High-Throughput Screening Reveals Alsterpaullone, 2-Cyanoethyl as a Potent p27Kip1 Transcriptional Inhibitor

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.

A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore

“…38 Using a stable cell line expressing this reporter, luminescent signals are generated in a dose-dependent fashion in the presence of splicing modulators (such as the sudemycins, herboxidiene and pladienolide B). 38 We have used this assay to screen a small molecule library (composed of 830 known drugs and 4,359 bioactive compounds) 39 and identified several active ‘hit’ compounds. These hits were then subjected to dose-response assays in the MDM2-Luc exon-skipping assay and those actives were then confirmed in an orthogonal PCR-based MDM2 alternative splicing assay in Rh-18 cells (see Supporting Information).…”

“…We have used our triple-exon skipping assay to screen a small molecule library (composed of 830 known drugs and 4,359 bioactive compounds 39 , see Supporting Information) and have identified initial hits. Using directed chemistry, which included a range of optically pure analogs, we also identified new more potent and selective CLK inhibitors and associated structure-activity relationships.…”

Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

“…Alternatively, in both sensory and non-sensory epithelial cells of the inner ear, p27 maintains cell cycle exit and terminal differentiation 28 and its inhibition resulted in their cell cycle reentry and regeneration for hearing restoration 29 30 . While small molecules that inhibit the transcription of p27 have been reported 31 , here we developed approaches to identify small molecules that bind directly to p27 and have potential to alter its function in the two cellular settings discussed above.…”

Cochlear hair cell regeneration after noise-induced hearing loss: Does regeneration follow development?

“…Screening for p27 kip1 inhibitors, for example, was undertaken via two different approaches. The first approach was performed at the transcriptional level by directly screening for p27 kip1 transcriptional inhibitors in Hela cells (Walters et al, 2014). By using a p27 kip1 promoter–driven luciferase reporter assay, Walters et al screened a library of 4,350 bioactive compounds, including 835 FDA-approved compounds, and discovered four inhibitors (Alsterpaullone, 2-Cyanoethyl or A2CE, Cerivastatin, Resveratrol, and Triacetylresveratrol) that can inhibit p27 kip1 transcription in various cell lines and cochlear explants.…”