SHAPE-enabled fragment-based ligand discovery for RNA.

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• The transcriptome, comprising all RNA molecules in a cell, offers a rich but underexplored target space for small-molecule drug discovery.
• Developing selective ligands for RNA structures is challenging due to RNA's complex nature and the limited tools available for structure-based ligand design.

Purpose of the Study:

• To create a novel technology for discovering small-molecule ligands that bind to specific RNA structures.
• To identify fragments and fragment pairs that bind to the thiamine pyrophosphate (TPP) riboswitch.
• To design and synthesize a high-affinity, druglike ligand for the TPP riboswitch that modulates its function.

Main Methods:

• Fragment-based screening combined with Selective Hydrolase Accepetability for Methylation and Primer extension (SHAPE-MaP) RNA structure probing.
• Structure-activity relationship (SAR) analysis to guide ligand design.
• Synthesis and characterization of linked-fragment ligands.

Main Results:

• Identified small-molecule fragments and cooperatively binding pairs targeting the TPP riboswitch with millimolar to micromolar affinities.
• Designed a novel linked-fragment ligand, distinct from the native ligand, with high ligand efficiency and druglike properties.
• Achieved high nanomolar binding affinity for the TPP thiM riboswitch and demonstrated modulation of RNA conformation during cotranscriptional folding.

Conclusions:

• The developed technology provides a powerful platform for discovering ligands targeting RNA structures.
• Leveraging fragment cooperativity and multisite binding enables the design of high-quality ligands for diverse RNA targets.
• This approach has broad applicability for RNA-targeted drug discovery and chemical biology research.