Discovery of a Novel Non‐Nucleoside Inhibitor of RNA‐Dependent RNA Polymerase Against Dengue Virus

Dengue virus (DENV) is an acute infectious pathogen worldwide, for which no effective therapeutics are available. The RNA‐dependent RNA polymerase (RdRp) displays an important role during DENV replication and is therefore a promising target in the development of antiviral drugs. However, there are still no clinically approved RdRp inhibitors available. In this study, we identified a natural small molecule, 12β‐hydroxydammar‐3‐one‐20(S)‐O‐β‐ d ‐glucopyranoside (PN‐1), using a surface plasmon resonance‐based screening assay. Biochemical and structural analyses revealed that PN‐1 selectively targets the RdRp of DENV NS5 protein by covalently modifying residues Glu255, Met387, Glu479, and Ala507. Mechanistic studies involving tryptophan scanning and hydrogen‐deuterium exchange mass spectrometry revealed that PN‐1 binding regulates RdRp conformational transitions. This allosteric mechanism leads to suppression of enzymatic activity and inhibition of DENV replication. Consequently, PN‐1 exhibited potent antiviral activity across various cell lines and conferred significant protection in both ICR suckling and AG129 mouse models. Taken together, our results show that PN‐1 functions as a novel non‐nucleoside inhibitor to suppress DENV replication by targeting RdRp. These findings highlight PN‐1 as a promising anti‐DENV lead compound, while revealing conserved RdRp residues as actionable targets for rational drug design.