title: 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.affiliation: Antibiotics Laboratory, RIKEN, Hirosawa, Wako-shi, Saitama, Japan; School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia.
authors.forename: Li-Ching
authors.initials: LC
authors.lastname: Ooi
authors.forename: Nobumoto
authors.initials: N
authors.lastname: Watanabe
authors.forename: Yushi
authors.initials: Y
authors.lastname: Futamura
authors.forename: Shaida Fariza
authors.initials: SF
authors.lastname: Sulaiman
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Darah
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints; D049109:Cell Proliferation / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000737:chemistry / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D005840:Gentian Violet / Q000737:chemistry / Q000494:pharmacology*; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010446:Peptide Fragments / Q000737:chemistry; D055550:Protein Stability; D059748:Proteolysis / Q000187:drug effects; D012717:Sesquiterpenes / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries; D054875:Ubiquitination / Q000187:drug effects*
chemical_list: D000970:Antineoplastic Agents; D010446:Peptide Fragments; D012717:Sesquiterpenes; D054852:Small Molecule Libraries; C587959:linichlorin A; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases; D005840:Gentian Violet
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