Disruption of Spike Priming in Virus Entry: Tetrandrine as a Pan‐Coronavirus Inhibitor

The emergence of novel and highly transmissible coronavirus (CoVs) highlights the urgent need for broad‐spectrum antiviral agents. In our pursuit of effective treatments for coronavirus, we identified tetrandrine, the traditional Chinese medicine, as a pan‐coronavirus inhibitor, exhibiting efficacy against HCoV‐229E, HCoV‐OC43, SARS‐CoV‐2, and its major variants of concern (VOCs), including alpha, beta, and omicron. Mechanistic investigations revealed that tetrandrine primarily targets the viral entry stage by binding to the Spike protein, disrupting its interaction with the host protease transmembrane serine protease 2 (TMPRSS2), and promoting Spike protein degradation, ultimately blocking the membrane fusion. Drug resistance selection study identified two mutations, G688R and D814Y, at S2 subunit of Spike, which reduced HCoV‐229E's sensitivity to tetrandrine, supporting its direct action on the viral fusion machinery. Molecular docking and molecular dynamic (MD) simulation together with co‐IP assay also verified the disruption of Spike‐TMPRSS2 complex formation by tetrandrine. Importantly, tetrandrine treatment reduced viral load and mitigated neuropathological damage in infected neonatal mice. These findings establish tetrandrine as a broad‐spectrum coronavirus entry inhibitor and offer mechanistic insights into its antiviral activity, providing a promising candidate for therapeutic development against current and future coronavirus threats.