Ligand‑Based Virtual Screening Discovery of Thienopyrimidinone Derivatives as Allosteric nSMase2 Modulators and Antivirals Against West Nile Virus

Orthoflaviviruses such as West Nile virus (WNV), Zika virus (ZIKV), or dengue virus (DENV) represent a growing global health threat, with millions of annual infections and no approved antiviral therapies. Targeting host pathways represents an alternative antiviral strategy that may overcome limitations associated with direct‐acting antivirals, including resistance and narrow antiviral spectra. Neutral sphingomyelinase‐2 (nSMase2) plays a central role in sphingolipid metabolism and has emerged as a host factor involved in the replication of several flaviviruses including WNV and ZIKV. Herein, we report a new family of thienopyrimidinone derivatives as nSMase2 inhibitors. A ligand‐based virtual screening (LBVS) campaign using DPTIP as a template ligand identified 13 candidates from over 5 million ZINC20 compounds, which were refined through ADMET‐properties prediction and molecular docking. Experimental evaluation identified compound 16 as a novel nSMase2 inhibitor. Rational design guided by computational affinity maps enabled the synthesis of several derivatives bearing a thienopyrimidinone core. These compounds inhibited nSMase2 and showed antiviral activity against WNV in cell‑based assays. These results identify thienopyrimidinone derivatives as a new scaffold for nSMase2 inhibition and support host‐directed modulation of sphingolipid metabolism as a promising strategy for the development of broad‐spectrum antivirals against flaviviruses.