Nanodisc-associated acetylcholinesterase as a novel model system of physiological relevant membrane-bound...

Acetylcholinesterase (AChE) plays a pivotal role in the cholinergic system, and its inhibition is sought after in a wide range of applications, from insect control to Alzheimer's disease treatment. While the primary physiological isoforms of AChE are membrane-bound proteins, most assays for discovering new, safer, and potent inhibitors are conducted using commercially available soluble isoforms, such as the electric eel AChE (eeAChE). In this study, we conducted a comparative analysis of the activity and selectivity to phenolic inhibitors of recombinant human AChE, eeAChE and a mutant variant of human AChE known as dAChE4. Despite numerous mutations, dAChE4 closely resembles its parental protein and serves as a suitable model for monomeric human AChE. We also established an in vitro system of membrane-bound AChE to create a model that closely mimics the physiological isoforms. This system ensures the proper work of the enzyme and allowed us to control the exact concentration of enzyme and lipids per assay.