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Development of a novel cell-based, In-Cell Western/ERK assay system for the high-throughput screening of agonists
Junaid Asghar1,2, Liaque Latif2, Stephen P H Alexander2
1Faculty of Pharmacy, Gomal University, Dera Ismail Khan, Pakistan.
Abstract:
Background: Extracellular signal-regulated kinases (ERKs) are important signaling mediators in mammalian cells and, as a result, one of the major areas of research focus. The detection and quantification of ERK phosphorylation as an index of activation is normally conducted using immunoblotting, which does not allow high-throughput drug screening. Plate-based immunocytochemical assays provide a cheaper and relatively high-throughput alternative method for quantifying ERK phosphorylation. Here, we present optimization steps aimed to increase assay sensitivity and reduce variance and cost using the LI-COR In-Cell Western (I-CW) system in a recombinant CHO-K1 cell line, over-expressing the human delta-opioid receptor (hDOPr) as a model. Methods: Cells cultured in 96-well microassay plates were stimulated with three standard/selective DOPr agonists (SNC80, ADL5859, and DADLE) and a novel selective DOPr agonist (PN6047) to elicit a phospho-ERK response as an index of activation. A number of experimental conditions were investigated during the assay development. Key results: Preliminary experiments revealed a clearly visible edge-effect which significantly increased assay variance across the plate and which was reduced by pre-incubation for 30 min at room temperature. ERK phosphorylation was detectable as early as 1 min after agonist addition, with a distinct peak at 3-5 min. Optimization of the cell seeding densities showed that 25,000 cells per well have the lowest basal phospho-ERK response and an optimal agonist ERK1/2 signal. Pre-incubation with apyrase (an ATPase) did not reduce the basal or agonist responses. All agonists produced concentration-dependent increases in phospho-ERK activation, and pertussis toxin was able to attenuate these ERK responses. Naltrindole, which is a selective DOPr antagonist, was able to antagonize the DOPr-mediated ERK activation of the ligands. Conclusion: We have developed an optimization protocol and highlighted a number of considerations when performing this high-throughput fluorescence immunocytochemical (ICC) assay measuring ERK phosphorylation in the human DOPr. The optimized protocol was found to be a more conducive option for the screening of delta agonists. This provides a basis for additional assay development to investigate opioid pharmacology. This protocol should be widely applicable for measuring ERK phosphorylation in any cell line and investigating other protein targets in GPCR drug discovery.
Insights
This study optimizes a high-throughput assay for measuring extracellular signal-regulated kinases (ERK) phosphorylation, a key indicator of cell signaling. The developed protocol enhances sensitivity and reduces costs for drug screening, particularly for delta-opioid receptor agonists.
Area of Science:
- Cellular signaling pathways
- Pharmacology and drug discovery
- Assay development and optimization
Background:
- Extracellular signal-regulated kinases (ERKs) are crucial signaling mediators in mammalian cells, making their activation a significant research focus.
- Traditional immunoblotting for detecting ERK phosphorylation is not suitable for high-throughput drug screening.
- Plate-based immunocytochemical assays offer a cost-effective, high-throughput alternative for quantifying ERK phosphorylation.
Purpose of the Study:
- To optimize a fluorescence-based immunocytochemical assay for quantifying ERK phosphorylation.
- To enhance assay sensitivity, reduce variance, and lower costs using the LI-COR In-Cell Western (I-CW) system.
- To establish a model system using a recombinant CHO-K1 cell line over-expressing the human delta-opioid receptor (hDOPr) for drug screening.
Main Methods:
- Utilized a 96-well microassay plate format with a recombinant CHO-K1 cell line expressing the hDOPr.
- Stimulated cells with selective delta-opioid receptor (DOPr) agonists to induce phospho-ERK response.
- Investigated and optimized various experimental conditions including cell seeding density and pre-incubation steps.
Main Results:
- Minimized plate edge-effects and reduced assay variance through a 30-minute room temperature pre-incubation.
- Detected ERK phosphorylation as early as 1 minute post-agonist stimulation, with a peak at 3-5 minutes.
- Identified optimal cell seeding density (25,000 cells/well) for low basal and optimal agonist-induced ERK phosphorylation.
Conclusions:
- Developed and optimized a high-throughput fluorescence immunocytochemical assay for measuring ERK phosphorylation in hDOPr-expressing cells.
- The optimized protocol is suitable for screening delta-opioid receptor agonists and investigating opioid pharmacology.
- This protocol is broadly applicable for measuring ERK phosphorylation in various cell lines and for other G protein-coupled receptor (GPCR) targets in drug discovery.
