Small Molecule Inhibitors of P27 Proteolysis in Carcinomas

描述(由申请人提供)：细胞周期蛋白依赖的激酶抑制剂p27是体内有效的细胞周期停滞所必需的。这种停滞通过泛素依赖的蛋白分解系统降解p27而得到缓解。蛋白过度降解导致的p27抑癌基因的缺失是晚期癌症的一个标志，它与存活率降低有关。P27的降解是由SCF介导的：Skp2依赖的多泛素化，而Skp2的过度表达足以导致异位p27的降解。因此，Skp2介导的蛋白分解途径的过度激活似乎是肿瘤中p27下调的主要机制，也是治疗干预的良好靶点。我们提出了一种化学生物学方法来鉴定能够干扰Skp2依赖的p27蛋白降解的合成分子。对这一途径的生化理解现在达到了可以设计特定抑制剂的筛选的地步。能够干扰Skp2依赖的p27降解的小分子预计会恢复肿瘤中p27的表达，从而抑制其不受抑制的生长。这一预测构成了这项赠款申请的主要假设。专门针对这种明显的缺陷进行治疗，以前从未尝试过。考虑到几乎所有正常分化的细胞都表达高水平的p27，这些化合物具有很高的肿瘤特异性，从而将严重的不良反应降至最低。该项目的目标是确定“SMIP”，即干扰Skp2介导的p27蛋白分解的小分子。为此，我们将使用以下方法：a.优化体外实验以筛选Skp2/p27蛋白相互作用抑制剂的合成化学文库。A.2.建立一种基于细胞的方法来筛选体内Skp2介导的p27降解的抑制物。A.3.在附加的体内和体外试验中，测试在初步筛选中确定的推定的SMIP，这涉及到它们抑制p27降解的特异性和有效性。

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 advanced carcinomas that correlates with decreased survival. P27 degradation is mediated by SCF:SKP2-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. We are proposing a chemical biology approach to identify synthetic molecules able to interfere with SKP2-dependent p27 proteolysis. The biochemical understanding of this pathway is now at a point where screens for specific inhibitors can be designed. 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 application. Specifically targeting this distinct defect for therapy was never before attempted. Considering that virtually all normal differentiated cells express high levels of p27, such compounds bear a high potential for tumor specificity, thus minimizing drastic adverse effects. The goal of this project is to identify "SMIPs", small molecules that interfere with SKP2-mediated p27 proteolysis. To this end, we will use the following approaches: A.1. Optimize an in vitro assay to screen synthetic chemical libraries for inhibitors of the SKP2/p27 protein interaction. A.2. Develop a cell-based assay to screen for inhibitors of SKP2-mediated p27 degradation in vivo. A.3. Test putative SMIPs identified in the primary screens in additional in vivo and in vitro assays, which address both their specificity and efficacy for inhibition of p27 degradation.