Screening Regulators Of Disease-associated Nucleic Acid Molecule-mediated Innate Immunity And The Underlying Mechanism

Background:The recognition of DNA and RNA by intracellular nucleic acid receptors constitutes the core element for the innate immune system to detect the invasion of exogenous pathogenic microorganisms and the appearance of endogenous abnormal nucleic acids.However,the ability of these sensors to distinguish between exogenous and endogenous nucleic acids is limited.Diseases usually occur and develop along with the production of related nucleic acid molecules.Whether in virus infection or autoimmune disease,it is usually accompanied by the production of a large number of abnormal DNA or RNA to activate the host innate immune response.The development of high-efficient and low-toxic regulators that inhibit antiviral immunity or autoimmunity is an important way to treat related diseases.In this study,SARS-Co V-2was used as the model for studying the intracellular RNA occurrence and the autoimmune disease Aicardi-Goutières syndrome(AGS)was used as the model for studying the intracellular DNA occurrence,the regulators were screened and the related mechanism studies were conducted.Virus infection is one of the most common reasons of exogenous nucleic acid into cells.Among them,Severe Acute Respiratory Syndrome Coronavirus 2(SARS-Co V-2)is a single-stranded RNA virus,which produces a large amount of viral genomic RNA during virus replication,and causes the antiviral immune response of host cells.SARSCo V-2 Nucleocapsid(N)protein is one of the key structural proteins of virus,which recognizes and encapsulates a large number of virus RNA in cytoplasm.Therefore,the mechanism between N protein and viral RNA needs to be deeply studied in order to develop specific antiviral inhibitors against SARS-Co V-2 from new mechanisms of N protein.One of the important defenses of host against the infection of pathogenic microorganisms is to product interferon and activate its downstream signaling pathway.In addition,cells also encode a series of nucleases involved in the normal metabolism of nucleic acids,which play a vital role in maintaining the metabolic homeostasis of nucleic acids in cells.However,loss-of-function mutations of some nucleases will destroy the homeostasis of nucleic acid metabolism in cells and excessive accumulation of endogenous nucleic acids will occur.Nucleic acids are immunogenic in cells,and excessive endogenous nucleic acids will continue to activate the expression of interferon and related genes,just like exogenous nucleic acids,and eventually lead to autoimmune diseases.The autoimmune disease AGS usually caused by single gene mutation is one of these autoimmune diseases.The three-prime nucleic acid exonuclease 1(TREX1)deletion in AGS patients will lead to the accumulation of abnormal self-DNA in the cytoplasm and causes the excessive autoimmune response.Therefore,it is particularly important to study how the host recognizes and responds to endogenous DNA and screen regulators that can inhibit the autoimmune response caused by endogenous DNA.Objective:(1)Screening of small molecular compounds that inhibit viral RNA-triggered phase separation of SARS-Co V-2 N protein.(2)Screening of non-steroidal anti-inflammatory drugs that inhibit endogenous DNA-mediated AGS-related autoimmune response.Methods:(1)Screening of small molecular compounds inhibiting SARS-Co V-2 N protein phase separation and its mechanism.Phase separation of SARS-Co V-2 N protein was detected and small molecular inhibitors that inhibit phase separation of N protein were screened by immunofluorescence.Ability of recognizing RNA and producing antiviral interferon in the presence of SARS-Co V-2 N protein was detected by q PCR.Interaction between candidate compound and N protein was verified by CNBr-agarose beads and EMSA.Inhibitory effect of candidate compound on N protein-mediated phase separation and virus replication was verified by live SARS-Co V-2 virus infection assay.(2)Screening of drugs inhibiting endogenous DNA-mediated autoimmune response in AGS and its mechanism.NSAIDs that can inhibit DNA-mediated autoimmune response were screened and tested in different cells with various types of DNA by q PCR.The mechanism of candidate drug was verified by Western Blot,CNBr-agarose beads,Native PAGE and immunofluorescence.The therapeutic effect of candidate drug on AGS was tested by using cell and animal models with TREX1 deletion.Results:GCG inhibits SARS-Co V-2 replication by disrupting the phase separation of N protein.(1)SARS-Co V-2 N protein undergo phase separation under the existence of RNA;(2)SARS-Co V-2 N protein inhibits the antiviral immune response;(3)GCG inhibits RNA-mediated phase separation of SARS-Co V-2 N protein;(4)GCG directly binds to SARS-Co V-2 N protein;(5)GCG suppresses SARS-Co V-2 replication by disrupting the phase separation of N protein;Meloxicam inhibits STING phosphorylation and alleviates intracellular DNAmediated autoimmune responses.(1)Meloxicam inhibits DNA-mediated autoimmune response;(2)Meloxicam inhibits different types of DNA-mediated autoimmune responses in various cells;(3)Meloxicam works by inhibiting the phosphorylation of STING;(4)Meloxicam does not directly target on STING;(5)Meloxicam alleviates the symptoms of AGS caused by TREX1 deletion.Conclusions:In this study,we screened small molecule compounds based on the pathogenic process of disease-related nucleic acid molecules,and aimed to develop novel regulators for the prevention and treatment of related diseases.We found that RNA triggers the phase separation of SARS-Co V-2 N protein.Using RNA-induced N protein phase separation as a model,small molecule compounds screening was conducted,and an inhibitor GCG inhibiting N protein phase separation was found to inhibit SARSCo V-2 assembly and replication.In addition,the non-steroidal anti-inflammatory drug meloxicam in inhibiting cytoplasmic DNA-induced autoimmune response was found and the related molecular mechanism was also revealed.We found that meloxicam inhibited the activation of c GAS-STING signaling pathway by inhibiting the phosphorylation of STING,which is a key molecule in the DNA recognition.Further experiments showed that the meloxicam could be used to relieve AGS-related autoimmune response induced by endogenous DNA.In summary,this study can provide more theoretical basis and potential drug treatment options for antiviral immunity and autoimmune diseases.