Development of Enterovirus D68 3C Protease Inhibitors with In Vivo Efficacy in a Neurological Infection Model

Abstract Enterovirus D68 (EV-D68) is a non-polio enterovirus that primarily causes respiratory illness but can also lead to severe disease, including acute flaccid myelitis. No approved vaccine or antiviral is available. Here, we report the rational design and structure−property optimization of dipeptidyl EV-D68 3C protease (3Cpro) inhibitors. Structure-guided optimization and prodrug modification identified Jun13133 as a potent antiviral active against multiple contemporary EV-D68 isolates and EV-A71, with high selectivity. Computational modeling supported a stable substrate-mimetic binding mode consistent with reported covalent 3Cpro inhibitors. Jun13133 also showed favorable in vivo pharmacokinetic properties. In a neonatal mouse model of EV-D68-induced neurological disease, Jun13133 mitigated paralysis, prevented weight loss, and reduced viral replication in spinal cord and muscle tissues. These results validate EV-D68 3Cpro as a druggable antiviral target and support Jun13133 as a promising lead for further development.