Post-translational modification sites are present in hydrophilic cavities of alpha-synuclein, tau, FUS, and TDP-43 fibrils: A molecular dynamics study.

Hydration plays a crucial role in the refolding of intrinsically disordered proteins into amyloid fibrils; however, the specific interactions between water and protein that may contribute to this process are still unknown. In our previous studies of alpha-synuclein (aSyn), we have shown that waters confined in fibril cavities are stabilizing features of this pathological fold; and that amino acids that hydrogen bond with these confined waters modulate primary and seeded aggregation. Here, we extend our aSyn molecular dynamics (MD) simulations with three new polymorphs and correlate MD trajectory information with known post-translational modifications (PTMs) and experimental data. We show that cavity residues are more evolutionarily conserved than non-cavity residues and are enriched with PTM sites. As expected, the confinement within hydrophilic cavities results in more stably hydrated amino acids. Interestingly, cavity PTM sites display the longest protein-water hydrogen bond lifetimes, three-fold greater than non-PTM cavity sites. Utilizing the deep mutational screen dataset by Newberry et al. and the Thioflavin T aggregation review by Pancoe et al. parsed using a fibril cavity/non-cavity definition, we show that hydrophobic changes to amino acids in cavities have a larger effect on fitness and aggregation rate than residues outside cavities, supporting our hypothesis that these sites are involved in the inhibition of aSyn toxic fibrillization. Finally, we expand our study to include analysis of fibril structures of tau, FUS, TDP-43, prion, and hnRNPA1; all of which contained hydrated cavities, with tau, FUS, and TDP-43 recapitulating our PTM results in aSyn fibril cavities.

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies.

OBJECTIVE: Understanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. BACKGROUND: Tauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. NEW/UPDATED HYPOTHESIS: We hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. MAJOR CHALLENGES FOR THE HYPOTHESIS: Our approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. LINKAGE TO OTHER MAJOR THEORIES: Our biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease-associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.

Correction to: α-Synuclein's Uniquely Long Amphipathic Helix Enhances its Membrane Binding and Remodeling Capacity.

The original version of the article unfortunately contained error in author group; two authors were not submitted and published in the original version. Also the funding information is erroneously omitted.

Membrane remodeling and mechanics: Experiments and simulations of α-Synuclein.

We review experimental and simulation approaches that have been used to determine curvature generation and remodeling of lipid bilayers by membrane-bending proteins. Particular emphasis is placed on the complementary approaches used to study α-Synuclein (αSyn), a major protein involved in Parkinson's disease (PD). Recent cellular and biophysical experiments have shown that the protein 1) deforms the native structure of mitochondrial and model membranes; and 2) inhibits vesicular fusion. Today's advanced experimental and computational technology has made it possible to quantify these protein-induced changes in membrane shape and material properties. Collectively, experiments, theory and multi-scale simulation techniques have established the key physical determinants of membrane remodeling and rigidity: protein binding energy, protein partition depth, protein density, and membrane tension. Despite the exciting and significant progress made in recent years in these areas, challenges remain in connecting biophysical insights to the cellular processes that lead to disease. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Polyunsaturated chains in asymmetric lipids disorder raft mixtures and preferentially associate with α-Synuclein.

Using molecular dynamics simulations, we have explored the effect of asymmetric lipids-specifically those that contain one polyunsaturated (PUFA) and one saturated fatty acid chain-on phase separation in heterogeneous membranes. These lipids are prevalent in neuronal membranes, particularly in synaptic membranes, where the Parkinson's Disease protein α-Synuclein (αS) is found. We have therefore explored the relationship between asymmetric, PUFA-containing lipids, and αS. The simulations show that asymmetric lipids partition to the liquid disordered (Ld) phase of canonical raft mixtures because of the highly disordered PUFA chain. In the case of a membrane built to mimic the lipid composition of a synaptic vesicle, the PUFA-containing asymmetric lipids completely disrupt phase separation. Because αS is positively charged, we show that it partitions with negatively charged lipids, regardless of the saturation state of the chains. Additionally, αS preferentially associates with the polyunsaturated fatty acid tails of both charged and neutral lipids. This is a consequence of those chains' ability to accommodate the void beneath the amphipathic helix. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

α-Synuclein's Uniquely Long Amphipathic Helix Enhances its Membrane Binding and Remodeling Capacity.

α-Synuclein is the primary protein found in Lewy bodies, the protein and lipid aggregates associated with Parkinson's disease and Lewy body dementia. The protein folds into a uniquely long amphipathic α-helix (AH) when bound to a membrane, and at high enough concentrations, it induces large-scale remodeling of membranes (tubulation and vesiculation). By engineering a less hydrophobic variant of α-Synuclein, we previously showed that the energy associated with binding of α-Synuclein's AH correlates with the extent of membrane remodeling (Braun et al. in J Am Chem Soc 136:9962-9972, 2014). In this study, we combine fluorescence correlation spectroscopy, electron microscopy, and vesicle clearance assays with coarse-grained molecular dynamics simulations to test the impact of decreasing the length of the amphipathic helix on membrane binding energy and tubulation. We show that truncation of α-Synuclein's AH length by approximately 15% reduces both its membrane binding affinity (by fivefold) and membrane remodeling capacity (by nearly 50% on per mole of bound protein basis). Results from simulations correlate well with the experiments and lend support to the idea that at high protein density there is a stabilization of individual, protein-induced membrane curvature fields. The extent to which these curvature fields are stabilized, a function of binding energy, dictates the extent of tubulation. Somewhat surprisingly, we find that this stabilization does not correlate directly with the geometric distribution of the proteins on the membrane surface.

α-Synuclein Reduces Tension and Increases Undulations in Simulations of Small Unilamellar Vesicles.

Using coarse-grained molecular dynamics simulations we have explored the effect of α-Synuclein (αSyn) on the structural and mechanical properties of small unilamellar vesicles in the fluid-phase. The study is motivated by observations that a high density of membrane-bound αSyn inhibits the fusion of synthetic small unilamellar vesicles. By combining three-dimensional pressure tensor calculations with our recently developed spherical harmonics fluctuation analysis approach, we show a reduction in membrane surface tension and increased membrane undulations when αSyn is bound to the vesicle's outer leaflet at a 200:1 L/P. The protein effects these changes by decreasing the negative pressure in the headgroup region of the outer leaflet and increasing the positive pressure throughout the hydrocarbon core.

Open and closed conformations of the isolated transmembrane domain of death receptor 5 support a new model of activation.

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer-sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers-undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.

α-Synuclein-induced membrane remodeling is driven by binding affinity, partition depth, and interleaflet order asymmetry.

We have investigated the membrane remodeling capacity of the N-terminal membrane-binding domain of α-synuclein (α-Syn100). Using fluorescence correlation spectroscopy and vesicle clearance assays, we show that α-Syn100 fully tubulates POPG vesicles, the first demonstration that the amphipathic helix on its own is capable of this effect. We also show that at equal density of membrane-bound protein, α-Syn has dramatically reduced affinity for, and does not tubulate, vesicles composed of a 1:1 POPG:POPC mixture. Coarse-grained molecular dynamics simulations suggested that the difference between the pure POPG and mixture results may be attributed to differences in the protein's partition depth, the membrane's hydrophobic thickness, and disruption of acyl chain order. To explore the importance of these attributes compared with the role of the reduced binding energy, we created an α-Syn100 variant in which we removed the hydrophobic core of the non-amyloid component (NAC) domain and tested its impact on pure POPG vesicles. We observed a substantial reduction in binding affinity and tubulation, and simulations of the NAC-null protein suggested that the reduced binding energy increases the protein mobility on the bilayer surface, likely impacting the protein's ability to assemble into organized pretubule structures. We also used simulations to explore a potential role for interleaflet coupling as an additional driving force for tubulation. We conclude that symmetry across the leaflets in the tubulated state maximizes the interaction energy of the two leaflets and relieves the strain induced by the hydrophobic void beneath the amphipathic helix.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor 5 networks that are highly organized.

Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.

Proteasomal Stimulation by MK886 and Its Derivatives Can Rescue Tau-Induced Neurite Pathology.

Proteasomal degradation of intrinsically disordered proteins, such as tau, is a critical component of proteostasis in both aging and neurodegenerative diseases. In this study, we investigated proteasomal activation by MK886 (MK). We previously identified MK as a lead compound capable of modulating tau oligomerization in a cellular FRET assay and rescuing P301L tau-induced cytotoxicity. We first confirmed robust proteasomal activation by MK using 20S proteasomal assays and a cellular proteasomal tau-GFP cleavage assay. We then show that MK treatment can significantly rescue tau-induced neurite pathology in differentiated SHSY5Y neurospheres. Due to this compelling result, we designed a series of seven MK analogs to determine if proteasomal activity is sensitive to structural permutations. Using the proteasome as the primary MOA, we examined tau aggregation, neurite outgrowth, inflammation, and autophagy assays to identify two essential substituents of MK that are required for compound activity: (1) removal of the N-chlorobenzyl group from MK negated both proteasomal and autophagic activity and reduced neurite outgrowth; and (2) removal of the indole-5-isopropyl group significantly improved neurite outgrowth and autophagy activity but reduced its anti-inflammatory capacity. Overall, our results suggest that the combination of proteasomal/autophagic stimulation and anti-inflammatory properties of MK and its derivatives can decrease tau-tau interactions and help rebalance dysfunctional proteostasis. Further development of MK to optimize its proteasomal, autophagic, and anti-inflammatory targets may lead to a novel therapeutic that would be beneficial in aging and neurodegenerative diseases.

pH Effects Can Dominate Chemical Shift Perturbations in 1H,15N-HSQC NMR Spectroscopy for Studies of Small Molecule/α-Synuclein Interactions.

1H,15N-Heteronuclear Single Quantum Coherence (HSQC) NMR is a powerful technique that has been employed to characterize small-molecule interactions with intrinsically disordered monomeric α-Synuclein (aSyn). We report how solution pH can impact the interpretation of aSyn HSQC NMR spectra and demonstrate that small-molecule formulations (e.g., complexation with acidic salts) can lower sample pH and confound interpretation of drug binding and concomitant protein structural changes. Through stringent pH control, we confirm that several previously identified compounds (EGCG, Baicalin, and Dopamine (DOPA)) as well as a series of potent small-molecule inhibitors of aSyn pathology (Demeclocycline, Ro90-7501, and (±)-Bay K 8644) are capable of direct target engagement of aSyn. Previously, DOPA-aSyn interactions have been shown to elicit a dramatic chemical shift perturbation (CSP) localized to aSyn's H50 at low DOPA concentrations then expanding to aSyn's acidic C-terminal residues at increasing DOPA levels. Interestingly, this CSP profile mirrors our pH titration, where a small reduction in pH affects H50 CSP, and large pH changes induce robust C-terminal CSP. In contrast, under tightly controlled pH 5.0, DOPA induces significant CSPs observed at both ionizable and nonionizable residues. These results suggest that previous interpretations of DOPA-aSyn interactions were conflated with pH-induced CSP, highlighting the need for stringent pH control to minimize potential false-positive interpretations of ligand interactions in HSQC NMR experiments. Furthermore, DOPA's preferential interaction with aSyn under acidic pH represents a novel understanding of DOPA-aSyn interactions that may provide insight into the potential gain of toxic function of aSyn misfolding in α-synucleinopathies.

Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein.

There is a critical need for small molecules capable of rescuing pathophysiological phenotypes induced by alpha-synuclein (aSyn) misfolding and oligomerization. Building upon our previous aSyn cellular fluorescence lifetime (FLT)-Förster resonance energy transfer (FRET) biosensors, we have developed an inducible cell model incorporating the red-shifted mCyRFP1/mMaroon1 (OFP/MFP) FRET pair. This new aSyn FRET biosensor improves the signal-to-noise ratio, reduces nonspecific background FRET, and results in a 4-fold increase (transient transfection) and 2-fold increase (stable, inducible cell lines) in FRET signal relative to our previous GFP/RFP aSyn biosensors. The inducible system institutes greater temporal control and scalability, allowing for fine-tuning of biosensor expression and minimizes cellular cytotoxicity due to overexpression of aSyn. Using these inducible aSyn-OFP/MFP biosensors, we screened the Selleck library of 2684 commercially available, FDA-approved compounds and identified proanthocyanidins and casanthranol as novel hits. Secondary assays validated the ability of these compounds to modulate aSyn FLT-FRET. Functional assays probing cellular cytotoxicity and aSyn fibrillization demonstrated their capability to inhibit seeded aSyn fibrillization. Proanthocyanidins completely rescued aSyn fibril-induced cellular toxicity with EC50 of 200 nM and casanthranol supported a 85.5% rescue with a projected EC50 of 34.2 µM. Furthermore, proanthocyanidins provide a valuable tool compound to validate our aSyn biosensor performance in future high-throughput screening campaigns of industrial-scale (million-compound) chemical libraries.

α-Synuclein induces both positive mean curvature and negative Gaussian curvature in membranes.

Using a combination of X-ray scattering, fluorescence correlation spectroscopy, coarse-grained molecular dynamics (MD) simulations and potential of mean force calculations, we have explored the membrane remodeling effects of monomeric α-synuclein (αS). Our initial findings from multiple approaches are that αS (1) causes a significant thinning of the bilayer and (2) stabilizes positive mean curvature, such that the maximum principle curvature matches that of synaptic vesicles, αS-induced tubules, and the synthetic lipid vesicles to which the protein binds most tightly. This suggests that αS binding to synaptic vesicles likely stabilizes their intrinsic curvature. We then show that αS induces local negative Gaussian curvature, an effect that occurs in regions of αS shown previously via NMR and corroborated by MD simulation to have significant conformational flexibility. The induction of negative Gaussian curvature, which has implications for all curvature-sensing and curvature-generating amphipathic α-helices, supports a hypothesis that connects helix insertion to fusion and fission of vesicles, processes that have recently been linked to αS function. Then, in an effort to explain these biophysical properties of αS, we promote an intrinsic curvature-field model that recasts long-range protein-protein interactions in terms of the interactions between the local curvature fields generated by lipid-protein complexes.

Threonine Cavities Are Targetable Motifs That Control Alpha-Synuclein Fibril Growth.

Recent high-resolution structures of alpha-synuclein (aSyn) fibrils offer promise for rational approaches to drug discovery for Parkinson's disease and Lewy body dementia. Harnessing the first such structures, we previously used molecular dynamics and free energy calculations to suggest that threonines 72 and 75─which line water-filled cavities within the fibril stacks─may be of central importance in stabilizing fibrils. Here, we used experimental mutagenesis of both wild-type and A53T aSyn to show that both threonine residues play important but surprisingly disparate roles in fibril nucleation and elongation. The T72A mutant, but not T75A, resulted in a large increase in the extent of fibrillization during primary nucleation, leading us to posit that T72 acts as a "brake" on run-away aggregation. An expanded set of simulations of five recent high-resolution fibril structures suggests that confinement of cavity waters around T72 correlates with this finding. In contrast, the T75A mutation led to a modest decrease in the extent of fibrillization. Furthermore, both T72A and T75A completely blocked the initial fibril elongation in seeded fibrillization. To test whether these threonine-lined cavities are druggable targets, we used computational docking to identify potential small-molecule binders. We show that the top-scoring hit, aprepitant, strongly promotes fibril growth while specifically interacting with aSyn fibrils and not monomer, and we offer speculation as to how such compounds could be used therapeutically.

Broad Tricyclic Ring Inhibitors Block SARS-CoV-2 Spike Function Required for Viral Entry.

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells requires binding of the viral spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor, which triggers subsequent conformational changes to facilitate viral and cellular fusion at the plasma membrane or following endocytosis. Here, we experimentally identified selective and broad inhibitors of SARS-CoV-2 entry that share a tricyclic ring (or similar) structure. The inhibitory effect was restricted to early steps during infection and the entry inhibitors interacted with the receptor binding domain of the SARS-CoV-2 spike but did not significantly interfere with receptor (ACE2) binding. Instead, some of these compounds induced conformational changes or affected spike assembly and blocked SARS-CoV-2 spike cell-cell fusion activity. The broad inhibitors define a highly conserved binding pocket that is present on the spikes of SARS-CoV-1, SARS-CoV-2, and all circulating SARS-CoV-2 variants tested and block SARS-CoV spike activity required for mediating viral entry. These compounds provide new insights into the SARS-CoV-2 spike topography, as well as into critical steps on the entry pathway, and can serve as lead candidates for the development of broad-range entry inhibitors against SARS-CoVs.

Interpretation of fluctuation spectra in lipid bilayer simulations.

Atomic resolution and coarse-grained simulations of dimyristoylphosphatidylcholine lipid bilayers were analyzed for fluctuations perpendicular to the bilayer using a completely Fourier-based method. We find that the fluctuation spectrum of motions perpendicular to the bilayer can be decomposed into just two parts: 1), a pure undulation spectrum proportional to q(-4) that dominates in the small-q regime; and 2), a molecular density structure factor contribution that dominates in the large-q regime. There is no need for a term proportional to q(-2) that has been postulated for protrusion fluctuations and that appeared to have been necessary to fit the spectrum for intermediate q. We suggest that earlier reports of such a term were due to the artifact of binning and smoothing in real space before obtaining the Fourier spectrum. The observability of an intermediate protrusion regime from the fluctuation spectrum is discussed based on measured and calculated material constants.

Determination of electron density profiles and area from simulations of undulating membranes.

The traditional method for extracting electron density and other transmembrane profiles from molecular dynamics simulations of lipid bilayers fails for large bilayer systems, because it assumes a flat reference surface that does not take into account long wavelength undulations. We have developed what we believe to be a novel set of methods to characterize these undulations and extract the underlying profiles in the large systems. Our approach first obtains an undulation reference surface for each frame in the simulation and subsequently isolates the long-wavelength undulations by filtering out the intrinsic short wavelength modes. We then describe two methods to obtain the appropriate profiles from the undulating reference surface. Most combinations of methods give similar results for the electron density profiles of our simulations of 1024 DMPC lipids. From simulations of smaller systems, we also characterize the finite size effect related to the boundary conditions of the simulation box. In addition, we have developed a set of methods that use the undulation reference surface to determine the true area per lipid which, due to undulations, is larger than the projected area commonly reported from simulations.

NaCl interactions with phosphatidylcholine bilayers do not alter membrane structure but induce long-range ordering of ions and water.

It is generally accepted that ions interact directly with lipids in biological membranes. Decades of biophysical studies on pure lipid bilayer systems have shown that only certain types of ions, most significantly large anions and multivalent cations, can fundamentally alter the structure and dynamics of lipid bilayers. It has long been accepted that at physiological concentrations NaCl ions do not alter the physical behavior or structure of bilayers composed solely of zwitterionic phosphatidylcholine (PC) lipids. Recent X-ray scattering experiments have reaffirmed this dogma, showing that below 1 M concentration, NaCl does not significantly alter bilayer structure. However, despite this history, there is an ongoing controversy within the molecular dynamics (MD) simulation community regarding NaCl/PC interactions. In particular, the CHARMM and GROMOS force fields show dramatically different behavior, including the effect on bilayer structure, surface potential, and the ability to form stable, coordinated ion-lipid complexes. Here, using long-timescale, constant-pressure simulations under the newest version of the CHARMM force field, we find that Na⁺ and Cl⁻ associate with PC head groups in a POPC bilayer with approximately equal, though weak, affinity, and that the salt has a negligible effect on bilayer structure, consistent with earlier CHARMM results and more recent X-ray data. The results suggest that interpretation of simulations where lipids interact with charged groups of any sort, including charged proteins, must be carefully scrutinized.

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

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.