Immobilized cholinesterases capillary reactors on-flow screening of selective inhibitors.

The discovery of selective inhibitors for acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) is extremely important for the development of drugs that can be used in the treatment of patients diagnosed with the Alzheimer's disease (AD). For this reason, there is a growing interest in developing rapid and effective assays techniques for cholinesterases (ChE) enzymes ligand screening. Herein is presented the results of selective screening assays of a coumarin derivatives library using BChE and AChE covalently immobilized onto silica fused capillaries (ICERs, 15 cm × 0.1 mm ID). The statistical comparison of the ICERs screening assay with that of the free enzymes is reported and highlights the advantages of the on-flow ICERs assay. Two out of 20 coumarin derivatives could be highlighted: compound 17 is more active toward BChE (IC50=109 ± 21 µM) and 19 showed activity against both enzymes (BChE IC50=128 ± 28 µM and hu-AChE IC50=144 ± 40 µM). The statistical evaluation of the results of the ICERs and free enzyme assays showed no difference between them, further validating the ICERs assay model. The ICERs ability to recognize selective ligands and its use for characterization of the inhibition mechanisms of the hits consolidates the approach here reported.

Acetylcholinesterase capillary enzyme reactor for screening and characterization of selective inhibitors.

The aim of the present work is to report on the optimized preparation of capillary enzyme reactors (ICERs) based on acetylcholinesterase (AChE, EC 3.1.1.7), for the screening of selective inhibitors. The AChE-ICERs were prepared by using the homobifunctional linker glutaraldehyde through Schiff base linkage. The enzyme was anchored onto a modified fused silica capillary and employed as an LC biochromatography column for online studies, with UV-vis detection. Not only did the tailored AChE-ICER result in maintenance of the activity of the immobilized enzyme, but it also significantly improved the stability of the enzyme in the presence of organic solvents. In addition, the kinetic studies demonstrated that the enzyme retained its activity with high stability, preserving its initial activity over 10months. The absence of non-specific matrix interactions, immediate recovery of the enzymatic activity, and short analysis time were the main advantages of this AChE-ICER. The use of AChE-ICER in the ligands recognition assay was validated by evaluation of four known reversible inhibitors (galanthamine, tacrine, propidium, and rivastigmine), and the same order of inhibitory potencies described in the literature was found. The immobilized enzyme was utilized in the screening of 21 coumarin derivatives. In this library, two new potent inhibitors were identified: coumarins 20 (IC(50) 17.14±3.50µM) and 21 (IC(50) 6.35±1.20µM), which were compared to the standard galanthamine (IC(50) 12.68±2.40µM). Considering the high inhibitory activities of these compounds, with respect to the AChE-ICER, the mechanism of action was investigated. Both coumarins 20 and 21 exhibited a competitive mechanism of action, furnishing K(i) values of 8.04±0.18 and 2.67±0.18µM, respectively. The results revealed that the AChE-ICER developed herein represents a useful tool for the biological screening of inhibitor candidates and evaluation of action mechanism.