Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition

Abstract

Background: Inhibition of acetylcholinesterase (AChE), a biomarker of organophosphorous and carbamate exposure in environmental and occupational human health, has been commonly used to identify potential safety liabilities. So far, many environmental chemicals, including drug candidates, food additives, and industrial chemicals, have not been thoroughly evaluated for their inhibitory effects on AChE activity. AChE inhibitors can have therapeutic applications (e.g., tacrine and donepezil) or neurotoxic consequences (e.g., insecticides and nerve agents). Objectives: The objective of the current study was to identify environmental chemicals that inhibit AChE activity using in vitro and in silico models. Methods: To identify AChE inhibitors rapidly and efficiently, we have screened the Toxicology in the 21st Century (Tox21) 10K compound library in a quantitative high-throughput screening (qHTS) platform by using the homogenous cell-based AChE inhibition assay and enzyme-based AChE inhibition assays (with or without microsomes). AChE inhibitors identified from the primary screening were further tested in monolayer or spheroid formed by SH-SY5Y and neural stem cell models. The inhibition and binding modes of these identified compounds were studied with time-dependent enzyme-based AChE inhibition assay and molecular docking, respectively. Results: A group of known AChE inhibitors, such as donepezil, ambenonium dichloride, and tacrine hydrochloride, as well as many previously unreported AChE inhibitors, such as chelerythrine chloride and cilostazol, were identified in this study. Many of these compounds, such as pyrazophos, phosalone, and triazophos, needed metabolic activation. This study identified both reversible (e.g., donepezil and tacrine) and irreversible inhibitors (e.g., chlorpyrifos and bromophos-ethyl). Molecular docking analyses were performed to explain the relative inhibitory potency of selected compounds. Conclusions: Our tiered qHTS approach allowed us to generate a robust and reliable data set to evaluate large sets of environmental compounds for their AChE inhibitory activity. https://doi.org/10.1289/EHP6993

Identification of environmental chemicals that activate p53 signaling after in vitro metabolic activation

“…We postulate that this reduction in bioactivity is most likely due to non-specific protein binding because similar results were observed with heat-attenuated microsomes (data not shown). Similar findings were also observed in our previously conducted screening for acetylcholinesterase inhibitors (Li et al 2021 ).…”

“…The enriched clusters mainly contain organophosphates, organothiophosphates, and organochlorines, chemical classes that are often used as pesticides. Results from an earlier study looking at inhibition of AChE activity in a cell-free system (Li et al 2021 ) confirmed the need for many organophosphates, such as chlorpyrifos, to undergo biotransformation for activity. Enhanced activity following biotransformation is not limited to pesticides, but extends to a number of other environmental compounds such as polycyclic aromatic hydrocarbons (Sen et al 2013 ).…”

“…The activity of identified metabolites can be measured in enzyme-based assays, which have high reproducibility but do not authentically recapitulate a normal physiological environment. (Li et al 2021 ). Therefore, translation of the results to human exposure scenarios is difficult.…”

Prediction Models for Agonists and Antagonists of Molecular Initiation Events for Toxicity Pathways Using an Improved Deep-Learning-Based Quantitative Structure–Activity Relationship System

“…The Toxicology in the 21st Century (Tox21) program is a US federal research collaboration between the US Environmental Protection Agency, the National Toxicology Program, the National Center for Advancing Translational Sciences, and the Food and Drug Administration that aims to develop toxicity assessment methods for commercial chemicals, pesticides, food additives/contaminants, and medical products using quantitative high-throughput screening [ 5 , 6 , 7 ]. The Tox21 10K library consists of approximately 10,000 (10K) chemicals, including nearly 100 million data points obtained by in vitro quantitative high-throughput screening, indicating the toxicological risk of chemical compounds obtained using an in silico approach [ 8 , 9 ]. Using Tox21 data, the quantitative structure–activity relationship (QSAR) competition organized by Merck—referred to as the Tox21 Data Challenge 2014—demonstrated high-performance prediction modeling using machine learning (ML) [ 10 ].…”

High-throughput screening for identifying acetylcholinesterase inhibitors: Insights on novel inhibitors and the use of liver microsomes

“…We recently implemented in vitro assays for measuring acetylcholinesterase (AChE) inhibition, a mechanism of toxicity, into Tox21 and published our study in Environmental Health Perspectives [4] . Chemicals that inhibit AChE activity block the metabolism of acetylcholine [5] , leading to overstimulation of cholinergic receptors and toxicologi- cal effects (e.g., salivation, lacrimation, urination, diaphoresis, gastrointestinal distress, emesis, and paralysis) [ 6 , 7 ].…”