Huntington's disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.
Bibliographical noteFunding Information:
This study was supported by U.S. National Institutes of Health (NIH) grant to R.L. (R01NS084345). C.H.L. was supported by a Doctoral Dissertation Fellowship from the University of Minnesota.
We thank Anthony Braun from the Sachs Group and Tory Schaaf and Samantha Yuen from the Thomas Group for technical support and discussions. Compound dispensing and surface plasmon resonance (SPR) (S10 Shared Instrument Grant 1S10OD021539-01 funded by the Office of Research Infrastructure Programs (ORIP)/National Institutes of Health (NIH)) at the UMN Institute of Therapeutic Drug Discovery and Development (ITDD) High-Throughput Screening Laboratory, and spectroscopy at the UMN Biophysical Technology Center. This research uses technology patented by the University of Minnesota, with an exclusive commercial license to Photonic Pharma LLC.
- Conformational change
- High-throughput screening
- Huntingtin aggregation
- Huntingtin induced cell cytotoxicity
- Small-molecule inhibitors
- Time-resolved FRET