Bruton's tyrosine kinase inhibitor BTKi-2 inhibits mpox virus and vaccinia virus infection

ABSTRACT The recent global outbreak of mpox virus (MPXV) infections underscores the urgent need for antiviral therapies against orthopoxviruses. In this study, using a high-content screening (HCS) platform based on a modified vaccinia virus Tiantan strain with GFP insertion (MVTT-GFP) under BSL-2 conditions, we screened 1,513 kinase inhibitors for antiviral activity. Among these, Bruton’s tyrosine kinase inhibitor BTKi-2 emerged as a potent candidate, exhibiting IC 50 of 0.535 μM against vaccinia virus (VACV) and 0.260 μM against MPXV in vitro , while maintaining low cytotoxicity. In a murine model of VACV-induced pneumonia, BTKi-2 treatment reduced lung viral loads by 90% and a significantly improved survival compared to vehicle-treated controls. Notably, mechanistic studies indicate that BTKi-2’s antiviral effects cannot be completely attributed to the inhibition of BTK or EGFR/ErbB2 signaling. These findings highlight BTKi-2 as a promising antiviral agent in vitro and in vivo , suggesting that BTKi-2 may offer a potential avenue for future therapeutic development against orthopoxvirus infections.