Advances in myotonic dystrophy type 1 drug discovery through design of novel ligands and mechanism establishment

Abstract

Myotonic dystrophy type 1 (DM1) is caused by an expanded CUG repeat (CUGexp) that sequesters muscleblind-like 1 protein (MBNL1), a protein that regulates alternative splicing. CUGexp RNA is a validated drug target for this currently untreatable disease. Herein, we describe the development of a bioactive small molecule (Chapter 2) and a small library of dimeric ligands (Chapter 3) leading to an optimized bivalent ligand. These novel ligands target CUGexp RNA and are able to inhibit the CUGexp⋅MBNL1 interaction in cells that model DM1. In a DM1 cell model these ligands were found to disperse CUGexp ribonuclear foci, release MBNL1, and partially reverse the mis-splicing of the insulin receptor pre-mRNA. Direct evidence for ribonuclear foci dispersion by this ligand was obtained in a live DM1 cell model using time-lapse confocal microscopy. In Chapter 4, We report a single-molecule approach to study the binding of MBNL1 to (CUG)n=4,6 and the effect of small molecules on this interaction. MBNL1 is able to bind to the (CUG)n･inhibitor complex indicating that the inhibition is not a straight forward competitive process. A simple bivalent ligand, shows a binding to (CUG)n almost 50-fold more tightly than the corresponding monomeric ligand and is more effective in destabilizing MBNL1･(CUG)4. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions. Chapter 5 includes a preliminary effort to solve the mystery of CUGexp unfolding/folding upon interaction with MBNL1. To approach this unanswered yet key structural question about how MBNL1 binds CUGexp, preliminary bulk FRET (Fluorescence Resonance Energy Transfer) studies, as well as single-molecule FRET studies are described