Cell-active dual specificity phosphatase inhibitors identified by high-content screening
Related Experiment Video
Updated: May 12, 2026
Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Andreas Vogt1, Kathleen A Cooley, Marni Brisson
1Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Abstract:
Phosphorylation of extracellular signal-regulated kinase (Erk) is tightly controlled by dual specificity phosphatases (DSPases), but few inhibitors of Erk dephosphorylation have been identified. Using a high-content, fluorescence-based cellular assay and the National Cancer Institute's 1990 agent Diversity Set, we identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences in intact cells. Three of the ten positive compounds inhibited the mitogen-activated protein kinase phosphatase-3 (MKP-3/PYST-1) in vitro without affecting VHR or PTP1B phosphatases. The most potent inhibitor of MKP-3 had an IC50 of < 10 microM and inhibited MKP-3 in a novel, fluorescence-based multiparameter chemical complementation assay. These results suggest that the phospho-Erk nuclear accumulation assay may be a useful tool to discover DSPase inhibitors with biological activity.
Insights
Researchers identified new compounds that inhibit mitogen-activated protein kinase phosphatase-3 (MKP-3), a key enzyme controlling extracellular signal-regulated kinase (Erk) phosphorylation. This discovery offers a novel approach for developing dual specificity phosphatase (DSPase) inhibitors with potential biological activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Extracellular signal-regulated kinase (Erk) phosphorylation is regulated by dual specificity phosphatases (DSPases).
- Few inhibitors targeting Erk dephosphorylation by DSPases have been discovered.
- Identifying novel DSPase inhibitors is crucial for understanding cellular signaling pathways and developing therapeutics.
Purpose of the Study:
- To discover novel inhibitors of DSPases, specifically targeting Erk dephosphorylation.
- To identify compounds that modulate phospho-Erk levels in intact cells.
- To validate the efficacy of identified compounds against specific DSPases like mitogen-activated protein kinase phosphatase-3 (MKP-3).
Main Methods:
- Utilized a high-content, fluorescence-based cellular assay to screen the National Cancer Institute's 1990 agent Diversity Set.
- Assessed compounds for their ability to alter phospho-Erk cytonuclear differences in intact cells.
- Performed in vitro inhibition assays to determine the specificity of active compounds against MKP-3, VHR, and PTP1B phosphatases.
Main Results:
- Identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences.
- Three of these compounds selectively inhibited MKP-3 (also known as PYST-1) in vitro.
- The most potent MKP-3 inhibitor exhibited an IC50 of < 10 microM and was characterized using a novel fluorescence-based assay.
Conclusions:
- The phospho-Erk nuclear accumulation assay is a valuable tool for discovering DSPase inhibitors.
- The identified compounds, particularly the MKP-3 inhibitors, show potential for further development.
- This study provides a foundation for developing new therapeutic strategies targeting DSPase-mediated signaling pathways.
