Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules

Abstract

The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)(exp)). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. A thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)(exp) and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)(exp). This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)(exp) and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of myotonic dystrophy type 1.

Fig. 1

Identification of small molecules that inhibit the formation of the r(CUG)₁₂-MBNL1 complex (A top) The biochemical outcome of a small molecule that targets r(CUG)^exp would be the improvement of DM1-associated pre-mRNA splicing defects. (A bottom) The biochemical outcome of a small molecule that targets MBNL1 would be the induction of DM1-associated pre-mRNA splicing defects. (B) The small molecules that inhibit formation of the r(CUG)₁₂-MBNL1 complex as identified by screening of the NIH’s MLPCN library.

Fig. 2

Compounds 1 – 4 improve translational defects caused by r(CUG)^exp. Improvement of translational could occur if a compound binds to r(CUG)^exp (compound 2) or MBNL1 (compound 1), as both modes of action disrupt the r(CUG)^exp-MBNL1 complex and could allow for more efficient nucleocytoplasmic export. (A, top) A schematic of the luciferase reporter system that models the DM1 translational defect. The presence of r(CUG)^exp in the 3’ UTR of firefly luciferase reduces nucleocytoplasmic transport and thereby suppresses luciferase expression. However, if a small molecule binds r(CUG)^exp and displaces proteins, nucleocytoplasmic transport is improved and luciferase activity increases. Likewise, if a small molecule binds proteins and displaces them from r(CUG)^exp, nucleocytoplasmic transport is also improved and luciferase activity increases. (A, bottom) Effects on luciferase activity when cells are dosed with 20 µM compound of interest. “+r(CUG)^exp” indicates that the cell line expresses luciferase with r(CUG)^exp in the 3′ UTR, “-r(CUG)^exp” indicates that the cell line expresses luciferase without r(CUG)^exp. Results are expressed as the percentage increase of luciferase activity relative to untreated cells, where a value of “0” denotes no change in activity. Experiments were completed in triplicate. Values shown in the plot are the averages of those experiments, and the errors reported are the standard deviations. (B), competition dialysis data for binding of 1 to BSA, MBNL1, and r(CUG)₁₂. The data clearly show that the preferred target is MBNL1 as there is no measureable binding to r(CUG)₁₂ or to BSA. Experiments were completed in duplicate. The values reported are the averages of those experiments, and the errors reported are the standard deviations.

Fig. 3

Compound 1 induces DM1-like splicing defects in HeLa cells. Briefly, HeLa cells were co-transfected with the mini-gene of interest and a plasmid that expresses r(CUG)^exp or an empty vector. Varying concentrations of 1 were added in growth medium post-transfection, and total RNA was harvested 16–20 h later. The percentage of each splicing isoform was determined by RT-PCR using a radioactively labeled forward PCR primer. (A, top) Schematic of the pre-mRNA splicing isoforms observed for the IR mini-gene. (A, middle) representative gel image of IR alternative splicing in the presence of varying concentrations of 1. (A, bottom) Quantification of exon inclusion for IR alternative splicing in the presence or absence of r(CUG)^exp and 1 (n ≥ 3). Error bars are the standard deviations in the measurements. (B, top) Schematic of the pre-mRNA splicing isoforms observed for the cTNT mini-gene. (A, middle) representative gel image of cTNT alternative splicing in the presence of varying concentrations of 1. (A, bottom) Quantification of exon inclusion for cTNT alternative splicing in the presence or absence of r(CUG)^exp and 1 (n ≥ 3). Error bars are the standard deviations in the measurements. “*” indicates p ≤ 0.05 and “**” indicates p ≤ 0.01 as determined by a two-tailed student t-test.

Fig. 4

Compound 1 induces an MBNL1-dependent DM1-like splicing shift in normal human fibroblasts. (A) Representative RT-PCR assays showing splicing changes in MBNL1-dependent exons in normal fibroblasts treated with increasing amounts of 1 (n=2). The DM1-like splicing shift is depicted as alternative exon inclusion (+alt. ex), while the normal splicing isoform is depicted as alternative exon exclusion (−alt. ex). As a control, untreated and DMSO-treated (DMSO) normal fibroblasts (n=5) and DM1 fibroblasts expressing 2000 CUG repeats (DM1 2000CUG) (n=4) were used. No RT lane refers to RT-PCR control without reverse transcriptase. (B–C) Quantification of alternative splicing shift towards the DM1-like phenotype (% of alternative exon inclusion) in normal fibroblasts treated with increasing concentrations of 1 (orange bars; n=2), untreated and DMSO treated normal fibroblasts (green bars; n=5), siMBNL1 treated normal fibroblasts (red bars; n=2) and DM1-affected human fibroblasts expressing 2000 r(CUG) repeats (blue bars; n=2). Splicing of MBNL1-dependent exons is shown in (B) while splicing of MBNL1-independent exons is shown in (C). Each sample was subjected to RT-PCR twice. The errors reported are the standard deviations derived from analysis of all samples. “*” indicates p ≤ 0.05; “**” indicates p ≤ 0.01; and “***” indicates p ≤ 0.001 as determined by a two-tailed student t-test.

Fig. 5

MBNL1 knock-down exacerbates DM1-like splicing shift induced by compound 1 in normal human fibroblasts. (A) Representative RT-PCR assays showing splicing changes in MBNL1-dependent exons of four genes in normal fibroblasts treated with siRNA against MBNL1 (siMBNL1) and increasing amounts of 1 (n=2). Mock controls are lipofectamine treated samples. (B) Quantification of alternative splicing shift towards the DM1-like phenotype (% of alternative exon inclusion) in four MBNL1-regulated exons (MBNL1, MBNL2, NCOR2, NFIX) upon combined treatment of normal human fibroblasts with an siRNA against MBNL1 and increasing concentrations of 1 (orange bars; n=2). For comparison, splicing shifts in siMBNL1-treated DM1-affected 500CUG fibroblasts are shown (blue bars; n=2). Mock controls represent splicing changes in lipofectamine treated cells (green bars - normal fibroblasts; violet bars - 500CUG fibroblast; n=2). “*” indicates p ≤ 0.05; “**” indicates p ≤ 0.01; and “***” indicates p ≤ 0.001 as determined by a two-tailed student t-test.

Fig. 6

Compound 2 improves DM1-like splicing defects induced in HeLa cells. (A) Compound 2 improves dysregulation of IR alternative splicing in a DM1 model cellular system. (A, top) Representative gel image of IR alternative splicing in the presence of varying concentrations of 2. (A, bottom) Quantification of exon inclusion for IR alternative splicing in the presence or absence of r(CUG)^exp and 2 (n ≥ 3). Error bars are the standard deviations in the measurements. (B) Compound 2 improves dysregulation of cTNT alternative splicing in a DM1 model cellular system. (B, top) Representative gel image of cTNT alternative splicing in the presence of varying concentrations of 2. (B, bottom) Quantification of exon inclusion for cTNT alternative splicing in the presence or absence of r(CUG)^exp and 2 (n ≥ 3). Error bars are the standard deviations in the measurements. The percentage of each isoform was determined by RT-PCR using a radioactively labeled forward PCR primer. “**” indicates p ≤ 0.01 as determined by a two-tailed student t-test.

Fig. 7

Docking studies support the binding of 1 to MBNL1 and 2 to r(CUG) repeats. (A) Extensive interactions are observed between MBNL1 and 1 at the RNA binding pocket. The ethenyl group and the phenyl and thiofuran rings of 1 are stabilized by extensive cation-π interactions with arginine residues. The cation-π interaction between 1 and Arg195 is reminiscent of the MBNL1-RNA interaction . The ligand binding pocket is shown as a transparent surface. Hydrogen bonds are shown as yellow dash lines. (B) Lowest free energy conformation of the 2-r(CUG) complex (binding mode p in Supplementary Table S4 and Supplementary Fig. S13). (B, top) RNA sequence used in MD simulations. Side (B, bottom left) and top (B, bottom right) views of the 2-r(CUG) complex. The yellow wireframe represents the molecular surface of compound 2. The RNA backbone is represented in light blue. For simplicity, hydrogen atoms are not shown. Note that in this binding mode, the interactions in the UU pair (green) are fully lost, and compound 2 stacks between the flanking GC base pairs (represented in red and blue).