Identification of Small-Molecule Inhibitors for EnterovirusA71 IRES by Structure-Based Virtual Screening

Structured RNAs play a crucial role in regulating gene expression, which includes both protein synthesis and RNA processing. Dysregulation of these processes is associated with various conditions, including viral and bacterial infections, as well as cancer. The unique tertiary structures of structured RNAs provide an opportunity for small molecules to directly modulate such processes, making them promising targets for drug discovery. Although small-molecule inhibitors targeting RNA have shown early success, <i>in silico</i> strategies like structure-based virtual screening remain underutilized for RNA-targeted drug discovery. In this study, we developed a virtual screening scheme targeting the structural ensemble of EV-A71 IRES SL II, a noncoding viral RNA element essential for viral replication. We subsequently optimized the experimentally validated hit compound IRE-03 from virtual screening through an “analog-by-catalog” search. This led to the identification of a more potent IRES inhibitor, IRE-03-3, validated through biochemical and functional assays with an EC₅₀ value of 11.96 μM against viral proliferation. Our findings demonstrate that structure-based virtual screening can be effectively applied to RNA targets, providing exciting new opportunities for future antiviral drug discovery.