Open AccessIdentification of small molecule inhibitors of p27 Kip1 ubiquitination by high‐throughput screening
Author(s) -
Ooi LiChing,
Watanabe Nobumoto,
Futamura Yushi,
Sulaiman Shaida Fariza,
Darah Ibrahim,
Osada Hiroyuki
Publication year - 2013
Publication title -
cancer science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.035
H-Index - 141
eISSN - 1349-7006
pISSN - 1347-9032
DOI - 10.1111/cas.12246
Subject(s) - ubiquitin , ubiquitin ligase , in vitro , protein subunit , proteolysis , biology , kinase , high throughput screening , carcinogenesis , small molecule , biochemistry , microbiology and biotechnology , chemistry , enzyme , gene
Dysregulation of p27 Kip1 due to proteolysis that involves the ubiquitin ligase ( SCF ) complex with S ‐phase kinase‐associated protein 2 ( S kp2) 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 S kp2 with fluorescent monomeric A zami G reen ( mAG ) and CDK subunit 1 ( C ks1) ( mAGS kp2– C ks1) to bind to p27 Kip1 phosphopeptides. We identified two compounds that inhibited the interaction between mAGS kp2– C ks1 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 H e L a cells. Notably, both compounds exhibited preferential antiproliferative activity against H e L a and ts FT 210 cells compared with NIH 3 T 3 cells and delayed the G 1 phase progression in ts FT 210 cells. Our approach indicates a potential strategy for restoring p27 Kip1 levels in human cancers.