Alzheimer's disease (AD) is a progressive neurodegenerative disease without known effective treatment. According to amyloid hypothesis, the main cause of AD is the excessive accumulation of the amyloid beta peptide (Aβ) in extracellular plaques. By binding to an allosteric site on α2A adrenergic receptor (α2AAR), Aβ oligomers redirect norepinephrine signaling to glycogen synthase kinase 3β (GSK3β) activation and tau hyperphosphorylation, resulting in formation of neurofibrillary tangles. Meanwhile, cholinergic hypothesis proposes that cognitive decline is caused by degeneration of the cholinergic neurons and reduced acetylcholine concentrations. In the scope of master's thesis, we evaluated the activity of novel multifunctional compounds A1112, A1130, A1134C, MSG-16 and A527-2, that are both butyrylcholinesterase inhibitors and α2AAR antagonists using the in vitro cell model of AD. Following the cytotoxicity evaluation of multifunctional compounds, determined by the spectrophotometry and flow cytometry on SH-SY5Y cells, the potential protective effect of the compounds was evaluated at concentrations that did not significantly reduce cell survival. An in vitro cell model of AB was established on the SH-SY5Y neuroblastoma cell line using the fibrillar form of Aβ peptide, where 5 µM concentration demonstrated an optimal toxic effect. By SDS-PAGE and western blot, we confirmed that Aβ induces GSK3β activation and tau phosphorylation. On the established cell model of AB, we showed that pre-treatment of SH-SY5Y cells with 5 and 10 µM concentrations of MSG-16 as well as 1 and 2,5 µM concentrations of A527-2 partially improved cell viability compared to Aβ-treated cells. Furthermore, 10 µM MSG-16 and 1 µM A527-2 reduced the increased activity of caspase-3 in Aβ-stimulated SH-SY5Y cells. Meanwhile, compounds A1112 and A1130 did not improve cell viability and caspase-3 activity in Aβ-treated cells. By western blot method, we determined that multifunctional compounds inhibit GSK3β activation and reduce the level of tau protein phosphorylation in Aβ-stimulated cells. To better evaluate the antagonistic effect of compounds on α2AAR, SH-SY5Y cells were differentiated into a noradrenergic phenotype using dbcAMP. All compounds reduced the level of protein tau phosphorylation, which is induced by Aβ binding to α2AAR and consequent GSK3β activation. Our experimental findings indicate that multifunctional compounds MSG-16 and A527-2 inhibit Aβ-promoted neurodegenerative processes, with MSG-16 representing the best potential for the development of new therapeutically useful agents for the treatment of AD due to its lower cytotoxicity and more potent effect on tau phosphorylation.
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