A novel peptide‐based tau aggregation inhibitor as a potential therapeutic for Alzheimer's disease and other tauopathies

Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers.

In a host of neurodegenerative diseases Tau, a microtubule-associated protein, aggregates into insoluble lesions within neurons. Previous studies have utilized cyanine dyes as Tau aggregation inhibitors in vitro. Herein we utilize cyanine dye 3,3'-diethyl-9-methyl-thiacarbocyanine iodide (C11) to modulate Tau polymerization in two model systems, an organotypic slice culture model derived from Tau transgenic mice and a split green fluorescent protein complementation assay in Tau-expressing cells. In slice cultures, submicromolar concentrations (0.001 microm) of C11 produced a significant reduction of aggregated Tau and a corresponding increase in unpolymerized Tau. In contrast, treatment with a 1 microm dose promoted aggregation of Tau. These results were recapitulated in the complementation assay where administration of 1 microm C11 produced a significant increase in polymerized Tau relative to control, whereas treatment of cells with 0.01 microm C11 resulted in a marked reduction of aggregated Tau. In the organotypic slice cultures, modulation of Tau aggregation was independent of changes in phosphorylation at disease and microtubule binding relevant epitopes for both dosing regimes. Furthermore, treatment with 0.001 microm C11 resulted in a decrease in both total filament mass and number. There was no evidence of apoptosis or loss of synaptic integrity at either dose, however, whereas submicromolar concentrations of C11 did not interfere with microtubule binding, higher doses resulted in a decrease in the levels of microtubule-bound Tau. Overall, a cyanine dye can dissociate aggregated Tau in an ex vivo model of tauopathy with little toxicity and exploration of the use of these type of dyes as therapeutic agents is warranted.

The aggregation of the microtubule-associated protein tau is a significant event in many neurodegenerative diseases including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activity. We have screened Aspergillus nidulans secondary metabolites containing aromatic ring structures for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol and the previously identified aggregation inhibitor emodin as a positive control. While several compounds showed some activity, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde were potent aggregation inhibitors as determined by both a filter trap assay and electron microscopy. In this study, these three compounds were stronger inhibitors than emodin, which has been shown in a prior study to inhibit the heparin induction of tau aggregation with an IC50 of 1-5 µM. Additionally, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde reduced, but did not block, tau stabilization of microtubules. 2,ω-Dihydroxyemodin and asperthecin have similar structures to previously identified tau aggregation inhibitors, while asperbenzaldehyde represents a new class of compounds with tau aggregation inhibitor activity. Asperbenzaldehyde can be readily modified into compounds with strong lipoxygenase inhibitor activity, suggesting that compounds derived from asperbenzaldehyde could have dual activity. Together, our data demonstrates the potential of 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde as lead compounds for further development as therapeutics to inhibit tau aggregation in Alzheimer's disease and neurodegenerative tauopathies.

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.

We would like to thank Dr Exley for his thoughts [1]. To address his comment regarding data presented in Fig. 1 of our recently published paper [4], which demonstrates that 0.2–20 mM AlCl3 inhibited heparin (10μM)-induced tau (10μM) aggregation, we repeated the heparin-induced tau aggregation experiment using a lower range of AlCl3 (50 nM–2 mM). This concentration range of Al did not show clear inhibition of heparininduced tau aggregation (see Supplementary Figure). Therefore, Dr Exley’s explanation that the excess of Al absorbed heparin and inhibited tau aggregation, may be a possibility. Another possibility as described in our discussion section [4] is that, Al may directly bind to tau, and inhibit heparin-induced tau aggregation. This suggestion of the direct binding of Al to tau is further supported by the presence of Al in neurofibrillary tangles found in Alzheimer’s disease (AD) brains as well as by previous in vitro studies that showed Al-induced tau aggregation in the absence of heparin [2]. In contrast to a previous study [2], our data showed that the addition of Al (50 nM–2 mM) to 10 μM tau produced a 0.05–200 Al/tau molar ratio that failed to evoke tau aggregation with increasing thioflavin T fluorescence. This discrepancy could reflect a difference in the concentration of tau used between the two studies, as Haase and colleagues used 80 μM tau triggered by 3 mM Al. So, while the molar ratio 34.5 of Al (3 mM)/tau (80 μM) may be appropriate for inducing tau aggregation, the same ratio using 10 μM tau may not, indicating a tau-concentration dependency of the aggregation-inducing properties of Al. It seems that a higher concentration of tau, such as 80 μM, is required for Al-induced β-sheet tau aggregation in an in vitro study. Thus, the physiological concentration of tau, which has been reported as being between 1–10 μM in neurons [3], cannot induce fibrilar tau aggregation. In our cellular study [4], Dr. Exley pointed out that N2a cells may not have been exposed to the indicated concentrations (50,100, and 200μM) of Al, in our fresh Al maltorate solution, prepared by mixing an equal volume of AlCl3 solution with Maltol solution (a two fold dilution). Even though exposed to a lower concentration of Al than anticipated (according to Dr Exley’s proposition), these cells clearly exhibited Al-induced SDS-insoluble tau aggregates. However, as suggested from our in vitro tau studies, the aggregates may be amorphous and not fibrilar as shown by AFM. Regarding our animal study [4], independent of the real concentration of the exogenously administrated Al, these animals showed an overall increase (up to ∼ 20 μM) in the total concentration of brain Al in comparison to controls. Acceleration of tau aggregation, however, was not detected by either biochemical or histopathological methods. Rather than tau-based brain pathology, the increased mortality of these animals could have been induced by the peripheral effect of Al on the animal’s physiology and homeostasis. A non-direct role for Al on AD pathogenesis is further supported by Pratico and colleagues (2002). Using an alternative method of Al administration in a different AD model (APP transgenic mouse), they report-

Carbon dots as dual inhibitors of tau and amyloid-beta aggregation for the treatment of Alzheimer's disease

Tau pathology, including neurofibrillary tangles, develops in Alzheimer's disease (AD). The aggregation and hyperphosphorylation of tau are potential therapeutic targets for AD. Administration of anti-tau antibodies reduces tau pathology in transgenic "tauopathy" mice; however, the optimal tau epitopes and conformations to target are unclear. Also unknown is whether intravenous immunoglobulin (IVIG) products, currently being evaluated in AD trials, exert effects on pathological tau. This study examined the effects of anti-tau antibodies targeting different tau epitopes and the IVIG Gammagard on tau aggregation and preformed tau aggregates. Tau aggregation was assessed by transmission electron microscopy and fluorescence spectroscopy, and the binding affinity of the anti-tau antibodies for tau was evaluated by enzyme-linked immunosorbent assays. Antibodies used were anti-tau 1-150 ("D-8"), anti-tau 259-266 ("Paired-262"), anti-tau 341-360 ("A-10"), and anti-tau 404-441 ("Tau-46"), which bind to tau's N-terminus, microtubule binding domain (MBD) repeat sequences R1 and R4, and the C-terminus, respectively. The antibodies Paired-262 and A-10, but not D-8 and Tau-46, reduced tau fibrillization and degraded preformed tau aggregates, whereas the IVIG reduced tau aggregation but did not alter preformed aggregates. The binding affinities of the antibodies for the epitope for which they were specific did not appear to be related to their effects on tau aggregation. These results confirm that antibody binding to tau's MBD repeat sequences may inhibit tau aggregation and indicate that such antibodies may also degrade preformed tau aggregates. In the presence of anti-tau antibodies, the resulting tau morphologies were antigen-dependent. The results also suggested the possibility of different pathways regulating antibody-mediated inhibition of tau aggregation and antibody-mediated degradation of preformed tau aggregates.

P4-347: Tau aggregation inhibitor (TAI) therapy with rember ™ arrests the trajectory of rCBF decline in brain regions affected by Tau pathology in mild and moderate Alzheimer's disease (AD)

Drug delivery of memantine with carbon dots for Alzheimer’s disease: blood–brain barrier penetration and inhibition of tau aggregation

The efficacy of current treatments is still insufficient for Alzheimer's disease (AD), the most common cause of Dementia. Out of the two pathological hallmarks of AD amyloid-β plaques and neurofibrillary tangles, comprising of tau protein, tau pathology strongly correlates with the symptoms of AD. Previously, screening for inhibitors of tau aggregation that target recombinant tau aggregates have been attempted. Since a recent cryo-EM analysis revealed distinct differences in the folding patterns of heparin-induced recombinant tau filaments and AD tau filaments, this study focused on AD seed-dependent tau aggregation in drug repositioning for AD. We screened 763 compounds from an FDA-approved drug library using an AD seed-induced tau aggregation in SH-SY5Y cell-based assay. In the first screening, 180 compounds were selected, 72 of which were excluded based on the results of lactate dehydrogenase assay. In the third screening with evaluations of soluble and insoluble tau, 38 compounds were selected. In the fourth screening with 3 different AD seeds, 4 compounds, lansoprazole, calcipotriene, desogestrel, and pentamidine isethionate, were selected. After AD seed-induced real-time quaking-induced conversion, lansoprazole was selected as the most suitable drug for repositioning. The intranasal administration of lansoprazole for 4 months to AD seed-injected mice improved locomotor activity and reduced both the amount of insoluble tau and the extent of phosphorylated tau-positive areas. Alanine replacement of the predicted binding site to an AD filament indicated the involvement of Q351, H362, and K369 in lansoprazole and C-shaped tau filaments. These results suggest the potential of lansoprazole as a candidate for drug repositioning to an inhibitor of tau aggregate formation in AD.

Aggregation of tau proteins underlies several neurodegenerative diseases, notably Alzheimer’s disease (AD). Protein aggregation is a complex multi-step process where normal soluble proteins self-assemble in an ordered fashion into higher order conglomerates of low solubility. Experimental evidence suggests that not only the large tau aggregates, known as neurofibrillary tangles (NFTs), are involved in neuronal death, but also small tau oligomers are more likely contributed to the neuronal toxicity.1-3 While in the recent years substantial progress has been made in the design of drugs that interfere with tau aggregation, there is a lack of methods available that allow rapid screening of potential drug candidates which is critical for the future successful clinical trial.4-6 The heterogeneous nature of protein aggregation is the key challenge for testing the aggregation inhibition.Our previous studies showed the capability of surface-based electrochemistry to monitor the conformational changes of tau film on the gold surface by interrogating the electrochemical properties of tau-modified surface using the ferro/ferricyanide redox couple.7 We also clearly demonstrated that electrochemistry is a highly useful tool to monitor tau-metal and tau-tau interactions.7 Thus, we proposed that this approach can be expanded to monitor changes in the current/impedance as a result of the interaction of surface-linked tau proteins with tau protein in solution or with drug candidates. The former will provide information about tau dimerization and aggregation, while the latter will give information about tau-drug interactions and can potentially be further developed into a drug screening tool by following two different strategies. The first approach examines the affinity of the drug to bind to tau, followed by determining the effectiveness of the drug to prevent the tau dimerization. The second strategy monitors tau oligomerization and aggregation in an attempt to examine the inhibitory activity of potential drug candidates.We utilized Screen printed gold electrodes to reduce the sample size and the preparation time. A commercially available tau aggregation inhibitor, Cpd16 (amino thienopyridazine) was chosen to evaluate the developed biosensor. Since the orientation of the protein molecules on the surface has a significant impact on the efficiency and robustness of the biosensor, two different approaches were employed to anchor tau proteins on the electrode surface. First, full-length tau protein was chemically linked to the gold surface using lipoic acid N-hydroxysuccinimide ester (Lip-NHS), in which tau molecules randomly immobilize on the gold electrode. Although, results obtained from this approach was promising, non-specific adsorption is one of the biggest challenges that might have contributed to the false positive results. Therefore, biotin-tagged tau proteins were immobilized on the gold surface through the anchor NeutrAvidin to provide an oriented self-assembled monolayer as a biorecognition element. The non-specific adsorption can be minimized by this approach due to highly specific interaction of biotin with NeutrAvidin. This is the first report of using biotin-tagged tau for modification of a protein-based electrochemical biosensor. The oriented film has the advantages of higher sensitivity and reproducibility compared to the random orientation film on the gold surface. Also, the time for surface modification was significantly reduce by this approach.For both surface modification, a range of different electrochemical techniques has been exploited to study tau-tau interaction as well as tau-Cpd16 interaction by monitoring the redox activities of ferro/ferricyanide redox couple. Also changes in the film resistance as a result of such biomolecular interactions were conveniently monitored by electrochemical impedance spectroscopy. Circular dichroism spectroscopy and transmission electron microscopy were utilized as supporting techniques to monitor tau aggregation kinetics. The IC50 of Cpd16 obtained from the developed biosensors is comparable with the reported IC50, which shows the ability of this biosensor to predict the IC50 of the drug candidate for the in vitro model of AD.This project represents a significant achievement that contributes to our understanding of tau protein aggregation and provides a highly sensitive analytical tool that makes it possible to rapidly screen drug candidates that target tau aggregation. It can be expected that such a tool will be critical to identify the efficient drug candidates for AD therapy.

BackgroundDA‐7503 is a potential first‐in‐class, orally available small‐molecule inhibitor of tau oligomerization, currently in clinical development for the treatment of Alzheimer’s disease and primary tauopathies. The neurobehavioral and biochemical findings from AD and tauopathy mouse models revealed that DA‐7503 improved memory and recognition, and reduced tau aggregation and hyperphosphorylation in the brain. This study aimed to elucidate the pharmacological properties of DA‐7503, focusing on its potency and specificity for pathological tau isoforms, as well as its therapeutic effects on motor impairments and tau aggregation in the JNPL3 mouse model.MethodThe inhibitory potency of DA‐7503 on aggregation of 4R and 3R tau isoforms was evaluated in tau‐BiFC HEK293 cells. For in vivo studies, JNPL3 transgenic mice, expressing human P301L‐mutant tau, were administrated with DA‐7503 orally once daily for 4.5 months from 8 months of age. Therapeutic effects were assessed through motor impairment evaluations and analysis of pathological tau aggregation. Non‐clinical toxicity and safety pharmacology studies, including 28‐day rat and dog toxicity assessments, were conducted according to GLP principles.ResultThis study reports the non‐clinical characteristics of DA‐7503, a novel tau aggregation inhibitor, in terms of efficacy and safety. DA‐7503 demonstrated comparable inhibitory potency against aggregation of both 4R tau and 3R tau under various tau aggregation‐inducing conditions in tau‐BiFC cells. In JNPL3 mice, DA‐7503 treatment significantly improved motor function in several behavioral tests, including the open field, coat hanger, vertical grid, rotarod, and gait pattern analysis. Notably, a dose of 15 mg/kg was sufficient to reduce pathological tau aggregates in the brainstem and spinal cord of JNPL3 mice. Non‐clinical toxicity studies revealed that DA‐7503 has a favorable safety profile, supporting its clinical development.ConclusionOur findings suggest that DA‐7503, a novel tau aggregation inhibitor with great potencies against pathological 3R and 4R tau isoforms and a favorable non‐clinical safety profile, effectively restored motor function and reduced tau aggregates in the JNPL3 mouse model of tauopathy. These results highlight DA‐7503’s potential as a promising therapeutic candidate for Alzheimer’s disease and primary tauopathies, including frontotemporal dementia, progressive supranuclear palsy and corticobasal degeneration. DA‐7503 is currently undergoing a phase 1 clinical study.

Nature's toolbox against tau aggregation: An updated review of current research

A variety of human diseases are suspected to be directly linked to protein misfolding. Highly organized protein aggregates, called amyloid fibrils, and aggregation intermediates are observed; these are considered to be mediators of cellular toxicity and thus attract a great deal of attention from investigators. Neurodegenerative pathologies such as Alzheimer's disease account for a major part of these protein misfolding diseases. The last decade has witnessed a renaissance of interest in inhibitors of tau aggregation as potential disease-modifying drugs for Alzheimer's disease and other "tauopathies". The recent report of a phase II clinical trial with the tau aggregation inhibitor MTC could hold promise for the validation of the concept. This Review summarizes the available data concerning small-molecule inhibitors of tau aggregation from a medicinal chemistry point of view.

Tau aggregation driven by microtubule‐associated protein tau (MAPT) mutations is central to frontotemporal dementia pathology, yet no disease‐modifying therapies effectively target mutant tau. Here, we identify purpurin (PUR) and oleocanthal (OLC) as selective inhibitors of mutant tau aggregation using peptide models spanning the R2R3 interface. Biophysical and cellular assays demonstrated that both compounds more effectively inhibit the aggregation of mutant tau peptides compared to wild‐type, with PUR preferentially targeting V287I and N279K variants, and OLC showing broader inhibitory activity. Surface plasmon resonance and docking analyses revealed more stable interactions and lower binding free energies with mutant tau, consistent with their enhanced inhibitory effects. Computational studies using monomeric and fibrillar tau structures supported the mutation‐specific binding profiles of PUR and OLC. Atomic force microscopy and confocal imaging confirmed reduced fibril formation, while post‐transduction treatment assays showed that both compounds significantly suppressed intracellular tau propagation. Additionally, OLC reduced tau phosphorylation and oligomerization in SY5Y‐TauP301L‐EGFP cells expressing mutant tau. These findings highlight the potential of PUR and OLC as structurally distinct, mutation‐targeted inhibitors of tau aggregation and propagation, providing a rationale for their further development as candidate therapeutics for frontotemporal dementia.