Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG) transcripts

Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG) exp ) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5’C A G/3’G A C motif found in r(CAG) exp hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG) exp . The compound was identified by first studying the binding of RNA 1×1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5’C A G/3’G A C motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate as small molecule capable of improving pre-mRNA splicing defects associated with the r(CAG) exp -MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG) exp and could serve as a useful chemical tool to dissect the mechanisms of r(CAG) exp toxicity. The approach used in these studies, defining the small RNA motifs that bind known nucleic acid binders and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in human genomic sequence.