Acetylcholinesterase capillary enzyme reactor for screening and characterization of selective inhibitors

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 73; pp. 44 - 52
• Main Authors: da Silva, Joyce Izidoro, de Moraes, Marcela Cristina, Vieira, Lucas Campos Curcino, Corrêa, Arlene Gonçalves, Cass, Quezia Bezerra, Cardoso, Carmen Lucia
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 25.01.2013; Elsevier
• Subjects: Acetylcholinesterase; Acetylcholinesterase - chemistry; Acetylcholinesterase - isolation & purification; Analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Bioreactors; Calibration; Cholinesterase Inhibitors - chemistry; Cholinesterase Inhibitors - pharmacology; Chromatography, High Pressure Liquid; Coumarins - chemistry; Coumarins - pharmacology; Coumarins derivatives; Dose-Response Relationship, Drug; Electrophorus; Enzyme Stability; Enzymes, Immobilized - chemistry; Fundamental and applied biological sciences. Psychology; General pharmacology; High-Throughput Screening Assays; Immobilization; Ligands; Medical sciences; Molecular Structure; Pharmacology. Drug treatments; Screening; Silica capillary; Silicon Dioxide - chemistry; Surface Properties; Immobilization; Screening; Acetylcholinesterase; Coumarins derivatives; Silica capillary; Capillary column; Enzyme; Coumarine derivatives; Lactone; Esterases; Carboxylic ester hydrolases; Characterization; Bioreactor; Hydrolases; Silica gel; Inhibitor
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2012.01.026

► Acetylcholinesterase from Electric eel was immobilized onto a fused silica capillary resulting AChE-ICER. ► AChE-ICER was employed as an LC biochromatography column in on line inhibitor studies. ► Selected compounds 20 (IC50=17.14±3.50μM and Ki=8.04±0.18) and 21 (IC50=6.35±1.20μM and Ki=2.67±0.18μM). ► Standard galanthamine (IC50=12.68±2.40μM and Ki=10.90±0.51μM). ► The obtained data suggest the applicability of the method developed herein for the identification of new ligands. 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 (IC50 17.14±3.50μM) and 21 (IC50 6.35±1.20μM), which were compared to the standard galanthamine (IC50 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 Ki 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.