RNA is an emerging new drug target for the treatment of human diseases. One promising strategy to target RNA is RNA-degrading chimeras which can selectively induce the cleavage of RNA of interest. RNA-degrading chimeras are bifunctional molecules that consist of a "guide arm", which functions to bind to the target RNA, and an "effector arm", which recruits an endogenous ribonuclease (e.g., RNase L) to cleave the bound RNA. RNase L is a latent ribonuclease that is dimerized and activated upon ligand binding. The endogenous ligand of RNase L, 2' ,5-oligoadenylates (2-5A) and a synthetic ligand have been used as the "effector arm" in the reported RNA-degrading chimeras. Antisense oligonucleotides (ASOs) that complementarily bind the primary RNA sequence or small molecules that bind the RNA secondary or tertiary structures can both work as the "guide arm". Particularly, the small molecule-based RNA-degrading chimeras (recently named ribonuclease targeting chimeras or RIBOTACs) have several advantages compared to the ASO-based ones such as better cellular uptake. One of the hallmarks of RNA viruses is that they usually contain highly structured untranslated regions (UTRs) in the RNA genomes, which are often essential to viral replication. We hypothesized that these RNA functional structural elements could be served as targets for RNA-degrading chimeras.<br/>By screening our RNA-focused small molecule library, we identified a small molecule (named C30) that binds to the 5'-UTR of the SARS-CoV-2 RNA genome with sub-micromolar affinity. The binding site of C30 was further determined to be located in a unique four-way junction RNA close to the start codon. Based on C30, the RIBOTAC strategy was used to make SARS-CoV-2 RNA degraders. The most active C30-RIBOTAC achieved a 70% decrease in the mRNA level of SARS-CoV-2 in a minigene-expressing assay. The antiviral activity of C30-RIBOTACs was further validated in a live virus assay. No toxicity was observed in host cells for tested RIBOTACs. We also compared the synthetic RNase L ligand with 2-5A in RIBOTACs. Interestingly, synthetic RNase L has better activity than 2-5A, even though 2-5A has an 80,000-fold better binding affinity to RNase L. Our results show the promise of RNA degrading strategy in antiviral drug development.

Development and evaluation of RNA-degrading chimeras as a new antiviral drug modality

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