Abstract
In Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic double-stranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2) and trigger other DM1-related pathogenesis pathway defects. In this paper, we characterize four d-amino acid hexapeptides identified together with abp1, a peptide previously shown to stabilize CUG RNA in its single-stranded conformation. With the generalized sequence cpy(a/t)(q/w)e, these related peptides improved three MBNL-regulated exon inclusions in DM1-derived cells. Subsequent experiments showed that these compounds generally increased the relative expression of MBNL1 and its nuclear-cytoplasmic distribution, reduced hyperactivated autophagy, and increased the percentage of differentiated (Desmin-positive) cells in vitro. All peptides rescued atrophy of indirect flight muscles in a Drosophila model of the disease, and partially rescued muscle function according to climbing and flight tests. Investigation of their mechanism of action supports that all four compounds can bind to CUG repeats with slightly different association constant, but binding did not strongly influence the secondary structure of the toxic RNA in contrast to abp1. Finally, molecular modeling suggests a detailed view of the interactions of peptide-CUG RNA complexes useful in the chemical optimization of compounds.
Highlights
In Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic doublestranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2) and trigger other DM1-related pathogenesis pathway defects
DM1-related phenotypes can be used as readouts to screen candidate therapeutics in vitro for effects on RNA toxicity associated with the DM1 mutation
We established three splicing events typically altered in DM1 as screening criteria for the 15 d-amino-acid hexapeptides previously identified (Supplementary Table 1)[22]: the inclusion of exon 5 of cardiac troponin T gene and the exclusion of exon 78 of the dystrophin gene (DMD; Entrez ID: 1756), both MBNL1-dependent and the exclusion of exon 23 of spectrin alpha non-erythrocytic gene (SPTAN1; Entrez ID: 6709), which is MBNL2-dependent[27]
Summary
In Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic doublestranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2) and trigger other DM1-related pathogenesis pathway defects. All peptides rescued atrophy of indirect flight muscles in a Drosophila model of the disease, and partially rescued muscle function according to climbing and flight tests Investigation of their mechanism of action supports that all four compounds can bind to CUG repeats with slightly different association constant, but binding did not strongly influence the secondary structure of the toxic RNA in contrast to abp[1]. We previously targeted the expression of 480 interrupted CTG repeats to the Drosophila mushroom bodies, which are a pair of brain structures in insects This generates a semi-lethal phenotype at the pupal stage used to screen a positional scanning synthetic combinatorial library of d-amino-acid hexapeptides that identified 16 candidate peptides, of which Abp[1] (ppyawe) was characterized in more detail[22]. Our current study addressed the characterization of the remaining 15 peptides in a secondary screen in a DM1-derived cell model of the disease and identified four closely related peptides that improved cell and Drosophila DM1 phenotypes by directly binding to the CUG RNA
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