Isolation and characterization of novel anti-mitotic age

有丝分裂激酶如Plk1、Cdk1、Akt1和Aurora-A活性的失调已在许多人类原发肿瘤和肿瘤细胞系中得到证实。因此，抑制这些激酶活性似乎对开发可能用于治疗癌症和其他超增生性疾病的药物很重要。在与Rexan公司合作的CRADA项目中，我们计划在96孔板中采用基于酵母的高通量液体生长试验来筛选和/或评估这些激酶的抑制剂。在酵母和培养的哺乳动物细胞中，非催化c端区域的polo-box结构域已被证明对Plk1的功能至关重要。因此，对polo-box功能的特异性抑制可能提供一种特异性抑制polo激酶功能的策略。芽殖酵母中polo-box的过度表达会导致严重的细胞动力学失效，导致细胞形态链化，生长速率显著降低。利用pdr5delta snq2delta（两种主要的多药泵）双突变体的这种表型，我们将筛选化学文库，以分离能够恢复polo-box依赖性生长缺陷和链式形态的化合物。由于内源性Cdc5的polo-box也可能被假定的polo-box抑制剂抑制，因此表达Cdc5deltaC- cnm67的细胞作为Cdc5的唯一来源将被用于筛选。Cdc5deltaC缺乏polo-box结构域，但其功能被Cdc5deltaC依赖cnm67定位到SPB所绕过。此外，我们发现过表达Cdk2严重抑制细胞生长。因此，该表型也将用于筛选新的抗Cdk抑制剂或评估BMI-1026和其他已存在的Cdk抑制剂（奥洛穆cine和罗斯科维汀，丁内酯I，黄吡醇等）的细胞毒性。另外提供一个活化的Cdc28/E12K或非抑制的Cdc28/Y19F等位基因，以消除Cdk抑制剂对内源性Cdc28活性的潜在抑制。

Deregulation of mitotic kinases such as Plk1, Cdk1, Akt1, and Aurora-A activities has been shown in a number of human primary tumors and tumor cell lines. Therefore, inhibition of these kinase activities appears to be important for the development of drugs that may be useful in the treatment of cancer and other hyper-proliferative diseases. In CRADA with Rexan Corp., Rockville, MD, we plan to employ yeast-based high-throughput liquid growth assays in 96-well plates to screen and/or evaluate inhibitors of these kinases. The polo-box domain in the non-catalytic C-terminal region has been shown to be essential for the function of Plk1 in both yeast and cultured mammalian cells. Thus, specific inhibition of the polo-box function may likely provide a strategy to specifically inhibit the polo kinase function. Overexpression of the polo-box in budding yeast induces a severe cytokinetic failure, leading to a chained cell morphology with substantially reduced growth rate. Using this phenotype in pdr5delta snq2delta (two major multi-drug pumps) double mutant, we will screen chemical libraries to isolate compounds that are capable of restoring the polo-box-dependent growth defect and chained morphology. Since the polo-box of endogenous Cdc5 may also be inhibited by the putative polo-box inhibitors, cells expressing Cdc5deltaC-Cnm67, which lacks the polo-box domain but the function of the polo-box is bypassed by the Cnm67-dependent localization of Cdc5deltaC to the SPB, as a sole source of Cdc5 will be used for the screen. In addition, we found that overexpression of Cdk2 severely inhibits cell growth. Thus, this phenotype will also be utilized to screen novel anti-Cdk inhibitors or evaluate cytotoxicity of BMI-1026 and other preexisting Cdk inhibitors (olomoucine and roscovitine, butyrolactone I, flavopiridol, etc). Either an activated Cdc28/E12K or non-inhibitable Cdc28/Y19F allele will be additionally provided to eliminate a potential inhibition of endogenous Cdc28 activity by Cdk inhibitors.