Dual-reporter assay for selective small molecule inhibition of expanded toxic huntingtin
Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion of CAG repeats in exon 1 of the human huntingtin gene (HTT). The resultant pathogenic allele (mHTT) produces a mutant huntingtin protein (mHtt) containing an expanded polyglutamine tract. Because mHtt drives neurodegeneration while wildtype huntingtin (Htt) remains critical for regulation of gene transcription and cell survival, an ideal therapeutic would specifically downregulate mHtt expression while preserving Htt. Small molecules are attractive candidates for HD given their ability to penetrate the blood-brain barrier and amenability to oral dosing, but there remains no high-throughput platform that reports both efficacy and allele selectivity. Here, we present a bacterial assay in which exon 1 of HTT and mHTT, each fused to spectrally distinct fluorescent proteins, are co-expressed in the same cell, allowing simultaneous quantification of their expression in the presence of small molecules. This high-throughput assay isolates the effect of small molecules on huntingtin expression without confounding factors present in mammalian cells, such as endogenous Htt or human RNA-binding proteins. To validate this assay, we evaluated two previously reported CAG repeat binding compounds and used circular dichroism spectroscopy and cell-free protein synthesis to investigate their underlying mechanisms. Furamidine exhibited reproducible, sequence-dependent effects, altering the structural stability of HTT RNA and selectively decreasing the wildtype Htt expression (a selectivity profile opposite to what is therapeutically required). In contrast, myricetin did not affect HTT RNA stability and its decreased signal in the dual-expression assay was due to fluorophore artifacts. This highlights the importance of complementary workflows in distinguishing genuine modulators from reporter-specific artifacts. Collectively, these findings establish the dual-expression assay as a screening platform for CAG repeat-targeting small molecules and demonstrate the importance of determining which allele reported compounds preferentially affect.