Cepharanthine inhibits enterovirus entry by endolysosomal deacidification and exhibits protective activity <i>in vivo</i>

Enteroviruses, which belong to the <i>Picornaviridae</i> family, are linked to a range of clinical illnesses that vary from mild to severe, including life-threatening diseases. Among these, enterovirus 71 (EV71) infections in infants and young children can lead to serious neurological conditions, posing a significant public health risk due to the absence of approved treatments. In this study, we assessed the anti-EV activities of four bisbenzylisoquinoline alkaloids (BBAs): tetrandrine (TET), cepharanthine (CEP), fangchinoline (FAN), and berbamine (BER), as well as their mechanisms of action. In all cases, we observed a dose-dependent decrease in EV71 protein levels and viral titers. TET and CEP exhibited lower 50% inhibitory concentrations and higher selectivity indexes among the tested BBAs. Therefore, we prioritized TET and CEP for mechanistic investigation and <i>in vivo</i> evaluation. Mechanistic studies revealed that TET and CEP inhibited EV71 infection primarily at the entry stage, without impacting viral binding, internalization, or post-entry processes. Further studies demonstrated that TET and CEP disrupted viral trafficking along the endolysosomal pathway. Both compounds were found to effectively neutralize low pH levels in endolysosomes, which corresponded to the reduced antiviral effects caused by the acidic replenishment of the medium. The antiviral effects of TET and CEP were observed against various serotypes of EV. Remarkably, administering CEP at a dose of 10 mg/kg provided complete protection to mice infected with EV71 from lethal challenges, significantly reducing viral titers, viral RNA levels, and pathological scores. Collectively, these findings highlight CEP as a promising candidate for the treatment of EV infections.