Rational Design of Broad‐Spectrum Anti‐Enteroviral Molecular Glues Targeting Enteroviral RNAi Suppressors

ABSTRACT Rational design of molecular glues (MGs) remains challenging, as most have been discovered serendipitously and have found limited application in antivirals. Previously, we identified the enteroviral 3A protein as a viral suppressor of RNAi (VSR) that functions through homodimerization to inhibit the antiviral RNA interference (RNAi) pathway. Herein, capitalizing on this homodimerization mechanism, we rationally designed 3A‐targeting broad‐spectrum anti‐enteroviral molecular glues targeting the dimeric interface to induce dysfunctional dimerization. The optimal compound, VTP‐32, exhibited good binding affinity with 3A (K _D = 0.29 µ m ), potent and pan‐enterovirus (groups A, B, D) antiviral effects (EC ₅₀ = 0.21–0.92 µ m ), and good safety (CC ₅₀ > 500 µ m ). VTP‐32 treatment (20 mg/kg) could effectively reduce viral load, alleviate clinical symptoms, and improve survival in EV‐A71‐infected mouse models. Mechanistic studies revealed that VTP‐32 stabilizes 3A protein into an abnormal dimer, promotes viral siRNA generation, and ultimately leads to RNAi‐mediated viral genome degradation. Overall, this study provides a promising countermeasure against enteroviral diseases and a rational design strategy for developing antiviral molecular glues.