Antiviral Activities And Involved Mechanism Of Action Of Novel Oleanolic Acid Derivatives Against Influenza A Viruses In Vitro

Influenza viruses is a common cause of seasonal influenza.Infected people show mild symptoms such as headache muscle pain and respiratory symptoms,as well as severe complications such as bronchitis and pneumonia.Influenza virus hosts are widespread and spread rapidly.Among them,influenza A virus（IAV）is highly easy to mutate and might lead to high mortality,which is a potential threat to global health.Vaccination has been extensively used to prevent and control influenza.In order to deal with the mutation of IAV,IAV vaccine must be continuously updated.Due to the lag in the development of influenza vaccines,commercial vaccines are usually difficult to prevent influenza outbreak induced by new emerging IAV strains.Anti-influenza drugs that target IAV neuraminidase（NA）,including oseltamivir,zanamivir,and peramivir have been used to prevent and treat influenza for many years.However,reduced efficacy of NA inhibtors has been observed due to increasing drug-resistance.Therefore,it is urgently required to develop novel anti-viral agents preferentially with novel mechanisms of action to combat influenza,especially the highly pathogenic influenza.Natural products have diverse and novel structures and are an important source of lead compounds for the development of new drugs,including antiviral drugs.In the past few years,pentacyclic triterpenoids have been identified as extensive antiviral activities against multiple viruses with novel mechanisms of actions,and therefore attracted increasing attentions.oleanolic acid belongs to pentacyclic triterpenoid compounds,which widely distributes in plants,and has a wide range of biological activities,such as antitumor,antiviral,antibacterial and anti-inflammatory activities.In this study,antiviral activity and cytotoxicity of a series of novel oleanolic acid derivatives was evaluated in A549 cells.Furthermore,the structure-activity relationship of these derivatives,and mechanism of action of representative compound OA-10 was investigated.In this study,the anti-influenza virus activity of a series of new oleanolic acid derivatives was determined by indirect immunofluorescence experiments on A549 cells to obtain EC₅₀,and their cytotoxicity was determined by MTT method to obtain CC₅₀.Among tested compounds,compound OA-10 was identified the most promising derivative with SI>45,and was selected as representative compound for further studies.Using indirect immunofluorescence,endpoint dilution method,and q RT-PCR to determine the antiviral activity of OA-10 on A549 cells for 48 h and the kinetics of virus inhibition kinetics at different time of 24 h,48 h and 72 h,respectively.Indirect immunofluorescence was used to investigate the antiviral activity of OA-10 against different influenza virus subtypes,and the inhibitory effects of OA-10 in different modes of action and different time periods in a single cycle were studied by endpoint dilution method and q RT-PCR.Hemagglutination inhibition,Surface plasmon resonance,Hemolysis inhibition,Computer-aided molecular docking analysis and other experiments were used to explore the anti-viral mechanism of OA-10.The results show that OA-10 exhibited significant antiviral activity against four different subtypes of IAV（H1N1,H5N1,H9N2 and H3N2）replications in A549 cell cultures with EC₅₀ ranging from 6.7μM to 19.6μM and a negligible cytotoxicity(CC₅₀>640μM).OA-10inhibited the virus in a dose-dependent manner from 20μM to 40μM at 48 hpi,and continuously inhibited virus replication from 24 hpi to 72 hpi.OA-10 exhibited significant inhibitory effect in the past-treatment,During the single life-cycle of virus replication,the addition of OA-10 in the period of 0～2 h and 2～4 h,showed reduced viral NP protein level reflected by IFA,which indicates that OA-10 interferes the early stage of IAV replicaton.Further studies showed that OA-10 inhibited acid-induced hemolysis in a dose-dependent manner,with an IC₅₀ of 26μM,and had a weak inhibition on the adsorption of H5 HA to chicken erythrocytes at higher concentrations（≥40μM）.Surface plasmon resonance analysis showed that OA-10 interacted with HA in a dose-dependent manner with the equilibrium dissociation constants（KD）of the interaction of 2.98×10^-12 M.Computer-aided molecular docking analysis suggested that OA-10 might bind to the cavity in the HA1-HA2 interface in the HA stem region.The present study showed that OA-10 has good anti-IAV activity against a variety of strains in vitro.OA-10 interfered the early stage of viral infection,including receptor binding and membrane fusion.It exertd antiviral effects,likely by blocking the conformational changes of the HA2 subunit required for virus fusion with the endosomal membrane and subsequently virus replication.