Dual-reporter assay for selective small molecule inhibition of expanded toxic huntingtin

Huntington's disease (HD) stems from an expansion of CAG repeats in the HTT gene, resulting in a mutant protein called mHtt that is neurotoxic, while the wildtype protein, Htt, is essential for gene transcription and cell survival. Ideally, a therapeutic for HD would selectively target mHtt without affecting Htt expression. Small molecules are promising candidates due to their ability to cross the blood-brain barrier and their oral dosing options, but a high-throughput platform that can simultaneously assess efficacy and allele selectivity has been lacking.

To address this gap, researchers developed a bacterial assay that co-expresses exon 1 of HTT and mHTT, each fused to uniquely fluorescent proteins. This setup allows for simultaneous measurement of expression levels of both proteins in the presence of small molecules, isolating the impact of these compounds on huntingtin expression without interference from endogenous Htt or human RNA-binding proteins. This method provides a highly efficient platform for screening small molecules targeting CAG repeat expansions.

The assay was validated using two previous compounds known to bind to CAG repeats. Circular dichroism spectroscopy and cell-free protein synthesis were employed to study the mechanisms behind their effects. Furamidine was shown to alter the structural stability of HTT RNA and selectively reduce the expression of wildtype Htt, indicating a selectivity that would be counterproductive for therapeutic purposes.

Conversely, myricetin did not affect HTT RNA stability and its diminished signal in the assay was attributed to fluorophore artifacts, underscoring the need for complementary assays to differentiate true modulators from false positives.

These results underscore the utility of the dual-expression assay as a screening tool specifically for targeting mHtt with CAG repeat-targeting small molecules. The findings also highlight the critical role of using multiple validation methods to accurately determine which compounds preferentially affect the toxic mHtt allele, ultimately guiding the development of safer and more effective HD therapies.