Potent α-Synuclein Aggregation Inhibitors, Identified by High-Throughput Screening, Mainly Target the Monomeric State

α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs’ membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula. Kurnik et al. developed an FRET-based high-throughput screen for inhibitors of α-synuclein aggregation, critical in development of Parkinson's disease. Out of 746,000 compounds, the 6 best hits are derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide, interact with α-synuclein's N-region and reduce oligomer-membrane interactions. We also identified compounds promoting aggregation; the 2 best share a phenyl-benzoxazol core.