Integrated in silico screening and biophysical evaluation of three plant flavonoids interacting with Newcastle...

Newcastle disease virus (NDV) represents a major threat to the worldwide poultry industry, and the absence of approved antiviral therapeutics highlights the need for alternative intervention strategies. In this study, an integrated computational and experimental methodology to assess the interaction of selected plant-derived flavonoids against NDV. Molecular docking was conducted to evaluate the interactions of karanjin, mammeigin, and 3, 5-dihydroxy-4',7-dimethoxyflavone (DHDM) with multiple NDV proteins, comprising hemagglutinin neuraminidase (HN), fusion protein (F), matrix protein (M), nucleoprotein (N), and phosphoprotein (P). Based on docking predictions, the NDV phosphoprotein was selected for experimental validation of biophysical interaction studies. Among the tested compounds, karanjin consistently exhibited the strongest binding affinities, with particularly favorable interactions observed for the phosphoprotein, a key regulator of viral transcription and replication. In silico pharmacokinetic analysis further supported the drug-likeness and oral absorption potential of karanjin. Based on docking predictions, the NDV phosphoprotein was selected for experimental validation. Recombinant phosphoprotein was expressed and purified, and flavonoid interactions were examined using UV-Vis absorption spectroscopy, fluorescence quenching, and isothermal titration calorimetry analysis. Karanjin displayed a clear and specific binding profile characterized by moderate-to-strong affinity and an enthalpy-driven interaction, whereas mammeigin and DHDM showed weak or nonspecific interactions. Spectroscopic analyses corroborated these findings, indicating stable complex formation without major structural perturbation. Collectively, these results identify karanjin as a promising flavonoid candidate targeting the NDV phosphoprotein.