Anthraquinones Inhibit Tau Aggregation and Dissolve Alzheimer's Paired Helical Filaments in Vitro and in Cells

Abstract

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC 50 values of 1-5 M and to disassemble preformed PHFs at DC 50 values of 2-4 M. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.

Synthesis and Multitarget Biological Profiling of a Novel Family of Rhein Derivatives As Disease-Modifying Anti-Alzheimer Agents

“…30,31 The structurally related compound rhein, 3 (Chart 1) is a natural product found in the traditional Chinese herbal medicine rhubarb, which is well tolerated in humans. 32 In the form of a doubly acetylated prodrug, diacerein, it is used for the treatment of arthritis.…”

High throughput screening for small molecule inhibitors of heparin-induced tau fibril formation

“…The fibrillization assay adopted here was modified from the one reported by Mandelkow et al [6] with the main difference that recombinant myc tagged truncated tau fragment K18 was employed rather than K19. The myc tag was added in order to evaluate antibody based assays, such as the DELFIA and Alpha Screen, in comparison with the thioflavine T (ThT) fluorescence assay.…”

“…Indeed efforts have been made to identify agents that could prevent tau aggregation and/or promote the dissolution of aggregates. [4][5][6][7] One of the major obstacles encountered in the study of tau fibrillization was the difficulty in effectively generating tau fibrils in vitro. However, the discovery that anionic cofactors (i.e.…”

“…heparin, arachidonic acid) efficiently induce the fibrillization process, and the observation that different truncated forms of tau are more prone to fibrillization compared to the full-length protein, enabled the development of in vitro assays amenable to high-throughput screening (HTS) of large compound libraries. This resulted in the identification of some structural classes of compounds including antraquinones, [6] polyphenols [7] and phenothiazines [4,7] that were found to inhibit tau fibrillization. However, despite these findings, clinical candidates have not yet been identified, and further screening as well as additional assay development, are clearly needed.…”

New directions for frontotemporal dementia drug discovery

“…The formation of the tau filaments in NFTs occurs in two steps, nucleation and extension so small molecules may be able to inhibit either step in this fibrillization process although the kinetics is often complex [9,[15][16][17]. For example, the rate limiting step for tau fibrillization is nucleation, and this can be exponentially increased by adding some kind of nucleation inducer, such as an anionic surfactant.…”