| Background:Influenza virus can cause an infection disease,which poses a tremendous threat to public health,society safety and economic growth.Additionally,according to annual influenza surveillance data,the peak of the influenza epidemic is between December and January of the following year duo to widely spread,rapid transmission,high morbidity and mortality.More effective prophylactic and therapeutic strategies to combat influenza virus are urgently required.In general,vaccination is the most effective strategy for prophylaxis and treatment influenza infections.However,inactivated and live attenuated influenza virus vaccines often show reduced or even loss of capacity and efficiency of productivity because of antigenic drift and shift.Another treatment strategy is the used anti-influenza viral drugs,which are divided into two classes according to their targets:M2 ion channel blockers and NA inhibitors.However,anti-influenza drugs are faced with the increasingly serious problem of drug resistance due to genomic mutation.Therefore,it is urgently needed to explore anti-influenza drugs with the new mechanism to encounter and conquer the drug resistance problem.Given that influenza viral RNA polymerase mediates viral genome replication and transcription,we want to validate the highly conserved RNA polymerase as drug target.Objectives:In this study,we explore the anti-influenza activity of 1,3-dihydroxy-6-benzo[c]chromenone(D715-2441)and expect to elucidate its mechanism.The purpose of this project is to discover small molecule compounds with independent intellectual property rights and offer a new lead compound for development of novel RNA polymerase inhibitors with broad-spectrum anti-IAV activity,which has great significance to the prevention and treatment of influenza infection in China.Methods:The MTT-based cell activity and plaque formation assays were executed to assess the cytotoxicity of D715-2441 to MDCK and A549 cells and evaluated its antiviral activities and safety index against multiple subtypes of influenza A viruses.Using immunofluorescence microscopy,quantitative real-time PCR(RT-PCR)and western blotting assays determined the inhibitory effect of D715-2441 on influenza A virus replication.H5N1 pseudovirus neutralization assay,haemagglutination(HA)inhibition assay,neuraminidase(NA)inhibition and time-of-addition assays were performed to explore the stages of influenza virus life cycle interfered by D715-2441.The methods of SPR and FP investigated the interaction between D715-2441 and PB2cap proteins.Confocal,mini-replicon assay and molecular docking method were implemented to affirm the target of D715-2441.Results:1.D715-2441 exerted an obvious inhibitory activities against multiple subtypes of influenza A viruses infection,including A/FM-1/47(H1N1),A/Aichi/2/68(H3N2),A/Puerto Rico/8/34(H1N1),Vietnam/1194/2004(H5N1),A/Anhui/1/2013(H7N9),A/PR/8/34(H1N1)with NA-H274Y and the influenza A viruses 690(H3 subtype)which was clinically relevant virus with IC50 value of 1.70±0.02,3.59±1.25,4.30±1.10,3.79±0.49,3.19±0.30,3.36±0.42 and 4.44±0.47 μM,respectively.The timeliness of antiviral activity was effective up to 72 h.2.D715-2441 had no influence on H5N1 pesudovirus entry and neuraminidase activity.3.D715-2441 notably restrained the expression level of influenza virus HA mRNA and NP protein in the 0-5 h post infection.4.The combination treatment of D715-2441 and zanamivir possessed remarkable synergistic antiviral effect.5.D715-2441 decreased influenza vRNP activity and affected PB2 protein localization by binding to influenza PB2cap protein.6.D715-2441 bound to PB2cap via the amino acids residues F323,E361 and K376 displayed>99%conservation,F404 have>98%conservation and H357>92%conservatio.7.D715-2441 increased the expression of antiviral factor Mxl.Conclusion:1.D715-2441 possessed antiviral activities against multiple subtypes of influenza Aviruses and inhibited multiple rounds of virus replication.2.D715-2441 disrupted the early stage of influenza A virus replication.3.D715-2441 suppressed influenza A virus infection by targeting viral PB2cap protein.4.The five amino acids of D715-2441 binding were conservative functional sites ofPB2cap.D715-2441 inhibited the viral transcription and replication by interfering with the combination of the PB2 cap-binding domain and the 5’-capped RNA fragments from host pre-mRNAs.5.D715-2441 up-regulated the expression level of antiviral protein Mxl resulting in the inhibition of virus replication.6.The combination of D715-2441 and zanamivir possessed remarkable synergistic antiviral effect... |
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