Fountain, M. A. | Search bioRxiv

bioRxiv2024

NMR structures and magnetic force spectroscopy studies of small molecules binding to models of an RNA CAG repeat expansion

RNA repeat expansions fold into stable structures and cause microsatellite diseases such as Huntingtons disease (HD), myotonic dystrophy type 1 (DM1), and spinocerebellar ataxias (SCAs). The trinucleotide expansion of r(CAG), or r(CAG)exp, causes both HD and SCA3, and the RNAs toxicity has been traced to its translation into polyglutamine (polyQ; HD) as well as aberrant pre-mRNA alternative splicing (SCA3 and HD). Previously, a small molecule, 1, was discovered that binds to r(CAG)exp and rescues aberrant pre-mRNA splicing in patient-derived fibroblasts by freeing proteins bound to the repeats. Here, we report the structures of single r(CAG) repeat motif (5CAG/3GAC where the underlined adenosines form a 1x1 nucleotide internal loop) in complex with 1 and two other small molecules via nuclear magnetic resonance (NMR) spectroscopy combined with simulated annealing. Compound 2 was designed based on the structure of 1 bound to the RNA while 3 was selected as a diverse chemical scaffold. The three complexes, although adopting different 3D binding pockets upon ligand binding, are stabilized by a combination of stacking interactions with the internal loops closing GC base pairs, hydrogen bonds, and van der Waals interactions. Molecular dynamics (MD) simulations performed with NMR-derived restraints show that the RNA is stretched and bent upon ligand binding with significant changes in propeller-twist and opening. Compound 3 has a distinct mode of binding by insertion into the helix, displacing one of the loop nucleotides into the major groove and affording a rod-like shape binding pocket. In contrast, 1 and 2 are groove binders. A series of single molecule magnetic force spectroscopy studies provide a mechanistic explanation for how bioactive compounds might rescue disease-associated cellular phenotypes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/608150v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@145398org.highwire.dtl.DTLVardef@7c37caorg.highwire.dtl.DTLVardef@132cbbeorg.highwire.dtl.DTLVardef@1de59d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

NMR structures of small molecules bound to a model of an RNA CUG repeat expansion

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntingtons disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntingtons disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1x1 nucleotide UU loops (5CUG/3GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5CUG/3GUC motifs in complex with three different small molecules, a diguandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5CUG/3GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

biochemistry↗