| Background and AimSevere acute respiratory syndrome coronavirus 2（SARS-CoV-2）is highly infectious,rapidly changing,and still mutating,and Corona Virus Disease 2019（COVID-19）caused by SARS-CoV-2 infection poses a huge threat to health and life and a heavy economic burden to the world.The multinational Delphi consensus suggests that the development of new therapies and a focus on vaccination could contribute to an effective response to the still raging epidemic.Vaccination significantly reduces the risk of SARS-CoV-2 infection,COVID-19 severity,and incidence of long COVID syndrome.However,as variants of concern continues to evolve in terms of transmissibility,virulence,and immune escape,the protective effect of infection or vaccination declines dramatically.Studies have shown that the vaccine efficacy（VE）of complete vaccination against Alpha,Beta,Gamma,Delta,and Omicron was 88.0%,73.0%,40.6%-63.0%,77.8%,and 50.12%,respectively.Notably,the enhanced immune escape of the variants rendered most of the previously developed monoclonal antibody against the original strains ineffective.Therefore,in order to effectively combat the widespread epidemic of the new mutant strain,it is crucial to actively develop effective and broad-spectrum direct antiviral agents against SARS-CoV-2variants,in addition to emphasizing vaccination and developing broad-spectrum neutralizing antibodies or variants specific monoclonal antibodies.The approved orally direct antiviral agents against SARS-CoV-2 are mainly divided into RNA-dependent RNA polymerase（Rd Rp）inhibitors（Azvudine,VV116 and Molnupiravir）and 3CL protease（3-CLpro）inhibitors(Paxlovid TM,SIM0417).However,these drugs have some shortcomings such as reduced effectiveness against variants,potential teratogenic effects,reversion to positive nucleic acid after conversion,and unknown adverse effects.In addition,other direct antiviral agents such as angiotensin-converting enzyme 2（ACE2）inhibitors（chloroquine and hydroxychloroquine）and Abidor,which inhibits the binding of viral lipid membranes to host cells,have been discontinued for the clinical treatment of COVID-19 due to their ineffectiveness in clinical trials.Therefore,safer,more effective,and more targeted antiviral drugs need to be developed in the face of the global SARS-CoV-2 epidemic.Novel drug development,which often requires a large budget and time investment,and drug repurposing can accelerate the drug development process by identifying new usages for approved drugs,starting with compounds with favorable pharmacological profiles and safety profiles,and significantly reducing the incidence of adverse reactions at the clinical stage.Drug repurposing has been used to screen therapeutic drug candidates for a variety of emerging pathogens.Therefore,in order to develop safe and effective direct antiviral agents,we evaluated the antiviral activity of selective estrogen receptor modulators（SERMs）and type Ⅲ interferons in vitro and in vivo using a drug repurposing strategy,and the role of SERMs in the life cycle of SARS-CoV-2 was explored by quantitative proteomics and the antiviral mechanism of type Ⅲ interferon in different cells were explored by transcriptomics,in order to further understand the pathogenic mechanism of SARS-CoV-2 and provide new targets for antiviral drug development.Part Ⅰ Study on the effect and mechanism of selective estrogen modulators against SARS-CoV-2 infectionSERMs are different from simple estrogen receptor（ER）agonists or antagonists in that they selectively act on ER in different tissues,producing hormone-like or anti-estrogenic effects in different target tissues,respectively.SERMs have been widely used in the clinic due to their excellent effects in the treatment of breast cancer and osteoporosis.Several SERMs have been reported to exhibit good antiviral activity,such as Cyclofenil（Cyclofenil）,which inhibits the replication of dengue and Zika viruses,Raloxifene hydrochloride（RLX）,Toremifene（TOR）and Clomifene citrate（CC）,which exhibited strong anti-Ebola virus activity.At the beginning of the pandemic,our group screened more than 90 small molecules from the Food and Drug Administration（FDA）approved drug library containing 2580 small molecules and they can effectively inhibit SARS-CoV-2 infection,including the selective estrogen receptor modulators（SERMs）Bazedoxifene Acetate（BZA）and Tamoxifen（TAM）,based on the principle of drug repurposing,which suggesting that SERMs may have good anti-SARS-CoV-2 infection activity.Therefore,we propose to investigate the anti-SARS-CoV-2 infection effects of different types of SERMs and evaluate the antiviral effects of SERMs in vivo and in vitro using cellular models as well as hamster infection models.And we also propose to clarify the key stages and target molecules of SERMs by exploring their role in the life cycle of SARS-CoV-2 and their effects on cell biological functions,and to elucidate their mechanism of action in inhibiting viral infection.Methods1.Evaluate the antiviral activity of SERMs in vitro using different cellular models of SARS-CoV-2 infection.2.Establish an animal model of SARS-CoV-2 infection.3.Evaluate the antiviral activity of SERMs in vivo using hamster infection model.4.Determine whether SERMs depend on the ER pathway to exert anti-SARS-CoV-2effects by examining the effects of estrogen receptor agonists（estradiol）and inhibitors（fulvestrant）on SARS-CoV-2 infection,and the effects of SERMs on ER expression.5.The effects of SERMs on the life cycle of SARS-CoV-2 were analyzed by time-of-drug-addition assay and time course analysis of drug.6.Analyze the role of SERMs in the early life stage of SARS-CoV-2 by pseudovirus-based inhibition assay.7.Determine the effects of BZA,CC,TAM and TOR on ACE2 expression in target cells.8.The effect of BZA,CC,TAM and TOR on the direct binding of ACE2 to S protein was determined by enzyme-linked immunosorbent assay（ELISA）and Biacore analysis.9.The effects of BZA,CC,TAM and TOR on specific stages of virus invasion were determined by virus attachment/penetration and drug blocking assays.10.Determine the effect of BZA on SARS-CoV-2 S-mediated membrane fusion by membrane fusion and drug blocking experiments.11.Quantitative proteomics were utilized to explore the pathways and specific targets on which BZA exerts its antiviral effects.12.The effect of BZA on cholesterol distribution was clarified by Filipin Ⅲ staining.13.The effect of BZA on endosomal acidification was clarified by acid lysosome assay.14.Knocking down the expression of ATG16L1 using small interfering RNA to detect the effect of ATG16L1 on the antiviral activity of BZA.15.To clarify the broad-spectrum antiviral activity of BZA by detecting its inhibition on infection of pseudoviruses（SARS-CoV,Delta and Omicron variants）,SARS-CoV-2Omicron,West Nile virus,yellow fever virus and chikungunya virus.Results1.TAM,CC,Lasofoxifene Tartrate（LAS）,BZA,TOR and Ospemifene（OSP）inhibited SARS-CoV-2 infection in a dose-dependent manner in Vero E6,Caco-2,Calu-3and Hela-ACE2 cells.2.Compared with the control group,hamsters in the infected group showed weight loss immediately after infection,viral nucleic acid and protein were positive in the lungs of hamsters,and a large number of inflammatory cell infiltrates could be detected in the lungs of hamsters after infection.3.BZA,CC,TAM and TOR reduced the viral load and relieved histopathological damage in the lungs of infected hamsters.4.Estradiol and fulvestrant had no effect on cells infected with SARS-CoV-2 original strain,and SERMs had no effect on ERαand ERβexpression in Vero E6,Caco-2,Calu-3and Hela-ACE2 cells.5.CC and TAM showed anti-SARS-CoV-2 activity at 0 hpi（hour post infection）,while TOR inhibited viral infection at all time points（0,2,5,8 hpi）,and BZA significantly inhibited viral replication at 0 and 2 hpi,but had no effect at 5 and 8 hpi.6.BZA,CC,TAM and TOR could inhibit SARS-CoV-2 pseudovirus infection in a dose-dependent manner.7.BZA,CC,TAM and TOR treatment had no effect on ACE2 expression in Vero E6,Caco-2,Calu-3 and Hela-ACE2 cells.8.The results of ELISA showed no significant difference in the affinity between ACE2 and S protein among SERMs groups,and Biacore showed TAM had some affinity with S protein due to its higher sensitivity(KD=8.01×10-5M).9.BZA did not affect the attachment of SARS-CoV-2 original strain to Vero E6 or Calu-3 cells,but could significantly inhibit its penetration into host cells.10.BZA pretreatment and full treatment groups both decreased the effect of SARS-CoV-2 S-mediated membrane fusion.11.The results of subcellular localization,GO analysis of cellular components and biological processes,and differential protein interaction network of regulated proteins between different groups showed that BZA was involved in lipid metabolism as well as autophagy and lysosome-related pathways during the infection of SARS-CoV-2 original strain on Calu-3 cells.And the regulated proteins included VPS39,LRP10,DHRS1,FAR1,CYP4F12 and ATG16L1.12.The distribution of cholesterol on the surface of the plasma membrane was significantly reduced after BZA treatment,while larger Filipin Ⅲ-positive vesicles were formed in the cytoplasm.13.The acidification process of intracellular endosomes was significantly reduced after BZA treatment of cells.14.The BZA-treated group could inhibit about 90%of SARS-CoV-2 infections,while after knockdown of ATG16L1 expression,BZA could only inhibit about 60%of viral infections.15.BZA had good inhibitory effect against infection of pseudovirus（SARS-CoV,Delta and Omicron variants）,Omicron BA.5 and BF.7,and chikungunya virus infection in vitro.ConclusionsBZA,CC,TAM and TOR inhibited SARS-CoV-2 infection in vitro in a dose-dependent manner,effectively relieved pulmonary inflammation and reduced viral load in the lungs of infected hamsters.BZA could exert antiviral effects by altering lipid distribution and endosomal acidification of host cells to affect viral membrane fusion processes and block viral penetration into host cells.In addition,BZA broadly inhibits the infection of pseudoviruses（SARS-CoV,Delta and Omicron variants）,Omicron BA.5 and BF.7,and chikungunya in vitro.Part Ⅱ 4D Study on the effect and mechanism of type Ⅲ interferon against SARS-CoV-2 infectionInterferons are a class of cytokines with broad-spectrum antiviral activity and can be classified as type I,type Ⅱ and type Ⅲ,with type Ⅲ interferons comprising four subtypes（interferonsλ1-4）.Type I and type Ⅲ interferons are the main antiviral cytokines produced by the host cells.Studies have shown that a sustained type I interferon response can lead to deleterious pro-inflammatory effects and is therefore risky for the treatment of SARS-CoV-2 infection,an acute respiratory disease that is prone to"cytokine storm",whereas type Ⅲ interferon response occurs in the epithelium and induces local antiviral immunity,inhibiting viral replication while preventing the development of cytokine storm,thus preventing patients from progressing to severe disease.Therefore,this study is intended to evaluate the antiviral effect and mechanism of type Ⅲ interferon in different target cells of SARS-CoV-2,to further investigate the pathogenic mechanism of SARS-CoV-2 infection,and to provide novel targets for antiviral drug research.Methods1.Evaluation of the inhibitory activity of type Ⅲ interferon against SARS-CoV-2original strain and Omicron BA.2 using different cellular models of SARS-CoV-2 infection.2.Evaluation of the preventive/protective effect of type Ⅲ interferon against Omicron BA.2 infection using a K18-h ACE2 transgenic mouse infection model.3.Exploring the targets and pathways of antiviral effects of type Ⅲ interferon in human cell lines through transcriptomic sequencing by treating Hela-ACE2,Huh 7 and Calu3 cells with interferon,respectively.4.Mice were treated with type Ⅲ interferon and infected with Omicron BA.2,and transcriptomic sequencing was used to explore the altered transcript levels in the nasal turbinates.5.Validation of the effect of type Ⅲ interferon on the expression of intracellular ISGs by qRT-PCR.6.Validation of the effect of type Ⅲ interferon on Isg15 expression in mouse tissues by qRT-PCR.Results1.Type Ⅲ interferon effectively inhibited the replication of the SARS-CoV-2 original strain and Omicron BA.2 in Hela-ACE2 cells while exhibited no inhibition on the infection of SARS-CoV-2 original strain on Calu-3 cells.2.Type Ⅲ interferon treatment significantly reduces the viral loads of SARS-CoV-2in the nasal turbinates,lungs and brain of the infected mice.3.Transcriptomic results showed that type Ⅲ interferon treatment upregulated the expression of various interferon-stimulated genes（including ISG15,IFIT1,MX1 and OAS3）in Hela-ACE2 and Huh 7 cells,but had no significant effect on Calu-3 cells.4.Transcriptomic results of mouse nasal turbinates showed that type Ⅲ interferon upregulated multiple interferon-stimulated genes,but only Isg15 was upregulated by Omicron BA.2 infection.5.Infection of SARS-CoV-2 original strain on Hela-ACE2 cells had no effect on ISGs expression,while type Ⅲ interferon treatment of Hela-ACE2 cells promoted transcription of ISG15 and IFIT1 genes.6.Calu-3 cells treated with type Ⅲ interferon had no effect on the expression of ISG15,IFIT1,MX1 and OAS3.7.Isg15 levels in the nasal turbinate,lung and brain tissues of mice were measured on day 5 after infection,and the results showed that compared with the infected control group,Isg15 levels in the nasal turbinate of mice treated with low/medium/high dosage of type Ⅲ interferon were significantly increased,while there were no changes in the lung and brain of the mice.ConclusionsType Ⅲ interferon treatment of Hela-ACE2 cells activates ISGs and inhibits infection of SARS-CoV-2 original strain and Omicron BA.2.Type Ⅲ interferon also protects K18-h ACE2 transgenic mice from Omicron BA.2 infection.Part Ⅲ SummaryIn this study,we evaluated the anti-SARS-CoV-2 activity of SERMs and type Ⅲ interferons in vitro and in vivo through a drug repurposing strategy,and further investigated their specific antiviral mechanisms through quantitative proteomics and transcriptomics,respectively.1.The anti-SARS-CoV-2 activity of BZA,CC,TAM and TOR were evaluated for the first time in vitro and in vivo,and BZA also inhibited the infection of pseudoviruses （SARS-CoV,Delta and Omicron variants）,Omicron BA.5 and BF.7 and chikungunya virus in vitro.2.The regulated proteins in Calu-3 cells under BZA treatment and SARS-CoV-2 original strain infection were systematically investigated in this study,and a more comprehensive understanding of the mechanism of anti-SARS-CoV-2 effect of BZA was obtained.3.We have confirmed for the first time that BZA can inhibit the cell membrane fusion process by altering the lipid distribution and endosomal acidification of host cells,and exert antiviral effects at the virus penetration into target cells,revealing related mechanism on broad-spectrum antiviral activity of BZA.4.In this study,we first investigated the transcriptomic changes induced by type Ⅲ interferon treatment in different cells and found that type Ⅲ interferon treatment upregulated the expression of various interferon-stimulated genes（including ISG15,IFIT1,MX1 and OAS3）in Hela-ACE2 and Huh7 cells,but had no significant effect on Calu-3 cells.This paper further deepens the understanding of the interaction relationship between SARS-CoV-2 infection and target cells by investigating the mechanism of BZA at the stage of SARS-CoV-2 invasion and the regulation of interferon-stimulated genes in different cells by type Ⅲ interferon,and also provides new strategies and directions for anti-SARS-CoV-2 drug research. |
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