Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility in patient-derived cells
| Autor: | Walter Englaro, Julia Frappier, Sabine Gratzer, Lisa M. Stanek, Barbara Calamini, Sandra Cristina Müller, Johannes H Wilbertz |
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| Rok vydání: | 2022 |
| Předmět: |
Huntingtin Protein
Huntingtin Flexibility (anatomy) Chemistry Mutant Nerve Tissue Proteins Neurodegenerative Diseases Small molecule Phenotype Biochemistry Cell biology Analytical Chemistry Exon Huntington Disease medicine.anatomical_structure Förster resonance energy transfer Mutation Fluorescence Resonance Energy Transfer medicine Humans Molecular Medicine Loss function Biotechnology |
| Zdroj: | SLAS Discovery. 27:219-228 |
| ISSN: | 2472-5552 |
| Popis: | Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1 encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets. |
| Databáze: | OpenAIRE |
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