Selective and Reversible Ligand Assembly on the DNA and RNA Repeat Sequences in Myotonic Dystrophy

Abstract

Small molecule targeting of DNA and RNA sequences has come into focus as a therapeutic strategy for diseases such as myotonic dystrophy type 1 (DM1), a trinucleotide repeat disease characterized by RNA gain-of-function. Herein, we report a novel template-selected, reversible assembly of therapeutic agents in situ via aldehyde-amine condensation. Rationally designed small molecule targeting agents functionalized with either an aldehyde or an amine were synthesized and screened against the target nucleic acid sequence. The assembly of fragments was confirmed by MALDI-MS in the presence of DM1-relevant nucleic acid sequences. The resulting hit combinations of aldehyde and amine inhibited the formation of r(CUG) exp in vitro in a cooperative manner at low micromolar levels and rescued mis-splicing defects in DM1 model cells. This reversible template-selected assembly is a promising approach to achieve cell permeable and multivalent targeting via in situ synthesis and could be applied to other nucleic acid targets.

Dynamic Covalent Template-Guided Screen for Nucleic Acid-Targeting Agents

“…Moving forward, it will be important to consider the cytotoxicity of the monomers. Although the monomers likely have lower selectivity than assembled multivalent targeting agents, we anticipate that these monomers, many of which carry a specific targeting agent such as melamine, will have some selectivity for the target sequence based on previous reports of similar compounds. ,, Several of the monomer hits are already known to have a low cytotoxicity at the low micromolar working concentration. Specifically, we have previously studied the toxicity of compounds A5 , N11 , and N20 and have studied similar structures to compounds N9 and N23 .…”

“…Although the monomers likely have lower selectivity than assembled multivalent targeting agents, we anticipate that these monomers, many of which carry a specific targeting agent such as melamine, will have some selectivity for the target sequence based on previous reports of similar compounds. ,, Several of the monomer hits are already known to have a low cytotoxicity at the low micromolar working concentration. Specifically, we have previously studied the toxicity of compounds A5 , N11 , and N20 and have studied similar structures to compounds N9 and N23 . Many of the other scaffolds have been reported and tested in the biological context, including the amilorides by Hargrove and Teramae , as well as adenine in the context of regular base-pairing.…”

“…20,31,40 Several of the monomer hits are already known to have a low cytotoxicity at the low micromolar working concentration. Specifically, we have previously studied the toxicity of compounds A5, 31 N11, 31 and N20 20 and have studied similar structures to compounds N9 and N23. 40 Many of the other scaffolds have been reported and tested in the biological context, including the amilorides by Hargrove 30 and Teramae 41,42 as well as adenine in the context of regular base-pairing.…”

“…Inspired by Rideout’s self-assembling therapeutic approach that used an aliphatic aldehyde and an N -aminoguanidine to form selectively a toxic hydrazone in cancer cells, , we sought to develop a library of compounds that could undergo reversible assembly on a nucleic acid target. Related efforts toward nucleic acid-templated reversible assembly have been reported by our and other groups using ligations such as amine-aldehyde condensation, disulfide chemistry, and native chemical ligation. − Our previously reported target-guided screening approach utilized proximity-induced, irreversible azide-alkyne cycloaddition as the ligation method. , We recently reported a proof-of-concept study where we achieved selective and reversible linkage of two compounds on disease-relevant nucleic acid templates to form larger dimeric or oligomeric products via a hydrolyzable imine bond . Herein, we report a method to screen a library of amines and aldehydes to discover those combinations that assemble on DM1- and HD-relevant nucleic acid templates (Figure ).…”

“…21,32 We recently reported a proof-of-concept study where we achieved selective and reversible linkage of two compounds on diseaserelevant nucleic acid templates to form larger dimeric or oligomeric products via a hydrolyzable imine bond. 31 Herein, we report a method to screen a library of amines and aldehydes to discover those combinations that assemble on DM1-and HD-relevant nucleic acid templates (Figure 1).…”