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Rational design of bioactive, modularly assembled aminoglycosides targeting the RNA that causes myotonic dystrophy type 1.

ABSTRACT: Myotonic dystrophy type 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)(exp)) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6'-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)(exp). However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was traced to suboptimal cellular permeability and localization. To improve these properties, second-generation compounds that are conjugated to a d-Arg(9) molecular transporter were synthesized. These modified compounds enter cells in higher concentrations than the parent compounds and are efficacious in cell-based DM1 model systems at low micromolar concentrations. In particular, they improve three defects that are the hallmarks of DM1: a translational defect due to nuclear retention of transcripts containing r(CUG)(exp); pre-mRNA splicing defects due to inactivation of MBNL1; and the formation of nuclear foci. The best compound in cell-based studies was tested in a mouse model of DM1. Modest improvement of pre-mRNA splicing defects was observed. These studies suggest that a modular assembly approach can afford bioactive compounds that target RNA.

SUBMITTER: Childs-Disney JL

PROVIDER: S-EPMC3528830 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Rational design of bioactive, modularly assembled aminoglycosides targeting the RNA that causes myotonic dystrophy type 1.

Childs-Disney Jessica L JL Parkesh Raman R Nakamori Masayuki M Thornton Charles A CA Disney Matthew D MD

ACS chemical biology 20121107 12

Myotonic dystrophy type 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)(exp)) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6'-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)(exp). However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was ...[more]

PMID: 23130637

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Project description:Myotonic muscular dystrophy types 1 and 2 (DM1 and DM2, respectively) are caused by expansions of repeating nucleotides in noncoding regions of RNA. In DM1, the expansion is an rCUG triplet repeat, whereas the DM2 expansion is an rCCUG quadruplet repeat. Both RNAs fold into hairpin structures with periodically repeating internal loops separated by two 5'GC/3'CG base pairs. The sizes of the loops, however, are different: the DM1 repeat forms 1 x 1 nucleotide UU loops while the DM2 repeat forms 2 x 2 nucleotide 5'CU/3'UC loops. DM is caused when the expanded repeats bind the RNA splicing regulator Muscleblind-like 1 protein (MBNL1), thus compromising its function. Therefore, one potential therapeutic strategy for these diseases is to prevent MBNL1 from binding the toxic RNA repeats. Previously, we designed nanomolar inhibitors of the DM2-MBNL1 interaction by modularly assembling 6'-N-5-hexyonate kanamycin A (K) onto a peptoid backbone. The K ligand binds the 2 x 2 pyrimidine-rich internal loops found in the DM2 RNA with high affinity. The best compound identified from that study contains three K modules separated by four propylamine spacing modules and is 20-fold selective for the DM2 RNA over the DM1 RNA. Because the modularly assembled K-containing compounds also bound the DM1 RNA, albeit with lower affinity, and because the loop size is different, we hypothesized that the optimal DM1 RNA binder may display K modules separated by a shorter distance. Indeed, here the ideal DM1 RNA binder has only two propylamine spacing modules separating the K ligands. Peptoids displaying three and four K modules on a peptoid scaffold bind the DM1 RNA with K(d)'s of 20 nM (3-fold selective for DM1 over DM2) and 4 nM (6-fold selective) and inhibit the RNA-protein interaction with IC(50)'s of 40 and 7 nM, respectively. Importantly, by coupling the two studies together, we have determined that appropriate spacing can affect binding selectivity by 60-fold (20- x 3-fold). The trimer and tetramer also bind approximately 13- and approximately 63-fold more tightly to DM1 RNAs than does MBNL1. The modularly assembled compounds are cell permeable and nontoxic as determined by flow cytometry. The results establish that for these two systems: (i) a programmable modular assembly approach can provide synthetic ligands for RNA with affinities and specificities that exceed those of natural proteins; and, (ii) the spacing of ligand modules can be used to tune specificity for one RNA target over another.

Project description:Herein, we describe the design of high affinity ligands that bind expanded rCUG and rCAG repeat RNAs expressed in myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 3. These ligands also inhibit, with nanomolar IC(50) values, the formation of RNA-protein complexes that are implicated in both disorders. The expanded rCUG and rCAG repeats form stable RNA hairpins with regularly repeating internal loops in the stem and have deleterious effects on cell function. The ligands that bind the repeats display a derivative of the bisbenzimidazole Hoechst 33258, which was identified by searching known RNA-ligand interactions for ligands that bind the internal loop displayed in these hairpins. A series of 13 modularly assembled ligands with defined valencies and distances between ligand modules was synthesized to target multiple motifs in these RNAs simultaneously. The most avid binder, a pentamer, binds the rCUG repeat hairpin with a K(d) of 13 nM. When compared to a series of related RNAs, the pentamer binds to rCUG repeats with 4.4- to >200-fold specificity. Furthermore, the affinity of binding to rCUG repeats shows incremental gains with increasing valency, while the background binding to genomic DNA is correspondingly reduced. Then, it was determined whether the modularly assembled ligands inhibit the recognition of RNA repeats by Muscleblind-like 1 (MBNL1) protein, the expanded-rCUG binding protein whose sequestration leads to splicing defects in DM1. Among several compounds with nanomolar IC(50) values, the most potent inhibitor is the pentamer, which also inhibits the formation of rCAG repeat-MBNL1 complexes. Comparison of the binding data for the designed synthetic ligands and MBNL1 to repeating RNAs shows that the synthetic ligand is 23-fold higher affinity and more specific to DM1 RNAs than MBNL1. Further studies show that the designed ligands are cell permeable to mouse myoblasts. Thus, cell permeable ligands that bind repetitive RNAs have been designed that exhibit higher affinity and specificity for binding RNA than natural proteins. These studies suggest a general approach to targeting RNA, including those that cause RNA dominant disease.

Dataset Information

Rational design of bioactive, modularly assembled aminoglycosides targeting the RNA that causes myotonic dystrophy type 1.

Publications

Rational design of bioactive, modularly assembled aminoglycosides targeting the RNA that causes myotonic dystrophy type 1.

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