Repurposing Nitazoxanide to target the expanded r(CGG)n repeat RNA for therapeutic intervention in fragile-X tremor/ataxia syndrome.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a debilitating neurodegenerative disorder linked to CGG trinucleotide repeat expansions in the FMR1 gene. These expanded repeats produce toxic FMR1poly-Glycine (FMR1polyG) proteins in neurons through mechanisms such as Repeat-Associated Non-AUG (RAN) translation and RNA foci formation, driving disease progression. In this study, we investigate the potential of Nitazoxanide (NTZ), a broad-spectrum antiparasitic and antiviral medication, as a therapeutic agent for FXTAS by targeting CGG repeat-associated toxicity. This comprehensive approach utilizing biophysical techniques, bioinformatic studies, cellular assays, and Drosophila models reveals NTZ's remarkable ability to bind specifically to toxic CGG repeat RNA, particularly GG mismatches, and to inhibit FMR1polyG aggregation. Biophysical methods, including Circular Dichroism (CD), Isothermal Titration Calorimetry (ITC), Electrophoretic Mobility Shift assays (EMSA), and Nuclear Magnetic Resonance (NMR) spectroscopy, accompanied with Molecular Docking, confirmed that NTZ effectively binds to CGG repeat RNA and mitigates its toxicity. Moreover, treatment with NTZ significantly reduced FMR1polyG-associated toxicity, corrected the splicing defects in FXTAS cell models, and improved different phenotypes in the Drosophila model of FXTAS. These compelling findings position NTZ as a promising candidate for neuroprotection in FXTAS, indicating its remarkable therapeutic potential and paving the way for future clinical applications to improve outcomes for the affected patients.