Influenza A virus (IAV) infection constitutes a major public health threat. Severe influenza virus infection can induce intense inflammatory responses and lung injury, leading to serious clinical symptoms or even death. The utility of current anti-influenza drugs is often limited by side effects and the emergence of drug-resistant strains. Based on the critical role of L-type voltage-gated calcium channels (L-VGCCs) in influenza virus replication, this study investigates the antiviral activity and mechanism of verapamil, a classic L-type calcium channel antagonist, against H1N1-UI182 virus. Verapamil, an L-type calcium channel blocker, is widely used in the treatment of cardiovascular diseases and has a well-established safety profile. Through molecular dynamics (MD) simulation and network pharmacology analysis, we predicted the stable binding mode of verapamil to the target protein (PDB id: 6JPA) and its potential multi-target network. In vitro, verapamil exhibited antiviral activity against H1N1-UI182 in MDCK cells, enhancing the survival rate of infected cells and reducing viral nucleoprotein (NP) expression. In a lethal H1N1-UI182 infection mouse model, verapamil treatment markedly improved survival rates, alleviated weight loss and lung pathological damage, exhibiting a dose-dependent protective effect. Lung tissue analysis showed that verapamil effectively reduced the lung index and viral load, suppressed the activation of the Nuclear factor kappa B (NF-κB) signaling pathway, and decreased the expression of key inflammatory factors, thereby mitigating the cytokine storm. A comparison of administration regimens indicated that pre-treatment yielded optimal efficacy, suggesting verapamil acts primarily during the early stage of the viral life cycle. This study systematically elucidates that verapamil exerts antiviral and immunomodulatory effects by regulating the NF-κB pathway. Network pharmacology analysis suggested the potential involvement of multiple targets and pathways, including EGFR, SRC, and phospholipase D signaling, providing hypotheses for future mechanistic investigation. This paper supports a drug repurposing strategy against drug-resistant influenza viruses and highlights its significant potential for clinical translation.

The influenza A virus (IAV) has been responsible for multiple seasonal epidemics and poses a pandemic threat, and the growing number of variant strains constitutes a persistent threat to humanity. This study aimed to identify anti-influenza compounds from a traditional Chinese medicine (TCM) monomer library using a machine learning approach, with calmodulin as a hypothesis-driven target. The antiviral efficacy of the compounds with the highest predicted binding scores from virtual screening was evaluated using integrated computational and experimental approaches. A pre-trained protein language model (ConPLex) was employed for virtual screening. Molecular docking was used to predict binding characteristics, and network pharmacology was applied to generate hypotheses on multi-target mechanisms. The cytotoxicity and anti-H1N1 activity of the selected compounds were assessed in vitro, followed by in vivo evaluation of survival, lung pathology, viral load, and inflammatory mediators in a lethal mouse infection model. Sodium deoxycholate (NaDC) and deoxycholic acid (DCA) were identified as promising lead compounds. Both exhibited dose-dependent inhibition of viral replication in vitro with low cytotoxicity. Treatment with NaDC and DCA significantly improved survival rates and reduced lung pathology in H1N1-infected mice. Treatment was associated with suppression of nuclear factor kappa-B (NF-κB) activation, reduced pro-inflammatory cytokines, and elevated interleukin-10 (IL-10) levels. Molecular docking predictions indicated that NaDC and DCA exhibit moderate binding affinity for calmodulin, with binding energies of −8.38 kcal/mol and −7.61 kcal/mol, respectively. Furthermore, network pharmacology analysis suggested that these compounds may modulate pathways related to viral infection, inflammation, and immune regulation. NaDC and DCA demonstrate anti-influenza activity both in vitro and in vivo, reducing viral replication and alleviating inflammatory lung injury. These findings position NaDC and DCA as promising lead compounds for anti-influenza drug development and provide a foundation for further mechanistic validation.