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High Throughput Screening for Modulators of STAT5 (RMI)

STAT5 调制器的高通量筛选 (RMI)

基本信息

批准号：
6879437
负责人：
DAVID A. FRANK
金额：
$ 8.55万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-30 至 2005-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6879437
关键词：
biological signal transduction biotechnology caseins cell line chemical registry /resource enzyme activity gene induction /repression genetic regulation high throughput technology luciferin monooxygenase neoplasm /cancer chemotherapy neoplastic cell nuclear factor kappa beta reporter genes robotics technology /technique development transcription factor

项目摘要

DESCRIPTION (provided by applicant): In identifying pathways for the targeted therapy of cancer, three requirements are paramount. The pathway must be activated in tumor cells, it must be amenable to pharmacological modulation, and inhibition of the pathway in normal cells must not lead to undue toxicity. STAT family transcription factors are targets that fulfill these requirements fully. In particular, STAT3 and STAT5 are activated inappropriately in a wide array of human tumors, and their inhibition can be tolerated in normal tissue with little detrimental effect. We have developed a system to screen for inhibitors of STAT5, and we have identified a number of promising compounds. However, STAT5, which is activated in many hematologic cancers, is generally not inhibited by the STAT3 modulators we have identified. Given the success we have had in identifying STAT3 inhibitors, and their lack of cross-reactivity with STAT5, the goal of this proposal is to develop a high throughput screening system to identify inhibitors of STAT5. We will create cell lines in which stable transfection is used to introduce a luciferase reporter gene driven by the beta-casein promoter. Luciferase activity is induced from this construct in response to activated STAT5, but not in response to other STATs or other transcription factors. We will use three cellular systems:T47D human breast cancer cells, in which STAT5 can be activated through the endogenous prolactin receptor; NKL human leukemia cells in which STAT5 is activated following IL-2 treatment; and, Ba/F3 cells, a hematopoietic cell line in which STAT5 is activated in response to IL-3. The screen will be conducted at the Institute of Chemistry and Cell Biology at Harvard Medical School, where we have a longstanding collaboration to identify modulators of other STATs. In the initial screen, we will identify compounds that inhibit or induce STAT5-dependent luciferase expression. Although the primary goal of this work is to identify STAT5 inhibitors, compounds that activate STATS may also have therapeutic benefit, particularly in breast cancer. These modulators will then be tested for specificity for the STAT pathway by performing a secondary screen in cells stably expressing NF-kappaB dependent luciferase constructs, which we had constructed previously for our earlier screens. Compounds that show specificity for STATS-dependent gene activation will then be evaluated for mechanistic properties, and will be studied in appropriate in vitro and animal models. Through this validated and structured approach, we plan to identify ten specific inhibitors and five specific activators of STAT5 by the end of the one year funding period. The ultimate goal is the development of compounds that will act as targeted therapies for human cancers.

描述（由申请人提供）：在确定癌症靶向治疗的途径时，三个要求是最重要的。该途径必须在肿瘤细胞中被激活，它必须服从药理学调节，并且在正常细胞中抑制该途径不得导致过度毒性。 STAT家族转录因子是完全满足这些要求的靶标。特别是，STAT 3和STAT 5在多种人类肿瘤中被不适当地激活，并且它们的抑制在正常组织中可以耐受，几乎没有有害影响。我们已经开发了一个系统来筛选STAT 5的抑制剂，我们已经确定了一些有前途的化合物。然而，在许多血液癌症中被激活的STAT 5通常不被我们已经鉴定的STAT 3调节剂抑制。鉴于我们在鉴定STAT 3抑制剂方面取得的成功，以及它们与STAT 5缺乏交叉反应性，本提案的目标是开发一种高通量筛选系统来鉴定STAT 5抑制剂。我们将创建细胞系，其中稳定转染用于引入由β-酪蛋白启动子驱动的荧光素酶报告基因。荧光素酶活性响应于活化的STAT 5而从该构建体诱导，但不响应于其它STAT或其它转录因子。我们将使用三种细胞系统：T47 D人乳腺癌细胞，其中STAT 5可以通过内源性催乳素受体激活; NKL人白血病细胞，其中STAT 5在IL-2处理后被激活;以及Ba/F3细胞，其中STAT 5响应于IL-3而被激活的造血细胞系。筛选将在哈佛医学院的化学和细胞生物学研究所进行，在那里我们有一个长期的合作，以确定其他STAT的调节剂。在最初的筛选中，我们将鉴定抑制或诱导STAT 5依赖性荧光素酶表达的化合物。虽然这项工作的主要目标是识别STAT 5抑制剂，但激活STAT 5的化合物也可能具有治疗益处，特别是在乳腺癌中。然后，通过在稳定表达NF-kappaB依赖性荧光素酶构建体的细胞中进行二次筛选，测试这些调节剂对STAT途径的特异性，我们之前已经为早期筛选构建了该构建体。然后将评估对STAT依赖性基因活化显示特异性的化合物的机械性质，并将在适当的体外和动物模型中进行研究。通过这种经过验证和结构化的方法，我们计划在一年的资助期结束前确定10种特定的STAT 5抑制剂和5种特定的STAT 5激活剂。最终目标是开发出能够作为人类癌症靶向疗法的化合物。