Identification of small molecule inhibitors of p27(Kip1) ubiquitination by high-throughput screening.

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.

• Authors: Li-Ching Ooi, Nobumoto Watanabe, Yushi Futamura, Shaida Fariza Sulaiman, Ibrahim Darah, Hiroyuki Osada
• Journal: Cancer science (Cancer Sci) Vol. 104 Issue 11 Pg. 1461-7 (Nov 2013) ISSN: 1349-7006 [Electronic] England
• PMID: 23910095 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2013 Japanese Cancer Association.

Chemical References

• Antineoplastic Agents
• Peptide Fragments
• Sesquiterpenes
• Small Molecule Libraries
• linichlorin A
• Cyclin-Dependent Kinase Inhibitor p27
• Cyclin-Dependent Kinases
• Gentian Violet

Topics

• Amino Acid Sequence
• Animals
• Antineoplastic Agents (chemistry, pharmacology)
• Cell Cycle Checkpoints
• Cell Proliferation (drug effects)
• Cyclin-Dependent Kinase Inhibitor p27 (chemistry, metabolism)
• Cyclin-Dependent Kinases (antagonists & inhibitors, metabolism)
• Drug Screening Assays, Antitumor
• Gentian Violet (chemistry, pharmacology)
• HeLa Cells
• High-Throughput Screening Assays
• Humans
• Mice
• Molecular Sequence Data
• NIH 3T3 Cells
• Peptide Fragments (chemistry)
• Protein Stability
• Proteolysis (drug effects)
• Sesquiterpenes (chemistry, pharmacology)
• Small Molecule Libraries
• Ubiquitination (drug effects)