Screening And Mechanisms Investigation Of Inhibitors Against Zika Virus

BackgroundZika virus(ZIKV)is a mosquito-borne transmission virus belonging to the genus Flavivirus.ZIKV is mainly transmitted by Aedes mosquitoes,and will usually causes asymptomatic infection.A few patients have mild clinical symptoms,such as mild fever,fatigue,headache,arthralgia and rash.ZIKV can rarely lead to serious diseases or complications,which attracted no attention for a long time.However,Since the recent outbreak of ZIKV infection reported in South America in May 2015,the infection of ZIKV is suspected to be associated with severe neurological diseases such as microcephaly in new-born babies and Guillain-Barre syndrome in adults.With the spread of the epidemic to more than 80 countries,territories or subnational areas,the epidemic caused widespread concern.Unfortunately,at present no vaccines or specific drugs are available to prevent or treat ZIKV infection.The non-structural proteins of ZIKV play an important role in viral replication.The C-terminal RNA-dependent RNA polymerase(RdRp)of NS5 mediates the ZIKV genomic RNA synthesis.While NS2B-NS3 protease is an important cleavage enzyme of viral polyprotein.Given these two protein sequences are highly conserved,it makes them ideal targets for developing drugs against ZIKV.ObjectivesIn this study,we will investigate the anti-ZIKV activity of 2-(4-bromophenyl)-7,7-dimethyl-1,3-diphenyl-2,3,4,6,7,8-hexahydroquinazoline-5(1H)one(Q63)and myricetin and explore antivirus mechanisms,which provided a new reference for the development of safe and effective drugs against ZIKV.Methods:1.Preliminary screen compounds of anti-ZIKV activity:The inhibition of Q63 and myricetin on the release of progeny ZIKV was detected by plaque assay CPE inhibition assay,and the toxicity of the compounds was evaluated by MTT test.2.RT-PCR was used to detect the inhibitory effect of candidate compounds on the ZIKV RNA accumulation in cells.ELISA and Western blot assay were applied to measure the inhibitory effect on protein synthesis level of ZIKV.3.Time of drug addition assay was used to investigate which stage of viral life cycle that Q63 interfered.RT-PCR,plaque assay and ELISA were utilized to validate which stage of virus life cycle that myricetin targeted in,as well as,to explore how soon the myricetin worked,respectively.4.ITC assay was used to detect the interaction between Q63 and NS5-RdRp.FRET assay was applied to measure the inhibitory effect of myricetin on NS2B-NS3 protease.ResultsQ63 and myricetin exhibited potential activity against ZIKV,with ICs0 value of 1.98 and 0.58 p.M,respectively.Q63 interfered the replicative stage of viral proliferation cycle,and myricetin inhibited the early stage of ZIKV life cycle.Q63 largely reduced the RNA and protein levels in ZIKV-infected cells with dose-dependent manner by interfered NS5-RdRp.Myricetin inactivated ZIKV quickly,and the inhibition rate was close to 100%.Myrncetin inhibited NS2B-NS3 protease activity with IC50 value of 0.58 ?M,which was consistent with that of myricetin inhibited viral activity.ConclusionsQ63,a small molecule compound of quinazolinone,targets NS5-RdRp to inhibit the activity of genomic RNA de novo synthesis during the replication stage of ZIKV life cycle,and thus exhibits antiviral activity.Myricetin,a traditional Chinese medicine monomer derived from natural plants,inhibits the early stage of ZIKV life cycle by interacting with NS2B-NS3pro of ZIKV.In conclusion,both of Q63 and myricetin are potential lead compounds against ZIKV.