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.

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.

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.

Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory and autoimmune diseases such as rheumatoid arthritis and Crohn's disease. The biological effects of TNF are mediated by binding to TNF receptors, TNF receptor 1 (TNFR1), or TNF receptor 2 (TNFR2), and this coupling makes TNFR1-specific inhibition by small-molecule therapies essential to avoid deleterious side effects. Recently, we engineered a time-resolved fluorescence resonance energy transfer biosensor for high-throughput screening of small molecules that modulate TNFR1 conformational states and identified zafirlukast as a compound that inhibits receptor activation, albeit at low potency. Here, we synthesized 16 analogues of zafirlukast and tested their potency and specificity for TNFR1 signaling. Using cell-based functional assays, we identified three analogues with significantly improved efficacy and potency, each of which induces a conformational change in the receptor (as measured by fluorescence resonance energy transfer (FRET) in cells). The best analogue decreased NF-κB activation by 2.2-fold, IκBα efficiency by 3.3-fold, and relative potency by two orders of magnitude. Importantly, we showed that the analogues do not block TNF binding to TNFR1 and that binding to the receptor's extracellular domain is strongly cooperative. Despite these improvements, the best candidate's maximum inhibition of NF-κB is only 63%, leaving room for further improvements to the zafirlukast scaffold to achieve full inhibition and prove its potential as a therapeutic lead. Interestingly, while we find that the analogues also bind to TNFR2 in vitro, they do not inhibit TNFR2 function in cells or cause any conformational changes upon binding. Thus, these lead compounds should also be used as reagents to study conformational-dependent activation of TNF receptors.

Indirubin Inhibits TRAIL-Induced Activation of Death Receptor 5 in Jurkat Cells.

Death receptor 5 (DR5) is an apoptosis-inducing membrane receptor that mediates cell death in several life-threatening conditions. There is a crucial need for the discovery of DR5 antagonists for the therapeutic intervention of conditions in which the overactivation of DR5 underlies the pathophysiology. DR5 activation mediates cell death in non-alcoholic fatty liver disease (NAFLD) and neurodegenerative processes including amyloid-beta (Aß) accumulation, spinal cord injury (SCI), and brain ischemia. In the current work, we used fluorescence resonance energy transfer (FRET) to monitor the conformational dynamics of DR5 that mediate death signaling. We used a time-resolved FRET screening platform to screen the Selleck library of 2863 U.S. Food and Drug Administration (FDA)-approved compounds. The high-throughput screen (HTS) identified 13 compounds that modulated the FRET between DR5 monomers beyond 5 median absolute deviations (MADs) from the DMSO controls. Of these 13 compounds, indirubin was identified to specifically inhibit tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 activity without modulating DR5 surface expression or TRAIL binding. Indirubin inhibited Fas-associated death domain (FADD) oligomerization and increased cellular FLICE-inhibitory protein (c-FLIP) expression; both are molecular mechanisms involved in inhibiting the DR5 signaling cascade. This study has elucidated previously unknown properties of indirubin that make it a promising candidate for therapeutic investigation of diseases in which overactivation of DR5 underlies pathology.

Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency That Does Not Affect TNFR2 Function.

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.

Nimesulide, a COX-2 inhibitor, sensitizes pancreatic cancer cells to TRAIL-induced apoptosis by promoting DR5 clustering †.

Nimesulide is a nonsteroidal anti-inflammatory drug and a COX-2 inhibitor with antitumor and antiproliferative activities that induces apoptosis in oral, esophagus, breast, and pancreatic cancer cells. Despite being removed from the market due to hepatotoxicity, nimesulide is still an important research tool being used to develop new anticancer drugs. Multiple studies have been done to modify the nimesulide skeleton to develop more potent anticancer agents and related compounds are promising scaffolds for future development. As such, establishing a mechanism of action for nimesulide remains an important part of realizing its potential. Here, we show that nimesulide enhances TRAIL-induced apoptosis in resistant pancreatic cancer cells by promoting clustering of DR5 in the plasma membrane. In this way, nimesulide acts like a related compound, DuP-697, which sensitizes TRAIL-resistant colon cancer cells in a similar manner. Our approach applies a time-resolved FRET-based biosensor that monitors DR5 clustering and conformational states in the plasma membrane. We show that this tool can be used for future high-throughput screens to identify novel, nontoxic small molecule scaffolds to overcome TRAIL resistance in cancer cells.

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.

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.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2ß2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

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.

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.

Fluorescence-Based TNFR1 Biosensor for Monitoring Receptor Structural and Conformational Dynamics and Discovery of Small Molecule Modulators.

Inhibition of tumor necrosis factor receptor 1 (TNFR1) is a billion-dollar industry for treatment of autoimmune and inflammatory diseases. As current therapeutics of anti-TNF leads to dangerous side effects due to global inhibition of the ligand, receptor-specific inhibition of TNFR1 signaling is an intensely pursued strategy. To monitor directly the structural changes of the receptor in living cells, we engineered a fluorescence resonance energy transfer (FRET) biosensor by fusing green and red fluorescent proteins to TNFR1. Expression of the FRET biosensor in living cells allows for detection of receptor-receptor interactions and receptor structural dynamics. Using the TNFR1 FRET biosensor, in conjunction with a high-precision and high-throughput fluorescence lifetime detection technology, we developed a time-resolved FRET-based high-throughput screening platform to discover small molecules that directly target and modulate TNFR1 functions. Using this method in screening multiple pharmaceutical libraries, we have discovered a competitive inhibitor that disrupts receptor-receptor interactions, and allosteric modulators that alter the structural states of the receptor. This enables scientists to conduct high-throughput screening through a biophysical approach, with relevance to compound perturbation of receptor structure, for the discovery of novel lead compounds with high specificity for modulation of TNFR1 signaling.

Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.

Fatty acid-induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor-receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.

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

Huntington's disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.

Conformational states of TNFR1 as a molecular switch for receptor function.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that plays a key role in the regulation of the inflammatory pathway. While inhibition of TNFR1 has been the focus of many studies for the treatment of autoimmune diseases such as rheumatoid arthritis, activation of the receptor is important for the treatment of immunodeficiency diseases such as HIV and neurodegenerative diseases such as Alzheimer's disease where a boost in immune signaling is required. In addition, activation of other TNF receptors such as death receptor 5 or FAS receptor is important for cancer therapy. Here, we used a previously established TNFR1 fluorescence resonance energy transfer (FRET) biosensor together with a fluorescence lifetime technology as a high-throughput screening platform to identify a novel small molecule that activates TNFR1 by increasing inter-monomeric spacing in a ligand-independent manner. This shows that the conformational rearrangement of pre-ligand assembled receptor dimers can determine the activity of the receptor. By probing the interaction between the receptor and its downstream signaling molecule (TRADD) our findings support a new model of TNFR1 activation in which varying conformational states of the receptor act as a molecular switch in determining receptor function.

How Do Ethanolamine Plasmalogens Contribute to Order and Structure of Neurological Membranes?

Ethanolamine plasmalogen (EtnPLA) is a conical-shaped ether lipid and an essential component of neurological membranes. Low stability against oxidation limits its study in experiments. The concentration of EtnPLA in the bilayer varies depending on cell type and disease progression. Here we report on mixed bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine (C18(Plasm)-18:1PE, PLAPE), an EtnPLA lipid subtype, at mole ratios of 2:1, 1:1, and 1:2. We present X-ray diffuse scattering (XDS) form factors F(qz) from oriented stacks of bilayers, related electron-density profiles, and hydrocarbon chain NMR order parameters. To aid future research on EtnPLA lipids and associated proteins, we have also extended the CHARMM36 all-atom force field to include the PLAPE lipid. The ability of the new force-field parameters to reproduce both X-ray and NMR structural properties of the mixed bilayer is remarkable. Our results indicate a thickening of the bilayer upon incorporation of increasing amounts of PLAPE into mixed bilayers, a reduction of lateral area per molecule, and an increase in lipid tail-ordering. The lateral compressibility modulus (KA) calculated from simulations yielded values for PLAPE similar to POPC.

The role of wild-type tau in Alzheimer's disease and related tauopathies.

Tau oligomers have recently emerged as the principal toxic species in Alzheimer's disease (AD) and tauopathies. Tau oligomers are spontaneously self-assembled soluble tau proteins that are formed prior to fibrils, and they have been shown to play a central role in neuronal cell death and in the induction of neurodegeneration in animal models. As the therapeutic paradigm shifts to targeting toxic tau oligomers, this suggests the focus to study tau oligomerization in species that are less susceptible to fibrillization. While truncated and mutation containing tau as well as the isolated repeat domains are particularly prone to fibrillization, the wild-type (WT) tau proteins have been shown to be resistant to fibril formation in the absence of aggregation inducers. In this review, we will summarize and discuss the toxicity of WT tau both in vitro and in vivo, as well as its involvement in tau oligomerization and cell-to-cell propagation of pathology. Understanding the role of WT tau will enable more effective biomarker development and therapeutic discovery for treatment of AD and tauopathies.

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.

Multiscale Computational Modeling of Tubulin-Tubulin Lateral Interaction.

Microtubules are multistranded polymers in eukaryotic cells that support key cellular functions such as chromosome segregation, motor-based cargo transport, and maintenance of cell polarity. Microtubules self-assemble via "dynamic instability," in which the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e., what is different between the GTP- and GDP-tubulin states that enables microtubule growth and shortening, respectively? More generally, a major challenge in biology is how to connect theoretical frameworks across length- and timescales, from atoms to cellular behavior. In this study, we describe a multiscale model by linking atomistic molecular dynamics (MD), molecular Brownian dynamics (BD), and cellular-level thermokinetic modeling of microtubules. Here, we investigated the underlying interaction energy when tubulin dimers associate laterally by performing all-atom MD simulations. We found that the lateral potential energy is not significantly different among three nucleotide states of tubulin, GTP, GDP, and GMPCPP and is estimated to be ≅ -11 kBT. Furthermore, using MD potential energy in our BD simulations of tubulin dimers confirms that the lateral bond is weak on its own, with a mean lifetime of ∼0.1 µs, implying that the longitudinal bond is required for microtubule assembly. We conclude that nucleotide-dependent lateral-bond strength is not the key mediator microtubule dynamic instability, implying that GTP acts elsewhere to exert its stabilizing influence on microtubule polymer. Furthermore, the estimated lateral-bond strength (ΔGlat0≅ -5 kBT) is well-aligned with earlier estimates based on thermokinetic modeling and light microscopy measurements. Thus, we have computationally connected atomistic-level structural information, obtained by cryo-electron microscopy, to cellular-scale microtubule assembly dynamics using a combination of MD, BD, and thermokinetic models to bridge from Ångstroms to micrometers and from femtoseconds to minutes.