LAUSR

A novel acetylcholinesterase (AChE) Nano LC capillary column (75μm i.d * 50 mm length) was developed for the fast screening of AChE inhibitors and the evaluation of their molecular recognition mechanism. Biotinylated AChE was immobilized on a streptavidin Nano LC capillary column. Because of the very strong streptavidin biotin interaction, the AChE immobilization step performed by frontal analysis is very fast (required less than 10 min) and the amount of immobilized AChE was in the microgram range (1μg). The yellow anion obtained from the enzymatic reaction detected at 412 nm was achieved within 60 s and the immobilized acetylcholinesterase retained 96% of the initial activity beyond 90 days. This column was successfully applied for the discrimination of weak affinity ligands to AChE from non-binders which is the heart of the Fragment Based Drug Design (FBDD). This column was used for the determination of the IC50 values of a series of inhibitor molecules. In addition, it was demonstrated that competitive experiments could be performed with our miniaturized system to confirm the existence and the binding pocket of a ligand to AChE contained in a methanol plant extract. The results revealed that our AChE Nano LC capillary column developed in this work represents a useful tool for the rapid screening of inhibitor candidates and evaluation of action mechanism. This article is protected by copyright. All rights reserved.