Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive - PubMed (original) (raw)

. 2012 May 18;7(5):856-62.

doi: 10.1021/cb200408a. Epub 2012 Mar 5.

Affiliations

Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive

Jessica L Childs-Disney et al. ACS Chem Biol. 2012.

Abstract

RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence.

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Figures

Figure 1

Figure 1

A schematic for the molecular mechanism of DM1. An expanded r(CUG) repeat (r(CUG)exp) in the 3′UTR of the DMPK mRNA folds into a hairpin that binds to muscleblind-like 1 protein (MBNL1), a pre-mRNA splicing regulator. Sequestration of MBNL1 by r(CUG)exp causes disregulation of alternative splicing of genes controlled by MBNL1, decreased translation of the DMPK pre-mRNA, and formation of nuclear foci. Designed, modularly assembled ligands targeting the repeating transcript have potential to improve these defects.

Figure 2

Figure 2

The structures of the optimal modularly assembled, nH-4 (13) compounds that inhibit formation of the r(CUG)exp-MBNL1 interaction in vivo. The syntheses of the compounds were previously reported.(13) The synthesis of the 2H-4 compound was optimized, and the details can be found in the Supporting Information.

Figure 3

Figure 3

nH-4 ligands improve DM1-associated pre-mRNA splicing defects. A, schematic of the pre-mRNA splicing pattern observed for the cTNT mini-gene (21) in the presence and absence of the DM1 mini-gene (21). B, representative gel autoradiogram to assess the effect of nH-4 compounds on the alternative splicing of the cTNT mini-gene. HeLa cells were transfected with either a DM1 mini-gene containing 960 interrupted CTG repeats and the cTNT mini-gene or a wild type (WT) mini-gene containing five CTG repeats and the cTNT mini-gene. After transfection, nH-4 compounds or water were added in growth medium to the cells. Total RNA was harvested 16–24 h later, and alternative splicing was assessed by RT-PCR using a radioactively labeled forward primer. The RT-PCR products were separated using a denaturing 5% polyacrylamide gel. The size of the RT-PCR products was confirmed using a radioactively labeled 100 bp DNA ladder. C, plot of data obtained from RT-PCR analysis. Statistically significant improvement of splicing is observed when cells are treated with 2H-4, 3H-4, and 4H-4 while only modest improvement is observed for 5H-4. Each experiment was completed in at least duplicate and the errors are the standard deviations from replicate measurements. (Please see the text for two tailed _p_-values.)

Figure 4

Figure 4

Designed small molecules targeting r(CUG)exp improve DM1-associated translational defects in a cell culture model. Top, a schematic of the model cell-based system that was used to study the efficacy of the compounds. Briefly, a stably transfected C2C12 line was created that expresses firefly luciferase mRNA with r(CUG)800 in the 3′ UTR. In the absence of a small molecule that targets r(CUG)800, the transcript is mostly retained in the nucleus and thus it is not efficiently translated. If, however, a small molecule binds to the r(CUG)800 and displaces or inhibits MBNL1 binding, then the transcript is more efficiently exported from the nucleus and translated in the cytoplasm. Bottom, 2H-4, 3H-4, and 4H-4 improve translational defects associated with DM1. No effect on translation of firefly luciferase is observed when 50 μM of each compound is tested in a model system lacking r(CUG) repeats. Each experiment was completed in at least triplicate and the errors are the standard errors from replicate measurements. Please note that untreated cells have a “Percentage Increase of Luciferase Activity” value of 0.

Figure 5

Figure 5

Disruption of nuclear foci by 2H-4, 3H-4, and 4H-4 as determined by fluorescence in situ hybridization (FISH). HeLa cells were transfected with a DM1 mini-gene containing 960 interrupted CTG repeats and then treated with the nH-4 compound.(21) After 16–24 h, the cells were fixed and the rCUG repeats were detected by FISH using DY547-2′OMe-(CAGCAGCAGCAGCAGCAGC). The cells were imaged by confocal microscopy. A, cells treated with 25 μM of 2H-4. B, cells treated with 25 μM of 3H-4. C, cells treated with 50 μM of 4H-4. D, untreated cells. For all panels: left, fluorescence in the DAPI channel indicating nuclei or nH-4 compound (nH-4 compounds have similar spectral properties as DAPI); middle, DY547 fluorescence indicating the presence of rCUG repeats; C, overlay of DY547 and DAPI/nH-4 images.

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