Identifying Inhibitors of the APC/C Using Yeast Mutants

本研究的长期目标是利用酿酒酵母的突变株来开发新型抗癌药物。研究的重点将是纺锤体检查点，它阻止处于有丝分裂状态的细胞对纺锤体和微管的扰动做出反应。微管蛋白是抗癌化疗的有效靶点。许多天然产品，如紫杉醇、长春花碱和长春新碱，单独或联合使用时，对广泛的肿瘤都有效。这些药物有一个共同的作用机制：它们将微管蛋白聚合调节为微管，从而激活纺锤体检查点。纺锤体检查点的靶点是Cdc20，这是一种辅助因子，将底物输送到后期促进复合体(APC/C)进行泛素化和破坏。我们已经产生了具有APC/C和CDC20功能缺陷的酵母菌株，并建议继续筛选专门抑制APCIC活性的化合物。我们建议进一步表征我们已经确定的六种化合物，并利用合理的药物设计来提高它们的有效性。我们将扩大分析范围，以确定它们是否影响培养的人类细胞的APC/C功能和有丝分裂。我们将制备非洲爪哇卵母细胞和HeLa细胞的提取物，以确定APC/C活性的哪一步受到影响。我们将使用定点突变的有序阵列来进行系统的遗传分析，并识别纺锤体检查点突变Bub3和所有缺乏非必需基因的突变之间的合成致死相互作用。这将为西雅图项目在Bub3屏幕上确定的化合物识别可能的细胞目标。我们将表达由相互作用的基因编码的蛋白质，并确定它们是否与化合物结合。

DESCRIPTION (provided by applicant)The long-term goals of this research are to use mutants of the yeast Saccharomyces cerevisiae to develop novel anti-cancer drugs. The focus of the research will be the spindle checkpoint, which arrests cells in mitosis in response to spindle and microtubule perturbation. Tubulin is a validated target for anti-cancer chemotherapy. A number of natural products, such as taxol, vinblastine and vincristine are effective against a broad spectrum of tumors when used individually or in combination therapy. The drugs have a common mechanism of action; they modulate tubulin polymerization into microtubuies, which activates the spindle checkpoint. The target of the spindle checkpoint is Cdc20, an accessory factor that delivers substrates to the anaphase-promoting complex (APC/C) for ubiquitination and destruction. We have generated strains of yeast defective for APC/C and Cdc20 function and propose to continue screening for compounds that specifically inhibit APCIC activity. We propose to further characterize the six compounds that we haven already identified and to improve their effectiveness using rational drug design. We will expand the analysis to determine if they affect APC/C function and mitosis in cultured human cells. We will prepare extracts from Xenopus oocytes and HeLa cells to determine which step of APC/C activity is affected. We will use ordered arrays of site directed mutants to perform systematic genetic analysis and identify synthetic lethal interactions between a spindle checkpoint mutant, bub3, and all mutants that lack non-essential genes. This will identify possible cellular targets for compounds identified in a bub3 screen by the Seattle Project. We will express the proteins encoded by the interacting genes and determine if they bind the compounds.