A novel electrochemical immunosensor fabricated from gold compact disc electrodes was designed for rapid evaluation of aggregation processes that lead to the formation of oligomeric and fibrillar states of amyloid-beta(1-42) (Aβ(1-42)) during Alzheimer's disease. Conformation-specific antibodies were immobilized on the surface of the gold electrode using a 3,3'-dithiobis (sulfosuccinimidyl) propionate (DTSSP) linker. Surface binding events were analyzed by electrochemical impedance spectroscopy (EIS) in which the formation of an antigen-antibody complex was quantified as a function of charge transfer resistance using a [Fe(CN)6](3-/4-) redox probe. The effectiveness of novel sym-triazine-derived aggregation modulators (TAE-1, TAE-2) to reduce the population of toxic oligomers was evaluated. Aβ fibril formation was validated by thioflavin T (ThT) fluorescence, whereas oligomer formation was investigated by MALDI. Antigen detection by EIS was further supported by immuno dot blot assays for oligomeric and fibrillar components. Docking simulations of the aggregation modulators TAE-1 and TAE-2 with Aβ(1-42) fibrils performed using Autodock Vina suggest a mechanism for the improved aggregation inhibition observed for TAE-2. The results demonstrate the utility and convenience of impedance immunosensing as an analytical tool for rapid and comprehensive evaluation of effective Aβ aggregation modulating agents.

Alzheimer's disease (AD) is a complex neurodegenerative disorder marked by numerous causative factors of disease progression, termed pathologies. We report here the synthesis of a small library of novel sym-triazine compounds designed for targeted modulation of multiple pathologies related to AD, specifically human acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and Aβ aggregation. Rational targeting of AChE was achieved by the incorporation of acetylcholine substrate analogues into a sym-triazine core in either a mono-, di-, or trisubstituted regime. A subset of these derivatives demonstrated improved activity compared to several commercially available cholinesterase inhibitors. High AChE/BuChE selectivity was characteristic of all derivatives, and AChE steady-state kinetics indicated a mixed-type inhibition mechanism. Further integration of multiple hydrophobic phenyl units allowed for improved β-sheet intercalation into amyloid aggregates. Several highly effective structures exhibited fibril inhibition greater than the previously reported β-sheet-disrupting penta-peptide, iAβ5p, evaluated by thioflavin T fluorescence spectroscopy and transmission electron microscopy. Highly effective sym-triazines were shown to be well tolerated by differentiated human neuronal cells, as demonstrated by the absence of adverse effects on cellular viability at a wide range of concentrations. Parallel targeting of multiple pathologies using sym-triazines is presented here as an effective strategy to address the complex, multifactorial nature of AD progression.

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