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

Abstract

The hexanucleotide repeat expansion GGGGCC [r­(G4C2)exp] within intron 1 of C9orf72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r­(G4C2)exp RNA gain- and loss-of-function mechanisms. The r­(G4C2)exp RNA folds into both a hairpin structure with repeating 1 × 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(G4C2)exp. Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r­(G4C2)exp hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 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.

Conformational Dynamics of RNA G4C2 and C2G4 Repeat Expansions causing ALS/FTD using NMR and Molecular Dynamics Studies

“…Our previous studies as well as those completed by others report that depending on the salt properties r(G4C2) exp prefers either a hairpin structure with repeating 1×1 G/G internal loops in syn-anti orientations (Figure 1AB) or a G-quadruplex structure 53 . Moreover, a small molecule, CB253, (Figure 1C) revealed formation of a minor and highly dynamic RNA structure, where the repeating 1×1 G/G loops transformed to 2×2 GG/GG due to strand slippage, which was detected by NMR spectroscopy 29 . Before investigating the structural properties of 2×2 GG/GG internal loops using T-REMD, we validated the RNA force field.…”

“…Recently, we showed that a small molecule, CB253 (Figure 1C), targeting r(G4C2) hexanucleotide repeat expansions associated with ALS/FTD can cause strand-slippage to transform the global RNA structure to an alternative form, which has 2×2 GG/GG internal loop motifs 29 . This internal loop motif is both transient and highly dynamic displaying rapid 1×1 G/G ↔ 2×2 GG/GG transformations, which prevents its characterization by traditional biophysical techniques.…”

“…This internal loop motif is both transient and highly dynamic displaying rapid 1×1 G/G ↔ 2×2 GG/GG transformations, which prevents its characterization by traditional biophysical techniques. 19 F NMR studies utilizing 19 F modified cytidine residues in closing base pairs indicate that 1×1 G/G and 2×2 GG/GG internal loop motifs formed 70% and 30%, respectively, in the absence of CB253 (Figure 1C) 29 . In vitro experimental studies also showed that 2×2 GG/GG was preferred by r(G4C2) exp in the presence of CB253 29 .…”

“…19 F NMR studies utilizing 19 F modified cytidine residues in closing base pairs indicate that 1×1 G/G and 2×2 GG/GG internal loop motifs formed 70% and 30%, respectively, in the absence of CB253 (Figure 1C) 29 . In vitro experimental studies also showed that 2×2 GG/GG was preferred by r(G4C2) exp in the presence of CB253 29 . This observation provides a new direction in targeting r(G4C2) exp with small molecules that can inhibit the pathobiological effects driven by this expanded repeat.…”

The cancer-associated RBM39 bridges the pre-mRNA, U1 and U2 snRNPs to regulate alternative splicing

“…A similar strategy was recently used to lower the level of huntingtin mRNA and develop an innovative therapeutic approach against Huntington’s disease (Bhattacharyya et al, 2021). The rapid expansion of the field of RNA therapeutics correcting splicing has shown the potential of antisense oligonucleotide and small molecule splicing switches to induce targeted splicing correction and has provided novel therapeutic strategies for genetic diseases (Campagne et al, 2019a; Centa et al, 2020; Hua et al, 2011; Lentz et al, 2013; Naryshkin et al, 2014; Palacino et al, 2015; Ursu et al, 2021). In the future, it could also benefit cancer therapy.…”