Small Molecule Inhibitors of P27 Proteolysis in Carcinomas

描述（由申请人提供）：周期蛋白依赖性激酶抑制剂p27是体内有效的细胞周期阻滞所必需的。这种阻滞通过泛素依赖的蛋白水解系统降解p27来缓解。高蛋白水解导致的p27肿瘤抑制因子的缺失是晚期癌症的一个标志，它与生存率降低有关。P27降解是由SCF介导的：SKP2依赖性多泛素化和SKP2过表达足以引起异位P27降解。因此，skp2介导的蛋白水解途径的过度激活似乎是肿瘤中p27下调的主要机制，也是治疗干预的一个良好靶点。我们提出了一种化学生物学方法来鉴定能够干扰skp2依赖性p27蛋白水解的合成分子。对这一途径的生物化学理解现在处于可以设计特定抑制剂的筛选的阶段。据预测，能够干扰skp2依赖性p27降解的小分子能够恢复p27在肿瘤中的表达，从而抑制其无限制的生长。这一预测构成了本拨款申请的主要假设。以前从未尝试过专门针对这种独特的缺陷进行治疗。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潜力，从而最大限度地减少剧烈的不良反应。该项目的目标是鉴定干扰skp2介导的p27蛋白水解的小分子“smip”。为此，我们将采用以下方法：优化体外实验筛选SKP2/p27蛋白相互作用抑制剂的合成化学文库。由信用证。开发一种基于细胞的实验来筛选skp2介导的p27降解抑制剂。出具。在额外的体内和体外试验中测试在初级筛选中发现的假定的smip，以确定其抑制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.