Targeted Degradation of Picornaviral 3C Protease via PROTACs Confers High Barrier to Viral Resistance and Broad‐Spectrum Antiviral Activity

ABSTRACT Diseases caused by picornaviruses pose a serious threat to society due to their high contagiousness and widespread prevalence. Beyond the poliovirus vaccine, antiviral therapeutics remain unavailable for most picornaviral infections. Moreover, existing inhibitors under development generally exhibit narrow‐spectrum activity and low barriers to resistance owing to the high mutability of RNA viral proteins. To address these issues, this study constructed a proteolysis‐targeting chimera (PROTAC) targeting the 3C protease (3C ^Pro ) — a key viral enzyme essential for picornavirus protein processing, host shutoff, and immune suppression. In enterovirus 71 (EV71), a representative member of the picornavirus family, experimental results showed that PROTAC molecule effectively inhibits viral replication through a dual mechanism of action: directly inhibiting the catalytic function of the 3C ^Pro and inducing its degradation via the ubiquitin‐proteasome pathway. Moreover, this PROTAC molecule not only demonstrated effective degradation of previously reported and AI‐predicted potential drug‐resistant EV71 mutants, but also exhibited degradation activity against 3C ^Pro from multiple picornaviruses, indicating its potential for broad‐spectrum antiviral activity. By integrating multidisciplinary approaches, this work demonstrates that targeted protein degradation can effectively induce the degradation of 3C ^Pro in picornaviruses, supporting the development of resistance‐resistant, broad‐spectrum antiviral agents and highlighting their potential to address evolving viral threats.