Inhibition Of Tau Aggregation Research Articles

Inhibition of tau aggregation using a naturally-occurring cyclic peptide scaffold

Disulfide-rich macrocyclic peptides are emerging as versatile scaffolds for the development of stable biochemical tools. This potential is due to the combination of their structural stability and range of bioactivities. Here, we explored the activity of these peptides on fibril growth of the hexapeptide Ac-VQIVYK-NH2 (AcPHF6), which is a tau-derived peptide that has been widely used to understand the pathological mechanism of numerous tauopathies, including Alzheimer's disease. Of the cyclic peptides tested, SFTI-1 and kB1 showed an inherent ability to inhibit AcPHF6 fibril formation. Using an end-capping strategy and combining it with a molecular grafting approach, we demonstrated that SFTI-1 could be used as a starting point to design more potent fibril inhibitors. We further identified chemical and structural features of SFTI-1 and its analogues that underpin their inhibitory activity. The ability to inhibit fibril growth using the strategy employed herein supports the 'steric zipper' model of AcPHF6 fibril formation and shows that naturally-occurring cyclic peptides have potential as drug leads or molecular probes for understanding fibril formation.

Tau-Centric Targets and Drugs in Clinical Development for the Treatment of Alzheimer's Disease.

The failure of several Phase II/III clinical trials in Alzheimer's disease (AD) with drugs targeting β-amyloid accumulation in the brain fuelled an increasing interest in alternative treatments against tau pathology, including approaches targeting tau phosphatases/kinases, active and passive immunization, and anti-tau aggregation. The most advanced tau aggregation inhibitor (TAI) is methylthioninium (MT), a drug existing in equilibrium between a reduced (leuco-methylthioninium) and oxidized form (MT+). MT chloride (methylene blue) was investigated in a 24-week Phase II clinical trial in 321 patients with mild to moderate AD that failed to show significant positive effects in mild AD patients, although long-term observations (50 weeks) and biomarker studies suggested possible benefit. The dose of 138 mg/day showed potential benefits on cognitive performance of moderately affected AD patients and cerebral blood flow in mildly affected patients. Further clinical evidence will come from the large ongoing Phase III trials for the treatment of AD and the behavioral variant of frontotemporal dementia on a new form of this TAI, more bioavailable and less toxic at higher doses, called TRx0237. More recently, inhibitors of tau acetylation are being actively pursued based on impressive results in animal studies obtained by salsalate, a clinically used derivative of salicylic acid.

Caspase-cleaved tau exhibits rapid memory impairment associated with tau oligomers in a transgenic mouse model

In neurodegenerative diseases like AD, tau forms neurofibrillary tangles, composed of tau protein. In the AD brain, activated caspases cleave tau at the 421th Asp, generating a caspase-cleaved form of tau, TauC3. Although TauC3 is known to assemble rapidly into filaments in vitro, a role of TauC3 in vivo remains unclear. Here, we generated a transgenic mouse expressing human TauC3 using a neuron-specific promoter. In this mouse, we found that human TauC3 was expressed in the hippocampus and cortex. Interestingly, TauC3 mice showed drastic learning and spatial memory deficits and reduced synaptic density at a young age (2–3months). Notably, tau oligomers as well as tau aggregates were found in TauC3 mice showing memory deficits. Further, i.p. or i.c.v. injection with methylene blue or Congo red, inhibitors of tau aggregation in vitro, and i.p. injection with rapamycin significantly reduced the amounts of tau oligomers in the hippocampus, rescued spine density, and attenuated memory impairment in TauC3 mice. Together, these results suggest that TauC3 facilitates early memory impairment in transgenic mice accompanied with tau oligomer formation, providing insight into the role of TauC3 in the AD pathogenesis associated with tau oligomers and a useful AD model to test drug candidates.

Diversification of edaravone via palladium-catalyzed hydrazine cross-coupling: Applications against protein misfolding and oligomerization of beta-amyloid.

N-Aryl derivatives of edaravone were identified as potentially effective small molecule inhibitors of tau and beta-amyloid aggregation in the context of developing disease-modifying therapeutics for Alzheimer's disease (AD). Palladium-catalyzed hydrazine monoarylation protocols were then employed as an expedient means of preparing a focused library of 21 edaravone derivatives featuring varied N-aryl substitution, thereby enabling structure-activity relationship (SAR) studies. On the basis of data obtained from two functional biochemical assays examining the effect of edaravone derivatives on both fibril and oligomer formation, it was determined that derivatives featuring an N-biaryl motif were four-fold more potent than edaravone.

Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1483-3) contains supplementary material, which is available to authorized users.

Prefibrillar Tau oligomers alter the nucleic acid protective function of Tau in hippocampal neurons in vivo

The accumulation of DNA and RNA oxidative damage is observed in cortical and hippocampal neurons from Alzheimer's disease (AD) brains at early stages of pathology. We recently reported that Tau is a key nuclear player in the protection of neuronal nucleic acid integrity in vivo under physiological conditions and hyperthermia, a strong inducer of oxidative stress. In a mouse model of tauopathy (THY-Tau22), we demonstrate that hyperthermia selectively induces nucleic acid oxidative damage and nucleic acid strand breaks in the nucleus and cytoplasm of hippocampal neurons that display early Tau phosphorylation but no Tau fibrils. Nucleic acid-damaged neurons were exclusively immunoreactive for prefibrillar Tau oligomers. A similar association between prefibrillar Tau oligomers and nucleic acid oxidative damage was observed in AD brains. Pretreatment with Methylene Blue (MB), a Tau aggregation inhibitor and a redox cycler, reduced hyperthermia-induced Tau oligomerization as well as nucleic acid damage.This study clearly highlights the existence of an early and critical time frame for hyperthermia-induced Tau oligomerization, which most likely occurs through increased oxidative stress, and nucleic acid vulnerability during the progression of Tau pathology. These results suggest that at early stages of AD, Tau oligomerization triggers the loss of the nucleic acid protective function of monomeric Tau. This study highlights the existence of a short therapeutic window in which to prevent the formation of pathological forms of Tau and their harmful consequences on nucleic acid integrity during the progression of Tau pathology.

Identification of Small Molecule Inhibitors of Tau Aggregation by Targeting Monomeric Tau As a Potential Therapeutic Approach for Tauopathies.

A potential strategy to alleviate the aggregation of intrinsically disordered proteins (IDPs) is to maintain the native functional state of the protein by small molecule binding. However, the targeting of the native state of IDPs by small molecules has been challenging due to their heterogeneous conformational ensembles. To tackle this challenge, we applied a high-throughput chemical microarray surface plasmon resonance imaging screen to detect the binding between small molecules and monomeric full-length Tau, a protein linked with the onset of a range of Tauopathies. The screen identified a novel set of drug-like fragment and lead-like compounds that bound to Tau. We verified that the majority of these hit compounds reduced the aggregation of different Tau constructs in vitro and in N2a cells. These results demonstrate that Tau is a viable receptor of drug-like small molecules. The drug discovery approach that we present can be applied to other IDPs linked to other misfolding diseases such as Alzheimer’s and Parkinson’s diseases.

Preventive methylene blue treatment preserves cognition in mice expressing full-length pro-aggregant human Tau.

IntroductionNeurofibrillary tangles (NFT) composed of Tau are hallmarks of neurodegeneration in Alzheimer disease. Transgenic mice expressing full-length pro-aggregant human Tau (2N4R Tau-ΔK280, termed TauΔK) or its repeat domain (TauRD-ΔK280, TauRDΔK) develop a progressive Tau pathology with missorting, phosphorylation, aggregation of Tau, loss of synapses and functional deficits. Whereas TauRDΔK assembles into NFT concomitant with neuronal death, TauΔK accumulates into Tau pretangles without overt neuronal loss. Both forms cause a comparable cognitive decline (with onset at 10mo and 12mo, respectively), which is rescued upon switch-off of transgene expression. Since methylene blue (MB) is able to inhibit Tau aggregation in vitro, we investigated whether MB can prevent or rescue Tau-induced cognitive impairments in our mouse models. Both types of mice received MB orally using different preventive and therapeutic treatment protocols, initiated either before or after disease onset. The cognitive status of the mice was assessed by behavior tasks (open field, Morris water maze) to determine the most successful conditions for therapeutic intervention.ResultsPreventive and therapeutic MB application failed to avert or recover learning and memory deficits of TauRDΔK mice. Similarly, therapeutic MB treatment initiated after onset of cognitive impairments was ineffective in TauΔK mice. In contrast, preventive MB application starting before onset of functional deficits preserved cognition of TauΔK mice. Beside improved learning and memory, MB-treated TauΔK mice showed a strong decrease of insoluble Tau, a reduction of conformationally changed (MC1) and phosphorylated Tau species (AT180, PHF1) as well as an upregulation of protein degradation systems (autophagy and proteasome). This argues for additional pleiotropic effects of MB beyond its properties as Tau aggregation inhibitor.Conclusions Our data support the use of Tau aggregation inhibitors as potential drugs for the treatment of AD and other tauopathies and highlights the need for preventive treatment before onset of cognitive impairments.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0204-4) contains supplementary material, which is available to authorized users.

Inhibitory effect of corcin on aggregation of 1N/4R human tau protein in vitro.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. One of the hallmarks of AD is an abnormal accumulation of fibril forms of tau protein which is known as a microtubule associated protein. In this regard, inhibition of tau aggregation has been documented to be a potent therapeutic approach in AD and tauopathies. Unfortunately, the available synthetic drugs have modest beneficial efficacy with several side effects. Therefore, pipeline drugs from natural sources with anti-aggregation properties can be useful in the prevention and treatment of AD. Among medicinal plants, saffron (Crocus sativus, L.), as a traditional herbal medicine has different pharmacological properties and can be used as treatment for several nervous system impairment including depression and dementia. Crocin as a major constituent of saffron is the glycosylated form of crocetin. In this study, we investigated the inhibitory effect of crocin on aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods and cell culture. Results revealed that tau protein under the fibrillation condition and in the presence of crocin had enough stability with low tendency for aggregation. Crocin inhibited tau aggregation with IC50 of 100 µg/ml. Furthermore, transmission electron microscopy images confirmed that crocin could suppress the formation of tau protein filaments. Inhibitory effect of crocin could be related to its interference with nucleation phase that led to increases in monomer species of tau protein. Based on our results, crocin is recommended as a proper candidate to be used in AD treatment.

Azaphilones inhibit tau aggregation and dissolve tau aggregates in vitro.

The aggregation of the microtubule-associated protein tau is a seminal 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 activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties in vitro. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in in vitro reactions to determine whether they interfere with tau's normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold.

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μM) required to reverse behavioral deficits and pathology in Tau transgenic mice.

PE859, a Novel Tau Aggregation Inhibitor, Reduces Aggregated Tau and Prevents Onset and Progression of Neural Dysfunction In Vivo

In tauopathies, a neural microtubule-associated protein tau (MAPT) is abnormally aggregated and forms neurofibrillary tangle. Therefore, inhibition of the tau aggregation is one of the key approaches for the treatment of these diseases. Here, we have identified a novel tau aggregation inhibitor, PE859. An oral administration of PE859 resulted in the significant reduction of sarkosyl-insoluble aggregated tau along with the prevention of onset and progression of the motor dysfunction in JNPL3 P301L-mutated human tau transgenic mice. These results suggest that PE859 is useful for the treatment of tauopathies.

Tau aggregation inhibitor therapy: an exploratory phase 2 study in mild or moderate Alzheimer's disease.

As tau aggregation pathology correlates with clinical dementia in Alzheimer's disease (AD), a tau aggregation inhibitor (TAI) could have therapeutic utility. Methylthioninium (MT) acts as a selective TAI in vitro and reduces tau pathology in transgenic mouse models. To determine the minimum safe and effective dose of MT required to prevent disease progression on clinical and functional molecular imaging outcomes. An exploratory double-blind, randomized, placebo-controlled, dose-finding trial of MT (69, 138, and 228 mg/day) was conducted in 321 mild/moderate AD subjects. The primary outcome was change on the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) at 24 weeks relative to baseline severity. Effect of treatment on regional cerebral blood flow decline was determined in a sub-study in 135 subjects. After 24 weeks, subjects were re-consented to enter sequential 6- and 12-month blinded extension phases. Registered with ClinicalTrials.gov (NCT00515333). At 24 weeks, there were significant treatment benefits in two independent populations at the 138 mg/day dose: in moderate subjects on the ADAS-cog scale (treatment effect: -5.42 units, corrected p = 0.047) and two other clinical scales; in mild subjects on the more sensitive regional cerebral blood flow measure (treatment effect: 1.97%, corrected p < 0.001). With continued treatment for 50 weeks, benefit was seen on the ADAS-cog scale in both mild and moderate subjects. The delivery of the highest dose was impaired due to dose-dependent dissolution and absorption limitations. The minimum safe and effective daily MT dose is 138 mg and suggests that further study of MT is warranted in AD.

Effects of tau domain-specific antibodies and intravenous immunoglobulin on tau aggregation and aggregate degradation.

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.

Structure and mechanism of action of tau aggregation inhibitors.

Since the discovery of phenothiazines as tau protein aggregation inhibitors, many additional small molecule inhibitors of diverse chemotype have been discovered and characterized in biological model systems. Although direct inhibition of tau aggregation has shown promise as a potential treatment strategy for depressing neurofibrillary lesion formation in Alzheimer's disease, the mechanism of action of these compounds has been unclear. However, recent studies have found that tau aggregation antagonists exert their effects through both covalent and non-covalent means, and have identified associated potency and selectivity driving features. Here we review small-molecule tau aggregation inhibitors with a focus on compound structure and inhibitory mechanism. The elucidation of inhibitory mechanism has implications for maximizing on-target efficacy while minimizing off-target side effects.

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337-342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.

TARGETING CHAPERONES TO TREAT FTLD

The microtubule-associated protein tau becomes neurotoxic in Alzheimer's disease and frontotemporal dementia. Finding therapeutics to remove this abnormal tau is needed. Small molecule-based drug discovery targeting tau is in its early stages, but major efforts are underway to accelerate this. Currently only one small molecule, a phenothiazine derivative, is in phase III clinical trials. The putative mechanism for this compound is direct inhibition of tau aggregation, but we found that this class of small molecules also blocks chaperones from preserving tau, leading to its clearance. This suggested that common structural properties govern tau aggregation and chaperone binding. We have used several techniques to evaluate the effects of chaperone inhibitors and their potential synergy with aggregation inhibitors. We have employed cell culture, biochemical, biophysical and ex vivo slice culture strategies to survey the efficacy of approaches that target chaperones alone or in combination with aggregation inhibitors. We have also employed electrophysiology to further explore the effect of this approach on restoring tau-mediated synaptic deficits. We recently discovered the molecular basis for tau interacting with Hsp90, providing the first demonstration of how a client interacts with this important chaperone. Through this and other work, we have determined that the same amino acid sequences in tau are required for both its aggregation and its binding to chaperones. Indeed compounds that inhibit tau aggregation and chaperone function are the most potent for reducing tau and rescuing synaptic deficits caused by tau accumulation. The basis for this mechanism of action is that chaperones have greater access to tau when it is unable to aggregate, creating more chaperone/tau complexes that can be targeted for degradation with inhibitors. We observed this same principle of competition for tau binding when we found that microtubule destabilization also enhanced tau binding to chaperones. While a chaperone/tau interface is potentially “druggable”, one of the biggest concerns with chaperone-based therapies is both the on- and off-target toxic liabilities. Perhaps by exploiting our understanding of tau aggregation and tau's interaction with chaperones, we can lower the doses required for efficacy of either drug, reducing the chances for detrimental side-effects.

Inhibition of tau aggregation by a rosamine derivative that blocks tau intermolecular disulfide cross-linking

Abnormal tau aggregates are presumed to be neurotoxic and are an important therapeutic target for multiple neurodegenerative disorders including Alzheimer’s disease. Growing evidence has shown that tau intermolecular disulfide cross-linking is critical in generating tau oligomers that serve as a building block for higher-order aggregates. Here we report that a small molecule inhibitor prevents tau aggregation by blocking the generation of disulfide cross-linked tau oligomers. Among the compounds tested, a rosamine derivative bearing mild thiol reactivity selectively labeled tau and effectively inhibited oligomerization and fibrillization processes in vitro. Our data suggest that controlling tau oxidation status could be a new therapeutic strategy for prevention of abnormal tau aggregation.

TauRx global phase 3 trial in Alzheimer's disease with tau aggregation inhibitor LMTX

TauRx global phase 3 trial in Alzheimer's disease with tau aggregation inhibitor LMTX

Pharmacophore-based Drug Design of Novel Potential Tau Ligands for Alzheimer's Disease Treatment

An intracellular hallmark of Alzheimer Disease (AD) is accumulation of hyperphosphorylated tau as tangles of paired helical filaments (PHF). A significant advance in understanding tau’s behaviour isolated came when it was recognized that the protein contains isolated short peptide motifs, embedded in an otherwise hydrophilic environment, which have a high tendency for beta-structure and aggregation, forming the core of the PHF. In a recent work, we used the smallest fragment responsible for aggregation, the hexapeptide 306VQIVYK311, in order to investigate with molecular dynamics simulations possible binding modes of the tau protein fragment with respect to an active flavonoid, which would be responsible for the inhibitory process of aggregation of tau. Considering such results, we have used in this work a selected pharmacophoric model and carried out a pharmacophore-based virtual screening with the purpose of designing novel potential Tau aggregation inhibitors. An initial set of 96 compounds was selected, of which 86 are unpublished regarding Tau anti-aggregation activity and the other 10 compounds are reported as Tau ligands. Prediction of biological activity and pharmaceutical properties indicated four tiophene derivatives as promising Tau aggregation inhibitors for Alzheimer’s disease treatment.