Discovery And Mechanism Investigation Of New Inhibitors Against RNA Viruses EV71 And DENV By Targeting Proteases

Two parts are included in this dissertation.The first part is focused on the discovery and mechanism study of the potent small molecule inhibitors against Enterovirus 71 protease(EV71 3C^pro),and the second part is about the related studies on Dengue virus by targeting NS2B/NS3^protease.In the first part,we accomplished the discovery and mechanism study of one potent small molecule inhibitor against EV71 3C^pro.EV71 is a single-stranded,positive-sense RNA virus,belonging to the Picornaviridae family.It is one of the major causative agents of hand,foot,and mouth disease?HFMD?,which is characterized by fever,vomiting,vesicular rashes on the hands,feet,buttocks,and oropharyngeal ulcers,with myocarditis,pneumonedema,aseptic meningoencephalitis and even death in severe cases.To date,there are no effective drugs against EV71 infection.Currently,the virus protease has been proved to be an effective antiviral target and 3C^pro performs multiple essential tasks in viral replication.Therefore,three in-house compound libraries were screened by docking-based virtual screening assay to discover the potent inhibitors against EV71 3C^pro.Finally,we found small molecule DC07090 showing efficient antiviral activity against EV71 and Coxsackievirus A16?CVA16?.Next,we also verified the direct binding of DC07090 and 3C^pro by fluorescence quenching and surface plasmon resonance assays.Our results have supplied useful lead compound structure information for anti-EV71 3C^pro inhibitor discovery.The second part is about the discovery and mechanism study of the small molecule inhibitors against DENV NS2B/NS3^pro.DENV is a kind of pathogens that are widespread in tropical and subtropical regions.It has four serotypes,including DENV-1,DENV-2,DENV-3 and DENV-4.Human infected with any type of these serotypes may cause serious infections,such as Dengue fever,Dengue hemorrhagic fever and Dengue shock syndrome.Currently,no effective drugs against DENV infection have been yet discovered.Given that NS2B/NS3^pro plays potently in viral replication?including the cleavage of the polymer precursor protein?,we screened out the antiviral inhibitors by targeting NS2B/NS3^pro.In the assay,we carried out the expression and purification of NS2B/NS3^pro in vitro,and constructed a set of enzyme-based HTS platform with docking-based virtual screening assay for screening NS2B/NS3^pro inhibitors.Finally,active compounds Y050,Y2,Y4 and Y5 were discovered.Structurally,these compounds share similar structure in common,showing us the key points on antiviral drug discovery against DENV.