The Exploration Of New Inhibitors For Sars-CoV-2 Envelope Protein

Envelope protein（E）is the smallest structural protein of coronavirus.E proteins of partial coronavirus participate in viral replication via forming viroporins.Knocking out E protein or using E channel inhibitors could reduce viral replication.SARS-CoV-2 envelope protein（2-E）has high homology with other coronavirus E proteins.It is reported that the transmembrane domain（TMD）of 2-E can form a pore structure.However,the properties and functions of 2-E remain unclear.In first part of this study,we demonstrated that 2-E protein forms a p H-sensitive cation channel in artificial lipid bilayer.Then,we found heterologous expression of2-E channel not only induced cell death,but also provoked robust immune responses in vitro and in vivo.In addition,we obtained two dominant negative mutations(2-E^F4Aand 2-E^T11A)that weakened SARS-CoV-2 replication and virulence by impairing 2-E channel function.Altogether,these results support that 2-E functions as an ion channel physiologically in SARS-CoV-2 pathogenicity.In second part of this study,we found the natural product BE-12（berberine）act as a channel blocker showing cell protection and anti-virus capability.Through cooperation with chemists,we obtained a series of BE-12 derivatives（BE-30-BE-33）.Among them,BE-33 exhibited exceptional antiviral activity against SARS-CoV-2infection,with an IC₅₀ 0.94μM and negligible cytotoxicity.It is noteworthy that the channel inhibition activity,the cell protection activity against 2-E-induced cell death and the antiviral activity in vitro of this class of compounds are positively correlated with each other.Collectively,these results support that inhibition of 2-E channel activity is likely to be a previous unrealized therapeutic strategy for SARS-CoV-2infection treatment.Under comprehensive consideration,we selected BE-33 for further exploring the antiviral activity in vivo.Results showed that BE-33 could not only reduce the virus titer,but also alleviate lung tissue damage and inflammation in vivo.Taken together,2-E channel is a promising antiviral drug target.At last,we built a cell based high-throughput screening assay targeting 2-E channels.Among the tested 4376 compounds,34 hits showing cell protection activity were identified in the primary screening.In the secondary antiviral assay,15compounds were found to reduce SARS-CoV-2 replication.After electrophysiology evaluation,three representatives,wortmannin,proanthocyanidins and veliparib,dose-dependently inhibited SARS-CoV-2 replication,displaying IC₅₀S 1.28,26.1 and92.0mM,respectively.These results further demonstrated that 2-E channel is a potential antiviral drug target and provided potential antiviral candidates against SARS-CoV-2.In conclusion,this study confirmed 2-E channel is a potential anti-SARS-CoV-2drug target and provided a series of antiviral candidates.Indeed,this study proposed a new strategy for COVID-19 treatment.