Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

Aim： Dengue is a severe epidemic disease caused by dengue virus （DENV） infection, for which no effective treatment is available. The protease complex, consisting of nonstructural protein 3 （NS3） and its cofactor NS2B, plays a pivotal role in the replication of DENV, thus may be a potential target for anti-DENY drugs. Here, we report a novel inhibitor of DENY2 NS2B/NS3 protease and its antiviral action. Methods： An enzymatic inhibition assay was used for screening DENY2 NS2B/NS3 inhibitors. Cytotoxicity to BHK-21 cells was assessed with MTT assay. Antiviral activity was evaluated in BHK-21 cells transfected with Rlu-DENV-Rep. The molecular mechanisms of the antiviral action was analyzed using surface plasmon resonance, ultraviolet-visible spectral analysis and differential scanning calorimetry assays, as well as molecular docking analysis combined with site-directed mutagenesis. Results： In our in-house library of old drugs （NIO00 compounds）, a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[（4-hydroxy- 2-methyl-5-sulfophenyl）methyl]-4-methyl-benzene-sulfonic acid （policresulen） was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with ICso of 0.48 pg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with ICso of 4,99 pg/mL, whereas its IC50 for cytotoxicity to BHK-21 cells was 459.45 pg/mL. Policresulen acted as a competitive inhibitor of the protease, and slightly affected the protease stability. Using biophysical technology-based assays and molecular docking analysis combined with site- directed mutagenesis, we demonstrated that the residues Gin106 and Arg133 of DENV2 NS2B/NS3 protease directly interacted with policresulen via hydrogen bonding. Conclusion： Policresulen is a potent inhibitor of DENY2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells. The bind- ing mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease.