Generalizing the Concept of Specific Compound Formulation Additives towards Non-Fluorescent Drugs: A Solubilization Study on Potential Anti-Alzheimer-Active Small-Molecule Compounds.

Tailor-made compound formulation additives enable the testing of potential drugs with undesirable pharmacological profiles. A combinatorial approach using Raman microscopy as the readout method is presented to select peptide sequences from large one-bead-one-compound libraries. The resulting peptide-PEG conjugates solubilize potential prophylactic and therapeutic anti-Alzheimer compounds and can be used as specific additives not only for fluorescent but also for non-fluorescent compounds.

Inhibition of tau filament formation by conformational modulation.

Antiaggregation drugs play an important role in therapeutic approaches for Alzheimer's disease. Although a large number of small molecules that inhibit the aggregation of the tau protein have been identified, little is known about their mode of action. Here, we reveal the mechanism and the nature of tau species that are generated by interaction of tau with the organic compound pthalocyanine tetrasulfonate (PcTS). We demonstrate that PcTS interferes with tau filament formation by targeting the protein into soluble oligomers. A combination of NMR spectroscopy, electron paramagnetic resonance, and small-angle X-ray scattering reveals that the soluble tau oligomers contain a dynamic, noncooperatively stabilized core with a diameter of 30-40 nm that is distinct from the core of tau filaments. Our results suggest that specific modulation of the conformation of tau is a viable strategy for reduction of pathogenic tau deposits.

Antimicrobial, antiproliferative, cytotoxic, and tau inhibitory activity of rubellins and caeruleoramularin produced by the phytopathogenic fungus Ramularia collo-cygni.

Photodynamically active anthraquinone derivatives produced by the phytopathogenic fungus Ramularia collo-cygni are known to cause a barley leaf-spot disease, but data about light-dependent and independent bioactivity have been sparse to date. We therefore conducted for the first time a broad bioactivity profiling of rubellins B, C, D, and E and caeruleoramularin. Antibacterial but not antiviral activity is reported with light-dependent increase. Furthermore, when tested without illumination, compounds exerted antiproliferative and cytotoxic activity in a series of human tumor cell lines. Inhibition of tau protein assembly was observed as well.

Development of tau aggregation inhibitors for Alzheimer's disease.

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.

Screening of a neuronal cell model of tau pathology for therapeutic compounds.

We have developed a cell-based phenotypic automated high-content screening approach for N2a cells expressing the pro-aggregant repeat domain of tau protein (tauRDΔK), which allows analysis of a chemogenomic library of 1649 compounds for their effect on the inhibition or stimulation of intracellular tau aggregation. We identified several inhibitors and stimulators of aggregation and achieved a screening reproducibility >85% for all data. We identified 18 potential inhibitors (= 1.1% of the library) and 10 stimulators (= 0.6% of the library) of tau aggregation in this cell model of tau pathology. The results provide insights into the regulation of cellular tau aggregation and the pathways involved in this process (e.g., involving signaling via p38 mitogen-activated protein kinase, histone deacetylases, vascular endothelial growth factor, rho/ROCK). For example, inhibitors of protein kinases (e.g., p38) can reduce tau aggregation, whereas inhibitors of deacetylases (histone deacetylases) can enhance aggregation. These observations are compatible with reports that phosphorylated or acetylated tau promotes pathology.

Structural Basis of Small Molecule Targetability of Monomeric Tau Protein.

The therapeutic targeting of intrinsically disordered proteins (IDPs) by small molecules has been a challenge due to their heterogeneous conformational ensembles. A potential therapeutic strategy to alleviate the aggregation of IDPs is to maintain them in their native monomeric state by small molecule binding. This study investigates the structural basis of small molecule druggability of native monomeric Tau whose aggregation is linked to the onset of Tauopathies such as Alzheimer's disease. Initially, two available monomeric conformational ensembles of a shorter Tau construct K18 (also termed Tau4RD) were analyzed which revealed striking structural differences between the two ensembles, while similar number of hot spots and small molecule binding sites were identified on monomeric Tau ensembles as on tertiary folded proteins of similar size. Remarkably, some critical fibril forming sequence regions of Tau (V306-K311, V275-K280) participated in hot spot formation with higher frequency compared to other regions. As an example of small molecule binding to monomeric Tau, it was shown that methylene blue (MB) bound to monomeric K18 and full-length Tau selectively with high affinity (Kd = 125.8 nM and 86.6 nM, respectively) with binding modes involving Cys291 and Cys322, previously reported to be oxidized in the presence of MB. Overall, our results provide structure-based evidence that Tau can be a viable drug target for small molecules and indicate that specific small molecules may be able to bind to monomeric Tau and influence the way in which the protein interacts among itself and with other proteins.

Inhibition of tau aggregation in cell models of tauopathy.

The pathological aggregation of tau into paired helical filaments is a hallmark of several neurodegenerative diseases, including Alzheimer's disease. We have generated cell models of tau aggregation in order to study mechanisms involving abnormal changes of tau. In the cell models the repeat domain of tau (tau(RD)) and some of its variants are expressed in a regulated fashion, e.g. the 4-repeat domain of tau with the wild-type sequence, the repeat domain with the deltaK280 mutation ("pro-aggregation mutant"), or the repeat domain with additional proline mutations ("anti-aggregation mutant"). The aggregation of tau(RD) is toxic to the cells, but aggregation and toxicity can be prevented by low molecular weight compounds identified by a screen for inhibitors. Thus the cell models are suitable for developing aggregation inhibitor drugs and testing their cellular roles.

Screening for inhibitors of tau protein aggregation into Alzheimer paired helical filaments: a ligand based approach results in successful scaffold hopping.

The aggregation of tau protein into paired helical filaments is one of the hallmarks of Alzheimer's disease and related dementias. We therefore continue our search for non-toxic, cell penetrating inhibitors of tau aggregation, which hold potential for brain penetration. Pickhardt et al. (2005) have reported a high throughput screen for tau aggregation inhibitors previously, which resulted in the identification of several hit classes. Here we report the identification of novel inhibitors which were not present in the initial high throughput assay. This was achieved by transformation of the high throughput screen data into the 3D relationships of virtual pharmacophores The pharmacophore models were utilized in a virtual screen of a Maybridge database. The virtual screen provided 136 hits; 19 representative hits were selected and assayed, this resulted in two novel leads with an IC(50) < 13 microM. These two leads feature a novel scaffold for tau aggregation inhibitors.

Inhibition of Tau Protein Aggregation by Rhodanine-based Compounds Solubilized Via Specific Formulation Additives to Improve Bioavailability and Cell Viability.

BACKGROUND: Anti-aggregation drugs play an important role in therapeutic approaches for Alzheimer's disease. We have previously developed a number of compounds that are able to inhibit the pathological aggregation of Tau protein. One common obstacle to application is the limited penetration across the plasma membranes into cells, where Tau aggregation occurs in the cytosol. We used an inducible N2a cell line which expresses the repeat domain of tau and develops tau aggregates. OBJECTIVE: Several peptide-polymer conjugates were synthesized to enhance the uptake of compounds into cells and thus to improve their biomedical application. The aim of this study was to test whether the peptide-inhibitor complexes still retain their inhibitory activity on Tau aggregation. METHOD: We screened peptide sequences with high binding capacity to a subset of aggregation inhibitors and identified them by fluorescence microscopy and MALDI MS/MS with regard to drug solubility and effective complexion. To explore whether the synthesized complexes can influence the aggregation propensity of Tau we performed in vitro and cellular assays. The effect on toxicity was investigated by measuring apoptosis markers. RESULTS/CONCLUSION: The tested peptide-compound complexes show no decrease in the total Tau levels but decreased ratios of soluble to pelletable Tau species. This indicates a conversion of insoluble Tau oligomers into soluble forms which appear to be less toxic than the insoluble ones, as seen by a decrease of apoptotic cells. Thus the peptide-compound complexes have a higher potency than the compounds alone due to improved bioavailability of the drug.

Gaining Insights into Specific Drug Formulation Additives for Solubilizing a Potential Anti-Alzheimer Disease Drug B4A1.

The pharmacological profiles of small molecule drugs are often challenged by their poor water solubility. Sequence-defined peptides attached to poly(ethylene glycol) (PEG) offer opportunities to overcome these difficulties by acting as drug-specific formulation additives. The peptide-PEG conjugates enable specific, noncovalent drug binding via tailored peptide/drug interactions as well as provide water solubility and drug shielding by well-solvated PEG-blocks. A systematic set of specific solubilizers for B4A1 as a potential anti-Alzheimer disease drug is synthesized and variations involve the length of the PEG-blocks as well as the sequences of the peptidic drug-binding domain. The solubilizer/B4A1 complexes are studied in order to understand contributions of both PEG and peptide segments on drug payload capacities, drug/carrier aggregate sizes, and influences on inhibition of the Tau-protein aggregation in an in vitro assay.

Time course of Tau toxicity and pharmacologic prevention in a cell model of Tauopathy.

The aggregation of Tau protein is a hallmark of neurodegenerative diseases including Alzheimer's disease. Previously, we generated a cell model of tauopathy based on the 4-repeat domain with the FTDP-17 mutation ΔK280 (Tau4RDΔK) which is expressed in a regulatable fashion (tet-on). The deletion variant ΔK280 is highly amyloidogenic and forms fibrous aggregates in neuroblastoma N2a cells staining with the reporter dye Thioflavin S. The aggregation of Tau4RDΔK is toxic, contrary to wildtype or anti-aggregant variants of the protein. Using a novel approach for monitoring in situ Tau aggregation and toxicity by combination of microscopic analysis with FACS and biochemical analysis of cells enabled the dissection of the aggregating species which cause a time-dependent increase of toxicity. The dominant initiating step is the dimerization of Tau4RDΔK which leads to further aggregation and induces a strong increase in reactive oxygen species (ROS) and cytoplasmic Ca2+ which damage the membranes and cause cell death. Tau-based treatments using Tau aggregation inhibitors reduce both soluble oligomeric and fully aggregated Tau species and decrease their toxicity.

Selection and Characterization of Tau Binding á´ -Enantiomeric Peptides with Potential for Therapy of Alzheimer Disease.

A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein, as well as toxic tau oligomers. Inhibitors of pathological tau aggregation, interrupting tau self-assembly, might be useful for the development of therapeutics. Employing mirror image phage display with a large peptide library (over 109 different peptides), we have identified tau fibril binding peptides consisting of d-enantiomeric amino acids. d-enantiomeric peptides are extremely protease stable and not or less immunogenic than l-peptides, and the suitability of d-peptides for in vivo applications have already been demonstrated. Phage display selections were performed using fibrils of the d-enantiomeric hexapeptide VQIVYK, representing residues 306 to 311 of the tau protein, as a target. VQIVYK has been demonstrated to be important for fibril formation of the full lengths protein and forms fibrils by itself. Here, we report on d-enantiomeric peptides, which bind to VQIVYK, tau isoforms like tau3RD (K19) as well as to full lengths tau fibrils, and modulate the aggregation of the respective tau form. The peptides are able to penetrate cells and might be interesting for therapeutic and diagnostic applications in AD research.

Screening for inhibitors of tau polymerization.

The histopathological diagnosis of Alzheimer's disease relies on two kinds of proteinaceous aggregates: the extracellular plaques built from filaments of the Abeta-peptide and the intracellular tangles consisting of tau polymerized into Paired Helical Filaments (PHFs). The order of aggregation events is still under debate, but it is well accepted that tau-related changes have an important impact on the viability of neurons. In neurons, early morphological changes are seen in axons which begin to loose and retract synapses. This process is accompanied by an increase of aggregated tau protein. Thus the prevention of tau aggregation seems to be a valuable target for therapy of Alzheimer's disease. Here we present a screening procedure by which we identified inhibitors of tau polymerization. In the primary screen we used a thioflavin-S based assay which detects PHF formation in solution. These initial hits were further analyzed for their capacity to depolymerize preformed PHFs. These results were confirmed by several secondary assays (tryptophan fluorescence, pelleting, filter trapping and electron microscopy). By this approach it is possible to identify small molecule compounds which prevent or reverse the aggregation of tau and thereby might improve the viability of neurons in a therapeutic approach.

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

INTRODUCTION: Neurofibrillary 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 Tau(ΔK)) or its repeat domain (TauRD-ΔK280, TauRD(ΔK)) develop a progressive Tau pathology with missorting, phosphorylation, aggregation of Tau, loss of synapses and functional deficits. Whereas TauRD(ΔK) assembles into NFT concomitant with neuronal death, Tau(ΔK) 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. RESULTS: Preventive and therapeutic MB application failed to avert or recover learning and memory deficits of TauRD(ΔK) mice. Similarly, therapeutic MB treatment initiated after onset of cognitive impairments was ineffective in Tau(ΔK) mice. In contrast, preventive MB application starting before onset of functional deficits preserved cognition of Tau(ΔK) mice. Beside improved learning and memory, MB-treated Tau(ΔK) 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.

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.

Progress and developments in tau aggregation inhibitors for Alzheimer disease.

Pharmacological approaches directed toward Alzheimer disease are diversifying in parallel with a growing number of promising targets. Investigations on the microtubule-associated protein tau yielded innovative targets backed by recent findings about the central role of tau in numerous neurodegenerative diseases. In this review, we summarize the recent evolution in the development of nonpeptidic small molecules tau aggregation inhibitors (TAGIs) and their advancement toward clinical trials. The compounds are classified according to their chemical structures, providing correlative insights into their pharmacology. Overall, shared structure-activity traits are emerging, as well as specific binding modes related to their ability to engage in hydrogen bonding. Medicinal chemistry efforts on TAGIs together with encouraging in vivo data argue for successful translation to the clinic.

"Lest we forget you--methylene blue...".

Methylene blue (MB), the first synthetic drug, has a 120-year-long history of diverse applications, both in medical treatments and as a staining reagent. In recent years there was a surge of interest in MB as an antimalarial agent and as a potential treatment of neurodegenerative disorders such as Alzheimer's disease (AD), possibly through its inhibition of the aggregation of tau protein. Here we review the history and medical applications of MB, with emphasis on recent developments.

N'-benzylidene-benzohydrazides as novel and selective tau-PHF ligands.

The structure activity relationship of N'-benzylidene-benzohydrazide (NBB) binding to tau and paired helical filament (PHF) proteins as well as amyloid-ß1₋42 fibrils indicate differential selectivity for these protein aggregates. The ability of the compounds to stain neurofibrillary tangles and senile plaques isolated from human AD brain was investigated histochemically. These studies resulted in several tau-PHF and amyloid-ß1₋42 fibril selective ligands respectively. Supported by these results, we rationalized a model for the design of selective ligands for tau, PHF, and amyloid-ß1₋42 fibrils.