Behavior of α‐synuclein–drug complexes during nanopore analysis with a superimposed AC field

Jakova, Elisabet; Lee, Jeremy S.

doi:10.1002/elps.201600253

Cited by 9 publications

(25 citation statements)

References 45 publications

(69 reference statements)

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“…MD simulations are very challenging due to the inherent flexibility of heterogeneous ensemble of the αS and Aβ monomers and oligomers and the impact of artificial or genetic mutations on the structures and thermodynamic properties of αS and Aβ in PD and AD. Computer-aided drug design that focuses on searching for potential inhibitors for the formation of αS and Aβ fibrils and aggregates is of great interest [ 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 ]. In his remarks at the Regulatory Affairs Professionals Society’s (RAPS) 2017 Regulatory Convergence Conference, U.S. Food and Drug Administration (FDA) Commissioner Dr. Scott Gottlieb, spent considerable time addressing how “seamless” clinical trials and more widespread use of modeling and simulation could help combat the costs of both drug development and new drugs (Available online: ).…”

Section: Introductionmentioning

confidence: 99%

Insights into the Molecular Mechanisms of Alzheimer’s and Parkinson’s Diseases with Molecular Simulations: Understanding the Roles of Artificial and Pathological Missense Mutations in Intrinsically Disordered Proteins Related to Pathology

Coskuner‐Weber

Uversky

2018

IJMS

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Amyloid-β and α-synuclein are intrinsically disordered proteins (IDPs), which are at the center of Alzheimer’s and Parkinson’s disease pathologies, respectively. These IDPs are extremely flexible and do not adopt stable structures. Furthermore, both amyloid-β and α-synuclein can form toxic oligomers, amyloid fibrils and other type of aggregates in Alzheimer’s and Parkinson’s diseases. Experimentalists face challenges in investigating the structures and thermodynamic properties of these IDPs in their monomeric and oligomeric forms due to the rapid conformational changes, fast aggregation processes and strong solvent effects. Classical molecular dynamics simulations complement experiments and provide structural information at the atomic level with dynamics without facing the same experimental limitations. Artificial missense mutations are employed experimentally and computationally for providing insights into the structure-function relationships of amyloid-β and α-synuclein in relation to the pathologies of Alzheimer’s and Parkinson’s diseases. Furthermore, there are several natural genetic variations that play a role in the pathogenesis of familial cases of Alzheimer’s and Parkinson’s diseases, which are related to specific genetic defects inherited in dominant or recessive patterns. The present review summarizes the current understanding of monomeric and oligomeric forms of amyloid-β and α-synuclein, as well as the impacts of artificial and pathological missense mutations on the structural ensembles of these IDPs using molecular dynamics simulations. We also emphasize the recent investigations on residual secondary structure formation in dynamic conformational ensembles of amyloid-β and α-synuclein, such as β-structure linked to the oligomerization and fibrillation mechanisms related to the pathologies of Alzheimer’s and Parkinson’s diseases. This information represents an important foundation for the successful and efficient drug design studies.

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Section: Introductionmentioning

confidence: 99%

Insights into the Molecular Mechanisms of Alzheimer’s and Parkinson’s Diseases with Molecular Simulations: Understanding the Roles of Artificial and Pathological Missense Mutations in Intrinsically Disordered Proteins Related to Pathology

Coskuner‐Weber

Uversky

2018

IJMS

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“…Based on the nanopore analysis, we suggest that the two A1R ligands CPA and DPCPX have different binding interactions with α-Syn. Therefore, as we previously suggested for 2-aminoindan [20,21], CPA could bind to the N-terminus of α-Syn similar to 2-aminoindan thereby leaving the NAC domain free to misfold and cause a higher chance of protein aggregation. In contrast, DPCPX, similar to 1-aminoindan, metformin and caffeine binding to α-Syn, appeared to bind to both the N-and C-termini of α-Syn causing the protein to create a "loop" conformation ( Fig.…”

Section: Discussionmentioning

confidence: 83%

“…In contrast, DPCPX, similar to 1-aminoindan, metformin and caffeine binding to α-Syn, appeared to bind to both the N-and C-termini of α-Syn causing the protein to create a "loop" conformation ( Fig. 10) [20,21]. This loop conformation has been suggested to block the NAC domain from misfolding and hence promote neuroprotection by stopping the misfolded α-Syn protein brils from aggregating and forming Lewy Bodies.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, some neuroprotective drugs have been found to bind to α-Syn and prevent further aggregation, including caffeine, nicotine, 1-aminoindan and metformin [20]. Additionally, there are other drugs such as methamphetamine, cocaine, 2-aminoindan and the herbicides, paraquat and rotenone, which appear to be neurotoxic because they increase α-Syn misfolding and can be correlated with a higher incidence of PD [20][21][22][23]. Chronic adenosine A1 receptor (A1R) stimulation has recently been reported to cause hippocampal and substantia nigra (SN) neuronal death, as well as increased α-Syn accumulation in dopaminergic SN neurons [24,25].…”

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confidence: 99%

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Adenosine A1 Receptor Ligands Bind to α-Synuclein: Implications for α-Synuclein Misfolding and α-Synucleinopathy in Parkinson’s Disease

Jakova¹,

Moutaoufik²,

Lee³

et al. 2020

Preprint

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Background:Accumulating α-synuclein (α-Syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson’s disease. Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-Syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-Syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, the A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) directly interact with α-Syn to modulate α-Syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN). Methods:Nanopore analysis and molecular dynamics simulation were used to test the binding properties of CPA and DPCPX with α-Syn in vitro. Sprague-Dawley rats were administered 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-Syn aggregation and neurodegeneration were examined in the substantia nigra pars compacta region. Results:Using nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-Syn, similar to 2-aminoindan, which is expected to promote a “knot” conformation and α-Syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-Syn, similar to 1-aminoindan, which is expected to promote a “loop” conformation which prevents α-Syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-Syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Histological and confocal imaging studies revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-Syn aggregation and neurodegeneration in SN pars compacta. In contrast, DPCPX and 1-aminoindan attenuated CPA-induced neurodegeneration but did not significantly reduce α-Syn aggregation.Conclusions:The results indicate A1R agonists and drugs promoting a “knot” conformation of α-Syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a “loop” conformation of α-Syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in Parkinson’s disease.

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“…Building on previous nanopore work that has shown the ability to study the effect of drug binding on protein aggregation 49 we applied the technology further to look at chemical and voltage induced protein unfolding and at drug binding to a monomeric species. Here, we have been able to study the effect of two different actinbinding drugs in solution and distinguish them from the monomer alone.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule nanopore sensing of actin dynamics and drug binding

Wang

Wilkinson

Lin

et al. 2019

Preprint

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Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.through different modes of action. Finally, we demonstrate the ability of nanopipette-based nanopores in drug discovery by measuring real-time drug-binding at a single molecule level, calculating the observed rate constant and the saturation concentration of Swinholide A dimer formation. Using these measurements, we propose a positively cooperative actin-binding model for the Swinholide A drug's mode of action. The ability to perform such studies, label-free and with single-molecule resolution demonstrates the potential of using quartz nanopipettes in both the direct probing of the unfolding of complex biomolecules but also for future molecular diagnostics and drug discovery.6 Results Experimental configurationNanopipettes were fabricated using laser-assisted pulling as reported previously 28 . The pulled nanopipettes terminated with a single nanopore with an average diameter of 25 ± 4 nm (N = 5), as measured by Scanning Electron Microscopy (Fig. 1a and Fig. S1). Linear IV curves were obtained in the monomeric actin buffer, and the nanopore conductance was determined to be 37.0 ± 3.9 nS (Fig. S2). Given the high potassium chloride concentration (1 M), we confirmed that actin remained monomeric in the nanopore buffer (Fig. S3). In all experiments, the analyte was introduced into an external reservoir (CIS) along with a reference/ground Ag/AgCl electrode. The pipette was filled only with buffer solution, and a patch/working Ag/AgCl electrode was inserted ( Fig. 1a). When an external electric field is applied, protein transport through the nanopore is a result of cooperative and competitive contributions from diffusion, electrophoretic (EP) and electroosmotic (EO) flows 29 . At pH 8.0, both the surface of the nanopipettes and actin (isoelectric point ~5.3) are negatively charged, meaning we see no translocation events at a negative voltage (Fig. S4). We used rel...

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Behavior of α‐synuclein–drug complexes during nanopore analysis with a superimposed AC field

Cited by 9 publications

References 45 publications

Insights into the Molecular Mechanisms of Alzheimer’s and Parkinson’s Diseases with Molecular Simulations: Understanding the Roles of Artificial and Pathological Missense Mutations in Intrinsically Disordered Proteins Related to Pathology

Insights into the Molecular Mechanisms of Alzheimer’s and Parkinson’s Diseases with Molecular Simulations: Understanding the Roles of Artificial and Pathological Missense Mutations in Intrinsically Disordered Proteins Related to Pathology

Adenosine A1 Receptor Ligands Bind to α-Synuclein: Implications for α-Synuclein Misfolding and α-Synucleinopathy in Parkinson’s Disease

Single-molecule nanopore sensing of actin dynamics and drug binding

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