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

We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (alpha SN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two alpha SN FRET biosensors provide complementary insight into alpha SN 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 alpha SN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of alpha SN pathology (phosphorylation of mouse alpha SN 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 alpha SN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate alpha SN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block alpha SN fibril assembly.