Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility...

Area of Science:

• Neuroscience
• Genetics
• Biochemistry

Background:

• Huntington's disease (HD) is a fatal monogenic neurodegenerative disorder.
• CAG repeat expansions in mutant Huntingtin (mHTT) cause polyglutamine (polyQ) stretches, influencing disease onset and severity.
• mHTT exhibits reduced N-terminal conformational flexibility compared to wild-type HTT, contributing to toxicity and loss-of-function phenotypes.

Purpose of the Study:

• To develop and validate a high-throughput screening (HTS) method for identifying small molecules that restore mHTT conformational flexibility.
• To apply this novel assay to human patient fibroblasts for drug discovery in Huntington's disease.

Main Methods:

• Utilized an antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay to measure endogenous HTT flexibility.
• Adapted the TR-FRET immunoassay to a high-throughput screening platform using human patient fibroblasts.
• Performed a small-scale compound screen to identify flexibility-modifying small molecules.

Main Results:

• Successfully miniaturized and automated the HTT TR-FRET immunoassay for compound screening.
• Identified several small molecules capable of partially rescuing mHTT inflexibility.
• These compounds are presumed to work by altering HTT post-translational modifications.

Conclusions:

• The developed HTT TR-FRET immunoassay is suitable for miniaturization and application in compound screening workflows.
• This automated assay can be employed in large-scale screening campaigns to discover novel Huntington's disease drugs.
• The identified small molecules represent potential therapeutic leads for altering mHTT conformation and mitigating disease pathology.