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.

Figure 1

Compounds cA, cB, and cC bind DNA (G4C2)₄ G4 and RNA (G4C2)₂ G4. (A) Imino region of 1D ¹H NMR spectra of DNA (G4C2)₄ G4 and that titrated with compounds cA (left), cB (middle), and cC (right), respectively, at different ratios from 1:1 (light brown) to 1:10 (pink). (B) Fluorescence intensity assay of 3′- FAM-labeled DNA (G4C2)₄ G4 titrated with cA, cB, and cC, respectively, with concentration from 0.1 to 10 mM for determining the K_D value. The curve was fitted by nonlinear regression (One site — Specific binding with Hill slope). (C) Imino region of 1D ¹H NMR spectra of RNA (G4C2)₂ G4 and that titrated with compound cA at 1:3 (green), 1:5 (blue), and 1:10 (pink) ratios. (D) Fluorescence intensity assay of 3′- FAM-labeled RNA (G4C2)₂ G4 titrated with cA with concentration from 0.1 to 10 mM for determining the K_D value. The curve was fitted by nonlinear regression (One site — Specific binding with Hill slope).

Figure 2

Compound structures. (A) Planar structures of compounds cA, cB, and cC. (B) Key HMBC, COSY, and NOESY correlations of compounds cA, cB, and cC. (C) Single-crystal X-ray diffraction confirms the NMR structure of cA—the open form (top) has exocyclic acetonyl and carboxylic acid groups; the cyclic form (bottom) has an α-hydroxylactone ring. Each form has 50% occupancy of molecular sites within the crystal.

Figure 3

Safety evaluations of compounds. (A) Permeability (cm/s) of memantine, nimodipine, DB1246, cA, cB, and cC. n = 5 batches of cell cultures; p values were determined by one-way ANOVA (Tukey’s multiple comparisons test). (B) Cytotoxicity of cA, cB, and cC was tested at concentrations of 0.1, 1, and 10 μg/mL (in molar units, cA: 0.16, 1.61, 16.13 μM; cB: 0.16, 1.58, 15.77 μM; cC: 0.16, 1.57, 15.67 μM) on HEK293T cells. n = 4 batches of cell cultures. (C) Hemolytic activity of cA, cB, and cC was tested at 0, 250, 500, and 1000 μg/mL (in molar units, cA: 0, 0.40, 0.81, 1.61 mM; cB: 0, 0.39, 0.79, 1.58 mM; cC: 0, 0.39, 0.78, 1.57 mM). Phosphate-buffered saline (PBS) and Triton X-100 served as negative and positive controls, respectively. n = 3 independent preparations. All data are mean ± s.e.m.

Figure 4

Compounds cA and cC rescue G4C2 EHR-related pathologies in cells. (A) Representative images of G4 antibody (green) labeled Neuro2a cells transfected with DNA (G4C2)₂₉ without (left) or with cA (1.61 μM, middle), cC (1.57 μM, right) treatments. DAPI was used as the counterstain (red). (B) Cell viability of cA, cB, or cC (1 μg/mL, in molar units, cA: 1.61 μM; cB: 1.58 μM; cC: 1.57 μM)-treated Neuro2a cells transfected with DNA (G4C2)₂₉. n = 8 batches of cell cultures; p values were determined by unpaired two-tailed t-tests. (C) Representative RNA fluorescent in situ hybridization (FISH) coupled hnRNP H (red) immunostaining images of Neuro2a cells transfected with DNA (G4C2)₂₉ and treated with cA (1.61 μM) or cC (1.57 μM). The RNA foci of G4C2 and CAGG repeats were detected with corresponding fluorescent probes (green). DAPI was used as the counterstain (blue). (D) Quantification of G4C2 and CAGG RNA foci numbers per cell in (G4C2)₂₉-expressing cells treated with cA (1.61 μM) or cC (1.57 μM). n = 17–25 coverslips from three batches of cell cultures; p values were determined by one-way ANOVA (Tukey’s multiple comparisons test).

Figure 5

Compounds cA and cC rescue G4C2 EHR-related pathologies in Drosophila. (A) Representative external eye images (top) and scanning electron microscopy (SEM) images (bottom) of 7-day-old WT and GMR-GAL4-(G4C2)₂₉Drosophila fed with DMSO (100 μM), compound cA (100 μM), or cC (100 μM) during the larval stage. (B) Quantification of the eye degeneration percentage of WT and GMR-GAL4-(G4C2)₂₉Drosophila fed with DMSO (100 μM), compound cA (100 μM), or cC (100 μM). n = 7–18 animals; p values were determined by one-way ANOVA (Tukey’s multiple comparisons test). (C) Climbing assay of 7-day-old WT and OK371-GAL4-(G4C2)₂₉Drosophila fed with DMSO (100 μM), compound cA (100 μM), or cC (100 μM) during the larval stage, showing the percentage of flies staying at the bottom (<2 cm) of the testing tubes at the testing time of 25 s. n = 8 batches of experiments; p values were determined by one-way ANOVA (Tukey’s multiple comparisons test). All data are mean ± s.e.m.

Figure 6

Structural models of DNA G4C2 G4 with cA, cB, and cC. Models of the predicted atomic interactions between DNA (G4C2)₄ G4 (PDB ID code: 2N2D; top: ribbon view, bottom: surface view) and cA (A, sticks view), cB (B, sticks view), cC (C, sticks view), were shown with intermolecular hydrogen bonds (red dashed lines), respectively.

Figure 7

ROS assay of cA, cB, and cC. (A) Cellular ROS levels in different treatment groups. n = 18–42 wells from three batches of cell cultures; p values were determined by one-way ANOVA (Tukey’s multiple comparisons test); # indicates the comparison to the control (ctrl) group; * indicates the comparison to E group. All data are mean ± s.e.m. (B) Dose–response curves (x: log concentration, y: normalized ROS fluorescence intensity) of cA, cB, and cC on scavenging cellular ROS tested on differentiated HT22 cell cultures pretreated with 5 mM l-glutamate. n = 3 independent preparations.