Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein
| Autor: | Anthony R. Braun, Noah Nathan Kochen, Samantha L. Yuen, Elly E. Liao, Razvan L. Cornea, David D. Thomas, Jonathan N. Sachs |
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| Jazyk: | angličtina |
| Rok vydání: | 2023 |
| Předmět: | |
| Zdroj: | ASN Neuro, Vol 15 (2023) |
| Druh dokumentu: | article |
| ISSN: | 1759-0914 17590914 |
| DOI: | 10.1177/17590914231184086 |
| Popis: | There is a critical need for small molecules capable of rescuing pathophysiological phenotypes induced by alpha-synuclein (aSyn) misfolding and oligomerization. Building upon our previous aSyn cellular fluorescence lifetime (FLT)-Förster resonance energy transfer (FRET) biosensors, we have developed an inducible cell model incorporating the red-shifted mCyRFP1/mMaroon1 (OFP/MFP) FRET pair. This new aSyn FRET biosensor improves the signal-to-noise ratio, reduces nonspecific background FRET, and results in a 4-fold increase (transient transfection) and 2-fold increase (stable, inducible cell lines) in FRET signal relative to our previous GFP/RFP aSyn biosensors. The inducible system institutes greater temporal control and scalability, allowing for fine-tuning of biosensor expression and minimizes cellular cytotoxicity due to overexpression of aSyn. Using these inducible aSyn-OFP/MFP biosensors, we screened the Selleck library of 2684 commercially available, FDA-approved compounds and identified proanthocyanidins and casanthranol as novel hits. Secondary assays validated the ability of these compounds to modulate aSyn FLT-FRET. Functional assays probing cellular cytotoxicity and aSyn fibrillization demonstrated their capability to inhibit seeded aSyn fibrillization. Proanthocyanidins completely rescued aSyn fibril-induced cellular toxicity with EC 50 of 200 nM and casanthranol supported a 85.5% rescue with a projected EC 50 of 34.2 μM. Furthermore, proanthocyanidins provide a valuable tool compound to validate our aSyn biosensor performance in future high-throughput screening campaigns of industrial-scale (million-compound) chemical libraries. |
| Databáze: | Directory of Open Access Journals |
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