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

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 noncovalent RNA-binding scaffold. In addition to known RNA-reactive
electrophiles such as N-acylimidazoles and bis(2-chloroethyl)amines,
covalent screening enabled the surprising discovery that common thiol-reactive
electrophiles (chloroacetamide) and rarely characterized electrophiles
(3-chloropivalamide) cross-linked to RNA. 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 bis-benzimidazole 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 differences in the RNA and DNA binding
modes, revealing that proper positioning of the electrophile toward
the nucleophilic guanine residue is important for efficient covalent
bond formation. Overall, this study highlights the potential of rationally
designing covalent RNA-targeting small molecules.