Using Modularly Assembled Ligands To Bind RNA Internal Loops Separated by Different Distances

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.

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“…The potency and specificity of the compounds could be improved using a modular assembly strategy. (13, 14, 16, 61)…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2012

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“…Previously, we have shown that dimeric compounds can be designed to bind adjacent motifs simultaneously in an RNA by using spacing rules to span different distances (16). Such a multivalent approach has been shown to improve avidity and potency to study and manipulate biology in many systems (17)(18)(19)(20)(21)(22)(23).…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies on spacing rules for targeting RNA internal loops separated by different distances showed that two to four propylamine spacing modules spanned the distance between two internal loops separated by 2 bp (16,20), the same distance separating the 1 × 1 GG internal loop targeted by 2 and the Drosha site targeted by 1. We screened the library of dimers for their ability to inhibit biogenesis of miR-96 in MDA-MB-231 cells, a model of TNBC (Fig.…”

Section: Resultsmentioning

confidence: 99%

Design of a small molecule against an oncogenic noncoding RNA

Velagapudi

Cameron

Haga

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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175

187

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The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of diseasecausing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-todrug paradigm.RNA | noncoding RNA | drug design | chemistry | nucleic acids

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“…Additionally, the ability to design small molecules with selectivities that are competitive with, if not exceed, those of siRNAs suggests that small-molecule targeting of RNA could be a sustained approach to affect biology for therapeutic benefit. Selectivity is derived from small-molecule recognition of a particular RNA 3D fold and could be further enhanced by designing compounds that interact with multiple motifs within an RNA target simultaneously (6,(73)(74)(75).…”

Section: Proteome-and Transcriptome-wide Studies To Evaluate Synucleozidmentioning

confidence: 99%

Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA

Zhang

Park

Zhang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson’s disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5′ untranslated region (5′ UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5′ UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound’s selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

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Using Modularly Assembled Ligands To Bind RNA Internal Loops Separated by Different Distances

Cited by 23 publications

References 31 publications

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

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

Design of a small molecule against an oncogenic noncoding RNA

Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA

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