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.

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.

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.

Phenylthiazolyl-hydrazide and its derivatives are potent inhibitors of tau aggregation and toxicity in vitro and in cells.

One of the key pathological features of Alzheimer's disease is the aggregation of tau protein. We are therefore searching for compounds capable of inhibiting this reaction. On the basis of an initial screen of 200000 compounds [Pickhardt, M., Gazova, Z., von Bergen, M., Khlistunova, I., Wang, Y., Hascher, A., Mandelkow, E. M., Biernat, J., and Mandelkow, E. (2005) Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells, J. Biol. Chem. 280, 3628-3635], we performed an in silico screen and predicted a new phenylthiazolyl-hydrazide (PTH) compound as a possible hit [Larbig, G., Pickhardt, M., Lloyd, D. G., Schmidt, B., and Mandelkow, E. (2007) Screening for inhibitors of tau protein aggregation into Alzheimer paired helical filaments: A ligand based approach results in successful scaffold hopping. Curr. Alzheimer Res. 4 (3), 315-323.]. Synthesis of this compound showed that it was indeed active in terms of inhibiting de novo tau aggregation and disassembling preformed aggregates (IC50 = 7.7 microM and DC50 = 10.8 microM). We have now synthesized 49 similar structures and identified the core of the PTHs to be crucial for activity, thus representing a lead structure. Analysis of the binding epitope by saturation transfer difference NMR shows strong interactions between the tau protein and the ligand in the aromatic regions of the inhibitor. By chemical variation of the core, we improved the inhibitory potency five-fold. The compounds showed a low toxicity as judged by an N2A cell model of tau aggregation and lend themselves for further development.

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.

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.