Abstract
p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 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 p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 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 p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.
Highlights
Creation and Validation of p27Kip1-luciferase Assay for High Throughput Screening
Cell viability was monitored during our high throughput screening (HTS), since inhibition of luciferase could be due to cell death and not repression of p27Kip1
Less p27Kip1 mRNA was detected in both HEK-293 and 3T3 cells when assayed by quantitative PCR normalized to the internal control, 18s (Fig. 4C, E). To understand if these cell lines were more sensitive to A2CE than HeLa cells, we measured cell viability by alamar blue, and detected a small but statistically significant reduction in viability at the highest doses in 3T3 cells but not in HEK293 cells indicating that 3T3 cells are slightly more sensitive to A2CE than HeLa cells
Summary
P27Kip ( known as Cdkn1B) is a member of the Cip/Kip family of cell cycle inhibitors which are characterized by their ability to bind and inhibit cyclin dependent kinases (CDK)/cyclin complexes, halting cell cycle progression in the G1 phase [1]. Multiple cell cycle inhibitors are upregulated in older cells [19], implying that a cocktail of cell cycle inhibitors, including p27Kip, may need to be developed to force proliferation to occur in older quiescent tissues These observations led us to pursue screening strategies for reduction of p27Kip. After the assay was validated, we screened our ‘‘bioactive’’ library of 8,904 (4,359 unique, 830 FDA approved) compounds and obtained 111 primary hits which inhibit p27Kip promoter activity These initial hits were narrowed down to 4 hits though our intensive secondary screens, and we chose to focus on our most potent compound, Alsterpaullone, 2-cyanoethyl (A2CE), to understand how p27Kip transcription was modulated by this compound. We established our p27Kip screening assay and validated it by screening our ‘‘bioactive’’ library Within this library, we discovered novel compounds that repress p27Kip transcription and mechanistically described how the most potent hit achieved this inhibition.
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