The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting.
There are few methods available for the rapid discovery of multi-target drugs. Herein, we describe the template-assisted, target-guided discovery of small molecules that recognize d(CTG) in the expanded d(CTG•CAG) sequence and its r(CUG) transcript that cause myotonic dystrophy type 1 (DM1). A positive cross-selection was performed using a small library of 30 monomeric alkyneand azide-containing ligands capable of producing more than 5000 possible di-and trimeric click products. The monomers were incubated with d(CTG) 16 or r(CUG) 16 under physiological conditions and both sequences showed selectivity in the proximity-accelerated azide-alkyne [3+2]cycloaddition click reaction. The limited number of click products formed in both selections and the even smaller number of common products suggests that this method is a useful tool for the discovery of single-target and multi-target lead therapeutic agents.
Disease intervention at the DNA level generally has been avoided because of off-target effects. Recent advances in genome editing technologies using CRISPR-Cas9 have opened a new era in DNA-targeted therapeutic approaches. However, delivery of such systems remains a major challenge. Here, we report a selective DNA-modifying small molecule that targets a disease-specific structure and mismatches involved in myotonic dystrophy type 1 (DM1). This ligand alkylates T−T mismatchcontaining hairpins formed in the expanded CTG repeats (d(CTG) exp ) in DM1. Ligand alkylation of d(CTG) exp inhibits the transcription of d(CAG•CTG) exp , thereby reducing the level of the toxic r(CUG) exp transcript. The bioactivity of the ligand also included a reduction in DM1 pathological features such as disease foci formation and misregulation of pre-mRNA splicing in DM1 model cells. Furthermore, the CTG-alkylating ligand may change the d(CAG•CTG) exp repeat length dynamics in DM1 patient cells. Our strategy of linking an alkylating moiety to a DNA mismatchselective small molecule may be generally applicable to other repeat expansion diseases such as Huntington's disease and amyotrophic lateral sclerosis.
The front cover picture shows small molecules (yellow) entering a diseased cell and then its nucleus. The molecules selectively bind to a toxic CCUG RNA transcript (purple) that causes myotonic dystrophy type 2. Binding by this potential therapeutic agent (structure shown in white) disrupts the MBNL1–rCCUGexp interaction and frees the key alternative splicing regulator MBNL (blue) to perform it normal function. For more details, see the Full Paper by Steven C. Zimmerman et al. on
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