Fangchinoline demonstrates efficacy against TGEV through pAPN binding, as determined by preliminary toxicity evaluation

Abstract Through structure-based virtual screening, this study identified the botanical compound fangchinoline (FAN) as an inhibitor of the porcine aminopeptidase N (pAPN) receptor and evaluated its anti-transmissible gastroenteritis virus (TGEV) activity, mechanism, and safety. FAN exhibited potent antiviral efficacy against TGEV in PK-15 and IPEC-J2 cells (EC 50 = 2.862 μM and 3.670 μM, respectively), significantly reducing viral RNA copies and N protein expression while suppressing the upregulation of the inflammatory cytokines IL-6 and TNF-α. It also showed broad-spectrum potential by inhibiting PEDV (EC 50 = 2.243 μM). Mechanistic studies using a cellular thermal shift assay (CETSA) and drug affinity-responsive target stability (DARTS) confirmed that FAN can bind to pAPN, affecting its thermal and enzyme stability. Time-of-addition assays indicated that FAN interferes with both the early (attachment and internalization) and late replication phases of TGEV. In acute toxicity tests, FAN showed low toxicity (LD 50 in mice: 2060.23 mg/kg for males, 1481.93 mg/kg for females) and no mutagenic potential in the Ames test. Favorable pharmacokinetic parameters were also observed in rats. In conclusion, FAN exerts its anti-TGEV effect by targeting the critical pAPN receptor. Its plant origin, potent and broad-spectrum antiviral activity, elucidated mechanism, and promising safety profile support its further development as a lead compound for anti-coronavirus strategies.