Acetylcholinesterase Inhibition Assays for High-Throughput Screening

Abstract

Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh), a vital neurotransmitter that regulates muscle movement and brain function, including memory, attention, and learning. Inhibition of AChE activity can cause a variety of adverse health effects and toxicity. Identifying AChE inhibitors quickly and efficiently warrants developing AChE inhibition assays in a quantitative, high-throughput screening (qHTS) platform. In this chapter, protocols for multiple homogenous AChE inhibition assays used in a qHTS system are provided. These AChE inhibition assays include a (1) human neuroblastoma (SH-SY5Y) cell-based assay with fluorescence or colorimetric detection; (2) human recombinant AChE with fluorescence or colorimetric detection; and (3) combination of human recombinant AChE and liver microsomes with colorimetric detection, which enables detection of test compounds requiring metabolic activation to become AChE inhibitors. Together, these AChE assays can help identify, prioritize, and predict chemical hazards in large compound libraries using qHTS systems.

1. Introduction

Acetylcholine (ACh) is a neurotransmitter associated with cognitive, autonomic, and neuromuscular functions. Acetylcholinesterase (AChE), an enzyme found at neuromuscular junctions and cholinergic synapses, is essential for maintaining normal function of the central and peripheral nervous systems by hydrolyzing ACh [1]. However, chemicals that inhibit AChE activity interfere with this process, leading to an accumulation of ACh and numerous adverse effects, such as smooth muscle contractions, skeletal muscle twitching, glandular secretions, and flaccid paralysis. Some examples of AChE inhibitors that cause adverse effects include pesticides (organophosphates and carbamates), chemical warfare agents (nerve agents), drugs, and phytochemicals.[2–4]

Given the toxicological effects due to AChE inhibition, in vitro AChE inhibition assays are important for identifying potential chemical hazards and prioritizing these chemicals for further toxicological testing. Additionally, AChE inhibition assays may facilitate development of predictive toxicology models such as quantitative structure-activity relationships (QSAR). Although AChE inhibition assays have been used extensively in manual, lower-throughput formats (e.g., 96-well plates), AChE inhibition assays used in quantitative high-throughput screening (qHTS) platforms [5] (e.g., 1536-well plates) will improve the ability to identify chemical hazards and develop predictive models when dealing with large chemical libraries. Tox21 (Toxicology in the 21st Century), a research consortium among multiple federal agencies uses a battery of high-throughput screening in vitro assays for determining toxicity.[6,7] The ultimate goals of Tox21 are identifying, prioritizing, and predicting chemical safety, which require high-throughput in vitro toxicology assays to screen a library containing 10,000 chemicals with human health relevance. The in vitro assays cover a variety of toxicological mechanisms and pathways, including AChE inhibition. [8–10]

In this chapter, protocols are provided for multiple in vitro AChE inhibition assays used in automated qHTS systems. Since a single AChE inhibition assay may be prone to interference, false positives or negatives, multiple complementary assay formats and detection schemes are provided to address these challenges. First, a protocol for a cell-based AChE inhibition assay is described. This method uses a human neuroblastoma cell line (SH-SY5Y) with a choice of Amplite Red fluorimetric (Fig. 1) or colorimetric (Fig. 2) assay. The cell-based qHTS assay is conducted in a homogenous format without reagent removal and washing steps, which improves assay performance [5,11–13]. The second protocol describes a cell-free, human recombinant AChE inhibition assay that uses a choice of colorimetric or Amplite Green fluorimetric (Fig. 3) assay. Finally, a third protocol is outlined, which uses a combination of human recombinant AChE and liver microsomes in a colorimetric assay. Many organophosphate pesticides themselves do not inhibit AChE activity but once transformed into metabolites, they become potent AChE inhibitors. The addition of liver microsomes in the AChE inhibition assay can detect many of these compounds that undergo bioactivation. An assay that incorporates metabolic activity may enhance the ability to detect AChE inhibitors such as organophosphates. Together these three AChE inhibition assays serve as high-throughput screening methods for identifying chemicals that may affect an important toxicological target.

Fig. 1.

Amplite Red fluorimetric AChE assay principle. AChE hydrolyzes acetylcholine to choline. Next, choline is oxidized to betaine aldehyde and hydrogen peroxide (H₂O₂) by choline oxidase. Finally, horseradish peroxidase converts H₂O₂ and Amplite Red to resorufin. The concentration of resorufin is quantified by its fluorescence intensity (excitation, 540 nm; emission, 590 nm), which reflects AChE activity.

Fig. 2.

Colorimetric AChE assay principle. AChE hydrolyses acetylthiocholine to thiocholine, which reacts with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) to produce 5-thio-2-nitrobenzoate (TNB). The concentration of TNB is quantified by its absorbance (405 nm), which reflects AChE activity.

Fig. 3.

Amplite Green fluorometric AChE assay principle. AChE hydrolyses acetylthiocholine to thiocholine, which reacts with a proprietary probe, Thiolite Green, to produce a fluorescent adduct. The concentration of the adduct is quantified by its fluorescence intensity (excitation, 490 nm; emission, 520 nm), which reflects AChE activity.

2. Materials

2.1. Equipment

1. Purifier Logic + Class II, Type A2 Biosafety Cabinet

2. Steri-Cult CO2 Incubator

3. BioRAPTR Flying Reagent Dispenser (FRD) workstation (Beckman Coulter Inc., Brea, CA)

4. Multidrop Combi 8-Channel Reagent Dispenser (Thermo Scientific, Waltham, MA)

5. Pintool workstation (Wako Automation, San Diego, CA)

6. EnVision Multilabel Plate Reader (Perkin Elmer, Shelton, CT)

7. ViewLux uHTS Microplate Imager (Perkin Elmer)

8. PHERAstar plate reader (BMG LABTECH, Cary, NC)

2.2. Reagents and Supplies

1. Human neuroblastoma cell line (SH-SY5Y) from American Type Culture Collection (ATCC, Manassas, VA)

2. Cell culture medium: A mixture of 45% F-12 and 45% Eagle’s minimum essential media from ATCC, supplemented with heat-inactivated 10% FBS (Hyclone Laboratories, Logan, UT) and 50 U/mL penicillin and 50 μg/mL streptomycin (Thermo Fisher Scientific, Grand Island, NY)

3. Cell assay medium: A customized formulation of DMEM/F-12 medium without choline and phenol red from Thermo Fisher Scientific, Inc. supplemented with 1% premium FBS (Thermo Fisher Scientific)

4. Positive control compounds: Chlorpyrifos-oxon from Chem Service (West Chester, PA), chlorpyrifos and 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) from Sigma-Aldrich (St. Louis, MO) (see Note 1)

5. Assay kits from AAT Bioquest (Sunnyvale, CA): Amplite Red fluorimetric acetylcholinesterase assay kit, Amplite Green fluorimetric acetylcholinesterase assay kit, Amplite colorimetric acetylcholinesterase assay kit, and Amplite Red fluorimetric peroxidase assay kit

6. 1536-well black wall/clear-bottom plates, cell culture treated, and sterile (Greiner Bio-One, Monroe, NC)

7. Recombinant human AChE from Sigma-Aldrich Co.

8. InVitroCYP 150-D human liver microsomes (HLM), prepared from 150 donor human liver tissue fraction pools with mixed gender, from BioIVT (Baltimore, MD)

9. Acroclor 1254-induced male Sprague-Dawley rat liver microsomes (RLM) from Molecular Toxicology (Boone, NC)

10. NADPH from Sigma-Aldrich

11. Cell culture flasks (225 cm²)

12. Test compound stock solutions in DMSO

3. Methods

3.1. Cell-Based Assays

1. Detach SH-SY5Y cells from flasks using 0.25% trypsin, followed by centrifugation for 4 min at 900 rpm.

2. Suspend SH-SY5Y cells (500,000 per mL) in customized cell assay media (without choline and phenol red).

3. Apply the cell suspension through a cell strainer prior to dispensing the cells at 2000 cells/4 μL/well in 1536-well plates using a Multidrop Combi 8-channel reagent dispenser.

4. Incubate SH-S5Y cells overnight (18 h) at 37°C and 5% CO₂.

5. Prepare detection solutions for colorimetric (see Note 2) or Amplite Red fluorimetric assays (see Note 3).

6. Transfer 23 nL of test compounds, negative controls (DMSO), and positive control compounds (chlorpyrifos oxon and BW284c51) to the assay plate via a Wako Pintool station. In addition to chlorpyrifos oxon and BW284c51, other known AChE inhibitors may also be used as positive controls (see Note 1). For an example of a 1536-well plate layout, see Fig. 4.

7. Incubate the assay plates for 1 h at 37°C, 5% CO₂.

8. Add 4 μL of Amplite Red detection solution or colorimetric detection solution to each well using BioRAPTR FRD and incubate assay plates for 40–90 min at room temperature.

9. For Amplite Red assay, measure fluorescence intensity (excitation, 544 nm; emission, 590 nm) using a ViewLux plate reader. For colorimetric assay, measure absorbance (405 nm) using a by an EnVison plate reader. To identify potential false positives for the Amplite Red fluorimetric assay, perform a counter-screen for peroxidase inhibitors (see Note 4).

10. Analyze fluorescence or absorbance measurements (see section 3.4, Data Analysis).

Fig. 4.

Plate layout with two positive control compounds (chlorpyrifos oxon and BW284c51) and negative controls (DMSO). For each of the two selected compounds, 1:2 dilution is performed with two replicates.

3.2. Human Recombinant AChE-Based Assays

1. Prepare detection solutions for colorimetric (see Note 2) or Amplite Green fluorimetric assays (see Note 5).

2. Dispense 4 μL human recombinant AChE (50 mU/mL for colorimetric assay or 20 mU/mL for Amplite Green assay) into 1536-well plates using a Multidrop Combi 8-channel dispenser.

3. Transfer 23 nL of test compounds, negative controls (DMSO), or positive controls immediately into the assay plates using a Wako Pintool station. The plate layout is similar to that of cell-based assays.

4. Incubate the assay plates for 30 min at room temperature.

5. Add 4 μL of colorimetric detection solution or Amplite Green detection solution using a BioRAPTR FRD.

6. Incubate the assay plates at room temperature for 10 to 30 min.

7. Measure absorbance (405 nm) for colorimetric assay or fluorescence intensity (excitation, 490 nm; emission, 520 nm) for Amplite Green assay using an EnVision plate reader.

8. Analyze absorbance or fluorescence measurements (see section 3.4, Data Analysis).

3.3. Human Recombinant AChE-Based Assays with Liver Microsomes

1. Prepare detection solutions for colorimetric (see Note 2) assay and NADPH solution.

2. Dispense 3 μL mixture containing human recombinant AChE (50 mU/mL) and liver microsomes (0.25 mg/mL) into 1536-well plates using a BioRAPTR FRD. Additionally, dispense a mixture containing AChE and heat-inactivated liver microsomes as a control for protein binding.

3. Transfer 23 nL of test compounds, negative controls (DMSO), or positive control compound (chlorpyrifos) into the assay plates using a Wako Pintool station. The plate layout is the same as those used for cell-based assays except the positive control compound.

4. Dispense 1 mg/mL NADPH (1 μL) into the assay plate using a BioRAPTR FRD for a final concentration of 0.25 mg/mL.

5. Incubate the assay plates for 30 min at room temperature.

6. Add 4 μL colorimetric detection solution using a BioRAPTR FRD.

7. Incubate assay plates at room temperature for 10 to 30 min.

8. Measure absorbance (405 nm) using an EnVision plate reader.

9. Analyze absorbance measurements (see section 3.4, Data Analysis).

10. Compare IC₅₀ values from assay incubations containing AChE and (1) liver microsomes, (2) heat-inactivated liver microsomes, and (3) no microsomes. The human recombinant AChE-based assay (see section 3.2) serves as a control for no microsomes.

3.4. Data Analysis

1. Construct concentration-response curves by plotting signal (fluorescence or absorbance) on y-axis and log of test compound concentration on the x-axis. Fit the concentration-response data to a nonlinear four parameter logistic regression using software such as GraphPad Prism. Examples of concentration-response curves are presented in Figs. 5 and 6.

2. Evaluate assay performance by calculating coefficient of variation (CV), signal-to-background ratio (S/B), assay quality (Z’-factor), and fold differences of IC₅₀ values for positive control compounds (see Note 6). For representative AChE assay performance data, see Table 1.

Fig. 5.

Concentration-response curves demonstrating AChE inhibition by (a) chlorpyrifos oxon and (b) BW284c51 in cell-based Amplite red fluorimetric assay. Each data point represents mean ± standard deviation from three experiments.

Fig. 6.

Concentration-response curves demonstrating AChE inhibition by (a) chlorpyrifos oxon and (b) BW284c51 using cell-based colorimetric assay. Each data point represents mean ± standard deviation from three experiments.

Table 1.

Representative assay performance parameters for cell-based AChE inhibition assays.

Readout	Amplite Red	Colorimetric
CV (%)	6.98 ± 0.03	1.32 ± 0.24
S/B	4.29 ± 0.14	1.96 ± 0.04
Z’ factor	0.69 ± 0.01	0.75 ± 0.02

Data were calculated from 32 wells per 1536-well plate across three independent experiments.

3.5. Confirmation of K_m and V_max for Colorimetric Recombinant AChE-Based Assay (Optional)

1. To ensure that AChE and acetylthiocholine substrate concentrations are appropriate for measuring AChE inhibition, this optional experiment can be carried out to confirm the substrate half-saturation concentration (K_m), maximum velocity (V_max), and linearity of absorbance over time.

2. Incubate a series of acetylthiocholine substrate concentrations (0.0–2.0 mM) with AChE and DTNB to measure the absorbance at multiple time points for each incubation.

3. Plot absorbance on the y-axis and time on the x-axis for each acetylthiocholine concentration tested. Fit each dataset by linear regression. The slope of each line is the enzyme velocity.

4. Plot the enzyme velocity on the y-axis and concentration of acetylthiocholine substrate on the x-axis. Fit the data to Michaelis-Menten equation by nonlinear regression using software such as GraphPad Prism (Fig. 7a). The K_m concentration for acetylthiocholine derived from the Michaelis-Menten equation should be used when performing AChE inhibition assays.

5. Confirm there is a linear relationship between acetylthiocholine substrate concentration and velocity up to the K_m concentration, which indicates initial velocity conditions (<10% of substrate depletion).

6. Incubate a series of AChE concentrations with acetylthiocholine substrate (K_m concentration) and DTNB and measure the absorbance.

7. Plot absorbance on the y-axis and AChE concentration on the x-axis. The AChE concentration used for inhibition assays should be within the linear range (Fig. 7b).

Fig. 7.

Kinetics of the colorimetric AChE inhibition assay. K_m and V_max were determined at various (a) acetylthiocholine substrate concentrations and (b) AChE enzyme concentrations. Each data point represents mean ± standard deviation from three experiments. Figure reproduced from reference [9].