Optimized High-Throughput Screen for Small Molecule Inhibitors of P27 Degradation

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DESCRIPTION (provided by applicant): The cyclin-dependent kinase inhibitor p27 is required for an effective cell cycle arrest in vivo. This arrest is relieved by degradation of p27 via the ubiquitin-dependent proteolysis system. Depletion of the p27 tumor suppressor resulting form hyperproteolysis is a hallmark of many advanced carcinomas that correlates with decreased survival. P27 degradation is mediated by SCFSKP2-dependent polyubiquitylation and SKP2 overexpression is sufficient to cause ectopic p27 degradation. Hyperactivation of the SKP2-mediated proteolysis pathway therefore appears to be the main mechanism of p27 downregulation in carcinomas and a good target for therapeutic intervention. Small molecules able to interfere with SKP2-dependent p27 degradation are predicted to restore p27 expression in carcinomas thus inhibiting their unrestrained growth. This prediction constitutes the principal hypothesis of this grant proposal. In preliminary work, 7368 compounds were screened in a 384-well plate format for their ability to upregulate p27 in prostate cancer cells overexpressing SKP2. 54 compounds (named SMIPs) were identified and verified, which were effective with a z score of >3 - 5. While this work has provided proof-of-principle for the feasibility of identifying SMIPs, the screening assay relied on detection of p27 by immunofluorescence and is therefore not suitable for high-throughput screening. In this application, an optimized, cell-based "mix-and-measure" assay for the identification of SMIPs is proposed. The assay will rely on stable expression of a YFPp27 fusion protein in HeLa cells. The addition of compounds that upregulate YFP-p27 will result in an increased fluorescence signal that will be measured by cellular imaging. The assay will also allow real-time measurement of SMIP activity. If validated, the assay will be used to perform a SMIP screen at the San Diego Center for Chemical Genomics, one of the Molecular Library Screening Centers in the NIH Roadmap Network.