Fragment-based discovery and optimization of Zika virus NS3 helicase ligands as antiviral hit candidates

Mosquito-borne flaviviruses, such as Zika virus (ZIKV), pose significant public health challenges, with no approved vaccines or therapeutics available. The ZIKV non-structural protein 3 (NS3) helicase domain (NS3Hel) is a key target due to its critical role in viral RNA replication. In this study, we used high-throughput crystallographic fragment screening to identify ligands binding to NS3Hel. Four fragments, including hydantoin, oxadiazole, and phenylurea moieties, were selected for synthesis, structural modification, fragment growing and linking strategies. Biophysical and biochemical assays revealed that enhancing affinity at the RNA binding site entry was challenging. However, a phenylurea derivative binding at the RNA binding site exit exhibited low micromolar affinity (K_d = 40 μM) and modest inhibition in a ZIKV replicon system. These results suggest that targeting the RNA binding site exit could provide a more effective approach for designing NS3Hel inhibitors. The phenylurea moiety thus emerges as a new molecular scaffold for the development of antiviral drugs targeting ZIKV.