Enterovirus D68: A Novel Inhibitor Reveals Underlying Molecular Mechanisms of Viral Entry and Uncoating

While enterovirus D68 (EV‐D68) has emerged as a significant pediatric pathogen causing severe respiratory illness and acute flaccid myelitis, no effective therapeutics exist. Through systematic screening of natural products, we identified geranyl‐ p ‐trans‐coumaric acid (GCA) as a potent EV‐D68 inhibitor with EC 50 values of 20‒40 μM against multiple clinical strains. Time‐of‐addition analysis revealed that GCA primarily targets early viral entry processes, with strongest inhibition during the adsorption phase. Mechanistic investigations demonstrated that GCA directly binds to EV‐D68 particles and prevents viral uncoating without interfering with initial cellular attachment. Thermal protection and PaSTRy assays confirmed GCA‐mediated capsid stabilization, increasing RNA release temperature from 51°C to 53°C. Receptor binding studies showed that GCA selectively inhibits virus interaction with heparan sulfate proteoglycans but not ICAM‐5, indicating specific interference with secondary receptor engagement. Resistance selection identified critical mutations in VP1 (T92N) and nonstructural protein 2 C (K6R), with resistance requiring both mutations simultaneously, suggesting cooperative dual‐targeting mechanisms. Molecular docking revealed GCA binding to the VP1 canyon region, and combination studies with pleconaril demonstrated synergistic effects (combination index = 0.83). Single mutations alone maintained drug sensitivity, but double mutants exhibited marked resistance (EC 50 > 200 μM), confirming dual targeting of viral capsid and cellular host factors. In vivo efficacy studies demonstrated dose‐dependent protection, with 71% survival at 12 mg/kg/day compared to 18% in controls ( p < 0.01) and significant viral burden reduction in brain and muscle tissues. These findings identify GCA as a promising therapeutic candidate against EV‐D68 operating through novel dual mechanisms.