Identification of Influenza H1N1 Endonuclease Inhibitor via Virtual Screening and In Vitro Validation

BACKGROUND: The H1N1 influenza pandemic remains a global health burden, causing acute respiratory illness and high mortality. The continuous emergence of therapeutically resistant strains necessitates the ongoing development of new, effective antiviral drugs. MATERIALS AND METHODS: This study employed a virtual screening approach to identify potential inhibitors from a fragment-based chemical library targeting the H1N1 polymerase acidic (PA) endonuclease, an enzyme crucial for the cap-snatching process and viral replication. Chemical fragments binding within the PA active site were identified through virtual screening, and the crystal violet assay, together with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, were subsequently used to experimentally validate inhibitory activity and cytotoxicity in Madin-Darby canine kidney (MDCK) cells. RESULTS: A new chemical scaffold of triazole pyridine derivative that binds within the PA active site was found as a result of a fragment-based screening approach. In the crystal violet assay, the compound exhibited substantial inhibitory activity, with an IC50 value of 1.392 μM/mL. This potent antiviral activity is consistent with the virtual screening results, which showed a lower binding energy than the control. In addition, the cytotoxicity assay yielded a CC₂⁽ value of 495 μmol/mL, indicating a favourable selectivity index. DISCUSSION: Compounds that inhibited the PA endonuclease in vitro decreased or abolished the influenza virus activity. This study identified a triazole-pyridine derivative as an inhibitor and characterised the key interacting residues involved in binding. The results indicate that binding affinity is determined by the nature of the interactions and the specific residues involved, rather than by the number of participating residues. The hit compound identified in this study demonstrated activity comparable to that of previously reported inhibitors, while exhibiting a more favourable safety profile, making it a promising candidate for further investigation as an antiviral agent. CONCLUSION: These findings demonstrate a successful virtual fragment-based screening method of novel inhibitors targeting the influenza polymerase endonuclease. This study suggests that triazole-pyridine derivatives can serve as promising lead compounds for the development of novel H1N1 influenza inhibitors, providing a valuable direction for next-generation influenza therapeutics.