MOLECULAR PHARM. AND BIOCHEM. OF NOVEL DUAL SPECIIFICITY PHOSPHATASE INHIBITORS

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This Project is a multidisciplinary effort to employ chemical diversity and smart assays to antitumor drug discovery. The overall goal of this Project is to examine small molecules obtained from chemical libraries and synthesized in other subprojects for their ability to disrupt dual specificity phosphatase (DSPase) activity and for their antiproliferative activity against human solid tumors. DSPases have emerged as important molecular targets for future therapeutic agents directed against cancer. The Cdc25 DSPases are major determinants of the phosphorylation state of cyclin-dependent kinases (Cdks), which control cell cycle checkpoints. Recently, with the support of this Program Project Grant, we identified the most potent and selective inihibitors of the Cdc25 phosphatases known. Although these compounds hold considerable promises as tool drugs, we propose to define more potent, selective and drug-like Cdc25 inhibitors. Thus, our working hypothesis continues to be that the phosphatase activity of oncogenic DSPases is responsible for the oncogenic properties of Cdc25 and that selective inhibitors of this catalytic activity are likely to generate medicinally useful and unique compounds. The biochemical assays in this Project have been explicitly designed to evaluate the novel, solid phase and solution-phase combinatorial chemistry strategies outlined in the other subprojects. Our Specific Aims are to (1) characterize the in vitro antiphosphatase activity of all novel library compounds, (2) assess the antiphosphatase, antiproliferative and cell cycle effects of prioritized compounds, (3) determine the ligand interactions and 3-dimensional structure of Cdc25B with novel analogs and, (4) develop a robust smart assay to evaluate new dynamic chemical libraries for ligands against Cdc25B. We will continue to use human breast, prostate and ovarian tumor cell lines. We propose to use nonmalignant cells as well as murine cells with a temperature sensitive Cdc25 substrate and fibroblasts from mice that lack functional Cdc25B or Cdc25C genes to examine the cellular activity of these compounds.