In Vitro Evaluation of Acetaminophen as a Repurposed Anti‐Influenza Virus Agent

Influenza remains a major global health concern, characterized by recurrent seasonal outbreaks and the persistent threat of pandemic strains. Although vaccines and antivirals are available, their effectiveness is often compromised by antigenic variation and the emergence of drug-resistant viruses. In this study, we investigated the drug repurposing potential of FDA-approved compounds. A total of 1120 FDA-approved antiviral drugs and inhibitors were selected from publicly available databases, including PubChem and DrugBank. The 2D structures of these ligands were imported and energy-minimized using Chem3D v16.0 to ensure geometrically optimized conformations suitable for docking. The 3D crystal structures of four key influenza proteins, nucleocapsid protein (Protein Data Bank [PDB]: 2IQH), neuraminidase (PDB: 2HU4), hemagglutinin HA1 (PDB: 3UBE), and the Influenza A H5N1 PB2 cap-binding domain (PDB: 4CB4), were retrieved from the RCSB PDB and carefully prepared for docking. Molecular docking identified acetaminophen, asparagine, and acyclovir as the top three scoring compounds, with acetaminophen showing the strongest interaction at the PB2 cap-binding site. Subsequent in vitro antiviral studies were performed for all three compounds; however, asparagine and acyclovir did not exhibit significant inhibitory effects, whereas acetaminophen demonstrated promising activity. These findings suggest that acetaminophen may interfere with some key steps in the life cycle of the influenza virus. Given its established safety profile and widespread availability, acetaminophen emerges as a promising candidate for therapeutic repurposing. Nevertheless, further mechanistic and in vivo validation is essential to substantiate its potential role in influenza treatment strategies.