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.

Keywords: drug design; myotonic dystrophy type 1; nucleic acids; small molecules; transcription inhibition.

Figure 1.. Myotonic Dystrophy Type 1 (DM1) Pathogenesis.

d(CTG·CAG)^exp is bidirectionally transcribed to form r(CAG)^exp and r(CUG)^exp. The r(CUG)^exp can form hairpin secondary structures that sequester MBNL1 proteins and lead to improper splicing of pre-mRNAs. Both RNA transcripts can undergo RNA translation to yield toxic homopolymeric peptides such as polyglutamine. The use of reactive small molecules to specifically target RNA and DNA and assemble on template could alleviate symptoms of DM1 by preventing bidirectional transcription of d(CTG·CAG)^exp or competitively binding the r(CUG)^exp hairpin structure to release MBNL1 and prevent RNA translation.

Figure 2.. Dynamic Covalent Chemistry Template-Selected Assembly Approach.

Cell-permeable monomers reversibly assemble, allowing for template-selected amplification of the potent multivalent targeting agent.

Figure 3.. MOE docking studies for ligands 2 and 3.

(a) Monomers 2 and 3 docked side by side. The fragments were manually ligated, and the structure minimized to form the dimeric product 2+3 on the r(CUG)₆ helix shown in (b). Uracil is green (light green are flipped in, dark green flipped out), cytosine is tan and guanine is grey. Hydrogen bonds between ligand and RNA are shown in red. R(CUG)₆ PDB ID: 3gm7.^[41] The distance between reactive atoms (from carbonyl carbon to aniline nitrogen) was measured to be 3.95 Å (purple dotted line in c.)

Figure 4.. MALDI-MS analysis of 2+3 assembly on and off template with reduction by NaCNBH₃.

(a) absence of template, (b) d(CTG)₁₆ template, (c) r(CUG)₁₆ template. Buffer: 2 mM each of KCl, MgCl₂, CaCl₂, and Tri-HCl, pH 7, incubated at 37 ^oC for 75 min before reduction and analysis. 100 μM compounds, 10 μM d(CTG)₁₆ or r(CUG)₁₆. See procedure S1 for details.

Figure 5.. High MW MALDI-MS analysis of 2+3 assembly on and off template with reduction by NaCNBH₃.

(a) absence of template, (b) d(CTG)₁₆ template, (c) r(CUG)₁₆ template, (d) r(CUG)₉₀ template. Buffer: 2 mM each of KCl, MgCl₂, CaCl₂, and Tri-HCl, pH 7, incubated at 37 ^oC for 24 h before reduction with NaCNBH₃, and analysis. Compound concentration 100 μM, 10 μM d(CTG)₁₆ and r(CUG)₁₆ or 500 nM r(CUG)₉₀. See Procedure S2 for details.

Figure 6.

in vitro Transcription Inhibition Assay with T7 RNA Polymerase and pSP72 plasmid containing 90 (CTG·CAG) repeats. Results are reported as the average of at least 3 independent experiments are error is reported as standard error of the mean.

Figure 7.. Insulin Receptor Mis-splicing Assay.

(a) Improper splicing patterns caused by MBNL1 sequestration by toxic RNA products. (b) Combination of ligands 2 and 3 rescued mis-splicing in the insulin receptor minigene in a dose-dependent manner. Results reported as the average of at least 3 independent replicates. Error bars indicate standard error. * indicates p<0.05 compared to negative control.

Scheme 1.

Synthesis of 2. See Supporting Information for detailed methods.

Scheme 2.

Synthesis of 3. See Supporting Information for additional details.