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High throughput screening for inhibitors of Akt plasma m

高通量筛选 Akt 血浆抑制剂

基本信息

批准号：
7168662
负责人：
Hongbo R Luo
金额：
$ 8.45万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-20 至 2007-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7168662
关键词：
HeLa cells NIH Roadmap Initiative tag antibody antineoplastics biotechnology cell membrane drug screening /evaluation enzyme activity green fluorescent proteins high throughput technology inhibitor /antagonist protein localization protein transport serine threonine protein kinase small molecule western blottings

项目摘要

DESCRIPTION (provided by applicant): A number of diseases, including cancer, are linked to perturbation of intracellular signaling pathways. One such pathway is the Ptdlns(3,4,5)P3/Akt (PKB) signaling pathway which is over activated in a wide range of tumor types. Deactivation of this pathway suppresses the growth of tumor cells and also reduces resistance of tumor cells to chemotherapeutic drug treatment. Akt is a serine/threonine protein kinase with oncogenic and anti-apoptotic activities. Its activation relies on Ptdlns(3,4,5)P3 (an inositol phospholipid localized on the plasma membrane) -mediated translocation of Akt from the cytoplasm to the plasma membrane where Akt gets phosphorylated and activated. Thus, Akt translocation provides an attractive target for anti-cancer drug discovery. Recently, we established an experimental system for visualizing Akt plasma membrane translocation in live cells and plan to utilize this system to conduct a high throughput screening for small- molecule inhibitors of Akt plasma membrane translocation. In this proposed research, we will adapt our cell- based system to a high-throughput format (Aim I). The selectivity and reproducibility of our high throughput screening assay will be examined by performing a pilot screening of a known bioactive compounds library (Aim II). An assay for the secondary screening is proposed to role out artifacts from the primary screen. In this assay, Akt plasma membrane translocation will be assessed by the level of Akt phosphorylation which can be monitored by western blot analysis using phospho-Akt specific antibody (Aim III). Finally, a plan to evaluate the biological activities of identified compounds is proposed. We will investigate whether blocking Akt membrane translocation by identified compounds is able to initiate the death of tumor cells and increase the efficacy of chemotherapeutic anticancer drugs (Aim IV). The ultimate goal of our research is to identify inhibitors of Akt plasma membrane translocation via performing a high through screening of small molecule libraries. Discovery of these inhibitors will greatly facilitate our research on Ptdlns(3,4,5)P3/Akt signaling. Moreover, the identified inhibitors can be directly utilized as starting chemical compounds for novel anti- cancer drug development.

描述（由申请人提供）：许多疾病，包括癌症，与细胞内信号传导途径的干扰有关。一种这样的途径是Ptdlns（3，4，5）P3/Akt（PKB）信号传导途径，其在广泛的肿瘤类型中被过度激活。该途径的失活抑制肿瘤细胞的生长，并且还降低肿瘤细胞对化疗药物治疗的抗性。 Akt是一种丝氨酸/苏氨酸蛋白激酶，具有致癌和抗凋亡活性。其活化依赖于Ptdlns（3，4，5）P3（位于质膜上的肌醇磷脂）介导的Akt从细胞质到质膜的易位，其中Akt被磷酸化和活化。因此，Akt易位为抗癌药物的发现提供了一个有吸引力的靶点。最近，我们建立了一个用于可视化活细胞中Akt质膜易位的实验系统，并计划利用该系统进行Akt质膜易位的小分子抑制剂的高通量筛选。在这项研究中，我们将我们的细胞为基础的系统，以高通量的格式（目标I）。我们的高通量筛选试验的选择性和重现性将通过进行一个已知的生物活性化合物库（目标II）的试点筛选检查。提出了一种用于二次筛选的测定法，以从一次筛选中排除伪影。在该测定中，将通过Akt磷酸化水平评估Akt质膜易位，所述Akt磷酸化水平可通过使用磷酸化-Akt特异性抗体（Aim III）的蛋白质印迹分析来监测。最后，提出了一个计划，以评估所确定的化合物的生物活性。我们将研究通过鉴定的化合物阻断Akt膜易位是否能够启动肿瘤细胞的死亡并增加化疗抗癌药物的功效（Aim IV）。本研究的最终目标是通过对小分子文库的高通量筛选来鉴定Akt质膜转位的抑制剂。这些抑制剂的发现将极大地促进Ptdlns（3，4，5）P3/Akt信号通路的研究。此外，所鉴定的抑制剂可直接用作用于新型抗癌药物开发的起始化合物。