Expanded DNA and RNA Trinucleotide Repeats in Myotonic Dystrophy Type 1 Select Their Own Multitarget, Sequence-Selective Inhibitors

Abstract

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.

Massively Parallel Optimization of the Linker Domain in Small Molecule Dimers Targeting a Toxic r(CUG) Repeat Expansion

“…36 These compounds were discovered by various approaches, such as a bead-based screening method called resin-bound dynamic combinatorial chemistry 35 and by fragment-based target-guided synthesis. 34 Our group has previously reported the modular assembly of RNA-binding modules targeting consecutive 5′CUG/GUC 1 × 1 internal loops to disrupt the toxic MBNL1−r(CUG) exp complex and improve DM1-associated defects (Figure 1B,C). 37 A dimeric compound named 2H-4 was built by a N-propylglycine peptoid bridge to separate the binding modules at a specific distance to enable simultaneous binding (Figure 1D).…”

“…These loops bind and sequester the pre-mRNA splicing regulator muscleblind-like 1 (MBNL1) protein as well as other RNA-binding proteins in nuclear foci (Figure A). Sequestration of MBNL1 by r­(CUG) exp prevents its interaction with its natural substrates, therefore leading to dysregulation of alternative pre-mRNA splicing and manifestation of disease. , Different types of modalities can bind to r­(CUG) exp and improve disease defects in DM1-affected cells, − including monomeric small molecules, − modularly assembled dimeric compounds that occupy multiple internal loops simultaneously, , pseudopeptides, and small molecules that cleave r­(CUG) exp . These compounds were discovered by various approaches, such as a bead-based screening method called resin-bound dynamic combinatorial chemistry and by fragment-based target-guided synthesis …”

Versatile Target-Guided Screen for Discovering Bidirectional Transcription Inhibitors of a Trinucleotide Repeat Disease

“…We previously described a one pot selection method using target-guided synthesis for discovering multitarget agents that selectively bind both d(CTG) exp and r(CUG) exp . 26 In this method, several RNA-and DNA-binding ligands with an azide or alkyne group were mixed and incubated with r(CUG) 16 or d(CTG) 16 to produce dimeric and trimeric ligands via template-assisted [3 + 2] cyclization. This selection method was effective in discovering selective inhibitors of DM1 RNA and DNA but was limited in the structural diversity of the dimers and trimers that were detected in the reaction.…”

“…The previously developed clickable fragment library for target-guided screening 26 contained compounds 1−5 (Figure 2). Click partner 1 contains a bisamidinium groove binder and 2 features an acridine intercalator, both linked to melamine recognition units.…”

“…Preliminary studies suggested that activated alkynes were necessary to afford triazole products at an appreciable rate. 26 As an initial screen, we used the four activated alkynecontaining and eight azide-containing compounds in Figure 2 to perform 32 parallel reactions, with the pairwise combinations run in the presence or absence of the target d(CTG) 16 or a random duplex. The short DNA target d(CTG) 16 was chosen for its ability to form a hairpin with a length that would limit click products to dimers and trimers.…”