The Hairpin Form of r(G ₄ C ₂ ) ^exp in c9ALS/FTD Is Repeat-Associated Non-ATG Translated and a Target for Bioactive Small Molecules

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G ₄ C ₂ repeat [(G ₄ C ₂ ) ^exp ] in C9ORF72. ALS/FTD-associated toxicity has been traced to the RNA transcribed from the repeat expansion [r(G ₄ C ₂ ) ^exp ], which sequesters RNA-binding proteins (RBPs) and undergoes repeat-associated non-ATG (RAN) translation to generate toxic dipeptide repeats. Using in vitro and cell-based assays, we identified a small molecule (4) that selectively bound r(G ₄ C ₂ ) ^exp , prevented sequestration of an RBP, and inhibited RAN translation. Indeed, biophysical characterization showed that 4 selectively bound the hairpin form of r(G ₄ C ₂ ) ^exp , and nuclear magnetic resonance spectroscopy studies and molecular dynamics simulations defined this molecular recognition event. Cellular imaging revealed that 4 localized to r(G ₄ C ₂ ) ^exp cytoplasmic foci, the putative sites of RAN translation. Collectively, these studies highlight that the hairpin structure of r(G ₄ C ₂ ) ^exp is a therapeutically relevant target and small molecules that bind it can ameliorate c9ALS/FTD-associated toxicity. The most common cause of ALS is an expanded RNA repeat [r(G ₄ C ₂ ) ^exp ] that folds into two forms in vitro, a G-quadruplex and a hairpin. Wang et al. show that the hairpin form is present in cells, undergoes aberrant translation that causes toxicity, and thus is a target for therapeutic development.