Until recently, the protein machinery of the cell cycle and the etiology of human cancer were considered separate topics. However, spectacular progress s been made in the identification of the molecules that drive and modulate the activity of the cell-cycle engine. With this increase in understanding of the fundamental process of cell division has come the realization that derangements in cell-cycle regulators may play crucial roles in both the uncontrolled growth and the intrinsic genomic instability of tumor cells. Central components of the cell-cycle engine are the cyclins and their associated cyclin-dependent kinases (cdks). The program in cell-cycle research has revealed a series of enzymes that serve as promising targets for the development of novel anticancer drugs. The overall objective of this program is to test the hypothesis that drugs targeted against specific components of the cell-cycle machinery will be effective as chemotherapeutic agents in the treatment of human cancer. The initial target enzymes selected for these drug discovery efforts are the cdc25 phosphatase and the cyclin D-associated protein kinase, cdk4. The cyclin D-cdk4 complex regulates a critical step in cell-cycle progression: the commitment of the G1-phase cell to enter S- phase, and, consequently, the decision to undergo mitosis. In contrast, the cdc25 phosphatase functions at a later point in the cell cycle as an essential trigger for the activation of the mitotic cdc2 kinase. Using Chinese traditional and other medicinal plants, bacterial and fungal fermentation broths, and natural product-based combinatorial libraries as sources of novel chemical structures, we will pursue the following specific aims: (1) to implement a multi-well-based screening assay for inhibitors of cdc25 phosphatase activity, and (2) to develop and place on-line a high-volume screen for inhibitors of the protein kinase activity of the cyclin D-cdk4 complex. Identified inhibitors of these cell-cycle targets will be subjected to mechanistic evaluations in experimental model systems in our laboratory, and will be tested for clinically-relevant anti-tumor activities by the core components of our drug discovery group. The objectives of this Core are to identify, from among compounds exhibiting activity in the biochemical target screens, those compounds that possess significant growth inhibitory activity against human tumor cells in vitro. This information will be used to determine which compounds require chemical modification to improve cell bioavailablity etc., and which compounds are taken for in vivo testing by Core C. We will routinely use a panel of 7 human tumor cell lines that include 2 breast, 2 colon, 2 prostate, an ovarian and a leukemia cell line. This will allow us to compare drug activity with a panel of generally intractable human solid tumor types and a generally responsive human tumor type (leukemia). The panel includes human solid tumors that will be studied as xenografts in vivo in Core C. We also have available panels of low passage human primary tumor cell lines, including breast and prostate, and cells engineered to overexpress the target of interest for specialized screening. Pharmacokinetic studies will be done on lead compounds to aid their preclinical development and to provide information for chemical modification to improve biological activity.
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