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The proteolysis targeting chimera (Protac^®), or protein degradation targeted chimera, is a bifunctional small molecule. Its one end is a ligand that binds the target protein, and the other end is a ligand that binds E3 ubiquitin ligase. The two ends are connected by a segment of linker. The target protein can be pulled closer to E3 enzyme in vivo, so that the target protein is labeled with ubiquitin, and then degraded through the ubiquitin-proteasome pathway.

RIBOTACs (ribonuclease targeting chimeras) is a promising new strategy for RNA degradation. RIBOTACs is based on the selective binding of small molecules to RNA, especially RNA with complex secondary and tertiary structures. The key innovation is to convert RNA-binding molecules into RNA-degrading molecules and develop the RIBOTACs concept by binding RNA-binding molecules to a small molecule and activating RNase L (a potential ribonuclease). This article mainly describes the use of this technique to target the novel coronavirus's RNA genome, inhibit the frameshift of RNA, and recruit cellular ribonuclease to kill novel coronavirus completely.

At the end of 2019, a respiratory disease quickly spread across the world. The COVID-19 is a global pandemic caused by SARS-CoV-2 virus with RNA genome. In this article “Targeting the SARS-CoV-2 RNA genome with small molecule binders and ribonuclease targeting chimera (RIBOTAC) degraders” [1], the authors’ overall idea is to first design the small molecules that bind or degrade the structural functional elements in novel coronavirus to inhibit virus reproduction, and then further process the ligand into a chimera (RIBOTAC) targeting ribonuclease to recruit cellular ribonuclease to destroy the virus genome.

Firstly, the structure of SARS-CoV-2 genome is analyzed, and the site of structural convergence is identified in the frameshift element (FSE) that controls the translation of pp1a and pp1ab polymers. This translation is very important for virus replication and pathogenesis. The FSE includes an Attenuator hairpin (AH), a Slippery Site (SS), and a three-stem pseudoknot, which together cause the ribosome to pause to initiate frameshift, thereby changing the protein coding content of the mRNA. Interestingly, enhancing the thermodynamic stability of FSE will damage the frameshift efficiency. Another way to suppress frameshift is to stabilize FSE through the binding of structure-specific ligands, which has been proved to be effective against other viral FSE. AH has a 1 × 1 nucleotide UU internal ring in its stem, and the conservation of this motif indicates that the UU ring is indeed a real structure, and its binding may inhibit the function of FSE. The authors screened 3271 compounds from the compound library by using AbsorbArray experiment, and obtained the following hit compounds that bind to RNA (figure below). And in the follow-up experiments, it was verified that C5 can significantly restrain the frameshift ability of SARS-CoV-2-FSE.

The authors then used their previous target fishing experiment-Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), specifically the proximity reaction, to make the small molecules bound by RNA covalently bind to the target protein. After modifying the structure of compound C5, the author obtained the small molecule C5-Chem-CLIP, the structure of which is shown in the following figure. Target fishing experiments can prove that small molecules of C5-Chem-CLIP can specifically bind to SARS-CoV-2 by incubating at the cellular level with SARS-CoV, which has a structure similar to SARS-CoV-2. In addition, the competitive experiment can also prove the target effect of C5-Chem-CLIP.

Finally, the authors speculated whether the efficiency and selectivity of compounds can be enhanced in a convenient way by converting simple binding small molecules (such as C5) to recruit endogenous nucleases to catalyze and substoichiometric cleave the target compounds or to target ribonuclease chimera (RIBOTAC). The authors wanted to modify the small molecules to bind heterocyclic RNase L-recruiting molecules to directionally degrade SARS-CoV-2 RNA. Through the structural modification of the compound, they obtained C5-RIBOTAC and C5Mae C14, of which the C5-C14 has a relatively weak activity. As expected, both C5-RIBOTAC and C5-C14 can reduce the frameshift efficiency of SARS CoV-2 FSE, and C5-RIBOTAC can specifically reduce SARS-CoV-2 FSE at the cellular level (figure below).

In short, from vaccine development to drug reuse and discovery, the scientific community is adopting a variety of strategies to deal with the ongoing SARS-CoV-2 pandemic. Through the in-depth study of novel coronavirus revealing the stable structure in the virus genome, the authors tested the drug availability of SARS-CoV-2 frameshift elements, and designed small molecules that selectively inhibit SARS-CoV-2 frameshift through two different modes of action. The simple use of small molecules and ligands that can recruit endogenous nucleases that play a role in the immune response of the virus has achieved the purpose of degrading the virus genome, which provides a reference for the development of COVID-19 drugs.

References:

1. Haniff, H. S., Tong, Y., Liu, X., Chen, J. L., Suresh, B. M., Andrews, R. J., ... & Disney, M. D. (2020). Targeting the SARS-CoV-2 RNA genome with small molecule binders and ribonuclease targeting chimera (RIBOTAC) degraders. ACS Central Science.
2. Dey, S. K., & Jaffrey, S. R. (2019). RIBOTACs: Small Molecules Target RNA for Degradation. Cell chemical biology, 26(8), 1047-1049.