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.

A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.

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.

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.

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.

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.

El cáncer es una enfermedad compleja de etiología desconocida. Factores genéticos y epigenéticos se asocian al incremento en el riesgo de desarrollar esta enfermedad.A pesar del avance en los tratamientos tradicionales contra el cáncer, el pronóstico de los pacientes no ha mejorado significativamente. Estudios en la patogénesis molecular del cáncer han evidenciado la existencia de dianas moleculares con potencial terapéutico que permiten trasladar los conocimientos de la investigación básica a la clínica implementando nuevas terapias para el beneficio del paciente.El conocimiento del genoma viral, su función, replicación y los mecanismos de infección a la célula tumoral han permitido el desarrollo de la terapia génica viral que puede ser la herramienta ideal para el tratamiento del cáncer.Este artículo revisa diferentes metodologías desarrolladas para el diseño de una terapia génica contra el cáncer, abordada desde diferentes contextos biológicos, y su aplicación clínica para el tratamiento del cáncer.Cancer is a complicated disease of unknown etiology. Genetic and epigenetic factors are associated with an increased risk for developing this disease.Despite the progress in the traditional cancer therapies, the prognosis of patients has not improved significantly. Studies on the molecular pathogenesis of cancer have demonstrated the existence of molecular targets with therapeutic potential. Furthermore, knowledge of the viral genome function and replication, as well as of the mechanisms of tumor cell infection, have made it possible to develop an ideal tool for gene therapy against cancer and thus, enable the transfer of knowledge from basic to clinical research for the benefit of patients.This article reviews different methodologies developed to design a cancer gene therapy and its clinical application for treating cancer, addressed from various biological contexts.

AbstractMaßgeschneiderte Additive für die Wirkstoff‐Formulierung ermöglichen die Testung potentieller neuer Wirkstoffe mit ungünstigen pharmakologischen Eigenschaften. Zur Identifizierung wirkstoffaffiner Peptidsequenzen wird die Verteilung eines Wirkstoffes in großen Peptidbibliotheken mittels Raman‐Mikroskopie verfolgt. Die Integration der Sequenzen in Peptid‐Polymer‐Konjugate ergibt spezifische Löslichkeitsvermittler, die prophylaktische und therapeutische Anti‐Alzheimer‐Wirkstoffe effektiv solubilisieren. Somit können maßgeschneiderte Additive nicht nur für fluoreszierende, sondern auch für nichtfluoreszierende Wirkstoffe gefunden werden.

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.

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.

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.

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.

Die Aggregation des Tau-Proteins ist ein Merkmal der Alzheimer-Krankheit, und Tau-Aggregationshemmer spielen eine Rolle als potenzielle krankheitsmodifizierende Medikamente. Das Phenothiazin Methylenblau hat einen einzigartigen Wirkmechanismus, welcher auf der gezielten Modifikation der Tau-Cysteinreste basiert. Diese Modifikation hält Tau in der monomeren, ungeordneten Konformation und verhindert die Bildung von Filamenten und deren toxischen Vorstufen. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.

Angewandte Chemie International EditionVolume 52, Issue 12 p. 3511-3515 Communication Mechanistic Basis of Phenothiazine-Driven Inhibition of Tau Aggregation† Elias Akoury, Elias Akoury Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen (Germany) These authors contributed equally to this work.Search for more papers by this authorDr. Marcus Pickhardt, Dr. Marcus Pickhardt DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) CAESAR Research Center, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) These authors contributed equally to this work.Search for more papers by this authorDr. Michal Gajda, Dr. Michal Gajda Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen (Germany)Search for more papers by this authorDr. Jacek Biernat, Dr. Jacek Biernat DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) CAESAR Research Center, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany)Search for more papers by this authorProf. Dr. Eckhard Mandelkow, Prof. Dr. Eckhard Mandelkow DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) CAESAR Research Center, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany)Search for more papers by this authorProf. Dr. Markus Zweckstetter, Corresponding Author Prof. Dr. Markus Zweckstetter markus.zweckstetter@dzne.de Max-Planck-Institut für Biophysikalische Chemie und Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Am Fassberg 11, 37077 Göttingen (Germany)Max-Planck-Institut für Biophysikalische Chemie und Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Am Fassberg 11, 37077 Göttingen (Germany)Search for more papers by this author Elias Akoury, Elias Akoury Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen (Germany) These authors contributed equally to this work.Search for more papers by this authorDr. Marcus Pickhardt, Dr. Marcus Pickhardt DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) CAESAR Research Center, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) These authors contributed equally to this work.Search for more papers by this authorDr. Michal Gajda, Dr. Michal Gajda Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen (Germany)Search for more papers by this authorDr. Jacek Biernat, Dr. Jacek Biernat DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) CAESAR Research Center, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany)Search for more papers by this authorProf. Dr. Eckhard Mandelkow, Prof. Dr. Eckhard Mandelkow DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany) CAESAR Research Center, Ludwig-Erhard-Allee 2, 53175 Bonn (Germany)Search for more papers by this authorProf. Dr. Markus Zweckstetter, Corresponding Author Prof. Dr. Markus Zweckstetter markus.zweckstetter@dzne.de Max-Planck-Institut für Biophysikalische Chemie und Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Am Fassberg 11, 37077 Göttingen (Germany)Max-Planck-Institut für Biophysikalische Chemie und Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Am Fassberg 11, 37077 Göttingen (Germany)Search for more papers by this author First published: 11 February 2013 https://doi.org/10.1002/anie.201208290Citations: 102 † We thank Dr. Andrei Leonov, Prof. Dr. Christian Griesinger, Dr. Eva-Maria Mandelkow, Prof. Dr. R. Heiner Schirmer, Dr. Bruno Bulic, and Dr. Dmitri Svergun for stimulating discussions and Ilka Lindner for sample preparation. This work was supported by the Cluster of Excellence and DFG Research Center “Nanoscale Microscopy and Molecular Physiology of the Brain”, the MPG consortium Toxic Protein Conformation, the Tau Consortium, and the DFG (ZW 71/2-2, 3-2 to M.Z.). Read the full textAboutRelatedInformationPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Blues brothers: Aggregation of the Tau protein is a key event in Alzheimer's disease and Tau aggregation inhibitors are important as potential drugs. Phenothiazines, such as methylene blue and its azure derivatives, have a unique mechanism, specifically modifying the Tau cysteine residues. The modification keeps Tau in a monomeric disordered conformation preventing the formation of filaments and their toxic precursors. Citing Literature Supporting Information As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Filename Description anie_201208290_sm_miscellaneous_information.pdf2.8 MB miscellaneous_information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume52, Issue12March 18, 2013Pages 3511-3515 RelatedInformation RecommendedTau‐tubulin kinase 1 (TTBK1), a neuron‐specific tau kinase candidate, is involved in tau phosphorylation and aggregationShinji Sato, Ronald L. Cerny, James L. Buescher, Tsuneya Ikezu, Journal of NeurochemistryTau Protein Associated Inhibitors in Alzheimer DiseaseQian-Qian Li, Ting-Ting Chu, Yong-Xiang Chen, Yan-Mei Li, Chinese Journal of ChemistryRelationship Between Tau Pathology and Neuroinflammation in Alzheimer's DiseaseMaria Jose Metcalfe MS, Maria E. Figueiredo-Pereira PhD, Mount Sinai Journal of Medicine: A Journal of Translational and Personalized MedicineDevelopment of Tau Aggregation Inhibitors for Alzheimer's DiseaseBruno Bulic Dr., Marcus Pickhardt Dr., Boris Schmidt Prof., Eva-Maria Mandelkow Dr., Herbert Waldmann Prof., Eckhard Mandelkow Prof., Angewandte Chemie International EditionSite-Specific Hyperphosphorylation Inhibits, Rather than Promotes, Tau Fibrillization, Seeding Capacity, and Its Microtubule Binding Mahmood Haj-Yahya, Pushparathinam Gopinath, Kolla Rajasekhar, Hilda Mirbaha, Marc I. Diamond, Hilal A. Lashuel, Angewandte Chemie International Edition

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.

The inhibition or reversal of tau aggregation is of potential therapeutic importance for Alzheimer's Disease and other tauopathies. We have previously generated a cell model of tauopathy based on the expression of an amyloidogenic tau construct in neuroblastoma N2a cells (Khlistunova et al., JBC 2006). Thus far, the quantitation of aggregation was based on fluorescence microscopy of cells stained with the dye thioflavin S. We developed a FACS-based assay of tau aggregation in cells for rapid evaluation of tau aggregation inhibitors and for separating cells in different stages of aggregation for biochemical analysis. Using FACS we have precisely counted N2a cells containing ThS-positive tau aggregates. Moreover FACS allowed us the measurement of the effect of inhibitory compounds on tau aggregation within N2a cells and the establishment of dose response relationships. The growing number of N2a cells containing aggregated tau repeat domain correlated very well with decreasing viability measured by MTT and increasing toxicity evaluated by LDH assays. Moreover, we have applied FACS for the preparative separation of cells containing tau aggregates from non-fluorescent cells without aggregates and will present a biochemical analysis of the protein modifications in both cases. The improvements in handling and separating of large cell numbers in a cell model of tauopathy has allowed us to speed up the design of cell-permeable compounds with low toxicity for the development of novel tau aggregation inhibitors.

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.

The structure activity relationship of N'-benzylidene-benzohydrazide (NBB) binding to tau and paired helical filament (PHF) proteins as well as amyloid-β₁₋₄₂ 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-β₁₋₄₂ fibril selective ligands respectively. Supported by these results, we rationalized a model for the design of selective ligands for tau, PHF, and amyloid-β₁₋₄₂ fibrils.