Identifying Inhibitors of the APC/C Using Yeast Mutants

使用酵母突变体鉴定 APC/C 抑制剂

• 批准号: 6744386
• 负责人: Daniel J Burke
• 金额: $ 27.88万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6744386
• 关键词: HeLa cells; Saccharomyces cerevisiae; Xenopus oocyte; antineoplastics; cell cycle; cell cycle proteins; combinatorial chemistry; drug design /synthesis /production; drug screening /evaluation; enzyme inhibitors; fungal genetics; fungal proteins; gene interaction; genetic strain; ligase; microtubules; mitotic spindle apparatus; mutant; neoplasm /cancer chemotherapy; protein structure function; site directed mutagenesis; tubulin; ubiquitin

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.

描述（申请人提供）本研究的长期目标是利用酿酒酵母突变体开发新型抗癌药物。研究的重点将是纺锤体检查点，它响应纺锤体和微管的扰动而阻止细胞有丝分裂。微管蛋白是抗癌化疗的有效靶点。许多天然产物，如紫杉醇、长春花碱和长春新碱，单独使用或联合治疗时，可有效对抗多种肿瘤。药物具有共同的作用机制；它们将微管蛋白聚合调节为微管，从而激活纺锤体检查点。纺锤体检查点的目标是 Cdc20，它是一种辅助因子，可将底物传递至后期促进复合物 (APC/C) 进行泛素化和破坏。我们已经产生了 APC/C 和 Cdc20 功能缺陷的酵母菌株，并建议继续筛选特异性抑制 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，并通过合理的药物设计来提高其有效性。我们将扩展分析以确定它们是否影响培养的人类细胞中的 APC/C 功能和有丝分裂。我们将从非洲爪蟾卵母细胞和 HeLa 细胞中制备提取物，以确定 APC/C 活性的哪一步受到影响。我们将使用定点突变体的有序阵列进行系统遗传分析，并识别纺锤体检查点突变体 bub3 和所有缺乏非必需基因的突变体之间的合成致死相互作用。这将确定西雅图项目在 bub3 筛选中发现的化合物的可能细胞靶标。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。