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

描述（由申请人提供）：细胞周期蛋白依赖性激酶抑制剂p27是体内有效细胞周期阻滞所必需的。这种停滞通过泛素依赖性蛋白水解系统降解p27而缓解。p27肿瘤抑制因子的过度蛋白水解导致的耗竭是许多晚期癌症的标志，其与存活率降低相关。P27降解由SCFSKP 2依赖性多泛素化介导，SKP 2过表达足以引起异位p27降解。因此，SKP 2介导的蛋白水解途径的过度活化似乎是癌中p27下调的主要机制，并且是治疗干预的良好靶点。预测能够干扰SKP 2依赖性p27降解的小分子， 恢复癌中p27的表达，从而抑制其不受限制的生长。这一预测构成了本拨款申请的主要假设。 在初步工作中，在384孔板格式中筛选了7368种化合物，以确定它们在过表达SKP 2的前列腺癌细胞中上调p27的能力。鉴定并验证了54种化合物（命名为SMIP），它们是有效的，z得分>3 - 5。虽然这项工作提供了可行性的原则证明， 鉴定SMIP时，筛选测定依赖于通过免疫荧光检测p27，因此不适合高通量筛选。 在本申请中，提出了一种用于鉴定SMIP的优化的、基于细胞的“混合和测量”测定。该测定将依赖于YFPp 27融合蛋白在HeLa细胞中的稳定表达。添加上调YFP-p27的化合物将导致荧光信号增加，这将通过细胞成像来测量。 该测定还将允许实时测量SMIP活性。如果经过验证，该检测试剂盒将用于在圣地亚哥化学基因组学中心（NIH路线图网络中的分子文库筛选中心之一）进行SMIP筛选。

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.