Application of gp130 inhibitor in preparation of medicine for treating virus infection

Abstract

The invention discloses application of a gp130 inhibitor in preparation of a medicine for treating virus infection, and relates to the technical field of medicines. Experiments prove that combination of IL-6 family cell factors and a gp130 receptor is inhibited, so that downward signal transmission of a JAK2 / STAT3 signal channel triggered by the IL-6 family cell factors is inhibited, the purposes of inhibiting cell fusion induced by SARS-CoV-2 spike protein and further influencing replication and propagation of SARS-CoV-2 are achieved, and a small-molecule inhibitor SC144 is an inhibitor of gp130, has a remarkable effect of inhibiting SARS-CoV-2 virus replication and has a good application prospect. Meanwhile, the traditional Chinese medicine composition also has a remarkable effect of inhibiting replication of viruses such as human immunodeficiency virus, herpes simplex virus and respiratory syncytial virus.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to gp130 inhibitors, and more specifically to the application of gp130 inhibitors in the preparation of drugs for treating viral infections. Background Technology

[0002] Cell fusion has multiple effects on viral transmission and pathological damage: 1) Syncytoids induced by cell fusion can enable direct viral transmission between cells, avoiding exposure to the extracellular environment and thus evading immune recognition and neutralizing antibodies; 2) Syncytoid formation leads to large-scale fusion and death of alveolar epithelial cells, releasing the virus and triggering an inflammatory storm, which is associated with severe pneumonia; 3) Syncytoids can phagocytose T / B cells (cell-in-cell structure), inducing apoptosis, which may lead to lymphopenia (lymphopenia); 4) Syncytoids may be involved in viral latency or persistent infection, especially in nerve tissue, and may be associated with neurological symptoms of "long-term COVID-19".

[0003] This invention reveals that IL-6 family factors, including IL-6, a representative of the cytokine storm induced by SARS-CoV-2, can promote spike protein-induced syncytial formation through the JAK2 / STAT3 signaling pathway mediated by the cell membrane receptor GP130, thereby enabling rapid replication and spread of SARS-CoV-2. Furthermore, it was found that the gp130 target is also crucial for the replication and spread of many other membrane fusion viruses, even playing a vital role in placental fusion during embryonic development.

[0004] Therefore, this invention proposes the application of a gp130 inhibitor in the preparation of drugs for treating viral infections. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of gp130 inhibitors in the preparation of drugs for treating viral infections.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] On the one hand, the present invention provides the application of gp130 inhibitors in the preparation of drugs for treating viral infections, including but not limited to SARS-CoV-2, HIV, herpes simplex virus and respiratory syncytial virus.

[0008] Preferably, the gp130 inhibitor includes, but is not limited to, the small molecule inhibitor SC144 and its derivatives, pharmaceutical hydrochloride compounds of SC144, LMT-28, and badoxifene acetate.

[0009] Preferably, the gp130 inhibitor can inhibit viral spike protein-induced syncytial formation and / or viral replication.

[0010] Preferably, the gp130 inhibitor inhibits the formation of viral spike protein-mediated syncytium by inhibiting the JAK2 / STAT3 signaling pathway triggered by the gp130 ligand or gp130 ligand receptor.

[0011] Preferably, the gp130 inhibitor inhibits viral replication by inhibiting STAT3 phosphorylation.

[0012] Preferably, the gp130 ligand is an IL-6 family cytokine.

[0013] Preferably, the gp130 ligand includes, but is not limited to, cytokines such as IL-6, IL-11, OSM, LIF, CNTF, CTF-1, and CLCF1.

[0014] Preferably, the effective concentration of the small molecule inhibitor SC144 is 10 nM to 10 μM.

[0015] Preferably, the dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, and syrups.

[0016] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0017] On the other hand, the present invention also provides a drug for treating COVID-19 infection, the drug comprising a gp130 inhibitor.

[0018] Preferably, the gp130 inhibitor is the small molecule inhibitor SC144 and its derivatives, the pharmaceutical hydrochloride compound of SC144, LMT-28, and badoxifene acetate.

[0019] Preferably, the virus is SARS-CoV-2, HIV, herpes simplex virus, or respiratory syncytial virus, wherein the SARS-CoV-2 virus is SARS-CoV-2 or a variant of SARS-CoV-2, and the variant of SARS-CoV-2 is a delta variant of SARS-CoV-2.

[0020] Compared with existing technologies, the beneficial effects of this solution are:

[0021] This invention experimentally demonstrates that inhibiting the binding of IL-6 family cytokines to gp130 and its ligand receptors inhibits the downward transmission of signals in the JAK2 / STAT3 signaling pathway triggered by these cytokines, thereby inhibiting SARS-CoV-2 spike protein-induced cell fusion and SARS-CoV-2 replication. Furthermore, experiments have shown that the small molecule inhibitor SC144 has a significantly greater effect on inhibiting SARS-CoV-2 viral replication than tocilizumab, and also exhibits significant inhibitory effects on the replication of viruses such as HIV-1, HSV-1, and RSV. This invention is the first to propose that gp130 can serve as an important target for the replication and spread of various membrane fusion viruses. Attached Figure Description

[0022] Figure 1 Most cytokines produced during SARS-CoV-2 infection are involved in the regulation of the JAK / STAT pathway (α is Caco-2 after 24 hours of infection with SARS-CoV-2-GFP / ΔNtrVLP). N RNA-seq sequencing results of cell samples (b is a schematic diagram of various JAK / STAT signal transduction);

[0023] Figure 2 The screening results for JAK2 / STAT3 molecules, which are crucial in the replication process of the novel coronavirus (a is the in vitro constructed novel coronavirus replicon infection system, which was obtained by infecting stably SARS-CoV-2-N positive cells and inducing the formation of GFP). + Schematic diagram of syncytium; b shows the statistical results of the inhibition rate of SARS-CoV-2 replicon by knockdown of all JAK / STAT groups; c shows the fluorescence image of the effect of JAK2 / STAT3 knockdown on SARS-CoV-2 replicon infection; d shows the quantitative analysis of the results in c.

[0024] Figure 3 The effects of JAK2 or STAT3 knockdown on viral invasion and early negative strand RNA synthesis mediated by the SARS-CoV-2 spike protein (a) Effect of STAT3 knockdown on SARS-CoV-2 pseudovirus replication, b) Effect of STAT3 knockdown on HIV pseudovirus replication, c) Effect of JAK2 knockdown on early RNA synthesis of SARS-CoV-2, d) Effect of STAT3 knockdown on early RNA synthesis of SARS-CoV-2.

[0025] Figure 4The effects of JAK2 or STAT3 knockdown on SARS-CoV-2 spike protein-mediated syncytial formation and spike protein membrane uptake efficiency (a is a fluorescence image of the effect of JAK2 or STAT3 knockdown on SARS-CoV-2 spike protein-mediated syncytial formation, b is a quantitative analysis of the results in a; c and d are the effects of JAK2 / STAT3 knockdown on SARS-CoV-2 spike protein membrane uptake efficiency, respectively).

[0026] Figure 5 The effect of gp130 on syncytial formation triggered by the SARS-CoV-2 spike protein (a shows the screening results of various cell receptors that can activate JAK2 / STAT3 for SARS-CoV-2 replicon infection, b shows the fluorescence image of the effect of gp130 knockdown on SARS-CoV-2 replicon replication, c shows the quantitative analysis of the results in b, d shows the fluorescence image of the effect of gp130 knockdown on SARS-CoV-2 spike protein-induced syncytial formation, and e shows the quantitative analysis of the results in d).

[0027] Figure 6 To evaluate the effect of gp130 on SARS-CoV-2 spike protein-induced syncytial formation using the Split-GFP system (a is a fluorescence image showing the effect of gp130 knockdown on SARS-CoV-2 spike protein-induced syncytial formation, and b is a quantitative analysis of the results in a);

[0028] Figure 7 Statistical results of cytokines transmitting signals via JAK2 in RNA-Seq sequencing analysis of Caco-2 cells infected with SARS-CoV-2 replicons;

[0029] Figure 8 The study investigated the effects of IL-6 protein on SARS-CoV-2 replicon replication and the influence of IL-6 family cytokines (IL-6 / IL-11 / OSM) on SARS-CoV-2 spike protein-induced syncytial formation (a: fluorescence image of the effect of IL-6 protein on SARS-CoV-2 spike protein-induced cell fusion; b: quantitative analysis of the results in a; c and d: the effect of IL-6 protein on SARS-CoV-2 replicon replication; e: Western blot analysis results for IL-6 protein activity verification; f and g: quantitative analysis of fluorescence images for simultaneously verifying the effects of knockdown of IL-6, IL-11, OSM and their receptors on SARS-CoV-2 spike protein-induced syncytial formation).

[0030] Figure 9 Fluorescent images and quantitative analysis of the effect of tocilizumab on syncytial formation induced by SARS-CoV-2 spike protein;

[0031] Figure 10 The effect of gp130 target inhibitor SC144 on SARS-CoV-2 replication (a is a schematic diagram of the mechanism of action of gp130 target inhibitor and tocilizumab; b and c are fluorescence images and quantitative analysis of the effect of different doses of SC144 on SARS-CoV-2 spike protein-induced syncytial formation; d and e are fluorescence images and quantitative analysis of the effect of SC144 and tocilizumab on SARS-CoV-2 replicon replication; f and g are the effects of SC144 and tocilizumab on the in vitro replication of SARS-CoV-2 delta variants, respectively).

[0032] Figure 11 The effects of the gp130 target inhibitor SC144 on HIV-1 replication are shown in the following figures: (a) Effect of SC144 on HIV-1 membrane protein-mediated cell fusion; (b) Effect of different doses of SC144 on SupT1 proliferation; (c) Effect of SC144 on HIV-1 viral replication; (d) Quantitative analysis of the grayscale value of p24 protein expression in c using ImageJ, followed by statistical analysis.

[0033] Figure 12 The effect of gp130 target inhibitor SC144 on herpes simplex virus HSV-1 replication (a) HSV-1 infection in HFF, comparing the effects of different drugs SC144 and tocilizumab on viral replication; b) Effect of SC144 on HSV-1 infection in HFF to induce syncytial formation).

[0034] Figure 13 To compare the effects of gp130 target inhibitor SC144 and tocilizumab on RSV-F-mediated syncytial formation. Detailed Implementation

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] The experimental methods used in the following embodiments are as follows:

[0039] 1. SARS-CoV-2 replicon (GFP / ΔN) infection experiment

[0040] Using SARS-CoV-2 GFP / ΔN virus, which can be handled in a biosafety level 2 (BSL-2) laboratory, rather than live virus, a stable HEK-293T strain expressing N and ACE2 was established. N-ACE2 and Caco-2 N-ACE2 Cell lines. SARS-CoV-2 GFP / ΔN virus can infect these cells and complete its entire life cycle. 3 × 10 4 HEK-293T N-ACE2 and Caco-2 N-ACE2 Cells were cultured overnight in 24-well plates. Different target genes were knocked down using siRNA, and replicons were infected after 48 hours. Cells were incubated at 37°C for 2 hours, then the viral medium was removed, and the cells were washed 1-2 times with PBS. Fresh medium was then used for further culture. Viral protein expression was detected approximately 48-72 hours after infection using fluorescence microscopy, flow cytometry, and Western blotting to investigate the infectivity of SARS-CoV-2 replicons under different experimental conditions. The life cycle of SARS-CoV-2 infection in target cells and the viral invasion mechanism mediated by the SARS-CoV-2 spike protein were investigated from multiple perspectives: an infection system was established using the NL4-3ΔEnv / GFP packaging vector to encapsulate the SARS-CoV-2 spike protein to detect the impact on the SARS-CoV-2 invasion stage; SARS-CoV-2 genome transcription: primers were designed using viral subgenomic RNA, and RT-qPCR was used to study changes in SARS-CoV-2 replication and transcription levels. The HiScript III first-strand cDNA synthesis kit (+DNA scavenger) was used to determine the replication level of SARS-CoV-2. The SARS-CoV-2 spike protein gene sequence-specific primers are F: 5'-CTGTTCTCTAAACGAACAATGTTTGTTTTT-3' and R: 5'-TA GCATGGAACCAAGTAACATTGG-3'; the probe (GSP) sequence is 5'-CAAACCAACC AACTTTCGATCTC-3'.

[0041] 2. Cell transfection

[0042] Empty plasmids or various plasmids using HieffTrans TM Liposome transfection reagent (catalog number: 40802ES08, Shanghai Sangon Biotech Co., Ltd.) was used for transfection according to the manufacturer's instructions. siRNA was synthesized by Ribobio (Guangzhou, China). siRNA was transfected using Lipofectamine RNAiMAX (Invitrogen, California, USA) according to the manufacturer's protocol.

[0043] 3. Western blotting

[0044] After collection, cells were washed twice with pre-cooled PBS and lysed at 4°C with lysis buffer (product number: P0013, Beyotime, Shanghai, China) and a solution containing intact protease inhibitor complex tablets (Roche, Basel, Switzerland) for 20 minutes. The lysate was then mixed with 5× loading buffer (product number: FD002, Fode, Zhejiang, China) and heated for 30 minutes. Cell lysates were centrifuged at 13,000 rcf for 5 minutes at 4°C. The supernatant was loaded and coagulated with sodium dodecyl sulfate-polyacrylamide.

[0045] Separation was performed by gel electrophoresis (SDS-PAGE). The separated proteins on the gel were transferred to a PVDF membrane (IPVH00010, Merck Millipore, Ireland) using a wet apparatus (Breed, California, USA). The membrane was incubated overnight with the primary antibody dilution. The following day, the membrane was washed three times with TBST and then incubated for 1 hour in secondary antibody dilution (1:5000, product number: HA1006 or HA1001, Huaan Biotechnology, Hangzhou, China). Exposure was performed using sensitive ECL chemiluminescence reagent (product number: PK10002, Proteintech, Wuhan, China) according to the manufacturer's protocol.

[0046] 4. Spike protein-mediated cell-cell fusion

[0047] Donor cells (HEK-293T) were knocked down with gp130 / JAK2 / STAT3. Forty-eight hours later, they were co-transfected with SARS-CoV-2 spike protein particles and intact green fluorescent protein. Approximately 24 hours later, HEK-293T cells were digested and then plated with recipient cells (Caco-2) from the previous day. N-ACE2 Co-culture was performed, and cell fusion was subsequently observed. After 24 hours, cell fusion was captured using fluorescence microscopy, and ImageJ was used to measure the number and area of ​​green-positive cell clusters (distinguished from individual HEK293T green fluorescent cells). Finally, GraphPad Prism9 software was used to statistically analyze the results and identify significant differences. The split GFP in vitro fusion system followed similar steps, with donor cells HEK-293T transfected with spike protein particles and GFP 1-10, and recipient cells transfected with Caco-2... N-ACE2 Change to HEK293T-2 N-ACE2 Cells transfected with GFP11 will only produce green fluorescence if cell fusion is mediated.

[0048] Example 1: The effect of the JAK2 / STAT3 pathway on the replication of SARS-CoV-2

[0049] This example involves Caco-2 cells infected with SARS-CoV-2-GFP / ΔNtrVLP (COVID-19 replicon) approximately 24 hours prior. N RNA-seq sequencing of cell samples revealed that most of the cytokines upregulated in SARS-CoV-2 are involved in the regulation of the JAK-STAT pathway. Figure 1 (a) Further systematic evaluation of the importance of all JAK / STAT components for SARS-CoV-2 replication ( Figure 1 (b)

[0050] Using the COVID-19 replication subsystem ( Figure 2 a) Filter all JAK / STAT, i.e., in Caco-2 N All JAK / STAT cells were knocked down sequentially, and the cells were infected with replicons 48 hours later. GFP was detected by flow cytometry approximately 48-72 hours after infection. + Cell number was determined using fluorescence microscopy to capture light microscopic images and corresponding fluorescence images, and infection results were quantified using ImageJ. The results indicated that JAK2 and STAT3 are the most critical JAK / STAT molecules during SARS-CoV-2 replication. Figure 2 (bd in the middle).

[0051] Since the initial steps of SARS-CoV-2 infection require spike protein-mediated receptor binding and viral fusion with the cell membrane, this study used HIV-NL4-3-GFP / Δenv to encapsulate Vsvg to simulate HIV pseudoviruses and SARS-CoV-2 spike proteins to simulate SARS-CoV-2 pseudoviruses, respectively, to investigate SARS-CoV-2 invasion. This was also done in Caco-2... N STAT3 was knocked down in cells, and after 48 hours, the cells were infected with the two pseudoviruses mentioned above. GFP was detected by flow cytometry. + The study examined cell counts and found that knocking down STAT3 had almost no effect on SARS-CoV-2 spike protein-mediated viral entry, similar to the effect on VSV-G-mediated viral entry. Figure 3 (a and b in the text).

[0052] After the virus enters, viral RNA synthesis begins. This embodiment further utilizes Caco-2... N JAK2 and STAT3 were knocked down in cells, and the cells were infected with replicons 48 hours later. Cell samples were collected every 2 hours from 2 to 12 hours to extract cellular RNA for the detection of negative-strand RNA synthesis of SARS-CoV-2. The results showed that knocking down JAK2 or STAT3 had only a slight effect on the early RNA synthesis of SARS-CoV-2. Figure 3 (c and d in the text).

[0053] Example 2: Effect of Gp130-mediated signal transduction on SARS-CoV-2 spike protein-induced cell fusion.

[0054] It is well known that SARS-CoV-2 infection can induce cell fusion both in vivo and in vitro (Asarnow D, et al., Cell, 2021). The cell fusion capability of SARS-CoV-2 is related to disease pathogenesis and immune escape (Qu P, et al., Cell Host Microbe, 2022). The SARS-CoV-2 spike protein alone can induce cell fusion (Braga L, et al., Nature, 2021). This invention utilizes a mature experimental system (Liu X, et al., Sci Signal, 2022), namely, constructing an in vitro SARS-CoV-2 spike protein-induced fusion system using HEK-293T cells as donor cells, explanting Spike and GFP plasmids, and Caco-2... ACE2 As recipient cells, they were co-cultured; only fused cells exhibited a green blotch (distinguishing them from single-point GFP in HEK-293T cells). JAK2 / STAT3 was knocked down in HEK-293T cells, and 48 hours later, spike proteins and GFP plasmids were transfected. 24 hours after transfection, the cells were then cultured with Caco-2... ACE2 After co-culturing for approximately 24 hours, cells were captured using a fluorescence microscope, and fluorescence images were taken. Then, ImageJ software was used to count GFP. + Cell cluster region; JAK2 / STAT3 was knocked down in HEK-293T cells, and Spike plasmid was transfected 48 hours later. Spike plasmid was detected by flow cytometry 24 hours after transfection. + Cell count. The results showed that SARS-CoV-2 spike protein-mediated syncytial formation was significantly affected by JAK2 or STAT3 expression. Figure 4 (ab); The expression levels of JAK2 or STAT3 proteins have little effect on the membrane uptake efficiency of the SARS-CoV-2 spike protein. Figure 4 (cd in the middle).

[0055] Because multiple cytokine receptors can activate the JAK2 / STAT3 pathway, this example investigates which pathways are crucial for syncytial formation triggered by the SARS-CoV-2 spike protein to determine which pathways are essential. N All JAK2 binding receptors were knocked down sequentially in cells, and the cells were infected with replicons 48 hours later. GFP was detected by flow cytometry approximately 48-72 hours after infection. + The number of cells was used to screen for various cellular receptors known to activate JAK2 / STAT3. Figure 5(a) In HEK-293T cells, gp130 was knocked down, and the cells were infected with replicons 48 hours later. Cells were captured using a fluorescence microscope and fluorescence images were taken 48-72 hours after infection. GFP was then counted using ImageJ. + The cell cluster region; and in HEK-293T cells, gp130 was knocked down, and 48 hours later, the spike plasmid was transfected. Approximately 20 hours after transfection, the HEK-293T cells were digested and counted at 80,000 cells / well compared to the 30,000 cells / well Caco-2 plated the previous day. ACE2 Mixed overnight, and after approximately 24 hours, images of the fluorescence were captured using a light microscope, and GFP was statistically analyzed using ImageJ. + The region of cell clusters. The results showed that gp130 had the most significant impact on SARS-CoV-2 replication. Figure 5 (bc in the text), and gp130 is also crucial for SARS-CoV-2 spike protein-induced syncytial formation ( Figure 5 (de in the middle).

[0056] This embodiment further evaluated the important role of gp130 in SARS-CoV-2 spike protein-induced syncytial formation using the Split-GFP system. A two-half GFP fusion system was constructed in vitro: HEK-293T was transfected with SARS-CoV-2 Spike and GFP 1-10 plasmids. ACE2 Transfect the cells with GFP 11 plasmid and co-culture them. Only the fused cells will turn green. Knock down gp130 in HEK-293T cells, and after 48 hours, transfect them with the Spike plasmid. About 20 hours after transfection, digest the HEK-293T cells and count 80,000 cells / well compared to the 100,000 cells / well HEK-293T cells seeded the previous day. ACE2 The mixture was incubated overnight and co-cultured for approximately 24 hours. Light microscopic and fluorescence images were captured using a microscope, and GFP was statistically analyzed using ImageJ. + The region of cell clusters. The results showed that gp130 knockdown indeed significantly inhibited SARS-CoV-2 spike protein-induced syncytial formation. Figure 6 (ab in the text)

[0057] Example 3: SARS-CoV-2 virus infection specifically upregulates the RNA expression levels of multiple IL-6 family cytokines and mediates SARS-CoV-2 spike protein-induced syncytial formation and viral replication in a gp130-dependent manner.

[0058] As is well known, IL-6 is an inflammatory cytokine upstream of gp130 and is often associated with severe SARS-CoV-2 infection (Moore JB, et al., Science, 2020). This embodiment uses Caco-2...N Cells were infected with the SARS-CoV-2 Delta strain (MOI = 0.01) and incubated at 37°C for 2 hours. After washing the cells 1-2 times with PBS and replacing with fresh culture medium, cell samples were collected 24 hours after infection for RNA-seq sequencing to detect differences in cytokine RNA levels. The results confirmed that SARS-CoV-2 virus infection promotes an increase in the expression levels of multiple IL-6 family cytokines, with a very significant upregulation fold. Figure 7 ).

[0059] HEK-293T cells were transfected with SARS-CoV-2 Spike and GFP plasmids. 24 hours later, HEK-293T cells were digested, and 80,000 cells / well were collected and plated with Caco-2 cells (30,000 cells / well) from the previous day. ACE2 Cells were co-cultured, with IL-6 protein added during mixing (final concentration 50 ng / well). After approximately 24 hours of co-culturing, light microscopic and fluorescence images were captured using a microscope, and GFP was statistically analyzed using ImageJ. + The region of cell clusters. The results showed that IL-6 protein significantly promoted syncytial formation induced by the spike protein of SARS-CoV-2. Figure 8 (ab) in the text, using Caco-2 N Cells were infected with SARS-CoV-2 replicons and incubated at 37°C for 2 hours. After washing the cells 1-2 times with PBS, the culture medium was replaced with fresh medium containing IL-6 protein, and the cells were cultured for another 48 hours. GFP was detected by flow cytometry. + Cell count. To investigate whether IL-6 protein affects Spike expression and protein activity, HEK-293T cells were treated with IL-6 protein (final concentration 50 ng / well) for 4 hours, then transfected with the SARS-CoV-2 Spike plasmid. Cell samples were collected 24 hours after transfection, and Spike expression and STAT3 phosphorylation were detected by Western blotting. The results showed that IL-6 active protein can promote the replication of the SARS-CoV-2 replicon, but not by affecting Spike expression. Figure 8 (ce)

[0060] In addition to IL-6, this embodiment also used the proteins of two other factors in the IL-6 family (IL-11 / OSM) to verify the syncytial formation function mediated by the SARS-CoV-2 spike protein. Specifically, HEK-293T cells were transfected with SARS-CoV-2 Spike and GFP plasmids, and 24 hours later, HEK-293T cells were digested and 80,000 cells / well were collected and plated with Caco-2 cells at 30,000 cells / well from the previous day. ACE2Cells were co-cultured, with IL-6, IL-11, and OSM protein added separately at the time of mixing (final concentration 50 ng / well). After approximately 24 hours of co-culturing, light microscopic and fluorescence images were captured using a microscope, and GFP was statistically analyzed using ImageJ. + The cell cluster region. IL-6, IL-11, and OSM receptors were knocked down in HEK-293T cells, and 48 hours later, the cells were transfected with SARS-CoV-2 Spike and GFP plasmids. 24 hours after transfection, 80,000 cells / well of HEK-293T cells were digested and plated with 30,000 cells / well of Caco-2 protein cells from the previous day. ACE2 Cells were co-cultured for approximately 24 hours, and light microscopic and fluorescence images were captured using a microscope. GFP was then statistically analyzed using ImageJ. + The cell cluster region. HEK-293T cells were transfected with SARS-CoV-2 Spike and GFP plasmids. 24 hours later, HEK-293T cells were digested, and 80,000 cells / well were collected and plated with Caco-2 cells (30,000 cells / well) from the previous day. ACE2 Cells were co-cultured with tocilizumab (final concentration 0.2 mg / ml) added during mixing, and co-cultured for approximately 24 hours. Light microscopic and fluorescence images were captured using a microscope, and GFP was statistically analyzed using ImageJ. + The region of cell clusters. The results showed that the addition of IL-11 and OSM proteins significantly promoted syncytial formation induced by the SARS-CoV-2 spike protein. Figure 8 The fact that f in the text inhibits the expression of its receptor also confirms this phenomenon. Figure 8 (g in the original text). Tocilizumab is controversial in the treatment of SARS-CoV-2, and the results of this example also confirm the ineffectiveness of this monoclonal antibody. Figure 9 (ab in the text)

[0061] Example 4: Effect of SC144 on inhibiting SARS-CoV-2 viral replication

[0062] This embodiment uses an in vitro fusion system and a COVID-19 replication subsystem to verify the function of IL-6 receptor target inhibitors or monoclonal antibodies. Figure 10 In step a), HEK-293T cells were transfected with SARS-CoV-2 Spike and GFP plasmids. Twenty-four hours later, HEK-293T cells were digested, and 80,000 cells / well were collected and plated with Caco-2 cells (30,000 cells / well) from the previous day. ACE2 Cells were co-cultured with gradient doses of SC144 (from MedChemexpress Biotechnology, USA) (0, 1 μM, and 10 μM) added during mixing. After approximately 24 hours of co-culturing, light microscopic and fluorescence images were captured using a microscope, and GFP was statistically analyzed using ImageJ. +The region of cell clusters. gp130 is an important target of the inflammatory factors screened in Example 2, and the results of this example show that SC144 has a surprisingly significant inhibitory effect. Figure 10 (b) in the text, and the small molecule drug also shows a certain dose dependence ( Figure 10 (c)

[0063] Based on the above results, we conducted a functional comparison of the two drugs (SC144 and tocilizumab (from Roche)) in the SARS-CoV-2 replication subsystem, specifically in Caco-2. N Cells were infected with replicons and incubated at 37°C for 2 hours. After washing the cells 1-2 times with PBS, the culture medium was replaced with fresh medium containing SC144 (10 μM) and tocilizumab (0.2 mg / ml). Cells were cultured for 48 hours after infection, and light and fluorescence images were captured using a microscope. GFP was then detected by flow cytometry. + Cell count. Caco-2 cells were further infected with the SARS-CoV-2 Delta variant (MOI = 0.01), incubated at 37°C for 2 hours, washed with PBS 1-2 times, and then cultured in fresh medium containing SC144 (10 μM) and tocilizumab (0.2 mg / ml) and their corresponding control solvents. Cell supernatant was collected 24 hours after infection, and RNA was extracted to detect differences in the transcriptional level of genomic membrane proteins. The results showed that SC144 significantly inhibited the replication of the SARS-CoV-2 replicon. Figure 10 This result was also verified in the delta variant of SARS-CoV-2. Figure 10 (fg in the middle).

[0064] Example 5: Effects of SC144 on syncytial formation and viral replication induced by various viral membrane proteins.

[0065] Currently, many viruses have evolved strategies to induce syncytial formation using viral glycoproteins, such as HIV-1 (Feng Y, et al, Science, 1996), HSV-1 (Atanasiu D, et al, ProcNatl Acad Sci USA, 2007), and RSV (Pastey MK, et al, Nat Med, 2000).

[0066] To further investigate the important role of the gp130 target in the formation of syncytia induced by various viral proteins, this embodiment first constructed an HIV-Env-mediated Split GFP fusion system in vitro. HEK-293T cells were transfected with HIV-1 envelope and GFP 1-10 plasmids, while TZM-bl cells were transfected with GFP 11 plasmid for 20 hours. A certain amount of cells was mixed with SC144 at a final concentration of 10 μM, and the fusion was observed using a fluorescence microscope. Furthermore, the GFP fusion was analyzed using ImageJ software. + Regional statistical analysis revealed that SC144 significantly affects HIV-Env-mediated syncytial formation. Figure 11 (a) Then, the HIV-1 NL4-3 genome was transfected into HEK-293T cells in vitro, the supernatant was collected and used to re-infect SupT1 cells to generate live HIV-1 virus, and the effect of different doses of SC144 (0, 10 nM, 100 nM and 1000 nM) on the proliferation of SupT1 cells was detected. Figure 11 (b) In this study, the expression level of HIV-1p24 protein in the supernatant of different doses of SC144 (10 nM and 100 nM) and the control group was detected by Western blotting. Figure 11 c) In the Western Blot analysis, the gray values ​​of the WB bands were quantified using ImageJ software, and the relative values ​​after drug treatment were statistically analyzed compared with the control group. The results showed that SC144 significantly inhibited HIV-1 viral replication. Figure 11 (d)

[0067] Secondly, in this embodiment, HFF cells were infected in vitro with HSV-1 virus (MOI=3). After incubation at 37°C for 2 hours, the virus-containing culture medium was removed, and the cell surface was washed with PBS. The cells were then cultured in fresh culture medium containing SC144 (10 μM) and tocilizumab (0.2 mg / ml), respectively. Cell samples were collected at three time points, and GFP was detected by flow cytometry. + Cells were analyzed, and the fusion index was calculated after 48 hours. The results showed that SC144 had a stronger and more significant inhibitory effect on viral replication compared to tocilizumab. Figure 12 (a) and SC144 has a significant inhibitory effect on the formation of syncytia in HFF. Figure 12 (b)

[0068] Finally, in this embodiment, an RSV-F membrane protein-mediated Split GFP fusion system was constructed in vitro. One group of HEK-293T cells was transfected with RSV-F and GFP 1-10 plasmids, while another group of HEK-293T cells was transfected with GFP 11 plasmid. After 24 hours, a certain amount of cells expressing GFP 1-10 and cells expressing GFP 11 were co-cultured in fresh culture medium containing SC144 and toliszumab. After 24 hours, the fusion phenomenon was observed using a fluorescence microscope, and ImageJ software was used to count fusion areas exceeding 500 μm. 2 GFP + Cell cluster number and GFP + Regional area. The results showed that SC144 had a very significant inhibitory effect on RSV-F membrane protein-mediated syncytial formation compared with tocilizumab. Figure 13 ).

[0069] Through the above three membrane fusion virus systems, we confirmed that the gp130 target is extremely important for the replication and spread of various membrane fusion viruses. Furthermore, we discovered that the gp130 target inhibitor SC144 possesses a novel broad-spectrum anti-membrane fusion virus function.

[0070] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. The application of gp130 inhibitors in the preparation of drugs for treating viral infections, characterized in that, The viruses mentioned include, but are not limited to, COVID-19, HIV, herpes simplex virus, and respiratory syncytial virus.

2. The application according to claim 1, characterized in that, The gp130 inhibitors include, but are not limited to, the small molecule inhibitor SC144 and its derivatives, the pharmaceutical hydrochloride compound of SC144, LMT-28, and badoxifene acetate.

3. The application according to claim 2, characterized in that, The gp130 inhibitor inhibits the expression of gp130, thereby suppressing viral spike protein-induced syncytial formation and / or viral replication.

4. The application according to claim 3, characterized in that, The gp130 inhibitor inhibits the formation of viral spike protein-mediated syncytia by inhibiting the JAK2 / STAT3 signaling pathway triggered by the gp130 ligand or gp130 ligand receptor.

5. The application according to claim 3, characterized in that, The gp130 inhibitor inhibits viral replication by suppressing STAT3 phosphorylation.

6. The application according to claim 4, characterized in that, The gp130 ligand is an IL-6 family cytokine.

7. The application according to claim 6, characterized in that, The gp130 ligands include, but are not limited to, IL-6 protein, IL-11 protein, OSM protein, LIF protein, CNTF protein, CTF-1 protein, and CLCF1 protein.

8. The application according to claim 2, characterized in that, The effective concentration of the small molecule inhibitor SC144 is 10 nM to 10 μM.

9. The application according to claim 1, characterized in that, The dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, and syrups.

10. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

11. A drug for treating viral infections, characterized in that, The drugs include gp130 inhibitors.

12. The method according to claim 10, characterized in that, The gp130 inhibitor is the small molecule inhibitor SC144 and its derivatives, the pharmaceutical hydrochloride compound of SC144, LMT-28, and badoxifene acetate.

13. The method according to claim 10, characterized in that, The virus is SARS-CoV-2, HIV, herpes simplex virus or respiratory syncytial virus, wherein the SARS-CoV-2 virus is SARS-CoV-2 or a variant of SARS-CoV-2, and the variant of SARS-CoV-2 is the delta variant of SARS-CoV-2.

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