High-Throughput Screening Reveals Alsterpaullone, 2-Cyanoethyl as a Potent p27(Kip1) Transcriptional Inhibitor

p27(Kip1) is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27(Kip1) is a known tumor suppressor, as the germline loss of p27(Kip1) results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27(Kip1) results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27(Kip1) inhibitors could be beneficial for tissue regeneration. Because p27(Kip1) is an intrinsically disordered protein, identifying direct inhibitors of the p27(Kip1) protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27(Kip1) transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27(Kip1) promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a “bioactive” library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27(Kip1). These hits were further refined using a battery of secondary screens. Here we report four novel p27(Kip1) transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC(50) = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27(Kip1) transcription by preventing FoxO3a from binding to the p27(Kip1) promoter. This screen represents one of the first attempts to identify inhibitors of p27(Kip1) and may prove useful for future tissue regeneration studies.