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

Abstract

Preventing or reversing the pathological misfolding and self-association of alpha-synuclein (aSyn) can rescue a broad spectrum of pathological cellular insults that manifest in Parkinson's Disease (PD), Dementia with Lewy bodies (DLB), and other alpha-synucleinopathies. We have developed a high-throughput, FRET-based drug discovery platform that combines high-resolution protein structural detection in living cells with an array of functional and biophysical assays to identify novel lead compounds that protect SH-SY5Y cells from aSyn induced cytotoxicity as well as inhibiting seeded aSyn aggregation, even at nanomolar concentrations.Our combination of cellular and cell-free assays allow us to distinguish between direct aSyn binding or indirect mechanisms of action (MOA). We focus on targeting oligomers with the requisite sensitivity to detect subtle protein structural changes that may lead to effective therapeutic discoveries for PD, DLB, and other alphasynucleinopathies. Pilot high-throughput screens (HTS) using our aSyn cellular FRET biosensors has led to the discovery of the first nanomolar-affinity small molecules that disrupt toxic aSyn oligomers in cells and inhibit cell death. Primary neuron assays of aSyn pathology (e.g. phosphorylation of mouse aSyn PFF) show rescue of pathology for two of our tested compounds. Subsequent seeded thioflavin-t (ThioT) aSyn aggregation assays demonstrate these compounds deter or block aSyn fibril assembly. Other hit compounds identified in our HTS are known to modulate oxidative stress, autophagy, and ER stress, providing validation that our biosensor is sensitive to indirect MOA as well. Author contributionsA.R.B. designed and conducted the experiments. E.E.L provided assistance with cell-based assays and western blot experiments. M.C.Y produce and purified recombinant protein. M.H. and K.L. performed primary neuron PFF assays. D.D.T. provided expertise on FRET and HTS, and provided comments and edits to the manuscript. M.E. and R.B. performed statistical model development and analysis on the PFF pathology model. E.E.L. provided comments and edits to the manuscript. A.R.B. and J.N.S. wrote the manuscript. Competing interestsDavid D. Thomas holds equity in and serves as executive officer for Photonic Pharma LLC, a company that owns intellectual property related to technology used in part of this project. These relationships have been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest polices.

Recent advances in cellular biosensor technology to investigate tau oligomerization

“…58 In sum, our new tau FRET biosensor and the fluorescence lifetime detection technology are well suited to identify novel compounds capable of remodeling heterogeneous tau oligomers and rescuing tau induced cytotoxicity, thus enabling therapeutic targeting of early-stage tau pathology. This HTS strategy has also been validated in other systems with α-synuclein 107 and huntingtin exon 1, 108 indicating its robustness in investigating intrinsically disordered proteins.…”

Discovery of Small Molecule Inhibitors of Huntingtin Exon 1 Aggregation by FRET-Based High-Throughput Screening in Living Cells

“…In conjunction with the fluorescence lifetime plate reader (FLT-PR) as a high-throughput screening (HTS) platform for drug discovery, the two biosensors can be used to identify small molecules capable of targeting and perturbing pathogenic Httex1 in different conformational states. The robustness and effectiveness of HEK293 cells transfected with FRET constructs as a HTS platform has been validated in our previous work with a wide range of protein targets − and has demonstrated high stability and low variability in its fluorescence signal for excellent data acquisition . Fluorescence lifetime detection increases the precision of FRET-based screening by a factor of 30 compared with conventional fluorescence intensity detection, and provides exquisite sensitivity to resolve minute structural changes within protein ensembles. , This sensitivity allows direct detection of Httex1 aggregate formation and its inhibition by small molecules.…”

“…14 Fluorescence lifetime detection increases the precision of FRET-based screening by a factor of 30 compared with conventional fluorescence intensity detection, 23 and provides exquisite sensitivity to resolve minute structural changes within protein ensembles. 15,16 This sensitivity allows direct detection of Httex1 aggregate formation and its inhibition by small molecules.…”