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.