Identification of small molecule inhibitors of p27^Kip1 ubiquitination by high-throughput screening

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.