Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. 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, thus repressing translation. This mechanism is 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.
Discoveries of RNA roles in cellular physiology and pathology are increasing the need for new tools that modulate the structure and function of these biomolecules, and small molecules are proving useful. In 2017, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND) and discovered distinguishing physicochemical properties of RNA-targeting ligands, leading us to propose the existence of an “RNA-privileged” chemical space. Biennial updates of the database and the establishment of a website platform () have provided new insights and tools to design small molecules based on the analyzed physicochemical and spatial properties. In this report and R-BIND 2.0 update, we refined the curation approach and ligand classification system as well as conducted analyses of RNA structure elements for the first time to identify new targeting strategies. Specifically, we curated and analyzed RNA target structural motifs to determine the properties of small molecules that may confer selectivity for distinct RNA secondary and tertiary structures. Additionally, we collected sequences of target structures and incorporated an RNA structure search algorithm into the website that outputs small molecules targeting similar motifs without a priori secondary structure knowledge. Cheminformatic analyses revealed that, despite the 50% increase in small molecule library size, the distinguishing properties of R-BIND ligands remained significantly different from that of proteins and are therefore still relevant to RNA-targeted probe discovery. Combined, we expect these novel insights and website features to enable the rational design of RNA-targeted ligands and to serve as a resource and inspiration for a variety of scientists interested in RNA targeting.
Discoveries of RNA roles in cellular physiology and pathology are raising the need for new tools that modulate the structure and function of these biomolecules, and small molecules are proving useful. In 2017, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND) and discovered distinguishing physicochemical properties of RNA-targeting ligands, leading us to propose the existence of an RNA-privileged chemical space. Biennial updates of the database and the establishment of a website platform (rbind.chem.duke.edu) have provided new insights and tools to design small molecules based on the analyzed physicochemical and spatial properties. In this report and R-BIND 2.0 update, we refined the curation approach and ligand classification system as well as conducted analyses of RNA structure elements for the first time to identify new targeting strategies. Specifically, we curated and analyzed RNA target structural motifs to determine properties of small molecules that may confer selectivity for distinct RNA secondary and tertiary structures. Additionally, we collected sequences of target structures and incorporated an RNA Structure Search algorithm into the website that outputs small molecules targeting similar motifs without a priori secondary structure knowledge. Cheminformatic analyses revealed that, despite the 50% increase in small molecule library size, the distinguishing properties of R-BIND ligands remained significantly different to that of proteins and are therefore still relevant to RNA-targeted probe discovery. Combined, we expect these novel insights and website features to enable rational design of RNA-targeted ligands and to serve as a resource and inspiration for a variety of scientists interested in RNA targeting.
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.
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