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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
High-throughput screening for inhibitors of the Cks1-Skp2 interaction
Kuo-Sen Huang1, Lyubomir T Vassilev
1Discovery Technologies, Hoffmann-La Roche, Inc., Nutley, New Jersey, USA.
Abstract:
The cyclin-dependent kinase inhibitor p27(Kip1) is a critical cell cycle regulator frequently altered in human cancer. The cellular level of p27 is controlled by ubiquitin-dependent degradation mediated by the E3 ligase SCF(Skp1). Decreased p27 level in cancer cells has been associated with enhanced ubiquitin-dependent degradation and linked to poor prognosis. Therefore, restoration of p27 by inhibiting SCF(Skp2) activity has been proposed as a novel therapeutic strategy. Recently, the small regulatory protein Cks1 has been found to bind Skp2 and dramatically increases the affinity of Skp2 to p27, thus facilitating its ubiquitylation and degradation. Here, we describe a high-throughput screening assay for inhibitors of the Cks1-Skp2 interaction. The assay measures the binding of recombinant human GST-Cks1 and His6-Skp2-Skp1 using a homogeneous time-resolved fluorescence format and permits a throughput in excess of 100,000 data points per day when implemented on the Zeiss uHTS system.
Insights
Researchers developed a high-throughput assay to find drugs that block the Cks1-Skp2 interaction. This interaction promotes cancer cell growth by degrading the p27 protein, a key cell cycle regulator.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- p27(Kip1) is a crucial cell cycle regulator frequently altered in human cancers.
- The E3 ligase SCF(Skp2) controls p27 levels via ubiquitin-dependent degradation.
- Reduced p27 levels in cancer correlate with poor prognosis, making its restoration a therapeutic target.
Purpose of the Study:
- To develop a high-throughput screening assay for identifying inhibitors of the Cks1-Skp2 interaction.
- To target the Cks1-Skp2 interaction as a novel therapeutic strategy for cancer.
Main Methods:
- A homogeneous time-resolved fluorescence (HTRF) assay was developed.
- The assay measures the binding between recombinant human GST-Cks1 and His6-Skp2-Skp1.
- The assay system achieved a throughput exceeding 100,000 data points per day on a Zeiss uHTS system.
Main Results:
- A robust high-throughput screening assay for the Cks1-Skp2 interaction was successfully established.
- The assay demonstrated high throughput, enabling rapid screening of potential inhibitors.
- This assay facilitates the discovery of compounds that disrupt the Cks1-Skp2 interaction, a key step in p27 degradation.
Conclusions:
- Inhibiting the Cks1-Skp2 interaction is a promising therapeutic strategy for cancers with altered p27 levels.
- The developed HTRF assay is a valuable tool for identifying novel inhibitors of this critical interaction.
- This screening platform can accelerate the development of new cancer therapeutics targeting cell cycle regulation.
