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

Abstract 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 in...

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Autores principales:	Anthony R. Braun, Elly E. Liao, Mian Horvath, Prakriti Kalra, Karen Acosta, Malaney C. Young, Noah Nathan Kochen, Chih Hung Lo, Roland Brown, Michael D. Evans, William C. K. Pomerantz, Elizabeth Rhoades, Kelvin Luk, Razvan L. Cornea, David D. Thomas, Jonathan N. Sachs
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Neurology. Diseases of the nervous system RC346-429
Acceso en línea:	https://doaj.org/article/ee6b8563f73c49f68804418a48d07270
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spelling	oai:doaj.org-article:ee6b8563f73c49f68804418a48d072702021-12-02T18:18:52ZPotent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors10.1038/s41531-021-00195-62373-8057https://doaj.org/article/ee6b8563f73c49f68804418a48d072702021-06-01T00:00:00Zhttps://doi.org/10.1038/s41531-021-00195-6https://doaj.org/toc/2373-8057Abstract 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.Anthony R. BraunElly E. LiaoMian HorvathPrakriti KalraKaren AcostaMalaney C. YoungNoah Nathan KochenChih Hung LoRoland BrownMichael D. EvansWilliam C. K. PomerantzElizabeth RhoadesKelvin LukRazvan L. CorneaDavid D. ThomasJonathan N. SachsNature PortfolioarticleNeurology. Diseases of the nervous systemRC346-429ENnpj Parkinson's Disease, Vol 7, Iss 1, Pp 1-17 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Neurology. Diseases of the nervous system RC346-429
spellingShingle	Neurology. Diseases of the nervous system RC346-429 Anthony R. Braun Elly E. Liao Mian Horvath Prakriti Kalra Karen Acosta Malaney C. Young Noah Nathan Kochen Chih Hung Lo Roland Brown Michael D. Evans William C. K. Pomerantz Elizabeth Rhoades Kelvin Luk Razvan L. Cornea David D. Thomas Jonathan N. Sachs Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors
description	Abstract 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.
format	article
author	Anthony R. Braun Elly E. Liao Mian Horvath Prakriti Kalra Karen Acosta Malaney C. Young Noah Nathan Kochen Chih Hung Lo Roland Brown Michael D. Evans William C. K. Pomerantz Elizabeth Rhoades Kelvin Luk Razvan L. Cornea David D. Thomas Jonathan N. Sachs
author_facet	Anthony R. Braun Elly E. Liao Mian Horvath Prakriti Kalra Karen Acosta Malaney C. Young Noah Nathan Kochen Chih Hung Lo Roland Brown Michael D. Evans William C. K. Pomerantz Elizabeth Rhoades Kelvin Luk Razvan L. Cornea David D. Thomas Jonathan N. Sachs
author_sort	Anthony R. Braun
title	Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors
title_short	Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors
title_full	Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors
title_fullStr	Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors
title_full_unstemmed	Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors
title_sort	potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved fret biosensors
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/ee6b8563f73c49f68804418a48d07270
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Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors

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