Discovery And Mechanism Investigation Of Novel Inhibitors Against DENV By Targeting NS5

Background:Transmitted by the bite of an infected mosquito,Dengue virus,an enveloped positive-sense RNA virus,belong to the Flavivirus genus,Flaviviridae family,is the pathogen of dengue infectious diseases.Infection of dengue virus causes diseases vary from a mild self-limiting dengue fever to severe dengue.It is estimated that there are about 50 million to 100 million cases of dengue fever,500 thousand cases of dengue hemorrhagic fever worldwide,and 25000 of them are incurable.In recent years,the number of dengue fever case is increasing rapidly.This unhealthful infectious disease has threatened the health and safety of 1/3 people worldwide.Prevention,cure and vector control are the major strategies to combat dengue.However,there are no effective antiviral drugs to date.It is urgent to develop potentially therapeutic antiviral drugs.The non-structural protein 5 of DENV is a key enzyme involved in the replication of the virus RNA genome.Its N-terminal domain encodes methyltransferase which involved in RNA cap formation.The C-terminal region comprises RNA-dependent RNA polymerase required for viral RNA synthesis.Both MTase and RdRp activities of dengue virus NS5 play an essential role in virus RNA replication.Objectives:NS5 targets-based screening were preformed to obtain novel non-nucleoside lead compound,which provides a new guide for the development of safe and effective anti-dengue drugs.Methods:(1)Detection of antiviral activity against DENY:Inhibitory effect of compounds on the release of progeny virus were performed by plaque assay;CPE suppression assay and LDH assay were employed to evaluate protective effect of infected cells;qPCR assay was used to explore the influence on viral RNA accumulation in DENV-infected cells incubated with candidate compounds;Western blotting and immunofluorescence assay was run to measure the effect on viral protein accumulation in DENV-infected cells treated with small molecular compounds(2)Validation of action target:SPR assay and Computer assisted molecular docking were utilized to test the interaction between small molecular compounds and NS5;immunofluorescence assay to evaluate replicative intermediates dsRNA in DENV-infected cells exposed to small molecular compounds;qPCR assay was used to explore the compounds influence on viral RNA accumulation;time point experiment and time-of-drug addition assay were applied to explore which stage of virus life cycle interfered.Results:Z1 and NS5 RdRp exhibited strongly binding affinities,while Diasarone-I binds to 2`O methyltransferase similar to s-adenosyl homocysteine,and both harbored significant antiviral activity against DENV2,with EC50 value of 4.75 and 4.50 μM respectively.Z1 and Diasarone-Ⅰ largely reduced viral RNA level,interfered the synthesis of replicative intermediates dsRNA in DENV2-infected cells and functioned on early replication stage of DENV2 life cycle.Z1 and Diasarone-I inhibited the production of viral structural proteins and unstructured proteins as well as restricted the spread of the infection correlation with concentration.Conclusions:Small molecule dihydropyrroles Z1 targets dengue virus NS5 C terminal RNA polymerase,inhibits the de novo synthesis of virus RNA and thus has antiviral activities.The role of Diasarone-I derived from traditional Chinese medicine water extract of Acorus tatarinowii Schott on dengue virus NS5 methyltransferase,inhibits viral RNA genome replication.Both molecules served as lead molecules for novel NS5 inhibitors.