Acetylcholinesterase Inhibition Assays for High-Throughput Screening

Methods Mol Biol. 2022:2474:47-58. doi: 10.1007/978-1-0716-2213-1_6.


Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh), a vital neurotransmitter that regulates muscle movement and brain function, including memory, attention, and learning. Inhibition of AChE activity can cause a variety of adverse health effects and toxicity. Identifying AChE inhibitors quickly and efficiently warrants developing AChE inhibition assays in a quantitative, high-throughput screening (qHTS) platform. In this chapter, protocols for multiple homogenous AChE inhibition assays used in a qHTS system are provided. These AChE inhibition assays include a (1) human neuroblastoma (SH-SY5Y) cell-based assay with fluorescence or colorimetric detection; (2) human recombinant AChE with fluorescence or colorimetric detection; and (3) combination of human recombinant AChE and liver microsomes with colorimetric detection, which enables detection of test compounds requiring metabolic activation to become AChE inhibitors. Together, these AChE assays can help identify, prioritize, and predict chemical hazards in large compound libraries using qHTS systems.

Keywords: AChE inhibitors; Acetylcholinesterase (AChE); Cell-based AChE inhibition assay; Enzyme-based AChE inhibition assay; High-throughput screening; Liver microsomes; Pesticides.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Acetylcholinesterase* / metabolism
  • Biological Assay
  • Cholinesterase Inhibitors* / pharmacology
  • High-Throughput Screening Assays* / methods
  • Humans


  • Cholinesterase Inhibitors
  • Acetylcholinesterase