Amyloid β(1-40) peptide was immobilized on an Au-colloid modified gold electrode and an electrochemical impedance spectroscopy (EIS) system was elaborated for determining the association constants, Ka, between small molecular ligands and the peptide. The changes in the resistance of the modified electrode layer with deposited Aβ(1-40) peptide were measured with EIS in relation to a series of concentrations of the ligands studied. The association constants were calculated from Langmuir isotherms. The method is sensitive, reproducible and consumes only very little amounts of interacting species. The method was applied to determine the affinity of a series of pyridine and piperidine derivatives, mainly alkaloids of a known ability, to cross the blood-brain barrier. Along with nicotine and its main metabolite cotinine, the following agents were taken for the study: anabasine, arecoline, coniine, lobeline, pseudopelletierine, trigonelline, as well as pyridine and piperidine themselves. For the sake of comparison, two vitamins were also subjected to the study: ascorbic acid and pyridoxine. There was no association of these vitamins, which were tested as a negative control. For the compounds studied, a strong association with Aβ(1-40) was determined with Ka ranging from 1.7 x 107 M-1 for (±)- anabasine to 2.3 x 108 M-1 for arecoline hydrobromide. As a positive control, a well known amyloid specific binder, Congo Red, was tested, displaying Ka equal to 3.7 x 108 M-1.
Keywords: Electrochemical impedance spectroscopy (EIS), Cyclic voltammetry, Ligand binding assay, Amyloid β peptide, Association constants, Alkaloids, Pyridine and piperidine ligands, Anti-Alzheimer's disease agents, Drug repositioning