title: Identification of acetylcholinesterase inhibitors using homogenous cell-based assays in quantitative high-throughput screening platforms.
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 serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell-based assays in high-throughput screening platforms are needed. In this study, 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). An enzyme-based assay using Amplex Red was also optimized and used to confirm the potential inhibitors. These three assays were used to screen 1368 compounds, which included a library of pharmacologically active compounds (LOPAC) and 88 additional compounds from the Tox21 program, 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 potential 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 that inhibit AChE activity.
authors.affiliation: Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.|Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China.
authors.forename: Shuaizhang
authors.initials: S
authors.lastname: Li
authors.affiliation: Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Ruili
authors.initials: R
authors.lastname: Huang
authors.affiliation: Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Solomon
authors.affiliation: Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD, USA.
authors.forename: Yitong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Santillo
authors.affiliation: Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism*; D045744:Cell Line, Tumor; D002800:Cholinesterase Inhibitors / Q000032:analysis*; D003584:Cytological Techniques / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results
chemical_list: D002800:Cholinesterase Inhibitors; D000110:Acetylcholinesterase
keywords: AChE inhibitors; Acetylcholinesterase (AChE); Cell-based AChE assay; Quantitative high-throughput screening (qHTS)