Rational Design of Capsid Protein VP1 Degraders to Overcome Pleconaril Resistance in Inhibiting Enterovirus D68

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen with pandemic potential, yet no vaccines or antivirals are available. Capsid inhibitors, such as pleconaril, that target the hydrophobic canyon on the viral capsid protein VP1, exhibit potent antiviral activity but have a low barrier to resistance. Here, we report a targeted protein degradation strategy to overcome antiviral resistance by selectively degrading the capsid protein VP1. Through a structure-based rational design, we developed a series of pleconaril-based PROTACs that recruit the cereblon (CRBN) E3 ligase to degrade the capsid VP1 protein. We established a single-cycle replication assay integrating immunofluorescence and Western blot to quantify VP1 protein levels when compounds were added postviral entry. Linker and CRBN ligand optimization led to the identification of Jun15702, a first-in-class VP1 degrader that displays potent antiviral activity against multiple wild-type EV-D68 strains and retains submicromolar potency against recombinant pleconaril-resistant variant, rMO-VP1 F159 V. Significantly, Jun15702 displayed a higher genetic barrier to drug resistance than pleconaril. Mechanistic studies demonstrate that Jun15702 not only inhibits viral entry, as does pleconaril, but also induces CRBN-dependent degradation of VP1 when added post viral entry. Collectively, this work demonstrates targeted degradation of a viral structural protein as a viable strategy and introduces a new paradigm for overcoming drug resistance in antiviral discovery against enteroviruses.