Chemical Genetic and Biochemical Studies of Mitotic Proteolysis

描述（由申请人提供）：微管抑制剂，如紫杉烷，是最广泛使用的癌症治疗方法之一。通过扰乱有丝分裂纺锤体，紫杉烷激活纺锤体组装检查点（SAC），随后诱导有丝分裂阻滞和细胞死亡。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阻滞的效率不同。由于SAC的唯一已知功能是抑制后期促进复合体/环小体（APC）的泛素连接酶活性，因此直接的APC抑制剂可能更有效地诱导有丝分裂停止，而没有微管扰动相关的毒性。利用爪蟾细胞周期提取物的表型筛选，结合生物化学研究来确定活性分子的靶点，我们已经确定了两个抑制APC依赖性蛋白水解的小分子。此奖助金申请包含四个主要目标。首先，在原子水平上理解TAME和cyclinal如何与它们的蛋白靶相互作用。其次，了解这些化合物干扰APC活性的详细机制。第三，了解这些化合物如何干扰SAC对APC的调节。第四，利用我们对这些分子的细胞效应的理解来识别新的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，并为开发APC抑制剂作为治疗癌症的药物奠定知识和技术基础。

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.