High-Throughput Screening Methodology to Identify Alpha-Synuclein Aggregation Inhibitors

Pujols, Jordi; Peña-Díaz, Samuel; Conde-Giménez, María; Pinheiro, Francisca; Navarro, Susanna; Sancho, Javier; Ventura, Salvador

doi:10.3390/ijms18030478

Cited by 62 publications

(70 citation statements)

References 39 publications

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“…Protein expression and purification of WT α-Syn and its variants (H50Q and A30P) were carried out as previously described (Pujols et al, 2017) and the resulting purified protein was lyophilized and kept at −80 • C until its use.…”

Section: Protein Purificationmentioning

confidence: 99%

“…After that, protein expression was induced for 4 h with 1 mM IPTG. Protein was purified as previously described (Pujols et al, 2017).…”

Section: Nuclear Magnetic Resonancementioning

confidence: 99%

“…We designed and optimized a screening protocol that allows to follow the aggregation kinetics of α-Syn by monitoring Th-T fluorescence emission for 32 h. This approach permitted us to study the inhibitory potential of more than 14,000 compounds (Pujols et al, 2017(Pujols et al, , 2018. The activity of molecules able to reduce significantly the final amount of Th-positive material and/or impact the nucleation or elongation rates of the reaction was further confirmed using light scattering and TEM measurements at the end of the reaction.…”

Section: Zpd-2 Reduces and Delays The Aggregation Of Human α-Synucleimentioning

confidence: 99%

“…A number of promising small molecules have been discovered with this approach, including anle138b (Levin et al, 2014), BIOD303 (Moree et al, 2015), fasudil (Tatenhorst et al, 2016), squalamine (Perni et al, 2017), or SynuClean-D (SCD) (Pujols et al, 2018). In this context, we have developed a robust screening and validation protocol to analyze large chemical libraries in the search for effective inhibitors of α-Syn aggregation (Pujols et al, 2017). The in vitro pipeline FIGURE 1 | Chemical structure of the compound ZPD-2.…”

Section: Introductionmentioning

confidence: 99%

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ZPD-2, a Small Compound That Inhibits α-Synuclein Amyloid Aggregation and Its Seeded Polymerization

Peña-Díaz

Pujols

Conde-Giménez

et al. 2019

Front. Mol. Neurosci.

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α-Synuclein (α-Syn) forms toxic intracellular protein inclusions and transmissible amyloid structures in Parkinson's disease (PD). Preventing α-Syn self-assembly has become one of the most promising approaches in the search for disease-modifying treatments for this neurodegenerative disorder. Here, we describe the capacity of a small molecule (ZPD-2), identified after a high-throughput screening, to inhibit α-Syn aggregation. ZPD-2 inhibits the aggregation of wild-type α-Syn and the A30P and H50Q familial variants in vitro at substoichiometric compound:protein ratios. In addition, the molecule prevents the spreading of α-Syn seeds in protein misfolding cyclic amplification assays. ZPD-2 is active against different α-Syn strains and blocks their seeded polymerization. Treating with ZPD-2 two different PD Caenorhabditis elegans models that express α-Syn either in muscle or in dopaminergic (DA) neurons substantially reduces the number of α-Syn inclusions and decreases synuclein-induced DA neurons degeneration. Overall, ZPD-2 is a hit compound worth to be explored in order to develop lead molecules for therapeutic intervention in PD.

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Section: Protein Purificationmentioning

confidence: 99%

“…After that, protein expression was induced for 4 h with 1 mM IPTG. Protein was purified as previously described (Pujols et al, 2017).…”

Section: Nuclear Magnetic Resonancementioning

confidence: 99%

Section: Zpd-2 Reduces and Delays The Aggregation Of Human α-Synucleimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

ZPD-2, a Small Compound That Inhibits α-Synuclein Amyloid Aggregation and Its Seeded Polymerization

Peña-Díaz

Pujols

Conde-Giménez

et al. 2019

Front. Mol. Neurosci.

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“…Previous high-throughput screening (HTS) strategies targeting aSyn aggregation have been predominately focused on inhibiting or disrupting aSyn fibrils [59][60][61] . The aSyn fibrillogenesis cascade (Fig.…”

Section: Introductionmentioning

confidence: 99%

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

Braun

Liao

Horvath

et al. 2020

Preprint

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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.

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Small Molecule Inhibitors for Amyloid Aggregation

Thomas

Kaur

Ali

et al. 2021

Biological Soft Matter

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High-Throughput Screening Methodology to Identify Alpha-Synuclein Aggregation Inhibitors

Cited by 62 publications

References 39 publications

ZPD-2, a Small Compound That Inhibits α-Synuclein Amyloid Aggregation and Its Seeded Polymerization

ZPD-2, a Small Compound That Inhibits α-Synuclein Amyloid Aggregation and Its Seeded Polymerization

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

Small Molecule Inhibitors for Amyloid Aggregation

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