-STAT2 (YFP-STAT2), and pCMV-N-CFP-TRIM54 and pCMV-C-YFP-STAT2 (CFP-TRIM54+YFP-STAT2). After culturing for 48 h, the cells were seeded into black 96-well cell culture plates, with three replicates per group and 5 × 10⁶ cells per well. 4Cells were used to calculate the binding rate of CFP and YFP in CFP+YFP cells; cells expressing CFP, YFP, and CFP-YFP were used to calculate the binding rate of CFP and YFP in CFP-YFP cells; and cells expressing CFP-TRIM54+His-STAT2, YFP-STAT2+Flag-TRIM54, and CFP-TRIM54+YFP-STAT2 were used to calculate the binding rate of CFP-TRIM54 and YFP-STAT2 in CFP-TRIM54+YFP-STAT2 cells. Fluorescence intensity was scanned in the CFP channel (ex: 430nm; em: 480nm), YFP channel (ex: 485nm; em: 530nm), and FRET channel (ex: 430nm; em: 530nm) using a microplate reader, and the efficiency of the interaction between the two fluorescently tagged proteins was calculated using a formula. The formula is: E%=1-(D DA / (D DA +(F DA -(D DA ×d)-(A DA ×a))×Q D / Q A )). (d=F D / D D , a = F A / A A D D : Readings of CFP protein expression detected in the donor channel; D DA : Readings of co-expressed CFP and YFP proteins detected in the donor channel; A A : Readings of YFP protein expression detected by the acceptor channel; A DA Fluorescence values ​​of CFP and YFP proteins detected in the acceptor channel; F A : Fluorescence value of YFP protein detected in the fret channel; F D : Fluorescence value of CFP protein detected in the fret channel; F DA : Fluorescence values ​​of simultaneously expressed CFP and YFP proteins detected in the fret channel; Q D =0.4 and Q A =0.61 (the quantum yields of the fluorescent donor and fluorescent acceptor are respectively). Figure 4 The results obtained by the enzyme-linked immunosorbent assay (ELISA) reader show that the binding rate of the negative control CFP+YFP is approximately 0%; the binding rate of the positive control CFP-YFP is approximately 50%; and the binding rate of CFP-TRIM54+YFP-STAT2 is approximately 30%.

[0053] Example 5: Screening small molecule compounds targeting the TRIM54 / STAT2 interaction

[0054] The small molecule library targeting protein interactions we selected was purchased from Med Chem Express and contained 167 small molecule compounds. The drugs were dissolved in their respective lyophilized powders using the appropriate solvents (DMSO, ethanol, and water) to a concentration of 10 mM and stored at -80°C for extended periods. The drugs in the small molecule drug library were diluted to 1 mM with DMEM at a ratio of 1:10 and then frozen at -20°C for subsequent experiments.

[0055] We transfected 293T cells with plasmids pCMV-N-CFP (CFP), pCMV-C-YFP (YFP), pCMV-N-CFP and pCMV-C-YFP (CFP+YFP), pCMV-N-CFP-YFP (CFP-YFP), pCMV-N-CFP-TRIM54 (CFP-TRIM54), pCMV-C-YFP-STAT2 and p3xflag-cmv-10-TRIM54 (YFP-STAT2), and pCMV-N-CFP-TRIM54 and pCMV-C-YFP-STAT2 (CFP-TRIM54+YFP-STAT2). After culturing for 48 h, the cells were seeded into black 96-well cell culture plates, with three replicates per group and 5 × 10⁶ cells per well. 4 Cells were cultured. Subsequently, the compound was added to cells co-expressing CFP-TRIM54 and YFP-STAT2 at a final concentration of 10 μM as the experimental group. DMSO of the same dilution was added to cells co-expressing CFP-TRIM54 and YFP-STAT2. After 24 h of culture, the cells were scanned using a microplate reader, and the fluorescence intensity was calculated using the formula E% = 1 - (Dt)t. DA / (D DA +(F DA -(D DA ×d)-(A DA ×a))×Q D / Q A The binding efficiency between CFP-TRIM54 and YFP-STAT2 after treatment with different compounds was calculated. The binding efficiency values ​​were then substituted into the formula "(control group - experimental group) / control group" to calculate the inhibitory efficiency of the compounds, so as to more intuitively demonstrate the inhibitory effect of the compounds.

[0056] Drugs with an inhibition rate greater than 20% screened in the FRET drug screening system are considered effective drugs. Initial screening results show ( Figure 5a) There are four compounds with inhibition rates greater than 20%, namely compounds 9, 64, 86 and 159, among which compound 159 has an inhibition rate of over 40%.

[0057] We then used flow cytometry to analyze the cells screened by the ELISA reader, obtaining the binding efficiency between CFP-TRIM54 and YFP-STAT2 after treatment with different compounds. The binding rate values ​​were then substituted into the formula: "(Control group - Experimental group) / Control group" to calculate the inhibitory efficiency of the compounds, providing a more intuitive representation of their inhibitory effects. Figure 5 As shown in b, six compounds exhibited inhibition rates higher than 20%, namely compounds 9, 64, 66, 86, 87, and 159. Among them, compounds 9 and 159 showed inhibition rates exceeding 40%.

[0058] Example 6: Re-screening of small molecule compounds

[0059] Initial screening of small molecule drugs showed that compounds 9, 64, 66, 86, 87, and 159 significantly inhibited the interaction between TRIM54 and STAT2. Since we were testing the fluorescence intensity of the FRET channel, compounds that inhibit cell viability would also inhibit FRET signaling, leading to their inclusion as false positives. Therefore, we investigated the inhibitory effect of compounds on the FRET channels of the CFP-YFP fusion protein group to exclude false positives. We added the above compounds to the experimental group (cells transfected with CFP-TRIM54 and YFP-STAT2 plasmids) to a final concentration of 10 μM, and simultaneously added the same compounds to the control group (cells transfected with the CFP-YFP fusion plasmid) to a final concentration of 10 μM. After culturing for 24 h, we read the fluorescence intensity of each channel using a microplate reader and calculated the effect of the compounds on the protein binding rates of the experimental and control groups. Figure 6 a). Further flow cytometry was used to detect the effect of the compound on the FRET channel positivity rate ( Figure 6 b). The results showed that the screened drug did not affect FRET signaling in the control group, and its inhibition of TRIM54 and STAT2 was specific.

[0060] Example 7: Performance of the compound in reversing the degradation of STAT2 by TRIM54

[0061] To verify whether the compounds could inhibit TRIM54-induced STAT2 degradation, we transfected 293T cells with a plasmid encoding Flag-TRIM54. Nine hours later, we added compounds (9, 64, 66, 86, 87, and 159) to a final concentration of 10 μM. After 24 hours, we collected and lysed the cells, and performed Western blot analysis on the cell lysates. We examined the intracellular STAT2 protein expression levels to verify the inhibitory effect of this drug on TRIM54-mediated STAT2 degradation. Figure 7 As shown, TRIM54 can induce the degradation of STAT2, while compounds 9, 64, 66, 87, and 159 can reverse the degradation of STAT2.

[0062] Example 8: Performance of the compound in blocking TRIM54 / STAT2 interaction

[0063] To verify the actual inhibitory effect of the compounds on the TRIM54 / STAT2 interaction, we used an immunoprecipitation assay. The plasmid encoding Flag-TRIM54 was transfected into 293T cells. After 24 hours, the cells were lysed, centrifuged, and the cell lysates were added with the compounds (9, 64, 66, 86, 87, 159) to a final concentration of 10 μM. The control group was treated with the same dilution of DMSO. All mixtures were incubated with the Anti-Flag Affinity gel at 4°C for 12 hours. Western blot analysis was then performed on the proteins attached to the gel. Figure 8 As shown, Flag-TRIM54 can bind to endogenous STAT2. When compounds targeting the TRIM54 / STAT2 interaction are added to the Co-IP system, the compounds can inhibit the interaction between TRIM54 and STAT2.

[0064] Example 9: Inhibitory effect of compounds on DENV2 and antibody 4G2 infection of K562.

[0065] K562 cells are a human monocyte cell line expressing FcγRIIa, which is suitable for establishing an ADE model. We infected monocytes with an immune complex formed by DENV2 and the monoclonal antibody 4G2 (a specific monoclonal antibody against DENV1 E protein). First, we incubated DENV2 and 4G2 antibodies (final concentration 1 μg / ml) at 25°C for 1 h to form a complex. K562 cells (5 × 10⁻⁶ cells) were then infected at an MOI of 1. 5In the control group, equal amounts of RPMI, 4G2, or DENV2 were added and incubated for 72 hours. Cells were then collected, fixed, permeabilized, and intracellular DENV2 was labeled with Alexa Fluor 647-labeled 4G2 antibody. The proportion of cells infected with DENV2 and the viral load were detected by flow cytometry. After cell removal and de-adhesion, the efficiency of viral infection of K562 cells was detected in the RL1 channel. Figure 9 As shown in Figure a, the infection rates in the control group (with RPMI, 4G2, or DENV2 added) were 1% (theoretically 0), 1% (theoretically 0), and 10%, respectively. The infection rate in the experimental group (DENV2+4G2) was 70%. These results indicate that the immune complex formed by DENV2 and 4G2 can promote DENV2 infection, and the ADE model was successfully constructed.

[0066] This invention investigated the inhibitory effect of a compound on ADE (anti-degenerative disorders) constructed in K562 cells. We added 10 μM of the compound to K562 cells, which formed an ADE model. After incubation for 72 h, we collected the cells. After fixation, perforation, and staining, we analyzed the cells by flow cytometry. Figure 9 As shown in b, the viral infection rate of K562 cells with the added compound showed a decreasing trend, suggesting that it may be a potential drug for inhibiting the ADE effect of the virus. Further investigation was conducted using compounds with stronger inhibitory effects (64, 86, 159) to examine the dose-dependent nature of their inhibitory effect on ADE. We incubated DENV2 and antibody 4G2 (final concentration 1 μg / ml) at 25°C for 1 h and then infected K562 cells (MOI = 1). Compounds (final concentrations of 1, 3, 10, and 30 μM) were added to the cells to investigate the inhibitory effect of different doses on the ADE effect. The control group received the same dilution of Med(DMSO). After fixation, permeabilization, and staining, the cells were analyzed by flow cytometry. The results are shown below. Figure 9 As shown in c, 64 (Baricitinib) and 159 (Rivaroxaban) showed significant inhibitory effects on the ADE effect, and these effects were dose-dependent.

[0067] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for screening antiviral ADE drugs targeting the inhibition of TRIM54 ubiquitination and degradation of STAT2, characterized in that, Includes the following steps: S1. Construct a first plasmid and a second plasmid respectively, introduce the first plasmid and the second plasmid into cells, and detect FRET signals; the first plasmid contains a gene encoding TRIM54 and a gene encoding a first fluorescent protein, the second plasmid contains a gene encoding STAT2 and a gene encoding a second fluorescent protein, and the first fluorescent protein and the second fluorescent protein can undergo fluorescence resonance energy transfer, wherein the nucleotide sequence of the gene encoding TRIM54 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding STAT2 is shown in SEQ ID NO.2; S2. Co-incubate the test compound with the cells described in step S1 as the experimental group, and co-incubate dimethyl sulfoxide with the cells described in step S1 as the control group. Detect FRET signals by flow cytometry or fluorescence enzyme labeling, and calculate the binding efficiency between TRIM54 and STAT2 in the experimental group and the control group, respectively. Substitute the value of the binding efficiency into the formula: (control group - experimental group) / control group to calculate the inhibition rate of the test compound. The test compounds with an inhibition rate greater than 20% are the drugs that inhibit the ADE effect of the virus.

2. The screening method according to claim 1, characterized in that: Cells in which a third plasmid was introduced were used as a positive control. The third plasmid contained genes encoding the first fluorescent protein and genes encoding the second fluorescent protein.

3. The screening method according to claim 1, characterized in that: Cells inoculated with the fourth and fifth plasmids were used as negative controls. The fourth plasmid contained the gene encoding the first fluorescent protein, and the fifth plasmid contained the gene encoding the second fluorescent protein.

4. The application of substances that inhibit the interaction between TRIM54 and STAT2 in the preparation of antiviral ADE-effect drugs, characterized in that: The substance is Baricitinib or Rivaroxaban, and the antiviral ADE effect is caused by DENV2 and monoclonal antibody 4G2 infection.

5. The application of substances characterizing the interaction between TRIM54 and STAT2 in the screening of antiviral ADE-effect drugs, characterized by: The substance is the first plasmid and the second plasmid as described in claim 1.

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