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.
SourceAvailable from: Kamil Jan Cygan[Show abstract] [Hide abstract]
ABSTRACT: Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions.01/2015; 5(2):893-909. DOI:10.3390/biom5020893
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ABSTRACT: DNA-templated self-assembly is an emerging strategy for generating functional supramolecular systems, which requires the identification of potent multi-point binding ligands. In this line, we recently showed that bis-functionalized guanidinium compounds can interact with ssDNA and generate a supramolecular complex through the recognition of the phosphodiester backbone of DNA. In order to probe the importance of secondary interactions and to identify side groups that stabilize these DNA-templated self-assemblies, we report herein the implementation of a dynamic combinatorial approach. We used an in situ fragment assembly process based on reductive amination and tested various side groups, including amino acids. The results reveal that aromatic and cationic side groups participate in secondary supramolecular interactions that stabilize the complexes formed with ssDNA.International Journal of Molecular Sciences 02/2015; 16(2):3609-3625. DOI:10.3390/ijms16023609 · 2.34 Impact Factor
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ABSTRACT: Fuchs endothelial corneal dystrophy (FECD) is an inherited, degenerative disease affecting the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects approximately 5% of middle-aged Caucasians in the United States and accounts for more than 14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG/CAG)n trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene, which is found in a majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1 (DM1), the poly (CUG)n RNA co-localizes with and sequesters the mRNA splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNASeq splicing data from the corneal endothelium of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of DM1 and epithelial-mesenchymal transition (EMT) as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common, non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG/CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 01/2015; 290(10). DOI:10.1074/jbc.M114.621607 · 4.60 Impact Factor