Screening for small molecule inhibitors of Stat5 (RMI)

描述(申请人提供)：信号转导和转录激活子(Stats)介导各种细胞外信号。作为对配体与其受体结合的反应，这些蛋白在位于C末端附近的不变酪氨酸残基上被磷酸化，要么是通过受体的激酶活性，要么是通过招募到受体的Janus激酶(JAK)。在酪氨酸磷酸化后，Stat蛋白通过一个单体的SH2结构域与另一个单体的pTyr相反的结构域进行二聚。它们也会在酪氨酸磷酸化残基附近的残基上被丝氨酸磷酸化，这可以调节其活性。然后，磷酸化的双齿连接的二聚体移位到细胞核，结合特定的DNA启动子序列，并启动指定反应元件的转录。在正常细胞中，Stats介导来自生长因子受体和免疫刺激的信号。然而，在几种癌症类型中，Stats被结构性激活，包括两个高度同源的蛋白Stat5a和Stat5b，它们存在于许多不同的乳腺癌、前列腺癌、白血病和淋巴瘤中。我们和其他人已经证明，Stat5a/b活性的破坏可以在一些肿瘤细胞系中诱导细胞死亡。因此，拥有高通量筛选(HTS)来识别Stat5的小分子抑制物以阻断它们对肿瘤促生存途径的异常激活将是有利的。本项目的目标是开发一种HTS来识别阻断Stat5寡聚和基因转录活性的小分子抑制剂。关于开发STAT5功能的小分子抑制剂的文献报道很少。我们的具体目标如下： 1.发现和优化小分子抑制剂，这些小分子抑制剂将来自a)Stat5 SH2结构域抑制剂的已知受体上的Stat5对接位点的组合磷肽库和b)筛选NCI多样性的小类药物化合物。这两个读数都将监测荧光偏振的变化。 2.通过在基于HTS细胞的确证报告实验中测试最有希望的化合物，以确定它们对基因转录的影响，来检测和确认潜在的抑制Stat5寡聚和DNA结合/功能的化合物。 在本研究的结论中，我们期望建立一种快速筛选和鉴定Stat5小分子抑制剂的方法。这一筛选应该允许更快地开发治疗各种疾病的治疗剂。

DESCRIPTION (provided by applicant): Signal transducers and activators of transcription (Stats) mediate a variety of extracellular signals. In response to ligand binding to its receptor, these proteins become phosphorylated on an invariant tyrosine residue located near the C-terminus, either by the kinase activity of the receptor, or by Janus kinases (Jaks) recruited to the receptor. Upon tyrosine phosphorylation, Stat proteins dimerize via opposing SH2 domain of one monomer with the pTyr of the other monomer. They also become serine phosphorylated on a residue proximal to the tyrosine phosphorylation residue that can regulate its activity. The phosphorylated bi-dentate ligated dimer then translocates to the nucleus, binds specific DNA promoter sequences and initiates transcription of designated response elements. In normal cells Stats mediate signaling from growth factor receptors and immune stimuli. However in several cancer types, Stats are constitutively activated, including two highly homologous proteins Stat5a and Stat5b, which are present in many different cancers of breast, prostate, leukemias and lymphomas. We and others have shown that disruption of Stat5a/b activity can induce cell death in some tumor cell lines. It would therefore be advantageous to have a high throughput screen (HTS) to identify small molecule inhibitors of Stat5 to block their aberrant activation of this tumor pro-survival pathway. The objective of this project is to develop a HTS to identify small molecule inhibitors that block oligomerization of Stat5 and gene transcriptional activity. There has been little effort reported in the literature on the development of small molecule inhibitors of Stat5 function. Our specific aims are as follows: 1. Discover and optimize small molecule inhibitors that will be derived from either a) Stat5 SH2 domain inhibitor combinatorial phosphopeptide library of known Stat5 docking sites on receptors and b) screening the NCI Diversity Set of small drug-like compounds. Both readouts will employ monitoring changes in fluorescence polarization. 2. Assay and confirm potential inhibitory compounds of Stat5 oligomerization and DNA binding/function by testing the most promising compounds in a confirmatory HTS cell based reporter assay to determine their effects on gene transcription. At the conclusion of this research we expect to have established a method to rapidly screen and identify small molecule inhibitors of Stat5. This screen should allow for the more rapid development of therapeutic agents to treat a variety of diseases.