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 EC₅₀ 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 (EC₅₀ > 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.