A novel small molecule screening platform for disrupting toxic tau oligomers in cells

Abstract

Tauopathies, including Alzheimer's disease, are a group of neurodegenerative disorders characterized by pathological aggregation of the microtubule binding protein tau. Recent studies suggest that toxic tau oligomers, which are soluble and distinct from insoluble beta-sheet fibrils, are central players in neuronal cell death. To exploit this new therapeutic window, we engineered two first-in-class FRET based biosensors that monitor tau conformations in cells. Because this new technology platform operates in cells, it enables high-throughput screening of small molecules that target tau oligomers while avoiding the uncertainties of idiosyncratic in vitro preparations of tau assemblies from purified protein. We found a small molecule, MK-886, that disrupts tau oligomers and reduces tau-induced cell cytotoxicity with nanomolar potency. Using SPR and an advanced single-molecule FRET technique, we show that MK-886 directly binds to tau and specifically perturbs the folding of tau monomer in the proline-rich and microtubule-binding regions. Furthermore, we show that MK-886 accelerates the tau aggregation lag phase using a thioflavin-T assay, implying that the compound stabilizes a non-toxic, on-pathway oligomer. The technology described here should generalize to the study and targeting of conformational ensembles within the aggregation pathways of most intrinsically disordered proteins.

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

“…Our approach to developing this drug discovery pipeline has been consistent to previous efforts with other biosensor systems (e.g. tau and TNFR1, amongst others) 84,[90][91][92][103][104][105] . At the core of any fluorescent assay is the potential of artifacts being introduced into the model system through the labeling of protein with synthetic fluorophores or fusion to large fluorescent XFPs.…”

“…Another concern with HTS campaigns is the physiological relevance of the cellular platform being deployed. The choice of HEK293 cells for our FRET HTS primary assay implementation is strongly rooted in the thorough vetting of this cell lines performance in multiple HTS campaigns that interrogated a wide range of protein targets 84,[90][91][92][103][104][105] . We acknowledge that not using neuronal cell lines in a primary HTS platform targeting neurodegeneration imparts a disconnect between the physiological and epigenetic link between cell type and disease environment.…”

“…Cell cultures were maintained in an incubator with 5% CO2 (Forma Series II Water Jacket CO2 Incubator, Thermo Scientific) at 37℃. The intermolecular (oligomer) and intra-molecular (monomer-conformation) aSyn FRET biosensors were generated by transiently transfecting HEK293 cells using Lipofectamine 3000 (Invitrogen) with GFP-aSyn and aSyn-RFP (1:8 DNA plasmid concentration ratio) or GFP-aSyn-RFP plasmid, respectively The effectiveness of HEK293 cells transfected with FRET constructs as a HTS platform has been demonstrated in our previous work 84, [90][91][92][103][104][105] .…”

Modulating disease-relevant tau oligomeric strains by small molecules

“…The conformational diversity of tau oligomers and the highly dynamic nature of these oligomeric strains, which can interconvert between many different assembly states or conformations, severely complicate efforts for developing therapeutic approaches for neurodegenerative diseases ( 16 , 17 ). Treatment options should consider small molecules that are able to target, modulate, and stabilize toxic tau oligomeric strains in a nontoxic conformation or enhance the cellular protein quality control mechanisms, including autophagy and proteasomal degradation ( 8 , 78 – 82 ). Modulating the conformations and depleting the disease-relevant structures using small molecules, including our novel curcumin analogs, could be an efficient therapeutic approach that targets their toxicity despite the many upstream factors that may be involved in the formation of tau oligomeric strains ( 48 , 79 , 80 , 83 , 84 ).…”

Development of fluorophores for the detection of oligomeric aggregates of amyloidogenic proteins found in neurodegenerative diseases

“…While numerous fluorescent probes have been developed to detect highly aggregated species, detecting these oligomeric species by fluorescence has proven more challenging. The difficulty in detecting these oligomers can be attributed to several factors: 1) heterogeneity and metastability of oligomers, 2) oligomers and higher-ordered aggregates sharing the same primary amino acid sequence, and 3) the lack of structural information of oligomers that enables reliable design of small molecules for targeting oligomers (Lo et al, 2019;Pilkington and Legleiter, 2019;Lo, 2022;Shea and Daggett, 2022;Vaikath et al, 2022;Wang et al, 2023).…”