Development of nano Bio LC columns for the search of acetylcholinesterase molecular targets.

Abstract

A novel acetylcholinesterase Nano liquid chromatography capillary column (75 μm i.d. × 50mm length) was developed for the fast screening of acetylcholinesterase inhibitors and the evaluation of their molecular recognition mechanism. Biotinylated acetylcholinesterase was immobilized on a streptavidin Nano liquid chromatography capillary column. Because of the very strong streptavidin-biotin interaction, the acetylcholinesterase immobilization step performed by frontal analysis is very fast (required less than 10min), and the amount of immobilized acetylcholinesterase was in the microgram range (1μg). The yellow anion obtained from the enzymatic reaction detected at 412nm 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 acetylcholinesterase from nonbinders, which is the heart of fragment-based drug design. 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 binding pocket of a ligand to acetylcholinesterase contained in a methanol plant extract. The results revealed that our acetylcholinesterase Nano liquid chromatography capillary column developed in this work represents a useful tool for the rapid screening of inhibitor candidates and evaluation of the action mechanism.