Cell surface receptors in the Notch family (Notch1 through Notch4) are expressed on numerous types of cells, including many types of stem cells and undifferentiated progenitor cells, either in the embryo or in self-renewing tissues after birth. A signal is induced when Notch binds to its specific ligands (Delta or Jagged in vertebrates), leading to transcriptional changes that subsequently specify lineage fate. In addition to this critical biological function, Notch signaling has also been implicated in a variety of human diseases, including autoimmunity, a heritable form of strokes, and multiple forms of cancer. Although much is known about the biochemistry and consequences of Notch signaling, a large number of questions remain. Identifying and/or developing small molecules (chemical compounds) that activate Notch signaling in stem/progenitor cells would greatly facilitate further study Notch function in both health and disease. ? Further, such Notch agonist compounds will provide a powerful platform with which to subsequently identify compounds that antagonize this signal, and thus have potential therapeutic uses. In the proposed project, we will exploit the fact that T lymphocyte progenitors have an absolute requirement for Notch signaling, and respond to Notch signaling by proliferation. We will use T progenitors to screen a library of 300,000 chemical compounds to identify those that activate Notch. The goals are to optimize conditions for an existing positive control assay for T progenitor proliferation, and to miniaturize this control assay into a format that is physically and economically compatible with a high-throughput screen. Subsequently, suitability of the miniaturized proliferation assay for compound screening will be validated using a smaller library of 1280 compounds with known pharmacologic activity (LOPAC), including compounds that either inhibit or activate proliferation. Once the assay is validated, we will screen the library of 300,000 compounds to identify those that induce proliferation in T lymphocyte progenitors. Since some compounds will almost certainly act as non-specific mitogens, the final step will be to validate activation of the Notch pathway in those compounds that induce proliferation in T progenitor cells. Ultimately, Notch agonists identified by this screen will be used as tools to study the mechanisms and effects of Notch signaling. In addition, these compounds will be used in future projects to re-screen compound libraries to identify Notch antagonists, which may be further developed for the treatment of Notch-associated diseases. ? ? ?
