Table 1_A high-throughput screening platform for acetylcholinesterase inhibitors using a genetically encoded acetylcholine fluorescent sensor.xlsx

Acetylcholinesterase (AChE) is a crucial hydrolytic enzyme in the central nervous system, responsible for the rapid degradation of the neurotransmitter acetylcholine (ACh) in the synaptic cleft, thereby maintaining the balance between neuronal excitation and inhibition. AChE is not only the primary target of neurotoxic agents and organophosphorus pesticides but its aberrant activity is also closely associated with various neurodegenerative diseases such as Alzheimer’s disease (AD) and myasthenia gravis. The efficient and rapid discovery and screening of AChE inhibitors hold urgent and significant value for chemical toxin detection, toxicological research, and drug development for neurodegenerative diseases. Addressing the limitations of existing methods, such as low biocompatibility, low detection throughput, relative operational complexity, and high cost, this study innovatively utilizes a genetically encoded biosensor to construct a stable cell line co-expressing the ACh probe and AChE, establishing a novel high-throughput screening method for AChE inhibitors. The results demonstrate that this method achieved to detect AChE inhibitors at micromole level. This method eliminates the need for purified enzymes and toxic chemical reagents (e.g., DTNB in Ellman’s assay), significantly reduces cost (by approximately two orders of magnitude), and offers a simplified, rapid, and high-throughput compatible workflow for applications in neurotoxin detection and neurotherapeutic drug discovery.