Isolation and characterization of novel anti-mitotic age

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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.