Identifying hypaphorine as a novel antiviral compound against dengue virus

Approximately 390 million individuals globally are infected with dengue virus annually. Notably, no specific therapeutic strategy has been clinically approved for dengue fever to date. In this study, molecular docking screening against the methyltransferase (MTase) domain of the dengue virus (DENV) NS5 protein unveiled hypaphorine as a high-affinity ligand, with calculated binding energies of −6.657 kcal/mol (DENV2) and −6.663 kcal/mol (DENV3). This computational prediction was subsequently validated via cellular thermal shift assay (CETSA) and surface plasmon resonance imaging (SPRi), collectively demonstrating direct target engagement with a dissociation constant (KD) of 2.19 × 10⁻⁹ M. Functional characterization revealed that hypaphorine exhibited concentration-dependent inhibition of MTase enzymatic activity (IC₅₀ = 29.9 μM). In antiviral assays, hypaphorine displayed dose-dependent suppression of viral replication in both BHK-21 (IC₅₀ = 18.85 μM) and Huh-7 cells (IC₅₀ = 15.7 μM), while maintaining low cytotoxicity (CC₅₀ = 605.8 μM and 617.3 μM, respectively). Time-course analyses indicated maximal antiviral efficacy when hypaphorine was administered either pre- or post-infection (<24 hpi), which is consistent with the role of MTase in viral RNA capping during successive rounds of virus replication. The reduction in viral titer was found to correlate with MTase inhibition, thereby establishing a clear structure-activity relationship. These findings systematically characterize hypaphorine as a first-in-class natural MTase inhibitor, highlighting the utility of structure-guided approaches in anti-flaviviral drug discovery.