Discovery And Mechanism Investigation Of Small Molecular Inhibitor Targeting Denguevirus NS3pro

Dengue virus(DENV) as one of the most widespread pathogens in tropical and subtropical areas is a member of flavivirus family which includes yellow fever virus(YFV), Japanese encephalitis virus(JEV), West Nile virus(WNV). There are four antigenically distinct viral serotypes for DENV, including DENV-1, DENV-2, DENV-3 and DENV-4, among which DENV-2 is the most prevalent. Patients infected with any of the four serotypes will result in severe epidemic diseases, such as dengue fever(DF) and even the fatal diseases including dengue hemorrhagic fever(DHF) and dengue shock syndrome(DSS). Currently, it is estimated that there are 390 million infections each year, of which 96 million become manifest. The antibody dependent enhancement effect(ADE) of DENV makes the vaccine study be a particularly hard work. Up to now, no clinically approved vaccines or therapeutics is available for DENV. Considering severity of situation, the discovery of an effective anti-DENV drug is now an urgent need.NS3 is a vital non-structural protein of DENV and its N-terminal is a trypsin-like protease which contains a serine protease catalytic triad(His51, Asp75, and Ser135). An important function of NS3 is to cleave DENV polyprotein precursor into individual proteins which is essential for viral replication and maturation. However, the proteolytic activity of NS3 requires the cofactor NS2 B. And the activity of NS3 could be enhanced by 3300-6600 folds in the presence of NS2 B. Due to the importance of component protease NS2B/NS3 pro for viral maturation and replication, NS2B/NS3 pro becomes a potential target for antiviral drug. There is no doubt that the discovery of NS2B/NS3 pro inhibitors will promote the development of DENV drugs.In our work, we utilized Escherichia coli BL21 transformed with recombinant plasmid p ET15b-DENV-2-NS2B/NS3 to express NS2B/NS3 pro. Then the protein was purified by Ni2+ affinity chromatography. The NS2B/NS3 pro inhibitor was obtained by the HTS(high-throughput screening) based on enzymatic tests. The cytotoxicity and antiviral activity of candidate inhibitors were evaluated by MTT assay and luciferase assay, respectively. For further investigating the inhibition mechanism of candidate compounds, enzymatic test, surface plasmon resonance(SPR) assay, ultraviolet-visible(UV) spectral analysis, differential scanning calorimetry(DSC) assay, molecular docking, and site-directed mutagenesis of protein were carried out. The results showed that compound MT02A3101 was a low cytotoxicity NS2B/NS3 pro inhibitor which could effectively inhibit replication of DENV-2 replicon. MT02A3101 was a competitive inhibitor against NS2B/NS3 pro, and it competed with the substrate thus reducing the stability of the protease. Besides, we verified that residues Q106, Q114, T132 and R133 of the protease were responsible for the H-bonding of policresulen to the protease, and residues of I109, I115 and V131 were involved in their hydrophobic bonding by molecular docking and bioassay.