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Identification of small molecule inhibitors of p27(Kip1) ubiquitination by high-throughput screening
Li-Ching Ooi1, Nobumoto Watanabe, Yushi Futamura
1Antibiotics Laboratory, RIKEN, Hirosawa, Wako-shi, Saitama, Japan; School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia.
Abstract:
Dysregulation of p27(Kip1) due to proteolysis that involves the ubiquitin ligase (SCF) complex with S-phase kinase-associated protein 2 (Skp2) as the substrate-recognition component (SCF(Skp2)) frequently results in tumorigenesis. In this report, we developed a high-throughput screening system to identify small-molecule inhibitors of p27(Kip1) degradation. This system was established by tagging Skp2 with fluorescent monomeric Azami Green (mAG) and CDK subunit 1 (Cks1) (mAGSkp2-Cks1) to bind to p27(Kip1) phosphopeptides. We identified two compounds that inhibited the interaction between mAGSkp2-Cks1 and p27(Kip1): linichlorin A and gentian violet. Further studies have shown that the compounds inhibit the ubiquitination of p27(Kip1) in vitro as well as p27(Kip1) degradation in HeLa cells. Notably, both compounds exhibited preferential antiproliferative activity against HeLa and tsFT210 cells compared with NIH3T3 cells and delayed the G1 phase progression in tsFT210 cells. Our approach indicates a potential strategy for restoring p27(Kip1) levels in human cancers.
Insights
Researchers identified small molecules, linichlorin A and gentian violet, that inhibit p27(Kip1) degradation by targeting the SCF(Skp2) complex. These compounds show potential for cancer therapy by restoring p27(Kip1) levels and inhibiting cell proliferation.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Dysregulation of p27(Kip1) protein degradation, mediated by the SCF(Skp2) ubiquitin ligase complex, is a key factor in tumorigenesis.
- Targeting p27(Kip1) degradation presents a potential therapeutic strategy for various cancers.
Purpose of the Study:
- To develop a high-throughput screening system for identifying small-molecule inhibitors of p27(Kip1) degradation.
- To discover novel compounds that can restore p27(Kip1) levels in cancer cells.
Main Methods:
- A screening system was established using fluorescently tagged Skp2-Cks1 (mAGSkp2-Cks1) to monitor the interaction with p27(Kip1) phosphopeptides.
- In vitro ubiquitination assays and cell-based degradation studies were performed.
- Antiproliferative activity and cell cycle progression were assessed in various cell lines.
Main Results:
- Two compounds, linichlorin A and gentian violet, were identified as inhibitors of the mAGSkp2-Cks1 and p27(Kip1) interaction.
- These compounds effectively inhibited p27(Kip1) ubiquitination in vitro and its degradation in HeLa cells.
- Linichlorin A and gentian violet demonstrated preferential antiproliferative effects on cancer cells (HeLa, tsFT210) and delayed G1 phase progression.
Conclusions:
- The developed high-throughput screening system successfully identified inhibitors of p27(Kip1) degradation.
- Linichlorin A and gentian violet represent promising lead compounds for developing novel cancer therapeutics.
- Restoring p27(Kip1) levels through inhibition of its degradation is a viable strategy for cancer treatment.
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