Regorafenib, a multitargeted kinase inhibitor, inhibits enterovirus 71 infection by suppressing the MAPK pathway

Objectives: To investigate the in vitro and in vivo Antiviral efficacy and underlying mechanism of regorafenib against Enterovirus 71 (EV71), as no licensed direct-acting antivirals are available for severe cases.

Methods: The inhibitory effect of regorafenib against EV71 was evaluated using qRT‒PCR, Western blot, IF, and CPE assays. The stage of the viral life cycle targeted by regorafenib was determined via time-of-addition, binding and entry assays. In vivo efficacy was assessed in an EV71-infected murine model by monitoring body weight, clinical scores, and viral RNA loads in tissues. The mechanism of action was elucidated by analyzing the effect of regorafenib on the MEK/ERK phosphorylation in the host mitogen-activated protein kinase (MAPK) pathway. A potential interaction with the EV71 capsid protein VP1 was investigated using molecular docking.

Results: Regorafenib demonstrated potent, dose-dependent Antiviral activity against EV71 in vitro and exhibited broad-spectrum efficacy against multiple human enteroviruses. In an EV71-infected murine model, oral administration of regorafenib at doses of 1.25 or 2.5 mg/kg/day significantly alleviated infection-associated symptoms and reduced viral loads in key organs. Mechanistic investigations revealed that regorafenib suppressed viral replication by inhibiting the phosphorylation of MEK and ERK in the MAPK pathway. Molecular docking studies further predicted that regorafenib binds to the viral capsid protein VP1, providing a potential structural basis for its Antiviral effect.

Conclusions: Our findings establish that regorafenib exhibits inhibitory activity against EV71 in vitro and in vivo, which warrants further preclinical evaluation as a candidate lead compound.

Capsid inhibitor; EV71; HFMD; MAPK pathway; Regorafenib.