Development and Validation of Cell-Based Assays in Quantitative High-throughput Formats for Identification of Acetylcholinesterase Inhibitors

Authors

• SZ Li
• RH Huang
• MF Santillo
• MH Xia

Abstract

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can cause neurotoxicity; therefore, high-throughput screening assays are needed to identify novel inhibitors. A fluorescent method using Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH-SY5Y), and compared with eel enzyme-based assay using Amplex Red. A total of 1368 compounds were tested at multiple concentrations in a quantitative, high-throughput screening (qHTS) format. All three assays exhibited exceptional performance characteristics including assay signal quality, precision, and reproducibility. A group of inhibitors were identified from this study, including known (e.g., physostigmine and neostigmine bromide) and novel AChE inhibitors (e.g., chelerythrine chloride and cilostazol). These results demonstrate that this platform is a promising means to profile large numbers of chemicals for AChE inhibitors in qHTS format.

Scientific Focus Area: Neuroscience

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