Title: Small Molecule Inhibits EV71 Replication via Allosteric Stabilization of a Ternary Complex

Abstract: Human Enterovirus 71 (EV71), the major etiological agent of the hand, foot, and mouth disease (HFMD), represents a serious global health threat against which there is no FDA-approved treatment. Enteroviruses utilize a highly structured type I Internal Ribosome Entry Site (IRES), to facilitate cap-independent translation and viral replication by recruiting multiple host RNA-binding proteins (ITAFs) that modulate these processes. Of particular interest, the stem-loop II (SLII) IRES domain has been shown to interact with several ITAFs through its bulge to effectively modulate viral translation. Further mutational studies revealed that modifications to the aforementioned bulge inhibit EV71 replication by offsetting IRES-driven translation. Altogether, these results provide a strong basis to pursue EV71-SLII as a promising therapeutic target. Here, we screened an RNA-focused small molecule (rfsm) library and identified DMA-135 as a dose-dependent inhibitor of viral translation and replication. Further structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure and stabilizes a ternary complex with AUF1; a complex known to downregulate viral replication. This pipeline has been applied to identify potential antivirals against SARS-CoV-2, and provides a blueprint for the identification of viral inhibitors with improved potency and the validation of their mechanism of action.