Targeting the r(CGG) repeats that cause FXTAS with modularly assembled small molecules and oligonucleotides
Tuan Tran1, Jessica L Childs-Disney, Biao Liu
1Department of Chemistry, The Scripps Research Institute, Scripps Florida , 130 Scripps Way #3A1, Jupiter, Florida 33458, United States.
Abstract:
We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)(exp)) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)(exp) toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)(exp) in vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)(exp)'s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2'-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.
Insights
Small molecules were designed to target the RNA causing fragile X-associated tremor ataxia syndrome (FXTAS). These compounds effectively reduce RNA toxicity in cellular models without impacting protein translation, offering a promising therapeutic strategy.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Fragile X-associated tremor ataxia syndrome (FXTAS) is an incurable neuromuscular disease.
- FXTAS pathogenesis involves an expanded r(CGG) repeat RNA (r(CGG)(exp)) that disrupts alternative pre-mRNA splicing.
- Current therapeutic strategies face challenges due to the structured nature of the target RNA.
Purpose of the Study:
- To design and evaluate small molecules capable of binding and modulating the toxicity of the r(CGG)(exp) RNA.
- To compare the efficacy and safety of small molecule binders versus oligonucleotide approaches for FXTAS therapy.
- To investigate the structural basis for RNA recognition and its implications for therapeutic design.
Main Methods:
- Design and synthesis of small molecules targeting structured RNA.
- In vitro and cellular assays to assess compound binding and modulation of r(CGG)(exp) toxicity.
- Pull-down experiments for in vivo target engagement validation.
- Comparative analysis of small molecules and modified oligonucleotides for RNA binding and functional effects.
Main Results:
- Designed small molecules successfully bind to r(CGG)(exp) in cellular models.
- Compound binding effectively modulates r(CGG)(exp) toxicity without inhibiting downstream open reading frame (ORF) translation.
- Oligonucleotides exhibit limited efficacy due to the energetic barrier of the structured r(CGG)(exp) RNA and can inhibit translation.
- Small molecules demonstrate superior targeting of structured RNAs compared to oligonucleotides.
Conclusions:
- Small molecules are a viable and potentially safer therapeutic modality for targeting structured RNAs like r(CGG)(exp) in FXTAS.
- The structural characteristics of r(CGG)(exp) present a challenge for oligonucleotide-based therapies but are amenable to small molecule recognition.
- Further development of small molecule therapeutics holds promise for treating FXTAS and similar RNA-mediated neurological disorders.
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