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.
Bibliographical noteFunding Information:
We thank Dr. Nagamani Vunnam from the Sachs Group; Dr. Tory Schaaf, Samantha Yuen, and Dr. Andrew R. Thompson from the Thomas Group; Benjamin Grant from Fluorescence Innovations; and Dr. Robyn T. Rebecck for technical support and discussions. Compound dispensing was performed at the UMN Institute of Therapeutic Drug Discovery and Development (ITDD) High-Throughput Screening Laboratory. Spectroscopy was performed at the UMN Biophysical Technology Center and at the adjacent Photonic Pharma laboratory. This research uses technology patented by the University of Minnesota, with an exclusive commercial license to Photonic Pharma LLC. The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported by U.S. National Institutes of Health (NIH) grants to J.N.S. and W.C.K.P. (NINDS 1R01NS117968 and NINDS R21 NS109505) and to J.N.S. and D.D.T. (NIA SBIR 1R43AG063675). This research was also supported by the NIH National Center for Advancing Translational Sciences, grant UL1TR002494. Support was also provided by T32GM132039 to K.A. and RF1AG053951 to E.R. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health’s National Center for Advancing Translational Sciences.
© 2021, The Author(s).
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