M. K. Wojciechowski, L. D. Goyzueta-Mamani, M. A. Chavez-Fumagalli, E. L. D'Antonio
Dengue virus serotype 2 is a human pathogenic flavivirus encoding non-structural protein 3 (DEN2-NS3), a superfamily-2 viral helicase. DEN2-NS3 contains an N-terminal protease domain and a C-terminal RNA helicase/nucleoside 5'-triphosphatase (NTPase) domain essential for replication. The enzyme utilizes energy from NTP hydrolysis to translocate 3'-to-5' along a duplex RNA substrate. Prompted by the high potency of (--)-epigallocatechin gallate (EGCG) against Zika virus, this study investigated if three catechins, EGCG, (--)-epicatechin gallate (ECG), and (--)-epigallocatechin (EGC), would be potent inhibitors of DEN2-NS3. Enzyme-inhibition assays demonstrated that the catalytic domain, DEN2-NS3(S171-K618), was strongly inhibited by the galloylated catechins (EGCG and ECG), verified by an enzyme-coupled confirmatory assay. Inhibition constants (Ki) and suggestive inhibition modes relative to NTPase activity were Ki = 400 {+/-} 86.6 nM for EGCG (mixed-mode), Ki = 550 {+/-} 250 nM for ECG (uncompetitive), and Ki = 18.3 {+/-} 4.2 {micro}M for EGC (mixed-mode). The coronavirus inhibitor SSYA10-001 inhibited DEN2-NS3 (Ki = 10.2 {+/-} 0.3 {micro}M, mixed-mode). Computational workflows using SiteMap identified a predicted druggable pocket associated with the RNA-binding region, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. Catechins were analyzed through 200-ns molecular dynamics simulations to evaluate binding stability. Computational results revealed that EGCG and ECG maintained high stability, forming highly persistent shared amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. Biochemical and computational data support these galloylated catechin leads, suggesting a plausible binding model within an amphipathic pocket. Future structural optimization into stable prodrug derivatives could yield promising antiviral candidates.