Abstract:

An economical model for rapid in vitro screening of acetylcholinesterase(AChE) inhibitors was developed, which used Ellman's reaction-based assay with reusable immobilized AChE. AChE-immobilized aminated silica gel microspheres were applied to develop new screening method. Firstly, the experimental conditions were optimized. And then, the inhibitory activity on immobilized AChE of tacrine and eserine(positive controls) was tested to validate the effectiveness of the new model. Multiple technical parameters of this new model were also evaluated. Finally, the inhibitory activity on AChE of monomeric compounds and natural products was evaluated by the proposed new model. The results show that the optimal conditions included the followings: 55 μL of immobilized AChE as the appropriate dose, 5 mmol/L acetylthiocholine iodide as substrate, methanol and ethanol as well as dimethyl sulfoxide which was under 6% as sample solvent. The obtained results also demonstrated that this new model was sensitive and specific for rapid screening of AChE inhibitors. Good model validation and multiple parameters evaluation results were obtained. The proposed model has many advantages: good applicability, continuous reuse of the immobilized AChE, with reliable results, etc. This model is a powerful tool for identification of AChE inhibitors from natural products or chemical libraries produced by combinatorial chemistry.

Key words: Immobilized enzyme, Acetylcholinesterase inhibitor, Target enzyme inhibitor screening