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.

Anti-Cholinesterase Combination Drug Therapy as a Potential Treatment for Alzheimer’s Disease

“…The reaction was undertaken for 30 min at room temperature, protected from the light, before the color change was read at 412 nm using a Multiskan Spectrum plate reader (Thermo Electron Corporation, Vantaa, Finland). Under these assay conditions, a final color change was reached (results not included) such that an end-point assay measurement was taken [ 29 ].…”

“…After 6 days, neurons were fully differentiated, and then treated with drugs across the broad concentration range of 0.1, 1, 10, 100, and 1000 µM; either as individual drugs or in two-drug combinations. The cell-based Ellman’s assay described by Li et al (2017) [ 29 ] was modified for 6-well plates. A final reaction volume of 400 µL was used, comprised of 190 µL of 0.01 M DTNB, 200 µL of 0.1 M (pH 8.0) phosphate buffer and 10 µL of 0.075 M ATCI substrate.…”

A novel in vitro assay model developed to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents

“…We consider that compared with previously reported in vitro models that evaluate a single component of the cholinergic system, the developed model makes it possible to more broadly screen lead compounds that have different strengths of activity and mechanisms of action by evaluating both extracellular and intracellular ACh. Although the development of cholinergic drugs has been highly focused on ACh inhibition and many AChE inhibitors have been developed [ 10 , 11 , 40 ], food constituents that are expected to counteract cognitive decline have various mechanisms besides AChE inhibition, as shown above [ 15 , 16 , 19 , 20 , 34 , 38 ]. Therefore, the developed assay model may be particularly effective to screen food constituents with mild cholinergic activities.…”

“…We, therefore, consider that an in vitro model capable of measuring intracellular ACh separately from extracellular ACh could serve as a promising tool to broadly screen cholinergic agents, especially food components with mild cholinergic activities. However, no such model exists, although a substantial number of in vitro models have been developed focusing on single cholinergic components such as AChE, ChAT, and AChRs [ 5 , 10 – 13 ].…”

Validation of an SH‐SY5Y Cell–Based Acetylcholinesterase Inhibition Assay for Water Quality Assessment

“…For example, matrix effects from diverse water samples were observed, and the co‐extracted dissolved organic carbon (DOC) contributed to reduced sensitivity of enzyme‐based AChE inhibition assays (Neale & Escher, 2013). Furthermore, cell‐based AChE inhibition assays may better reflect in vivo physiology considering cellular environment and localization of AChE (Li et al, 2017, 2021). Given that localization of AChE anchored to the cell membrane or in the cytoplasm can prevent the direct interaction between DOC and AChE (Hicks et al, 2013; Thullbery et al, 2005), a cell‐based assay could provide an alternative screening tool for environmental samples; but this has not been explored yet.…”