Structure-Guided Design of a Subnanomolar Dengue Virus NS4B Inhibitor

Dengue virus nonstructural protein 4B (NS4B) is an essential regulator of viral replication but remains structurally unresolved, limiting drug discovery. Here, we combine modeling, ligand-based design, and experimental validation to enable structure-guided inhibition of NS4B. Comparative models across all four serotypes reveal a conserved transmembrane binding pocket consistent with resistance mutations. Docking of JNJ-series inhibitors defines key interaction features that guide the development of predictive field-based 3D-QSAR models (R = 0.91, Q = 0.89). Prospective screening of bioisosteric and in-house compounds identifies BNJ-495 as a candidate broad-spectrum inhibitor with predicted activity across all four dengue serotypes. Experimental validation confirms potent activity against dengue virus serotype 2 (IC50 = 0.17 nM) with a high selectivity index (∼37,000). Conserved binding interactions across serotypes support broad-spectrum potential. This study establishes a general framework for targeting structurally elusive viral membrane proteins.