Structural Insights into DENV-2 NS2B-NS3 Protease and Inhibition by Glutathione-Coated Gold Nanocluster

Dengue virus (DENV) continues to pose a significant global health threat, with increasing infection rates and limited treatment options. The viral NS2B-NS3 protease (NS2B-NS3pro), a highly conserved two-component enzyme essential for polyprotein processing and replication, is a key target for antiviral drug development. Here, we report the 2.25 Å-resolution crystal structure of DENV serotype 2 (DENV-2) NS2B-NS3pro, in which a PEG fragment is bound within a solvent-exposed pocket between β 10, β 11, β 14 and β 15 of NS3. This structure reveals a previously unidentified potential allosteric site, suggesting a novel pocket for inhibitor design. We also show that the glutathione-coated gold nanocluster (GSH-AuNC) directly inhibits DENV-2 NS2B-NS3pro. Bio-layer interferometry and fluorescence-based protease assays indicate that the nanocluster binds NS2B-NS3pro with a dissociation constant of 15.64 µM and inhibits its catalytic activity with an IC ₅₀ of 16.04 µM, consistent with a direct inhibition mechanism. Molecular dynamics simulations further suggest that GSH-AuNC interacts with the catalytic triad of NS2B-NS3pro, forming stable electrostatic and van der Waals interactions that block substrate binding. Collectively, these findings provide a structural and mechanistic basis for the development of inhibitors targeting DENV-2 NS2B-NS3pro, offering new strategies for antiviral therapy using small molecules or nanomedicines.