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.
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