Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). 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, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies.
Wojciechowski, M. K., Goyzueta-Mamani, L. D., Chavez-Fumagalli, M. A., D'Antonio, E. L.
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