Study On The Activity And Mechanism Of Anthraquinone Compound D1996 Against Zika Virus

Zika virus is an arbovirus belong to the Flaviviridae family.Infected with Zika virus may cause serious life-threatening neurological diseases,such as meningitis,Guillain-Barré syndrome and congenital Zika virus syndrome,etc.Until now,Zika virus has spread in 87 countries and regions in the tropics and subtropics.The World Health Organization declared the Zika virus outbreak as a public health emergency of international concern in February 2016.However,there are currently no antiviral drugs against Zika virus in clinic.Therefore,with the purpose of discovering drug precursors of anti-Zika virus,we screened the small molecule targeted inhibitors with anti-Zika virus activity,and explained the mechanism of anti-Zika virus.It has important theoretical and clinical significance for the further development of anti-Zika virus targeted drugs.In this study,the compounds with anti-Zika virus were screened by plaque reduction assay,and the cytotoxicity of the compounds with anti-Zika virus was detected by tetramethylazolyl blue colorimetry.As a result,the optimized compounds with strong anti-Zika virus activity and low cytotoxicity were obtained.This study found that 24 new structural compounds have anti-Zika virus activity,and 19 optimized compounds with strong activity of anti-Zika virus and low cytotoxicity were obtained,of which 13 compounds belonged to anthraquinone derivatives.The inhibitory effect of D1996 on Zika virus infection rate,titer,protein and RNA copy number was detected by immunofluorescence assay,plaque reduction assay,western blotting assay and real-time fluorescence quantitative polymerase chain reaction assay,respectively.The inhibitory effect of D1996 on pyroptosis-related genes and proteins was detected by real-time fluorescence quantitative polymerase chain reaction assay and western blotting assay,respectively.The inhibitory effect of D1996 on the release of lactate dehydrogenase from cells was detected by lactate dehydrogenase detection assay.D1996 combined with RNA-dependent RNA polymerase were simulated by molecular operating environment software and verified by surface plasmon resonance assay.The inhibitory effect of D1996 on the release of nitric oxide from cells was detected by Nitric oxide assay.This study found that D1996 could inhibit Zika virus infection,and inhibit the expression of Zika virus non-structural protein 5(NS5)and Zika virus genome RNA replication,and had weak cytotoxicity and significant anti-Zika virus activity in vitro.In the study of the mechanism against Zika virus,D1996 can directly bind to the Rd Rp of Zika virus.It is speculated that D1996 may cause the conformational change of Rd Rp,thereby inhibiting the replication of Zika virus RNA,thus showing the activity of inhibiting Zika virus.At the same time,this study found that D1996 can not only inhibit the up-regulation of inflammatory factors and pyroptosis-related genes caused by Zika virus infection,but also inhibit a series of pyroptosis phenomena caused by Zika virus infection,including inhibiting the activation of Caspase-1 and Gasdermin D,the release of lactate dehydrogenase from cells,indicating that D1996 has the effect of inhibiting pyroptosis caused by Zika virus.In addition,this study also found that D1996 can also inhibit the release of nitric oxide from cells,suggesting that D1996 can inhibit cell inflammation.In summary,24 new structural compounds were found to have anti-Zika virus activity for the first time,among which the anthraquinone compound D1996 was the one with the strongest anti-Zika virus activity and less cytotoxicity.Further studies have found that the anthraquinone compound D1996 inhibits Zika virus activity by directly binding to Zika virus NS5,and can inhibit the pyroptosis and inflammation caused by Zika virus.D1996 is expected to be a candidate drug against Zika virus.This study provides a theoretical basis for the in-depth research on druggability of D1996.