Min Chen,Wenhuan Chen,Xinyi Jiang,Shaomei Liang,Yingqiao Qin,Jiayi Tang,Yifei Li,Qifan Wu,Xue Liang,Pengcheng Wei,Kunpeng Liu,Sulan Luo
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 protei. Here, we report the discovery of Ajmaline, an antiarrhythmic alkaloid, as a broad-spectrum anti-IAV agent through virtual screening targeting the conserved transmembrane domain of the M2 protein. Molecular docking and 100-ns dynamics simulations revealed stable binding across multiple IAV subtypes (H1N1, H2N2, H5N1, H7N9). Cellular Thermal Shift Assay confirmed direct binding to wild-type and N31S mutant M2 protein. Ajmaline exhibited potent antiviral activity through dual mechanisms: direct inhibition of viral replication and upregulation of interferon-related genes (IFN-β, 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.
Min Chen,Huifang Jiao,Dandan Lu,Wenhuan Chen,Zhen Yang,Xue Liang,Pengcheng Wei,Kunpeng Liu
Influenza viruses generate new strains or subtypes through antigenic drift and antigenic shift, leading to the failure of existing antiviral drugs and potentially causing widespread viral infection and influenza epidemics. However, during evolution, certain influenza virus proteins remain unchanged or undergo only minor mutations. Therefore, drugs targeting these evolutionarily conserved viral protein regions may exhibit broad-spectrum antiviral activity across subtypes and against mutations. The purpose of this study is to target the evolutionarily conserved domain of influenza A virus M2 protein and screen for drugs with cross-strain, mutation-resistant, and broad-spectrum antiviral activity. This study used AlphaFold3 to predict the M2 conserved domain structure and employed AutoDock to perform virtual screening of the NMPA database, identifying seven potential candidate drugs. Molecular dynamics simulations indicated that pamiparib exhibits strong affinity for the M2 conserved domain. It demonstrates excellent physiological activity in vitro in inhibiting the replication of H1N1 replication, while showing no effect on other viruses. CETSA experiments confirmed that pamiparib has the potential to bind to the M2 proteins of various influenza virus subtypes. In addition, pamiparib suppresses effectively viral infection in vivo. Ultimately, this study verifies that antiviral drugs designed to target the evolutionarily conserved regions of the influenza virus M2 protein indeed possess broad-spectrum antiviral activity across different strains.
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