Assay Development and Screening for Notch Agonists

描述(申请人提供)：Notch家族(Notch1至Notch4)的细胞表面受体在多种类型的细胞上表达，包括多种类型的干细胞和未分化的前体细胞，无论是在胚胎中还是在出生后的自我更新组织中。当Notch与其特定的配体(脊椎动物中的Delta或锯齿状)结合时，信号被诱导，导致转录变化，随后决定谱系命运。除了这一重要的生物学功能外，Notch信号还与多种人类疾病有关，包括自身免疫、一种可遗传形式的中风和多种形式的癌症。虽然对Notch信号的生物化学和后果已知很多，但仍有大量的问题存在。鉴定和/或开发激活干/祖细胞中Notch信号的小分子(化合物)将极大地促进进一步研究Notch在健康和疾病中的作用。 此外，这种Notch激动剂化合物将提供一个强大的平台，用于随后识别拮抗这一信号的化合物，从而具有潜在的治疗用途。在拟议的项目中，我们将利用T淋巴细胞前体细胞对Notch信号的绝对需求，并通过增殖来响应Notch信号。我们将使用T祖细胞来筛选300,000种化合物的文库，以识别那些激活Notch的化合物。我们的目标是优化现有的T祖细胞增殖阳性对照试验的条件，并将该对照试验小型化，使其在物理和经济上与高通量筛选兼容。随后，将使用1280种具有已知药理活性(LOPAC)的化合物(包括抑制或激活增殖的化合物)的较小文库来验证小型增殖试验对化合物筛选的适用性。一旦测试得到验证，我们将筛选30万种化合物的文库，以确定那些诱导T淋巴细胞前体细胞增殖的化合物。由于一些化合物几乎肯定会作为非特异性有丝分裂原，最后一步将是验证那些诱导T祖细胞增殖的化合物中Notch途径的激活。最终，通过这一筛选确定的Notch激动剂将被用作研究Notch信号转导机制和作用的工具。此外，这些化合物将用于未来重新筛选化合物文库以识别Notch拮抗剂的项目，这些化合物可能会进一步开发用于治疗Notch相关疾病。

DESCRIPTION (provided by applicant): 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.