Enzyme inhibition

Inhibition of CEs was determined in 96 well plates using 3mM o-nitrophenyl acetate (o-NPA) as a substrate.[10] Briefly, inhibitors (dissolved in DMSO) were aliquoted into individual wells containing 50mM Hepes, pH7.4 and o-NPA, and the reaction was initiated by the addition of enzymes. All data points assays were run in triplicate, at least 8 concentrations of inhibitor were used and samples were compared to DMSO-alone treated controls. Results were fitted to a multifactorial equation[29] and then analyzed using Akaike’s information criteria[30, 31] to assess the best model for CE inhibition. Once established, data were plotted using Prism (GraphPad, La Jolla, CA) and K_i values were determined from derived curve fits.

A determination of the inhibition of hAChE or hBChE was undertaken in a similar fashion except that acetylthiocholine or butyrylthiocholine were used as a substrates, respectively.[32, 33] Inhibitors were assayed at a single concentration (5μM).