Selective C9orf72 G-Quadruplex-Binding Small Molecules Ameliorate Pathological Signatures of ALS/FTD Models

Abstract

The G-quadruplex (G4) forming C9orf72 GGGGCC (G4C2) expanded hexanucleotide repeat (EHR) is the predominant genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Developing selective G4-binding ligands is challenging due to the conformational polymorphism and similarity of G4 structures. We identified three first-in-class marine natural products, chrexanthomycin A (cA), chrexanthomycin B (cB), and chrexanthomycin C (cC), with remarkable bioactivities. Thereinto, cA shows the highest permeability and lowest cytotoxicity to live cells. NMR titration experiments and in silico analysis demonstrate that cA, cB, and cC selectively bind to DNA and RNA G4C2 G4s. Notably, cA and cC dramatically reduce G4C2 EHR-caused cell death, diminish G4C2 RNA foci in (G4C2)29-expressing Neuro2a cells, and significantly eliminate ROS in HT22 cells. In (G4C2)29-expressing Drosophila, cA and cC significantly rescue eye degeneration and improve locomotor deficits. Overall, our findings reveal that cA and cC are potential therapeutic agents deserving further clinical study.

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes

“…Furthermore, we demonstrated how the G4-ligand PDS can diminish mG4-induced aggregation of (GGGGCC) 10 by driving the system to a lower molecularity state. In previously reported studies, G4-ligands have been shown to prevent aggregation events in ALS/FTD phenotypes 26,24 . It was hypothesised that the ligands interfere with the downstream biological functions of the gene, specifically in relationship to the toxic translated protein 52 , or displace proteins bound to the repeats.…”

“…Indeed, the candidacy of these structures as potential therapeutic targets is demonstrated by using small-molecule probes that bind to and stabilise their secondary structures, leading to amelioration of disease phenotypes 20,21,22,23,24,25 . In particular, the use of ligands to bind G4s has been shown to ameliorate ALS phenotypes in neuronal cells 20,24,26,27 and targeting of the hairpin with a small molecule inhibited repeat-associated non-ATG (RAN) translation and subsequent generation of toxic dipeptide repeats from the C9orf72 gene mutation 21 . However, none of these studies have fully clarified the role of the nucleic acid structures in the regulation of pathological aggregation, which is a pre-requisite to devising optimised therapeutic agents.…”

“…r(GGGGCC) n , in particular, has previously been shown to arrange into hairpin and G- quadruplex (G4) structures, both of which have exhibited potential involvement in disease progression 20 . Indeed, the candidacy of these structures as potential therapeutic targets is demonstrated by using small-molecule probes that bind to and stabilise their secondary structures, leading to amelioration of disease phenotypes 20,21,22,23,24,25 . In particular, the use of ligands to bind G4s has been shown to ameliorate ALS phenotypes in neuronal cells 20,24,26,27 and targeting of the hairpin with a small molecule inhibited repeat-associated non-ATG (RAN) translation and subsequent generation of toxic dipeptide repeats from the C9orf72 gene mutation 21 .…”

Pathogenic CANVAS-causing but not nonpathogenic RFC1 DNA/RNA repeat motifs form quadruplex or triplex structures

“…Ligands that bind to G-quadruplexes or triplex structures are known. Ligands specific to the structures formed by disease-specific DNA and RNA repeat motifs have been shown to inhibit gene expression, alter gene splicing, or alter repeat instability ( 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ). Understanding the disease-specific DNA and RNA structures formed by the CANVAS-causing repeat motifs in RFC1 , relative to the nonpathogenic motifs, is a beginning to opening such therapeutic paths.…”

G-Quadruplexes Formation by the C9orf72 Nucleotide Repeat Expansion d(GGGGCC)n and Conformation Regulation by Fangchinoline

“…A wide-spectrum GQ ligand, TMPyP4, was reported to be able to mitigate neurotoxicity in Drosophila models by altering the structure of C9-HRE RNA GQ, which causes an inhibition of the toxic repeat-associated non-AUG (RAN) translation [ 30 , 31 , 32 ]. Zhu [ 33 ] and other groups [ 34 , 35 ] have reported that small-molecule ligands targeting C9-HRE RNA GQ can significantly reduce RNA foci and DPRs levels, and thus ameliorate C9ALS/FTD pathology in vitro and in vivo. Although the pathological efficacy of small-molecule ligands targeting C9-HRE DNA GQs still needs to be addressed, it is posited that modulating the stability or topology of C9-HRE DNA GQ structures might contribute to the impediment of transcription within the C9-HRE locus, and thus reduce the overall C9-HRE-linked gain-of-function pathogenic mechanisms, such as ribonucleoprotein sequestration by RNA foci or the RAN translation of toxic DPRs [ 36 ].…”