Novel Protein Kinase Inhibitors Related to Tau Pathology Modulate Tau Protein-Self Interaction Using a Luciferase Complementation Assay

Abstract

The current number of drugs available for the treatment of Alzheimer's disease (AD) is strongly limited and their benefit for therapy is given only in the early state of the disease. An effective therapy should affect those processes which mainly contribute to the neuronal decay. There have been many approaches for a reduction of toxic Aβ peptides which mostly failed to halt cognitive deterioration in patients. The formation of neurofibrillary tangles (NFT) and its precursor tau oligomers have been suggested as main cause of neuronal degeneration because of a direct correlation of their density to the degree of dementia. Reducing of tau aggregation may be a viable approach for the treatment of AD. NFT consist of hyperphosphorylated tau protein and tau hyperphosphorylation reduces microtubule binding. Several protein kinases are discussed to be involved in tau hyperphosphorylation. We developed novel inhibitors of three protein kinases (gsk-3β, cdk5, and cdk1) and discussed their activity in relation to tau phosphorylation and on tau⁻tau interaction as a nucleation stage of a tau aggregation in cells. Strongest effects were observed for those inhibitors with effects on all the three kinases with emphasis on gsk-3β in nanomolar ranges.

Keywords: AD drug discovery; derivatives; lead structure; structure-activity; synthesis.

Scheme 1

Synthesis of the benzofuropyridines 3a–e and 4a–c: (a) KOH, nBu₄NBr, alkyl Br, THF, 12 h reflux; (b) CuI, Me₂S, MeCOCl, THF, 15 min −20 °C; (c) PhMgCl, THF, 15 min −20 °C; (d) p-benzoquinone, HClO₄, dioxane; (e) 1,4-naphthoquinone, HClO₄, dioxane; and (f) H₂, Pd/C, MeOH, 2 bar.

Figure 1

Quantification of threonine 231 und serine 235 tau phosphorylation after 24 h inhibitor treatment at 8 µM concentration of 2N4R-tau transfected COS-7 cells by Western blot analysis. Phospho-dependent immunoreactivity was normalized to total tau immunoreactivity and depicted as mean ± SD.

Figure 2

Modulation of tau interaction by protein kinase inhibitors. Tau protein self-interaction was assessed using a split-luciferase assay. Luminescence readings were related to vehicle control (Co) and normalized to the full length constitutively-active NanoLuc luciferase and depicted as mean ± SD. (a) Inhibitor concentration of 1 µM and (b) inhibitor concentration of 10 µM. * p < 0.05; ** p < 0.01, *** p < 0.001.