# A clinical SARS-CoV-2 Mpro inhibitor blocks replication of multiple enteroviruses and confers oral in vivo protection in animal models

## TL;DR

A drug originally developed for SARS-CoV-2 shows broad antiviral activity against multiple enteroviruses and protects mice from infection.

## Contribution

Bofutrelvir, a clinical-stage SARS-CoV-2 Mpro inhibitor, is shown to inhibit enterovirus replication and provide oral protection in animal models.

## Key findings

- Bofutrelvir inhibits enterovirus 3Cpro and suppresses replication of EV71, CA16, and other enteroviruses.
- Oral administration of bofutrelvir reduces viral load and disease severity in neonatal mice infected with EV71 and CA16.
- Crystallographic analysis reveals bofutrelvir binds to the conserved active site of EV71 3Cpro.

## Abstract

Enteroviruses, which belong to the family Picornaviridae, cause hand, foot, and mouth disease (HFMD), respiratory symptoms, and severe neurological complications in children. Since vaccines cannot provide cross-protection against different serotypes of enteroviruses, the development of broad-spectrum anti-enteroviral drugs is imperative. The viral 3C protease (3Cpro), which is essential for polyprotein processing represents a validated target for therapeutic intervention. Importantly, enterovirus 3Cpro shares conserved structural and catalytic features with coronavirus main protease (Mpro, also known as 3C-like protease, 3CLpro), providing a rationale for cross-target inhibitor repurposing. Through targeted screening of peptidomimetic protease inhibitors, a clinical-stage SARS-CoV-2 Mpro inhibitor was identified as a potent inhibitor of enterovirus A71 (EV71) 3Cpro. Bofutrelvir displayed nanomolar antiviral activity in multiple cell lines and demonstrated broad-spectrum efficacy against several enteroviruses including coxsackievirus B5, coxsackievirus A16 (CA16) and echovirus 11. In EV71 infected neonatal mice, intraperitoneal administration of bofutrelvir markedly reduced viral loads in brain, spinal cord, and muscle, alleviated clinical symptoms, and suppressed tissue inflammation. Oral administration of bofutrelvir also provided therapeutic benefits in neonatal mice models of both EV71 and CA16. Crystallographic analysis revealed that bofutrelvir binds in the conserved substrate-binding cleft of EV71 3Cpro, elucidating its molecular mechanism of inhibition. These findings identify bofutrelvir as a broad-spectrum peptidomimetic 3Cpro inhibitor with strong antiviral efficacy against enteroviruses and highlight its potential for repurposing as a promising antiviral candidate for the treatment of enteroviral infections.

Hand, foot and mouth disease (HFMD) caused by enteroviruses remains a major public health concern, yet no approved antiviral therapy is currently available. Although SARS-CoV-2 Mpro and EV71 3Cpro share low sequence similarity, they are highly conserved in overall three-dimensional fold, catalytic cysteine-based mechanism, and strict specificity for P1-glutamine substrates. This structural and functional homology provides a rationale for repurposing coronavirus protease inhibitors to target enteroviral proteases. In this study, we identify bofutrelvir, a clinical-stage protease inhibitor originally developed for SARS-CoV-2, as a potent and broad-spectrum inhibitor of enterovirus. We show that bofutrelvir effectively blocks the activity of the enteroviral 3Cpro, a key enzyme required for viral replication. Using structural, cellular, and animal models, we demonstrate that bofutrelvir suppresses viral replication, reduces disease severity, and provides protection against EV71 and CA16 infections in neonatal mice following oral administration. Importantly, bofutrelvir exhibits favorable pharmacokinetic properties and has previously demonstrated good safety and tolerability in human clinical trials. Together, our findings highlight bofutrelvir as a promising candidate for repurposing as an orally available antiviral therapy for enterovirus-associated diseases.