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An ultra-high throughput cell-based screen for wee1 degradation inhibitors
Franck Madoux1, Scott Simanski, Peter Chase
1Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, Jupiter, FL 33458, USA.
Journal of Biomolecular Screening
|July 28, 2010
Summary
Researchers developed a new assay to find small molecules that stabilize Wee1 protein, crucial for cell cycle control. This ultra-high-throughput screening identified compounds that prevent Wee1 degradation, impacting cell cycle progression.
Area of Science:
- Cell Biology
- Biochemistry
- Drug Discovery
Background:
- Wee1 tyrosine kinase regulates entry into mitosis by inhibiting CDK1.
- Wee1 activity is primarily controlled by its phosphorylation and subsequent degradation via the ubiquitin-proteasome pathway.
- Identifying small molecules that prevent Wee1 degradation is key for understanding cell cycle control.
Purpose of the Study:
- To develop a homogeneous, ultra-high-throughput screening (uHTS) compatible assay for identifying Wee1 stabilizers.
- To discover novel small molecules that prevent Wee1 degradation.
- To assess the utility of this uHTS approach for discovering inhibitors of the ubiquitin-proteasome pathway.
Main Methods:
- A homogeneous cell-based assay using HeLa cells with a Wee1-luciferase fusion protein was developed and miniaturized to a 1536-well format.
- Bulk transfection and cryopreservation of cells enabled uHTS compatibility.
- Counterscreens measuring N-cyclin B-luciferase stabilization and cell viability were employed for compound triage.
Main Results:
- The assay demonstrated robust performance with a Z' factor of 0.65 +/- 0.05.
- Screening of approximately 218,000 compounds identified 4 unrelated cell-permeable small molecules that selectively stabilized Wee1-luciferase with micromolar potency.
- One identified compound, SID4243143 (ML 118), inhibited cell cycle progression.
Conclusions:
- The developed uHTS assay is suitable for identifying selective chemical probes that prevent Wee1 degradation.
- The findings highlight the importance of Wee1 degradation in cell cycle regulation.
- This approach is generally applicable for discovering inhibitors of the ubiquitin-proteasome pathway.
