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

Prakriti Kalra,David D Thomas,Noah Nathan Kochen,Kelvin Luk,Malaney C Young,Razvan L Cornea,Karen Acosta,Michael D Evans,Mian Horvath,Elly E Liao,Anthony R Braun,Jonathan N Sachs,Chih Hung Lo,Elizabeth Rhoades,Roland Brown,William C K Pomerantz

doi:10.1038/s41531-021-00195-6

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.

Highlights

Preventing or reversing the pathological misfolding and selfassociation of α-synuclein can rescue a broad spectrum of pathological cellular insults that manifest in Parkinson’s disease (PD), dementia with Lewy bodies, multiple systems atrophy, and other alpha-synucleinopathies[1,2,3,4,5,6,7,8,9]
We tested the efficacy of hit compounds in SH-SY5Y cells overexpressing unlabeled wild-type (WT) αSN, and we report that two small molecules, Demeclocycline HCl (DEM) and Ro 90–7501 (RO), completely protected SH-SY5Y cells from αSN-induced death
We explored whether the fluorescence resonance energy transfer (FRET) observed in our biosensors is derived from soluble oligomers or fibrillar αSN by treating αSN biosensor sensitivity control (CTRL) and biosensor (Gα:αR αSN) transfected HEK cells As a first test of the sensitivity of this platform, we developed with pre-formed fibril (PFF) or vehicle (phosphate-buffered saline (PBS)) for 24 h

Summary

INTRODUCTION

Preventing or reversing the pathological misfolding and selfassociation of α-synuclein (αSN) can rescue a broad spectrum of pathological cellular insults that manifest in Parkinson’s disease (PD), dementia with Lewy bodies, multiple systems atrophy, and other alpha-synucleinopathies[1,2,3,4,5,6,7,8,9]. Many questions remain regarding these heterogeneous oligomers, including what their exact macromolecular constituency is in cells (i.e., what co-mingles with αSN1,15–17), whether they possess any well-defined structural motifs—various forms, including a tetramer, have been described in the literature18–24—and how variations in their molecular properties contribute to toxicity These unknowns complicate drug discovery, where it remains to be seen whether a unique small-molecule binding site or epitope even exists, as has been suggested recently for fibrils[25,26,27]. The same inhibitor protected neuron loss in a Caenorhabditis elegans model of PD but again in less than half the worms[27] Another recent HTS study used Förster or fluorescence resonance energy transfer (FRET) to monitor sodium dodecyl sulfoxide (SDS)-induced fibrillization of recombinant αSN37. Prevented the growth of pre-formed fibril (PFF) seeds with a single 25 μM inhibitor dose required for cytoprotection[25]

RESULTS

DISCUSSION

METHODS

Findings

CODE AVAILABILITY