Cell-active dual specificity phosphatase inhibitors identified by high-content screening

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.