High throughput screening for inhibitors of Akt plasma m

描述（由申请人提供）：许多疾病，包括癌症，与细胞内信号通路的扰动有关。其中一个通路是Ptdlns(3,4,5)P3/Akt （PKB）信号通路，它在多种肿瘤类型中被过度激活。该通路的失活抑制了肿瘤细胞的生长，也降低了肿瘤细胞对化疗药物治疗的耐药性。Akt是一种丝氨酸/苏氨酸蛋白激酶，具有致癌和抗凋亡活性。它的激活依赖于Ptdlns(3,4,5)P3（一种定位于质膜上的肌醇磷脂）介导的Akt从细胞质到质膜的易位，Akt在质膜上被磷酸化和激活。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，我们建立了一个在活细胞中可视化Akt质膜易位的实验系统，并计划利用该系统对Akt质膜易位的小分子抑制剂进行高通量筛选。在本研究中，我们将把基于细胞的系统调整为高通量格式（Aim I）。我们的高通量筛选试验的选择性和可重复性将通过对已知生物活性化合物文库（Aim II）进行中试筛选来检验。提出了一种用于二次筛选的分析方法，以从初级筛选中剔除伪影。在本实验中，Akt质膜易位将通过Akt磷酸化水平来评估，Akt磷酸化水平可以通过使用phospho-Akt特异性抗体（Aim III）进行western blot分析来监测。最后，提出了评价鉴定化合物生物活性的方案。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提高化疗抗癌药物的疗效（Aim IV）。我们研究的最终目标是通过对小分子文库进行高通量筛选来鉴定Akt质膜易位的抑制剂。这些抑制剂的发现将极大地促进我们对Ptdlns(3,4,5)P3/Akt信号的研究。此外，所鉴定的抑制剂可直接作为新型抗癌药物开发的起始化合物。

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.