Structural Features of Small Molecules Targeting the RNA Repeat Expansion That Causes Genetically Defined ALS/FTD

Abstract

Genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide GGGGCC repeat expansions [r­(G4C2)exp] within the C9orf72 gene. In these diseases, neuronal loss occurs through an interplay of deleterious phenotypes, including r­(G4C2)exp RNA gain-of-function mechanisms. Herein, we identified a benzimidazole derivative, CB096, that specifically binds to a repeating 1 × 1 GG internal loop structure, 5′CGG/3′GGC, that is formed when r­(G4C2)exp folds. Structure–activity relationship (SAR) studies and molecular dynamics (MD) simulations were used to define the molecular interactions formed between CB096 and r­(G4C2)exp that results in the rescue of disease-associated pathways. Overall, this study reveals a unique structural feature within r­(G4C2)exp that can be exploited for the development of lead medicines and chemical probes.

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

“…37 In addition to the previously identified benzimidazole derivative CB096 29 (Figure 1A), the 2,4-diaminoquinazoline CB253 (Figure 1B) dose dependently displaced TO-PRO-1 from the r(G 4 C 2 ) 8 hairpin. 29 A direct binding assay by microscale thermophoresis (MST) was used to measure CB253′s affinity for 5′-labeled Cy5-r(G 4 C 2 ) 8 , yielding an EC 50 of 30(±4) μM (Figures 1C and S1A). In contrast, no saturable binding was observed for the fully paired RNA r(GGCCC) 10 , d(G 4 C 2 ) 8 , and triplet repeating RNAs 38 These studies afforded an on-rate (k on ) of ∼3 × 10 4 M −1 × s −1 , an off-rate (k off ) of ∼2.3 × 10 −1 s −1 , and a K d of 21(±4) μM (Figure S3A).…”

“…We previously have shown that model r(G 4 C 2 ) 2 duplexes recapitulate the 1D spectra of r(G 4 C 2 ) 4 and r(G 4 C 2 ) 8 and can allow the identification of a small molecule's binding site. 29 To precisely determine if 1 × 1 GG internal loop sites within the r(G 4 C 2 ) exp hairpin are targeted by CB253, two model duplexes were employed featuring either a single 5′CGG/3′GGC (Figure S8A) or 5′GGC/3′CGG (Figure S8B) motif. Surprisingly, no change was observed in the 1D imino spectra of either construct upon dose-dependent addition of CB253 (Figure S8).…”

“…11 Various small molecules have been reported to inhibit RAN translation, including the ellipticine derivative that stabilizes both 5′CGG/3′GGC and 5′GGC/3′CGG loops 11 selectively interacts with 5′CGG/3′GGC (Figure 1A). 29 In addition, small molecules targeting the G-quadruplex form have also been reported. 12 Collectively, these studies suggest that the r(G 4 C 2 ) exp RNA conformational ensemble 20,30−32 exhibits different structural features that can be stabilized via conformational selection by distinct chemotypes 33 in vitro and in cellulis.…”

“…Notably, the hairpin form is a biologically relevant fold that undergoes RAN translation in vitro and in cellulis . Various small molecules have been reported to inhibit RAN translation, including the ellipticine derivative that stabilizes both 5′C G G/3′G G C and 5′G G C/3′C G G loops and the benzimidazole CB096 that selectively interacts with 5′C G G/3′G G C (Figure A) . In addition, small molecules targeting the G-quadruplex form have also been reported .…”

Supramolecular Polymorphism of (G4C2)n Repeats Associated with ALS and FTD

“…The molecular mechanisms through which these repeat expansions and their quadruplex structures cause the neurodegenerative modifications are not yet known. Nevertheless, some potential therapeutics targeting them are already being investigated [ 9 , 10 ].…”