Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors.

Autor:	Braun AR; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Liao EE; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Horvath M; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA., Kalra P; Department of Chemistry, University of Minnesota, Minneapolis, MN, USA., Acosta K; Biochemistry & Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA, USA., Young MC; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Kochen NN; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Lo CH; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Brown R; Clinical and Translational Science Institute, University of Minnesota, Minneapolis, MN, USA., Evans MD; Clinical and Translational Science Institute, University of Minnesota, Minneapolis, MN, USA., Pomerantz WCK; Department of Chemistry, University of Minnesota, Minneapolis, MN, USA., Rhoades E; Biochemistry & Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA, USA.; Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA., Luk K; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA., Cornea RL; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.; Photonic Pharma LLC, Minneapolis, MN, USA., Thomas DD; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.; Photonic Pharma LLC, Minneapolis, MN, USA., Sachs JN; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA. jnsachs@umn.edu.
Jazyk:	angličtina
Zdroj:	NPJ Parkinson's disease [NPJ Parkinsons Dis] 2021 Jun 28; Vol. 7 (1), pp. 52. Date of Electronic Publication: 2021 Jun 28.
DOI:	10.1038/s41531-021-00195-6
Abstrakt:	We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson's disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.
Databáze:	MEDLINE
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