Chemical Correction of Pre-mRNA Splicing Defects Associated with Sequestration of Muscleblind-like 1 Protein by Expanded r(CAG)-Containing Transcripts

Abstract

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’CAG/3’GAC 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’CAG/3’GAC 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.

A Small Molecule That Targets r(CGG)exp and Improves Defects in Fragile X-Associated Tremor Ataxia Syndrome

“…Those searches identified compounds that are similar to the bis-benzimidazole Hoechst 33258, 4',6-diamidino-2-phenylindole (DAPI), and pentamidine. This RNA-focused collection of small molecules contained two small molecules that improve defects that are associated with r(CAG) exp and r(CUG) exp in cell culture models of HD and DM1, respectively (12, 13). Thus, Hoechst-, pentamidine-, and DAPI-like compounds were screened to identify inhibitors of the r(CGG)-DGCR8Δ protein complex.…”

Methods to enable the design of bioactive small molecules targeting RNA

“…50, 52 The small molecule lead for r(CAG) exp was identified by studying known nucleic binders (DAPI-like small molecule D6; Figure 3). 51 For all three cases, the small molecule lead was inactive and/or toxic in cellular model systems. Chemical similarity searching successfully identified a bioactive compound that mitigates toxicity of each repeat in cellular model systems and in a mouse model of DM1.…”

“…This approach has been employed to develop lead compounds that target expanded repeating RNAs, including r(CUG) exp (DM1), 50 r(CAG) exp (HD), 51 and r(CGG) exp (FXTAS), 52 and telomerase RNA 53 (please see section entitled, “ Identification of small molecules that bind telomerase RNA using docking and chemical similarity searching” (Figures 3 & 4). For r(CUG) exp and r(CGG) exp , the lead small molecule that was subjected to chemical similarity searching was identified from 2DCS and StARTS (Hoechst 33258, affording H1 and 1a, respectively; Figure 3).…”

PubChem applications in drug discovery: a bibliometric analysis

“…Utilization or adaption of experimental protocols deposited in PubChem BioAssay by biologists for devising their own experiments [48]. …”