Cholinesterase Inhibition Assay Protocol

Cholinesterase activity was measured by Ellman’s method [47 (link)] adapted for microtiter plates, as previously described by Ristovski et al. [9 (link)]. A complex and its ligands were first screened for the IC₅₀ determination and then the inhibitory constants (K_i) were determined. A stock solution of C1 (1 mg/mL) was prepared in 5% v/v DMSO in deionized water, while a stock solution of chlorido analogue C1′ (1 mg/mL) was prepared in 100% methanol (MeOH). Stock solutions of ligands L1 and pta (1 mg/mL) and the positive control (neostigmine methyl sulphate, 1 mg/mL, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were prepared in 100% MeOH. These solutions were added to the wells, and gradually diluted in 100 mM of potassium phosphate buffer (pH 7.4) to the final volume of 50 µL. Acetylthiocholine chloride and 5,5′-dithiobis-2-nitrobenzoic acid were then dissolved in the same buffer to the respective final concentrations of 1 and 0.5 mM and added (100 μL) to the wells of the microtiter plates. C1 was screened against a suite of ChEs of human and animal origin (eeAChE, hrAChE, hsBChE) (all three from Sigma-Aldrich, St. Louis, MO, USA), hrBChE (gift from the research group of Professor Stanislav Gobec, Faculty of Pharmacy, University of Ljubljana), and csBChE. All enzymes were dissolved in the 100 mM potassium phosphate buffer (pH 7.4) to 0.0075 U/mL. Fifty μL of each ChE was added to start the reactions, which were followed spectrophotometrically at 405 nm and 25 °C for 5 min using a kinetic microplate reader (Dynex Technologies Inc., Chantilly, VA, USA). The blank reactions without the inhibitors were run with the appropriate dilutions of the solvents, in which the tested compound and positive control were initially diluted (5% aqueous DMSO or 100% MeOH), and the readings were corrected according to the appropriate blanks. At the end of the experiments, the concentrations of compounds causing 50% inhibition of ChE activity (IC₅₀) were determined. To determine C1 inhibition constants (K_i), the kinetics were monitored using three different final substrate concentrations (0.125, 0.25, 0.5 mM). Each measurement was repeated at least three times. Data were analyzed using OriginPro software (OriginPro 2020, OriginLab Corporation, Northampton, MA, USA).