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.