Structure-Guided Design of a Subnanomolar Dengue Virus NS4B Inhibitor

Abstract 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.