Small Molecule Targeting IRES Domain Inhibits Enterovirus 71 Replication via an Allosteric Mechanism that Stabilizes a Ternary Complex

Abstract

We herein report an RNA-targeting antiviral small molecule that reduces replication of the human enterovirus 71 (EV71) via stabilization of an inhibitory small molecule-RNA-protein ternary complex. The EV71 virus poses serious threats to human health, particularly in regions of Southeast Asia, and no FDA approved drugs or vaccines are available. We first screened an RNA-biased small molecule library using a peptide-displacement assay to identify ligands for the stem loop II structure of the EV71 internal ribosomal entry site, which was previously shown to impact viral translation and replication. One ligand, DMA-135, decreased viral translation and replication in cell-based studies in a dosedependent manner with no significant toxicity. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein that then represses translation. This mechanism was further supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

“…We pursued targeting of the 5'-end stem loop structures with the DMA scaffold, which has been previously reported as an RNA binding scaffold that can be successfully optimized for selectivity for distinct RNA elements. 31 To quickly assess potential CoV antiviral activity, human OC43 betacoronavirus was used due to its lower virulence and thus suitability for use in standard cell culture facilities. 3 Vero E6 cells were infected with human coronavirus OC43 at an MOI=1.…”

“…The successes in DMA exploration highlight the scaffold's potential for tuning as well as broad applicability as an antiviral scaffold. 31 Herein we report DMA analogues that show promising antiviral properties by reducing SARS-CoV-2 viral titer in a dose-dependent manner in infected Vero E6 cells. In addition, dual luciferase reporter assays confirmed the antiviral activity of the small molecules to be dependent on the 5'-UTR and proximal region of the SARS-CoV-2 genome.…”

“…The DMA scaffold had been previously reported to be poised for tuning for specific RNA constructs via a facile three-step synthesis. 31 Further investigation and functionalization of the scaffold resulted in a bioactive antiviral analogue that formed a repressive ternary complex with IRES stem loop II RNA and the human AUF1 protein, ultimately inhibiting translation and compromising viral replication. The successes in DMA exploration highlight the scaffold's potential for tuning as well as broad applicability as an antiviral scaffold.…”