A broadly active flavivirus NS4B targeting compound that blocks NS4B-NS3 binding and replication organelle formation

Mosquito-borne orthoflaviviruses, including West Nile virus (WNV), dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV), cause substantial global morbidity and mortality, yet no broadly effective direct-acting antivirals are available. The viral nonstructural protein NS4B has emerged as a promising antiviral target, although the compounds described to date are largely DENV-specific. Here we characterize SRI-38841, a xanthine analog derived from hit-to-lead optimization of a previously described WNV inhibitor, which inhibits WNV replication with an IC90 of 0.87 µM and low cytotoxicity, and retains activity against DENV, ZIKV, and JEV. Serial passage of WNV under compound selection yielded resistant viruses bearing mutations in NS4B, and reverse genetics was used to confirm the role of individual amino acid substitutions. Several of these residues map to regions analogous to those implicated in resistance to the DENV NS4B inhibitors JNJ-A07 and NITD-688. Docking of SRI-38841 to an AlphaFold2 model of WNV NS4B predicted a binding pocket within a cytosolic-facing funnel, and mutation of the pocket residues A45 and K56 likewise conferred resistance, while changes at Q170 and T220 did not. Together, these data favor a topology model in which the compound engages a cytosolic-facing pocket over one placing these residues in the ER lumen. Mechanistically, SRI-38841 disrupts the NS4B–NS3 interaction and abolishes replication organelle formation in infected cells. Unlike JNJ-A07 and NITD-688, which were inactive against WNV and ZIKV, SRI-38841 inhibits all four viruses tested, identifying this xanthine class as a starting point for broadly active orthoflavivirus antivirals.