Virtual screening targeting the conserved domain of the IAV M2 protein reveals the potential broad-spectrum anti-IAV activity of ajmaline.

Influenza A virus (IAV) evolves rapidly through antigenic variation, leading to drug resistance and pandemic potential. To circumvent mutational escape, we employed a drug repurposing strategy targeting the evolutionarily conserved transmembrane domain (TMD) of the IAV M2 protein. Here, we report the discovery of Ajmaline, an antiarrhythmic alkaloid, as a broad-spectrum anti-IAV agent identified via virtual screening. Molecular docking and 100-ns dynamics simulations revealed stable binding across multiple IAV subtypes (H1N1, H2N2, H5N1, H7N9). The Cellular Thermal Shift Assay (CETSA) confirmed direct binding to both wild-type and N31S mutant M2 proteins. Ajmaline exhibited potent antiviral activity through dual mechanisms: direct inhibition of viral replication and upregulation of interferon-related genes (IFNB1, ISG56) coupled with suppression of apoptosis (Bax downregulation). In vivo, Ajmaline (15 mg/kg) improved survival to 83%, reduced pulmonary viral load, and alleviated lung pathology in H1N1-infected mice. These findings identify Ajmaline as a repurposed broad-spectrum antiviral candidate capable of overcoming existing drug resistance, providing a proof-of-concept for conserved-domain-targeting strategies in antiviral drug development.