Chemical Genetic and Biochemical Studies of Mitotic Proteolysis

描述(申请人提供)：微管抑制剂，如紫杉烷，是最广泛使用的癌症治疗方法之一。通过干扰有丝分裂纺锤体，紫杉烷激活纺锤体组装检查点(SAC)，从而导致有丝分裂停滞和细胞死亡。然而，癌症对紫杉烷的敏感性差异很大，可能是由于检查点的强度和有丝分裂抑制效率的不同。由于SAC唯一已知的功能是抑制后期促进复合体/环体(APC)的泛素连接酶活性，直接APC抑制剂可能更有效地诱导有丝分裂停止，而没有与微管扰动相关的毒性。利用非洲爪哇细胞周期提取物的表型筛选，结合生化研究来鉴定活性分子的靶标，我们已经鉴定出两个抑制APC依赖的蛋白分解的小分子。这项赠款申请包括四个主要目标。首先，在原子水平上，了解温顺和环状化合物如何与它们的蛋白质靶标相互作用。第二，了解这些化合物扰乱APC活性的详细机制。第三，了解这些化合物如何扰乱SAC对APC的调节。第四，利用我们对这些分子的细胞效应的了解来确定新的APC抑制剂类别。通过我们的研究，我们希望对这些化合物如何抑制APC活性建立一个明确的机制理解，并为APC抑制剂作为治疗癌症药物的开发奠定智力和技术基础。

DESCRIPTION (provided by applicant): Microtubule inhibitors, such as taxanes, represent one of the most widely used treatments for cancer. By perturbing the mitotic spindle, taxanes activate the spindle assembly checkpoint (SAC), which subsequently induces mitotic arrest and cell death. However, cancers vary widely in their sensitivity to taxanes, perhaps due to differences in the strength of the checkpoint and efficiency of mitotic arrest. Because the only known function of the SAC is to inhibit the ubiquitin ligase activity of the Anaphase-Promoting Complex/Cyclosome (APC), direct APC inhibitors might be more effective at inducing mitotic arrest, without toxicities associated with microtubule perturbation. Using phenotypic screens in Xenopus cell cycle extracts, combined with biochemical studies to identify targets of active molecules, we have identified two small molecules that inhibit APC dependent proteolysis. This grant application encompasses four major goals. First, to understand, at the atomic level, how TAME and cyclinal interact with their protein targets. Second, to understand the detailed mechanisms by which these compounds perturb APC activity. Third, to understand how these compounds perturb regulation of APC by the SAC. Fourth, to leverage our understanding of the cellular effects of these molecules to identify new classes of APC inhibitors. Through our studies, we hope to establish a definitive mechanistic understanding of how these compounds inhibit APC activity and lay the intellectual and technical groundwork for development of APC inhibitors as drugs to treat cancer.