Abstract

Background Viruses continue to threaten global health, highlighting the urgent need for antiviral strategies with broad-spectrum activity. The nucleocapsid (N) protein undergoes liquid–liquid phase separation (LLPS), a conserved process essential for viral assembly. However, pharmacological targeting of this process remains largely unexplored. Purpose This study aimed to establish a high-content screening platform to discover small-molecule inhibitors of the conserved LLPS of the SARS-CoV-2 nucleocapsid protein and to evaluate their broad-spectrum antiviral activity and anti-inflammatory effects. Methods We established a high-content imaging–based screening platform to identify small-molecule inhibitors of SARS-CoV-2 nucleocapsid protein LLPS. Candidate compounds were evaluated for antiviral activity using immunofluorescence microscopy, quantitative RT–PCR, and virus-like particle systems. Antiviral efficacy and immunomodulatory effects were further validated in SARS-CoV-2–infected cell and mouse models. Results We identified tannic acid (TA) as a potent inhibitor that disrupts nucleocapsid protein condensation by binding to multiple regions of nucleocapsid and interfering with nucleocapsid–RNA interactions. Tannic acid robustly suppressed SARS-CoV-2 replication in vitro and in vivo . In parallel, tannic acid attenuated virus-induced inflammatory responses by interacting with G3BP1 and suppressing NF-κB signaling. Importantly, tannic acid maintained inhibitory activity against nucleocapsid proteins from major SARS-CoV-2 variants and exhibited broad-spectrum antiviral effects against multiple human coronaviruses, influenza A virus, and vesicular stomatitis virus. Conclusions These findings demonstrate that targeting nucleocapsid protein LLPS represents a conserved antiviral strategy resilient to viral evolution and identify tannic acid as a promising lead compound w