Abstract

Influenza A viruses subvert lysosomal function to evade host degradation mechanisms. Using Connectivity Map (CMap) screening and transcriptomic analysis, we identified fangchinoline (Fan)—a bisbenzylisoquinoline alkaloid—as a potent enhancer of lysosomal gene expression. Owing to its alkaline properties, Fan accumulates within lysosomes, elevates luminal pH, and induces TFEB nuclear translocation, thereby restoring lysosomal biogenesis and initiating a TFEB-driven antiviral response. Concurrently, Fan disrupts autophagosome–lysosome fusion and impairs autophagic flux, further enhancing its antiviral activity. Time-resolved functional assays demonstrate that Fan primarily inhibits H1N1 infection at the entry stage by obstructing endolysosomal trafficking. Together, these results identify Fan as a novel TFEB-mediated lysosomal modulator that antagonizes influenza infection by counteracting viral lysosomal evasion strategies and highlighting the therapeutic potential of lysosome-targeted compounds in influenza treatment.