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

Abstract

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.

Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions

“…Although our most potent anti-aggregation compound, 576755, has quinone potential, two additional misfolding blocking compounds without this liability were found in this screen using novel oligomerization assays. The biochemical bioluminescent protein complementation αSyn oligomerization assay, in particular, is a highly useful and novel assay in that it is amenable to high-throughput screening and does not rely on shaking or detergents to accelerate αSyn misfolding as is the case for a recently published αSyn highthroughput oligomerization assay 44 . The ability to identify anti-aggregation compounds through a fundamentally different screening paradigm -that of SPRbased screening using monomeric αSyn -allowed us to obtain alternative compounds inhibiting aggregation.…”

Chemical Kinetic Strategies for High‐Throughput Screening of Protein Aggregation Modulators

“…The scope of light scattering techniques is also expanding from proof‐of‐concept to high‐throughput applications as in the cases of dynamic light scattering (DLS) for size distribution monitoring, and small angle X‐ray scattering (SAXS) for characterizing the oligomeric equilibrium of non‐aggregated samples . In another approach, a time‐resolved fluorescence resonance energy transfer (FRET) assay was developed to screen compounds and identify 56 hits that markedly inhibit α‐synuclein and several phenyl‐benzoxazol compounds that promote α‐synuclein aggregation (proaggregators) . Important for subsequent phases of hit to lead optimization, cell‐based assays based on phenotypic and cytotoxicity readouts have also been used for compound screening purposes in the identification of IAAP aggregation inhibitors …”

“…[61] In another approach, atime-resolvedf luorescence resonance energy transfer (FRET)a ssay was developed to screen 7:46 Â 10 5 compounds and identify 56 hits that markedly inhibit a-synucleina nd severalp henyl-benzoxazol compounds that promote a-synuclein aggregation (proaggregators). [62] Important for subsequentp hases of hit to lead optimization, [5,18] cell-based assays based on phenotypica nd cytotoxicity readouts have also been used for compound screening purposes in the identification of IAAP aggregation inhibitors. [63]…”

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

“…Numerous groups have employed FRET to study aSyn aggregation and monomer confirmation in both cell-free and cellular contexts 28,[70][71][72][73][74] Recombinantly expressed, purified, and fluorescently labeled aSyn has been used in traditional FRET as well as single-molecule FRET experiments to elucidate oligomer and fibril formation under a wide range of conditions 71,72,[74][75][76][77] . These systems have also been adopted for use in HTS campaigns to identify inhibitors of oligomer/fibril formation 78 . The simplicity and purity afforded by these recombinant protein systems are well suited for biophysical characterization of direct compound-to-aSyn mechanism of action (MOA).…”