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.

Figure 1.

Screening and compound prioritization workflow. Cell-based and enzyme-based assays were developed for screening AChE inhibitors, and enzyme-based assay with metabolic activation was also included to screen inhibitors. After primary concentration–response screenings, in which each compound was tested at 15 concentrations, 187 compounds were identified based on potency and efficacy. A total of 187 compounds were tested in the follow-up studies. Based on efficacy and ${\text{IC}}_{50}$, 111 compounds were selected for further studies.

Figure 2.

Structure clusters of the AChE inhibitors identified from enzyme-based assay (left) and cell-based assay (right). The Tox21 10K compound collection was clustered based on structural similarity. In the heat maps, each hexagon represents a cluster of structurally similar compounds. The color gradient is indicative of the enrichment of AChE inhibitors in that specific cluster [negative logarithmic scale of the p-value, -log (p-value)]. Clusters enriched with active inhibitors are closer to a maroon color, whereas clusters deficient of active inhibitors are colored in shades of blue or green. A light gray color indicates that the fraction of active inhibitors in that cluster is close to the library average. Empty clusters with no compounds in them are in a darker shade of gray. Each cluster was evaluated for its enrichment of active AChE inhibitors by comparing the fraction of actives in the cluster with the fraction of actives not in the cluster. The significance of enrichment was determined by the Fisher’s exact test ($p<0.01$).

Figure 3.

Concentration response curves of representative parent compounds in AChE assay with or without human liver microsomes (MS). (A) pyrazophos; (B) phosalone; (C) triazophos; (D) parathion; (E) bromophos-ethyl; (F) carbophenothion; (G) isocarbophos; and (H) Phoxim. Each value represents the $\text{mean}\pm \text{standard deviation\hspace{0.17em}}\left(\text{SD}\right)$ of three independent experiments.

Figure 4.

Molecular docking result of aldicarb (carbon atoms in magenta) and aldicarb sulfoxide (carbon atoms in cyan) (A) and inactive hit 2, 3-dihydro-2, 2-dimethyl-7-benzofuranol (B) in the active site of AChE (gray, PDB: 4EY7). Key residues of AChE that interact the sulfoxide oxygen atom were shown in stick. The residues that potentially interact with aldicarb sulfoxide but not aldicarb were highlighted in green. Note: PDB, Protein Data Bank.