Preclinical evaluation of GW406108X, a broad-spectrum therapeutic blocking ULK1/2-mediated autophagy to inhibit enterovirus replication.

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen, with severe cases developing into acute flaccid myelitis, a paralytic condition. Despite growing concern, no approved antivirals currently exist for EV-D68, underscoring the urgent need for therapeutic discovery. Here, we report the development of a high-throughput, phenotypic-based assay to screen 7986 compounds across nine diverse compound libraries for EV-D68 antivirals, on two EV-D68-susceptible cell lines (RD and H1299). The screen identified GW406108X as a potent post-entry inhibitor of EV-D68 infection (EC50: 1.804 μmol/L). Mechanistic studies using luciferase replicon assays, siRNA knockdowns, and drug-resistant mutant generation suggest that GW406108X targets the autophagy pathway through ULK1/2 inhibition. Transmission electron microscopy and fluorescence bioimaging demonstrate a significant reduction in autophagosome formation in treated, infected cells. This disruption likely impairs the virus's ability to exploit autophagy, which may in turn hinder replication organelle formation and non-lytic virion release, leading to reduced viral replication. Preclinical evaluations showed that GW406108X demonstrates strong antiviral efficacy without detectable cytotoxicity. These findings reveal a previously uncharacterized antiviral mechanism and position GW406108X as a promising candidate for antiviral therapeutic development. Findings from this study expand the current antiviral landscape for enteroviruses and represent a significant step toward clinical intervention for EV-D68 and potentially related viral pathogens.