A Small Molecule Exploits Hidden Structural Features within the RNA Repeat Expansion That Causes c9ALS/FTD and Rescues Pathological Hallmarks

The hexanucleotide repeat expansion GGGGCC [r(G(4)C(2))(exp)] within intron 1 of C9orf 72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD., the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(G(4)C(2))(exp) RNA gain- and loss-of-function mechanisms. The r(G(4)C(2))(exp) RNA folds into both a hairpin structure with repeating 1 x 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r(G(4)C(2))(exp). Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 x 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r(G(4)C(2))(exp) hairpin interconverts between 1 x 1 and 2 x 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 x 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.