DEVELOPMENT OF SMART ASSAY SYSTEMS TO TARGET CELL CYCLE CONTROL

细胞周期检查点在恶性细胞中经常异常，并且 因此是特定化疗的目标。在细胞中的进展 细胞周期受细胞周期蛋白依赖性蛋白激酶的相互作用控制 (CDKs)激活细胞周期蛋白，失活CDK抑制剂。虽然 一些已知的CDK催化活性的药物抑制剂， 识别可阻断特定相互作用的化合物 有CDK的自行车没有被追查。这样做的长期目标是 该计划项目拨款的组成部分是完善无细胞和细胞- 基于智能分析，用于鉴定存在于 加速细胞周期蛋白解离的组合文库 CDK。该项目的具体目标是：(1)开发一种体外 用于筛选抑制分子相互作用的检测系统 Cyclins与相应的CDKs，通过评估溶出度 夹心ELISA法预制细胞周期蛋白D1-CDK4和细胞周期蛋白E-CDK2复合体 使用针对每个成分的抗体；以及(2)完善细胞 基于转录开关分析，可以区分特定的 CDK-Cyclin相互作用的拮抗剂和非特异性的CDK-Cyclin抑制剂 细胞存活率和转录。这项化验将利用Pilot 实验表明，组蛋白脱乙酰基酶的募集 启动子的作用主要是抑制记者正在进行的转录 吉恩。这一观察将作为发展的基础 一种确定的蛋白质-蛋白质相互作用的抑制物 导致阻遏物的解离和随后的诱导 抄写。基于细胞的分析系统将结合使用 以特定目标1中描述的体外系统作为平行初级系统 对来自ELISA的阳性进行二次筛查。使用 总而言之，这些化验应该是识别 能够针对特定蛋白质-蛋白质相互作用的分子 控制恶性细胞的细胞周期进展，同时避免 特别是有毒分子。

Cell cycle check-points are frequently abnormal in malignant cells, and thus are targets for specific chemotherapy. Progression through the cell growth cycle is controlled by the interaction of cyclin dependent kinases (CDKs) with activating cyclins and inactivating CDK inhibitors. Although some pharmacologic inhibitors of CDK catalytic activity are known, the identification of compounds that block the specific interactions of cyclins with CDKs have not been pursued. The long-term goal of this component of the program project grant is to perfect cell-free and cell- based "smart assays" for the identification of compounds present in combinatorial libraries that accelerate the dissociation of cyclins from CDKs. The specific aims of the project are: (1) to develop an in vitro assay system to screen for molecules that inhibit the interaction of cyclins with the corresponding CDKs, by assessing the dissolution rate of preformed cyclin D1-CDK4 and cyclin E-CDK2 complexes in a sandwich ELISA employing antibodies against each component; and (2) to perfect a cell based transcriptional switch assay that can distinguish between specific antagonists of CDK-cyclin interactions, and non-specific inhibitors of cell viability and transcription. This assay will take advantage of pilot experiments showing that the recruitment of a histone deacetylase to a promoter acts to dominantly suppress ongoing transcription of a reporter gene. This observation will serve as the basis for the development of assays in which the inhibitors of a defined protein-protein interaction results in the dissociation of the repressor and subsequent induction of transcription. The cell based assay system will be used in conjunction with the in vitro system described in Specific Aim 1 as a parallel primary screen and a secondary screen of positives derived from the ELISA. Used together, these assays should represent powerful tools to identify molecules capable of targeting specific protein-protein interactions controlling cell cycle progression in malignant cells, while avoiding non- specifically toxic molecules.