Small Molecule Screening Discovers Compounds that Reduce FMRpolyG Protein Aggregates and Splicing Defect Toxicity in
Arun Kumar Verma1, Eshan Khan1, Subodh Kumar Mishra1
1Department of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol, Indore, 453552, India.
Abstract:
Expansion of CGG trinucleotide repeats in 5' untranslated region of the FMR1 gene is the causative mutation of neurological diseases such as fragile X syndrome (FXS), fragile X-associated tremor/ataxia syndrome (FXTAS), and ovarian disorder such as fragile X-associated primary ovarian insufficiency (FXPOI). CGG repeats containing FMR1 transcripts form the toxic ribonuclear aggregates, abrupt pre-mRNA splicing, and cause repeat-associated non-AUG translation, leading to the disease symptoms. Here, we utilized a small molecule library of ~ 250,000 members obtained from the National Cancer Institute (NCI) and implemented a shape-based screening approach to identify the candidate small molecules that mitigate toxic CGG RNA-mediated pathogenesis. The compounds obtained from screening were further assessed for their affinity and selectivity towards toxic CGG repeat RNA by employing fluorescence-binding experiment and isothermal calorimetry titration assay. Three candidate molecules B1, B4, and B11 showed high affinity and selectivity for expanded CGG repeats RNA. Further, NMR spectroscopy, gel mobility shift assay, CD spectroscopy, UV-thermal denaturation assay, and molecular docking affirmed their high affinity and selectivity for toxic CGG RNAs. Next, these lead compounds selectively improved the pre-mRNA alternative splicing defects with no perturbation in global splicing efficacy and simultaneously reduced the FMR1polyG protein aggregate formation without affecting the downstream expression of the gene. Taken together these findings, we addressed compound B1, B4, and B11 as potential lead molecules for developing promising therapeutics against FXTAS. Herein, this study, we have utilized shape similarity approach to screen the NCI library and found out the potential candidate which improves the pre-mRNA splicing defects and reduces FMR1polyG aggregations.
Insights
Researchers identified three small molecules (B1, B4, B11) that target toxic CGG repeat RNA, improving splicing defects and reducing protein aggregates in fragile X-associated tremor/ataxia syndrome (FXTAS) models.
Area of Science:
- Neurogenetics
- RNA Biology
- Pharmacology
Background:
- Fragile X gene (FMR1) CGG repeat expansion causes FXS, FXTAS, and FXPOI.
- Toxic ribonuclear aggregates, splicing defects, and non-AUG translation drive disease pathology.
Purpose of the Study:
- Identify small molecules to mitigate CGG RNA-mediated pathogenesis.
- Develop potential therapeutics for FXTAS.
Main Methods:
- Screened a 250,000-member NCI library using shape-based similarity.
- Assessed compound affinity and selectivity for CGG repeat RNA via fluorescence binding and ITC.
- Validated lead compounds using NMR, gel shift, CD, UV-thermal denaturation, and molecular docking.
Main Results:
- Identified B1, B4, and B11 with high affinity and selectivity for expanded CGG repeat RNA.
- These compounds selectively corrected pre-mRNA splicing defects without affecting global splicing.
- Reduced FMR1polyG protein aggregate formation without altering downstream gene expression.
Conclusions:
- Compounds B1, B4, and B11 are promising lead molecules for FXTAS therapeutics.
- Shape similarity screening effectively identified modulators of toxic RNA aggregates and splicing defects.
