Covalent Probes Reveal Small-Molecule Binding Pockets in Structured RNA and Enable Bioactive Compound Design

Area of Science:

• RNA structural biology
• Antiviral drug discovery
• Molecular virology

Background:

• The SARS-CoV-2 frameshift stimulation element (FSE) is crucial for viral replication and a potential antiviral target.
• Understanding RNA-ligand interactions is key for developing effective antiviral therapies.

Purpose of the Study:

• To identify small-molecule binding pockets within the SARS-CoV-2 FSE.
• To develop a ligandability map for the FSE.
• To design and synthesize novel bioactive compounds with antiviral activity.

Main Methods:

• Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP) for covalent target validation and binding site mapping.
• Dimethyl sulfate (DMS) probing combined with Deconvolution of RNA Alternative Conformations (DRACO) to assess ligand-induced structural changes.
• Structure-based design, synthesis, and competitive displacement assays.
• In silico pocket analysis of cryo-EM derived FSE structures.

Main Results:

• Chem-CLIP precisely mapped the binding pocket of the fluoroquinolone merafloxacin at nucleotide resolution.
• Development of bioactive compounds with antiviral activity through structure-based design and synthesis.
• DMS-DRACO revealed ligand-induced alterations in local RNA folding, distinguishing direct binding from structural changes.
• In silico analysis identified recurring cavities in the FSE, corroborating experimental findings.

Conclusions:

• Combining Chem-CLIP and DMS profiling is essential for accurate RNA-ligand interaction studies.
• The study provides a strategy for designing small molecules that target RNA structures.
• Findings advance the understanding of RNA-ligand interactions and support FSE as an antiviral target.