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.

Keywords: NMR spectroscopy; RNA; amyotrophic lateral sclerosis; bistable RNA; frontotemporal dementia; microsatellite disorders; quinazoline; repeat associate non-ATG (RAN) translation; repeat expansion; small molecules.

Figure 1.

Identification and validation of CB253, a lead molecule targeting r(G₄C₂)₈ hairpin. (A) Chemical structures of established small molecules that target r(G₄C₂)^exp. Ellipticine-based derivative compound 4 and benzimidazole CB096, selectively bind 1 × 1 GG internal loops, where CB253 targets 2 × 2 GG internal loops (B). (C) CB253 selectively binds the r(G₄C₂)₈ hairpin (EC₅₀ = 30(±4) μM) over the fully base paired r(GGCC)₁₀, as determined by microscale thermophoresis (MST). Data points include the mean values ± SD and are representative of two independent experiments, each performed in duplicate. (D) Binding isotherms for CB253 and 5′ Fl-r(G₄C₂)₄, where Fl indicates fluorescein. Data are reported as the mean ± SD and are representative of two independent experiments, each performed in duplicate.

Figure 2.

Binding of CB253 to the r(G₄C₂)₈ hairpin investigated by 1D imino proton NMR spectroscopy. Representative 1D imino proton spectra recorded upon dose-dependent addition of CB253 to the r(G₄C₂)₈ hairpin. Upon addition of two equivalents of CB253, imino proton peaks (purple circles) emerge in slow exchange with the apo r(G₄C₂)₈ hairpin RNA population showcased by green circles. Signal saturation occurs at a 6:1 ratio, indicating the stoichiometry of the complex. 1D NMR spectrum is representative of two independent experiments.

Figure 3.

Modeling of r(G₄C₂)₈’s structure by free-energy minimization suggests multiple stable conformations. (A) Secondary structures of the four lowest energy folds predicted by free-energy minimization. The lowest free-energy (LFE) structure, Fold 1, comprises five 1 × 1 GG internal loops (highlighted in orange and blue) and a 2 × 1 GG/G asymmetric internal loop (highlighted in purple). Fold 2 features six 1 × 1 GG internal loops (orange and blue) and an extended heptaloop. Folds 3–5 all feature 2 × 2 GG internal loops (highlighted in yellow and green). (B) 1D imino proton spectrum of a model RNA duplex featuring a single 2 × 2 GG internal loop in the presence and absence of CB253. The addition of CB253 triggered the emergence of imino proton peaks highlighted in purple circles. 1D NMR spectrum is representative of two independent experiments. (C) CB253 binds an RNA model that folds into a single structure with three 2 × 2 GG internal loops (highlighted in orange) with an EC₅₀ of 37(±4) μM, as assessed via MST. Data are reported as the mean ± SD and are representative of two independent experiments, each performed in duplicate.

Figure 4.

CB253 binds 2 × 2 GG internal loops formed by r(G₄C₂)^exp, either by conformational selection or induced fit. (A) Modeling of folds formed by a r(G₄C₂)₂ duplex by free-energy minimization. (B) Monitoring the slow exchange equilibrium between the 1 × 1 GG and 2 × 2 GG internal loop populations by means of replacing cytosine residue at position 6 with 5-fluoro C (^5FC) via 1D imino proton and ¹⁹F NMR spectroscopy. (C) Addition of CB253 to ^5FC6-r(G₄C₂)₂ RNA duplex recapitulates the imino proton peaks specific for the 2 × 2 GG internal loop (purple circles). Saturation of the NMR signal is indicative of the stoichiometry of the complex (2:1 small molecule:RNA). 1D NMR spectrum is representative of two independent experiments. (D) Addition of CB253 to ^5FC6-r(G₄C₂)₂ RNA duplex selectively affects the ¹⁹F NMR peak at −166.75 ppm (highlighted in purple). Signal saturation indicates a 2:1 CB253:RNA ratio, as observed in C. Each ¹⁹F NMR spectrum is representative of two independent experiments.

Figure 5.

Major conformational transitions observed in r(G₄C₂)₂ from MD simulation (t = 65 μs). (A) A model RNA system, r(GGGGCCGGGGCC), which initially contained two 2 × 2 GG internal loops in anti-conformation, was designed to study the transformation from 2 × 2 GG to 1 × 1 GG internal loops. The dynamic nature of this construct produced various conformations including base flipping at the terminal sites (B) and internal loops (C). (D) First major conformational transformation, slippage at one end of the RNA duplex occurred around 10 μs. (E) Next, a series of transformations in the loop regions show G₄:G₂₂ adopted the anti-syn orientation. Reshuffling of the bases created slippage at both ends of the RNA helix around 20 μs, resulting in three 1 × 1 GG internal loops (F) in which G₄:G₂₂ and G₁₀:G₁₆ adopted the anti-syn configuration, while G₇:G₁₉ adopted the anti-anti configuration. (G) Middle loop switched to syn-anti conformation around 33 μs, creating an RNA duplex with three 1 × 1 GG internal loops adopting anti-syn, syn-anti, and anti-syn orientations, respectively. Time stamps are provided below each figure corresponding to conformational transformations observed in the MD trajectory (see Movies S1 and S2).

Figure 6.

CB253 ameliorates various cellular c9ALS/FTD-associated pathologies by directly binding r(G₄C₂)^exp. (A) CB253 inhibits RAN translation (mCherry) of r(G₄C₂)^exp selectively in transfected HEK293T cells. Canonical translation corresponds to ATG-GFP. Data are reported as the mean ± SD of three independent measurements each performed in triplicate. ****p < 0.0001. (B) CB253 does not alter r(G₄C₂)₆₆ mRNA levels in transfected HEK293T cells. Data are reported as the mean ± SD (n = 3). n.s. indicates not significant as determined by an ordinary one-way ANOVA multiple comparison test. (C) CB253 reduces the levels of the toxic dipeptide repeat poly(GP) upon 24 h incubation in transfected HEK293T cells. Poly(GP) levels were measured using an electrochemiluminescent sandwich immunoassay. Data are reported as the mean ± SD (n = 2 independent experiments, each with two biological replicates). *p < 0.05, as determined by a two-tailed Student’s t-test. (D) ASO-Bind-Map in r(G₄C₂)₆₆-No ATG-GFP transfected HEK293T cells reveals that CB253 directly engages r(G₄C₂)^exp. Data are reported as the mean ± SD (n = 2 independent experiments, each containing four biological replicates). n.s. = not significant; **p = 0.005 as determined by a two-tailed unpaired t- test with Welch’s correction. (E) HEK293T cells stability expressing Lentiviral-S-tdTomato cotransfected with (G₄C₂)₆₆-Nluc (disease), SV40-Luc (canonical translation for normalization), and ATG-GFP (imaging control) demonstrate that r(G₄C₂)₆₆-transfection causes mislocalization of the S-tdTomato marker in the cytoplasm. Additional cells were cotransfected with SV40-Luc and ATG-GFP to control for background mislocalization due to transfection. Treatment of r(G₄C₂)₆₆-expressing HEK293T cells with CB253 rescue S-tdTomato mislocalization. KPT-335 (“KPT”) inhibits nuclear export and was used as a positive control. The ratio of cytoplasmic:nuclear S-tdTomato signal was quantified from n = 3 biological samples for each treatment group and reported above. ****p < 0.0001, as determined by a one-way ANOVA with multiple comparisons. (F) HEK293T cells stably expressing Lentiviral-S-tdTomato cotransfected with (G₄C₂)₆₆-No ATG-NLuc [(G₄C₂)₆₆-induced stress granules], SV40-Luc, and ATG-GFP or transfected with ATG-GFP and treated with 0.5 mM NaAsO₂ [chemically induced stress granules] treated with 25 μM CB253 or vehicle (0.1% DMSO). Ataxin-2 (ATXN2) was imaged by immunohistochemistry as a marker for stress granule formation, and the cells were imaged by confocal microscopy. The number of ATXN-2-positive stress granules per nucleus was quantified from n = 3 biological replicates per treatment group. ****p < 0.0001, as determined by an unpaired t-test with Welch’s correction. (G) CB253 dose dependently lowers the toxic dipeptide repeat poly(GP) levels in a patient-derived lymphoblastoid cell line (LCL). Poly(GP) levels were measured by an electroluminescent sandwich immunoassay. Data are reported as the mean ± SD (n = 3 biological replicates). **p < 0.05, as determined by a one-way ANOVA with multiple comparisons.