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， 因此是特异性化疗的靶点。通过细胞 生长周期受细胞周期蛋白依赖性激酶的相互作用控制 （CDK）与活化细胞周期蛋白和失活CDK抑制剂。虽然 已知一些CDK催化活性的药理学抑制剂， 鉴定阻断特定相互作用的化合物 细胞周期蛋白与CDK的结合尚未被研究。长期目标是 该计划的项目赠款的组成部分是完善无细胞和细胞- 基于"智能分析"的化合物鉴定存在于 组合文库，其加速细胞周期蛋白从 CDK。本项目的具体目标是：（1）开发一种体外 检测系统筛选抑制相互作用的分子 细胞周期蛋白与相应的CDK，通过评估的溶解速率， 在夹心ELISA中预先形成的细胞周期蛋白D1-CDK4和细胞周期蛋白E-CDK2复合物 使用针对每种成分的抗体;以及（2）完善细胞 基于转录开关分析，可以区分特定的 CDK-细胞周期蛋白相互作用的拮抗剂，以及 细胞活力和转录。本试验将利用中试 实验表明，组蛋白去乙酰化酶的招募， 启动子的作用是显性抑制报告基因的持续转录 基因这一观察结果将作为发展的基础， 其中确定的蛋白质-蛋白质相互作用的抑制剂 导致阻遏物的解离和随后的诱导 转录。基于细胞的检测系统将与 与特定目标1中描述的体外系统作为平行主要 筛选和来自ELISA的阳性的二次筛选。使用 总之，这些分析应该是识别 能够靶向特定蛋白质-蛋白质相互作用的分子 控制恶性细胞中的细胞周期进程，同时避免非恶性细胞的发生。 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.