Characterization of reversible and pseudo-irreversible acetylcholine sterase inhibitors by means of an immobilized enzyme reactor

The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective lmeraction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver-Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak a ap area. AChE-CIM-IMER kinetic parameters (K-m(app) and v(max)(app)) were derived as well as inhibitory constants (K-i(app)) of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbarnoylation and decarbarnoylation steps in pseudo-irreversible binding of carbarnate derivatives (physostigmine and rivastigmine). AChE-CIM-lMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbarnates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials. (c) 2006 Elsevier B.V. All rights reserved.