Targeting Dengue Virus NS3 Helicase: Biochemical and Computational Evaluation of Catechins from Camellia sinensis as Potential Therapeutic Leads

Dengue virus serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3), a superfamily-2 viral helicase. DEN2-NS3 contains an N-terminal serine protease domain and a C-terminal RNA helicase/nucleoside 5’-triphosphatase (NTPase) domain that is essential for viral replication. In the helicase/NTPase domain, DEN2-NS3 utilizes energy derived from NTP hydrolysis and has a translocation polarity from the 3’-to-5’ end with preference of a duplex RNA substrate. A galloylated catechin, (−)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika virus NS3 helicase/NTPase, with an IC ₅₀ value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, EGCG, (−)-epicatechin gallate (ECG), and (−)-epigallocatechin (EGC), would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays performed in this study revealed that the helicase/NTPase catalytic domain, DEN2-NS3(S171-K618), was strongly inhibited by these galloylated catechins. This inhibition was verified by the insignificant impact observed by the compounds on our enzyme-coupled confirmatory assay, at the concentrations tested. The observed inhibition constant (K _i ) values and apparent modes of inhibition (relative to NTPase activity) of the tested compounds were K _i = 400 ± 86.6 nM for EGCG (mixed-mode inhibition), K _i = 550 ± 250 nM for ECG (uncompetitive inhibition), and K _i = 18.3 ± 4.2 µM for EGC (mixed-mode inhibition). The coronavirus nsp13 helicase reference inhibitor SSYA10-001 also revealed inhibition against DEN2-NS3 NTPase activity with a K _i = 10.2 ± 0.3 µM (mixed-mode inhibition). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, our biochemical and computational data support EGCG and ECG as galloylated catechin leads and suggest a plausible binding model involving an amphipathic pocket associated with the nucleic acid binding region of DEN2-NS3. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral candidates.