Small molecule-based approach for targeting CGG repeat expansion in fragile X-associated tremor/ataxia syndrome

Abstract

Fragile X-associated tremor ataxia syndrome is a result of the expansion of 55 to 200 CGG nucleotide repeats in 5’UTR of Fragile X mental retardation 1. This expansion of CGG nucleotide repeats causes neuronal toxicity by two main pathogenic mechanisms. In the first case splicing alterations and formation of ribonuclear inclusions takes place as a result of the sequestration of specific RNA regulatory proteins by mRNA. While in the second pathogenic mechanism FMRPolyG which is a toxic homopolymeric protein is formed as a result of repeat-associated non-canonical translation (RANT). In this study, we have used a small molecule-based approach for targeting the CGG repeat expansion. FDA-approved drugs were screened against RNA library having a 1*1 internal nucleotide loop by fluorescence intercalator displacement assay. After performing screening of these molecules lead molecules were identified. These lead molecules were then checked for binding affinity with the target CGG repeat by fluorescence binding assay, Isothermal calorimetry titration, and electrophoretic mobility shift assay. Finally, to check the potency of these molecules in cell-based assay effect of drugs on protein aggregation and protein accumulation in the FXTAS cell model was studied. A significant decrease in protein aggregates was found in one of the lead molecules while the remaining lead molecules also showed a decrease in protein aggregation. These lead molecules showed positive results in cell-based assay further studies have to be done for these lead molecules.