SARS-CoV-2 Antivirals Identified from Small Molecule Modulators of Programmed −1 Ribosomal Frameshifting

Remaining SARS-CoV-2 vulnerable populations and the threat of viral resistance to existing immunity and COVID-19 vaccines highlight the need to expand treatment options. While the number of SARS-CoV-2 viral proteins is limited for therapeutic targeting, the RNA genome offers a wide range of conserved functional structures. Previous studies suggest that modulating the RNA-regulated programmed -1 ribosomal frameshift (-1 PRF) step in the coronavirus life cycle can inhibit viral replication. In this work, we aimed to identify small molecules that target the highly conserved SARS-CoV-2 frameshift site pseudoknot. An RNA-biased synthetic small molecule library screen against the RNA pseudoknot structure identified several candidate molecules. Of these, seven showed evidence of dose-dependent activity in in vitro and in cellulo frameshift assays and inhibited SARS-CoV-2 replication in cell culture. While binding was required for activity, affinity did not correlate with frameshift inhibition. Similarly, small molecule impacts to pseudoknot stability, as measured with an RT-qPCR assay, did not predict impacts on the frameshift efficiency. Comparison of the hits, as well as previously published inhibitors, across several assays supports the need for multiple characterization methods in small molecule-RNA targeting and the possibility of multiple modes of action for -1 PRF modulation. This work underscores the complexity of targeting dynamic RNA structures and the benefits of screening early for function and later optimizing for mode of action. The use of orthogonal techniques led to the identification of several promising hits and a lead antiviral compound.