Discovery of RNA-Reactive Small Molecules Guides Design of Electrophilic Modules for RNA-Specific Covalent Binders

Abstract

RNA is a key drug target that can be modulated by small molecules, however covalent binders of RNA remain largely unexplored. Using a high-throughput mass spectrometry screen of 2,000 electrophilic compounds, we identified ligands that react with RNA in a binding-dependent manner. RNA reactivity was influenced by both the reactive group and the RNA-binding scaffold. Electrophilic modules such as 3-chloropivalamide, bis(2-chloroethyl)amine, chloroacetamide, and N-acylimidazole that react with proteins also cross-linked to RNA, especially when paired with aromatic heterocycles, particularly those with a thieno[3,2-c]pyridinium core. These results suggest that electrophiles commonly used for protein targeting can also covalently modify RNA, potentially contributing to both on- and off-target effects. This insight enabled the design of an RNA-specific covalent compound by modifying a Hoechst scaffold, originally identified to bind DNA, to react selectively with the expanded triplet repeat RNA, r(CUG)exp, that causes myotonic dystrophy type 1 (DM1). Selectivity appears to arise from binding to the RNA major groove near the reactive site. Overall, this study highlights the potential of rationally designing covalent RNA-targeting small molecules. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/649986v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@2049c7org.highwire.dtl.DTLVardef@1cbc49aorg.highwire.dtl.DTLVardef@d88363org.highwire.dtl.DTLVardef@99fef8_HPS_FORMAT_FIGEXP M_FIG C_FIG