Application of substance aiming at VDAC1 target spot in preparation of product and medicine for preventing and treating diseases caused by RSV

Abstract

The invention discloses application of a substance aiming at a VDAC1 target spot in preparation of a product and a medicine for preventing and treating diseases caused by RSV, and relates to the technical field of respiratory syncytial virus resistance. A novel RSV cell receptor VDAC1 (voltage-dependent anion channel 1) independent of NCL (nucleolin) is discovered, a novel RSV infected cell receptor is identified, and discovery of the receptor provides a molecular basis for development of novel antiviral drugs and is crucial for development of novel drugs. The substance aiming at the VDAC1 target spot, provided by the invention, has a good application prospect in prevention and treatment of diseases caused by RSV, and the resistance of a subject to RSV infection can be greatly improved.

Description

Technical Field

[0001] This invention relates to the field of respiratory syncytial virus (RSV) technology, and more specifically, to the use of substances targeting VDAC1 in the preparation of products and drugs for the prevention and treatment of diseases caused by RSV. Background Technology

[0002] Human respiratory syncytial virus (RSV) is a major pathogen causing severe lower respiratory tract infections in infants and young children worldwide, resulting in significant morbidity and mortality [1]. Although significant progress has been made in the research of RSV vaccines and antiviral drugs recently, their clinical efficacy remains limited, and the continuous emergence of viral variants poses a major challenge [2].

[0003] Nucleolin (NCL) is one of the key host cell receptors in RSV infection. In 2011, a team from the University of Alberta, Canada, first discovered that this protein plays a functional cell receptor role in RSV invasion, promoting infection by mediating the initial binding of the virus to the host cell [3]. NCL is usually located in the nucleolus, but during RSV infection, it is relocated to the cell membrane surface, forming a "gateway" for viral invasion. Specifically, during the stage of viral adsorption to the host cell, RSV binds to NCL through its surface G / F protein [3, 4], triggering the signal transduction process before virus-cell membrane fusion. Subsequent studies have found that the activation of NCL requires the participation of insulin-like growth factor 1 receptor (IGF1R) and protein kinase Cζ (PKCζ), which form a complex receptor system. IGF1R / PKCζ acts as a signal transduction pathway, recruiting NCL to the virus binding site on the cell membrane. This mechanism explains the phenomenon that RSV stays on the cell surface for several hours before infection (compared to only a few minutes for influenza virus) [5].

[0004] The potential of NCL as a therapeutic target lies in the fact that blocking the NCL-RSV interaction may completely inhibit infection. This discovery provides a molecular basis for the development of novel antiviral drugs. However, AS1411, as an NCL antagonist that has entered phase II clinical trials, still has limited resistance to RSV infection. It can only partially reduce the viral titer in the lungs, and the drug is highly toxic [6]. Moreover, there is no specific drug for RSV worldwide. Therefore, the identification of RSV cell receptors is still ongoing.

[0005] There is an urgent need for a highly effective and safe drug to treat RSV and reduce the social burden caused by RSV infection.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide the application of substances targeting VDAC1 in the preparation of products and drugs for the prevention and treatment of diseases caused by RSV, in order to solve the above-mentioned technical problems.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides the application of a substance targeting VDAC1 in the preparation of a product, wherein the product functions as at least one of A1) to A5): A1) Prevention of diseases caused by respiratory syncytial virus (RSV); A2) Treatment of diseases caused by RSV; A3) Inhibits RSV virus replication; A4) Enhances the cell viability of hosts infected with RSV; A5) Screen for drugs to prevent or treat diseases caused by RSV.

[0009] Secondly, the present invention also provides the application of VDAC1 protein in the preparation of respiratory syncytial virus infection models.

[0010] Thirdly, the present invention also provides a double-stranded RNA molecule that inhibits VDAC1 expression, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0011] Fourthly, the present invention also provides a medicament for the prevention or treatment of diseases caused by RSV, comprising the aforementioned double-stranded RNA molecule that inhibits VDAC1 expression.

[0012] The present invention has the following beneficial effects: This invention discovers a novel RSV cellular receptor, VDAC1 (voltage-dependent anion channel 1), independent of NCL (nucleolin), and identifies a new cellular receptor for RSV infection. This discovery provides a molecular basis for the development of novel antiviral drugs and is crucial for their development. Cellular experiments show that the specific inhibitors of VDAC1, DIDS, effectively inhibit RSV infection in a dose-dependent manner, suggesting that the anti-RSV mechanism may be achieved by targeting host factors active in the early stages of infection. Specific knockout or downsampling of VDAC1 significantly inhibited RSV titers in a dose-dependent manner, while overexpression of VDAC1 in cells significantly enhanced RSV titers. Immunofluorescence-laser confocal microscopy experiments show that VDAC1 binds to the viral F protein but not to NCL.

[0013] This invention also conducted experiments on the effects of the VDAC1-specific inhibitor DIDS on RSV infection in animals, showing a protective effect of 30%-40%. Therefore, the substance targeting VDAC1 provided by this invention has good application prospects in the prevention and treatment of diseases caused by RSV, and can significantly improve the resistance of subjects to RSV infection. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The results of in vitro anti-RSV assays using the VDAC1 inhibitor DIDS are shown, along with the results of in vitro anti-RSV assays using DIDS at different time points. (AB) HEp-2 or A549 cells were co-incubated with DIDS for 48 h, and total cellularity (TC) was measured using the MTT assay. 50 Cells were then infected with RSV at an MOI of 0.1 for 1 hour, washed with PBS, and treated with different concentrations of DIDS. The supernatant was collected 48 hours post-infection (hpi) and analyzed using TCID50. 50 Viral titers were determined using the method shown. The dashed lines in the left and right panels represent the detection limit and 50% level, respectively. (CD) 160 μM, 320 μM, and 640 μM DIDS were pre-incubated with RSV at 37°C for 1 h (viral pretreatment) or added at 1, 12, or 24 h post-infection. The cells were HEp-2, and the virus titer was determined by TCID at 48 hpi. 50 Viral titers were determined by a method. Quantitative data are expressed as mean plus standard deviation (n=4 per group). (D) The dashed line in the figure represents TCID. 50 The detection limit of the method. **p<0.01, **p<0.001, ns indicates no significant difference; Figure 2 VDAC1 is a proviral host factor in RSV infection. (A) After infecting HEp-2 cells with RSV at different MOIs (0.05 or 0.1) for 48 h, the expression level of VDAC1 in the cell lysate was analyzed by Western blot. (B) Different concentrations of VDAC1-specific siRNA ( si-VDAC1-1778 ) or negative control siRNA ( si-NC HEp-2 cells were transfected for 24 hours, then infected with RSV at 0.1 MOI for 48 hours. VDAC1 knockdown was confirmed by Western blot and analyzed using TCID50. 50RSV titer was assessed using the following methods: (C) HEp-2 cells were transfected with 2.5 μg of VDAC1 expression plasmid (pcDNA3.1-VDAC1) or empty vector (Vehicle). After 48 hpi, the degree of VDAC1 overexpression was confirmed by Western blot. (D) RSV was infected with 0.1 MOI for 48 h. The level of VDAC1 overexpression protein was verified by Western blot and measured by TCID. 50 RSV titer was determined by the method. β-actin was used as an internal control for protein expression. Quantitative data are expressed as mean + standard deviation (n=3 per group). *p<0.05, **p<0.01, ***p<0.001; Figure 3 The results of the immunofluorescence assay for VDAC1 and RSV F proteins; Figure 4 VDAC1 cannot bind to nucleolin (NCL); Figure 5 Treatment with DIDS significantly inhibited lethal respiratory syncytial virus (RSV) infection in mice. (A) Percentage change in body weight and survival rate of mice in the control group (Mock), GZ08-18 + saline group (n=9), GZ08-18 + 12.5 mg / kg DIDS group (n=9), GZ08-18 + 25.0 mg / kg DIDS group (n=9), and GZ08-18 + 25.0 mg / kg ribavirin group (n=9); (B) Viral titers in the GZ08-18 + saline group, GZ08-18 + 12.5 mg / kg DIDS group, GZ08-18 + 25.0 mg / kg DIDS group, and GZ08-18 + 25.0 mg / kg ribavirin group. Detailed Implementation

[0016] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0018] Definition of noun As used herein, the term "prevention" means reducing the risk of disease in an individual or group, or mitigating the severity of disease or delaying its progression, through a series of proactive interventions. Specifically, in this application, "prevention" refers to administering the drug of this application prior to respiratory syncytial virus (RSV) infection, which can alleviate lung tissue damage and / or alveolar structural destruction following RSV infection. Disease prevention is a proactive health management concept; by "intervening" to avoid or mitigate the harm of disease, its value far exceeds that of treatment after disease onset.

[0019] As used herein, the term "treatment" refers to a series of medical interventions, techniques, or methods used to address an existing disease, with the aim of eliminating the cause, alleviating symptoms, controlling disease progression, promoting tissue repair, restoring function, or reducing pain. Specifically, in this application, "treatment" refers to the administration of the drug of this application following respiratory syncytial virus infection, which can improve lung tissue damage and / or alveolar structural destruction.

[0020] In a first aspect, the present invention provides the application of a substance targeting VDAC1 in the preparation of a product, wherein the product functions as at least one of A1) to A5): A1) Prevention of diseases caused by respiratory syncytial virus (RSV); A2) Treatment of diseases caused by RSV; A3) Inhibits RSV virus replication; A4) Enhances the cell viability of hosts infected with RSV; A5) Screen for drugs to prevent or treat diseases caused by RSV.

[0021] Substances targeting VDAC1 include, but are not limited to: proteins, peptides, oligopeptides, oligonucleotides and nucleotide analogs, and small (chemical) molecules.

[0022] The inventors discovered that substances that inhibit VDAC1 protein activity and / or expression, or substances that inhibit VDAC1 gene expression, can prevent and treat diseases caused by RSV. Cellular experiments showed that the VDAC1-specific inhibitors DIDS effectively inhibited RSV infection in a dose-dependent manner, and the anti-RSV mechanism may be achieved by targeting host factors active in the early stages of infection. Specific knockout or downsampling of VDAC1 significantly inhibited RSV titers, while overexpression of VDAC1 in cells significantly enhanced RSV titers.

[0023] Therefore, substances targeting VDAC1 can prevent and treat diseases caused by RSV. In addition, it can be expected that drugs that prevent or treat RSV-related diseases can be screened based on the VDAC1 target. The ability of candidate drugs to prevent or treat RSV-related diseases can be determined based on their inhibitory effect on the VDAC1 target. For example, if a candidate drug has extremely high activity in inhibiting the VDAC1 target, it can be preliminarily classified as a drug that can treat RSV-related diseases.

[0024] Diseases caused by RSV include, but are not limited to, bronchiolitis, pneumonia, laryngotracheobronchitis, otitis media, and upper respiratory tract infections in infants and children. It can cause the common cold in adults and pneumonia and severe bronchitis in the elderly.

[0025] In a preferred embodiment of the present invention, the substance targeting VDAC1 is selected from substances that inhibit the activity and / or expression level of VDAC1 protein or substances that inhibit the expression of the VDAC1 gene.

[0026] In a preferred embodiment of the present invention, the substance that inhibits VDAC1 gene expression is selected from: substances that inhibit VDAC1 transcription, substances that degrade VDAC1 transcripts, or substances that inhibit VDAC1 translation.

[0027] In a preferred embodiment of the present invention, the substance that inhibits VDAC1 gene expression is selected from at least one of small molecule compounds, proteins, peptides, nucleic acid molecules, and recombinant vectors, recombinant cells, and recombinant bacteria containing nucleic acid molecules, and the nucleic acid molecules are selected from at least one of miRNA, siRNA, sgRNA, shRNA, and nucleic acid aptamers.

[0028] In a preferred embodiment of the present invention, the substance targeting VDAC1 is selected from VDAC1 inhibitors, which are selected from DIDS, VBIT-4, VBIT-12, superoxide dismutase, cyclosporine A, TRO19622, Bcl-x(L)BH4(4-23), isolated double-stranded RNA molecules that inhibit VDAC1 expression, or any random phosphate-thionucleotide.

[0029] In a preferred embodiment of the present invention, the nucleotide sequence of one strand of the isolated double-stranded RNA molecule that inhibits VDAC1 expression is as shown in any one of SEQ ID NO: 1-4.

[0030] In a preferred embodiment of the present invention, the drug also includes pharmaceutically acceptable excipients.

[0031] In a preferred embodiment of the present invention, the excipients include, but are not limited to, conventional pharmaceutical excipients, carriers, or diluents.

[0032] Drugs also include pharmaceutically acceptable carriers, such as water, saline, sugars, polysaccharides, buffers, excipients, biodegradable polymers, liposomes, stabilizers, and other substances.

[0033] Pharmaceutically acceptable excipients include, but are not limited to, fillers, lubricants, disintegrants, binders, and flow aids.

[0034] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, hydroxypropyl methylcellulose, starch and its derivatives, polyethylene glycol and its derivatives, lactose, lactose-starch complex, lactose-cellulose complex, sucrose, mannitol, mannitol-starch complex, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, povidone, copovidone, calcium hydrogen phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, low-substituted hydroxypropyl cellulose, croscarmellose sodium cellulose, croscarmellose, magnesium stearate, sodium stearate fumarate, talc, and stearic acid, or a combination thereof.

[0035] In an alternative embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, and gelling agents (e.g., methylcellulose), as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the reagent selected.

[0036] In a preferred embodiment of the present invention, the dosage form of the drug is tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection, spray or injection.

[0037] In one embodiment, a substance targeting VDAC1 may be contained in a gel.

[0038] Secondly, the present invention also provides the application of VDAC1 protein in the preparation of respiratory syncytial virus infection models.

[0039] In one embodiment, a respiratory syncytial virus (RSV) infection model that significantly enhances RSV titers can be obtained by overexpressing the VDAC1 protein in host cells and then infecting the VDAC1-overexpressing cells with RSV virus. Those skilled in the art can also screen for corresponding anti-RSV drugs based on this RSV infection model.

[0040] Thirdly, the present invention also provides a double-stranded RNA molecule that inhibits VDAC1 expression, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0041] Fourthly, the present invention also provides a medicament for the prevention or treatment of diseases caused by RSV, comprising the aforementioned double-stranded RNA molecule that inhibits VDAC1 expression.

[0042] In a preferred embodiment of the present invention, the drug also includes pharmaceutically acceptable excipients.

[0043] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0044] Example 1 This embodiment provides a cell experiment demonstrating the high efficacy of the VDAC1-specific inhibitor DIDS in inhibiting RSV infection in HEp-2 and A549 cells. DIDS (CAS No. 67483-13-0, HY-D0086, purity = 99.13%) was purchased from MedChemExpress Co., Ltd., Shanghai, China.

[0045] HEp-2 or A549 cells were co-incubated with DIDS, a specific inhibitor of VDAC1, for 48 h, and total cellularity (TC) was measured by the MTT assay. 50 Cells were then infected with RSV at an MOI of 0.1 for 1 hour, washed with PBS, and treated with different concentrations of DIDS. The supernatant was collected 48 hours post-infection (hpi) and analyzed using TCID50. 50 The viral titer was determined by a method.

[0046] To determine the cytotoxic concentration of DIDS in our experiments, we first used the MTT assay to determine the half-maximal cytotoxic concentration (TC) of DIDS in HEp-2 and A549 cells. 50 )value. Figure 1 The results showed that DIDS inhibited TC in HEp-2 and A549 cells. 50 The values ​​were 6109.65 μM and 4641.02 μM, respectively. Figure 1 (The top right corner of diagrams A and B).

[0047] Based on TC 50As a result, this embodiment detected the virus via TCID 48 hours after infection (hpi). 50 The anti-RSV infection activity of DIDS was evaluated experimentally. The results showed that DIDS significantly inhibited RSV infection in both cell types in a dose-dependent manner, with a half-maximal inhibitory concentration (IC50) of [missing value]. 50 The concentrations were 17.85 μM and 26.41 μM in HEp-2 and A549 cells, respectively. Figure 1 (See the bottom left figures for A and B). Therefore, the anti-RSV treatment index (SI) of DIDS in HEp-2 and A549 cells were 342.28 and 175.73, respectively. The TC of DIDS... 50 With IC 50 The results are reliable (R 2 All are greater than 0.95).

[0048] Pre-incubate 160 μM, 320 μM, and 640 μM DIDS with RSV at 37°C for 1 h (virus pretreatment) or add ( ) 1, 12, or 24 h post-infection. Figure 1 (Figure C in the image), the cells are HEp-2, and at 48 hpi, via TCID... 50 The viral titer was determined by a method.

[0049] Figure 1 The results of the D-plot suggest that DIDS exerts its antiviral effect in the early stages of viral infection. This suggests that its anti-RSV mechanism may be achieved by targeting host factors active in the early stages of infection.

[0050] Example 2 Given the significant anti-RSV infection effect of the VDAC1-specific inhibitor DIDS, we hypothesize that VDAC1 is a proviral host factor in RSV infection. To verify this hypothesis, this study investigated the changes in VDAC1 expression over 48 hours at different multiplici of infection (MOIs) (0.05 and 0.1).

[0051] The specific steps are as follows: HEp-2 cells were infected with RSV at different MOIs (0.05 or 0.1) for 48 h, and the expression level of VDAC1 in the cell lysate was analyzed by Western blot. Figure 2 Figure A in the results shows that VDAC1 expression exhibits a significant viral dose-dependent upregulation.

[0052] To elucidate the role of VDAC1 in RSV infection, this example utilizes siRNA ( si-VDAC1-1778 Knock down VDAC1 expression.

[0053] Table 1. si-VDAC1 sequences

[0054] HEp-2 cells were transfected with different concentrations of VDAC1-specific siRNA (si-VDAC1-1778) or negative control siRNA (si-NC) for 24 hours, followed by infection with 0.1 MOI RSV for 48 hours. The VDAC1 knockdown effect was confirmed by Western blot and analyzed using TCID50. 50 Methods for assessing RSV titers.

[0055] This silencing treatment significantly suppressed RSV titers in a dose-dependent manner. Figure 2 (B)

[0056] In addition, this embodiment constructed a VDAC1 eukaryotic expression plasmid (pcDNA3.1-VDAC1) carrying a Flag tag.

[0057] HEp-2 cells were transfected with 2.5 μg of VDAC1 expression plasmid (pcDNA3.1-VDAC1) or empty vector (Vehicle). VDAC1 overexpression was confirmed by Western blot after 48 hpi. RSV was then infected at 0.1 MOI for 48 h, and the cells were analyzed using TCID45. 50 The method for determining RSV titers revealed that VDAC1 is an important factor in RSV infection.

[0058] Figure 2 The results in Figures C and D show that overexpression of VDAC1 in HEp-2 cells significantly enhances RSV titer.

[0059] Example 3 In RSV infection, viral membrane protein fusion (F) proteins play an important role in the binding of the virus to the host cell receptor in the early stage of infection, forming a syncytia body. Therefore, we performed immunofluorescence-laser confocal microscopy experiments 24 hours after transfecting HEp-2 cells with pcDNA3.1-VDAC1.

[0060] The experimental steps are as follows: HEp-2 cells were transfected with 2.5 μg pcDNA3.1-VDAC1. After 24 h, cells were infected with 0.1 MOI RSV. After 48 h, cells were fixed with PFA, permeabilized with 0.02% Triton X-100, and then blocked using immunofluorescence blocking solution. Cells were stained with VDAC1 antibody (red fluorescence) and RSV F protein antibody (green fluorescence), respectively, and observed and imaged using a laser confocal microscope. Scale bar = 10 μm.

[0061] The results showed that VDAC-1 could co-localize with F protein on the outer side of the cell nucleus. Figure 3 This result indicates that VDAC-1 can bind to the viral F protein, suggesting that VDAC-1 may be the receptor protein for RSV infection of host cells.

[0062] In previous literature, Tayyari et al. reported that nucleolin (NCL) is a functional receptor for RSV infection of cells. Therefore, we hypothesized that VDAC-1 may bind to NCL and mediate RSV infection of cells. Thus, we conducted a co-immunoprecipitation (Co-IP) experiment in cells.

[0063] The experimental steps are as follows: HEp-2 cells were transfected with 2.5 μg pcDNA3.1-VDAC1-Flag for 24 h. Cells were lysed and protein samples were collected. A portion of the samples were used to verify the expression levels of VDAC1 and NCL by Western blot, with β-actin as an internal control. The other portion of the samples were enriched with magnetic beads to collect proteins carrying the Flag tag, and the expression levels of VDAC1 and NCL were verified by Western blot. An empty plasmid transfection group (Vehicle) was set up as a control group.

[0064] Figure 4 The results indicate that VDAC-1 and NCL did not bind.

[0065] Based on the above results, VDAC1 may be a novel RSV cell receptor independent of NCL.

[0066] Example 4 Animal anti-RSV infection experiments of DIDS.

[0067] The percentage change in body weight and survival rate of mice in the control group (Mock), GZ08-18 + saline group (n=9), GZ08-18 + 12.5 mg / kg DIDS group (n=9), GZ08-18 + 25.0 mg / kg DIDS group (n=9), and GZ08-18 + 25.0 mg / kg ribavirin group (n=9) were recorded. Mice in the GZ08-18 + saline group, GZ08-18 + 12.5 mg / kg DIDS group, GZ08-18 + 25.0 mg / kg DIDS group, and GZ08-18 + 25.0 mg / kg ribavirin group were inoculated intratracheally (it) with GZ08-18 virus (1×10⁻⁶ dose per inoculation) every 10-12 hours for 2 consecutive days. 10 Half-maximum tissue culture infection dose, TCID50 The mice were vaccinated a total of 3 times. One hour after challenge with GZ08-18, they were treated with DIDS or ribavirin via intraperitoneal injection (ip), also with a 3-dose regimen. Daily changes in mouse body weight were recorded. Figure 5 (Left of Figure A) and survival rate ( Figure 5 (The right image of Figure A in the middle), and statistical analysis was performed using the Log-rank test. **p<0.01, *p<0.001.

[0068] Viral titers were determined in the GZ08-18 + saline group, GZ08-18 + 12.5 mg / kg DIDS group, GZ08-18 + 25.0 mg / kg DIDS group, and GZ08-18 + 25.0 mg / kg ribavirin group. Mice were sacrificed 3 days post-infection (dpi), and bronchoalveolar lavage fluid (BALF) samples were collected and analyzed using TCID45. 50 The experiment quantitatively analyzed viral load. The dashed line represents TCID. 50 The lower limit of detection for the experiment. Data are expressed as mean + standard deviation (mean + SD) (n=3 per group).

[0069] The results showed that DIDS treatment significantly inhibited lethal respiratory syncytial virus (RSV) infection in mice.

[0070] This invention provides a highly effective and safe drug for treating RSV, alleviating the social burden caused by RSV infection. The significance of this invention lies in the original identification of a novel cellular receptor for RSV infection. This discovery provides the molecular basis for the development of novel antiviral drugs and is crucial for the development of new drugs.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0072] [1] Agac A, Kolbe SM, Ludlow M, Osterhaus A, Meineke R, RimmelzwaanGF. 2023. Host Responses to Respiratory Syncytial Virus Infection. Viruses15. [2] Savic M, Penders Y, Shi T, Branche A, Pirçon JY. 2023.Respiratory syncytial virus disease burden in adults aged 60 years and olderin high-income countries: A systematic literature review and meta-analysis.Influenza Other Respir Viruses 17:e13031. [3] Tayyari F, Marchant D, Moraes TJ, Duan W, Mastrangelo P, HegeleRG. Identification of nucleolin as a cellular receptor for human respiratorysyncytial virus. Nat Med. 2011 Aug 14;17(9):1132-5. [4] Mastrangelo P, Chin AA, Tan S, Jeon AH, Ackerley CA, Siu KK, LeeJE, Hegele RG. Identification of RSV Fusion Protein Interaction Domains onthe Virus Receptor, Nucleolin. Viruses. 2021 Feb 8;13(2):261. [5]Griffiths CD, Bilawchuk LM, McDonough JE, Jamieson KC, Elawar F,Cen Y, Duan W, Lin C, Song H, Casanova JL, Ogg S, Jensen LD, Thienpont B,Kumar A, Hobman TC, Proud D, Moraes TJ, Marchant DJ. IGF1R is an entryreceptor for respiratory syncytial virus. Nature. 2020 Jul;583(7817):615-619. [6]Mastrangelo P, Norris MJ, Duan W, Barrett EG, Moraes TJ, HegeleRG. Targeting Host Cell Surface Nucleolin for RSV Therapy: Challenges andOpportunities. Vaccines (Basel). 2017 Sep 19;5(3):27。

Claims

1. The application of substances targeting VDAC1 in the preparation of products, characterized in that, The product has at least one of the functions of A1) to A5): A1) Prevention of diseases caused by respiratory syncytial virus (RSV); A2) Treatment of diseases caused by RSV; A3) Inhibits RSV virus replication; A4) Enhances the cell viability of hosts infected with RSV; A5) Screen for drugs to prevent or treat diseases caused by RSV.

2. The application according to claim 1, characterized in that, The substances targeting VDAC1 are selected from substances that inhibit the activity and / or expression of VDAC1 protein or substances that inhibit the expression of the VDAC1 gene.

3. The application according to claim 2, characterized in that, The substance that inhibits VDAC1 gene expression is selected from substances that inhibit VDAC1 transcription, substances that degrade VDAC1 transcripts, or substances that inhibit VDAC1 translation.

4. The application according to claim 1, characterized in that, The substance targeting VDAC1 is selected from VDAC1 inhibitors, which are selected from DIDS, VBIT-4, VBIT-12, superoxide dismutase, cyclosporine A, TRO19622, Bcl-x(L)BH4(4-23), isolated double-stranded RNA molecules that inhibit VDAC1 expression, or any random phosphate-thionucleotide.

5. The application according to claim 4, characterized in that, The nucleotide sequence of one strand of the isolated double-stranded RNA molecule that inhibits VDAC1 expression is shown in any one of SEQ ID NO: 1-4.

6. The application according to any one of claims 1-5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. Application of VDAC1 protein in the preparation of respiratory syncytial virus infection model.

8. A double-stranded RNA molecule that inhibits VDAC1 expression, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

9. A medicine for preventing or treating diseases caused by RSV, characterized in that, It includes the double-stranded RNA molecule that inhibits VDAC1 expression as described in claim 8.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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