Probing Diabetes: Development of a HTS-compatible TORC2 Redistribution Assay.

描述（由申请人提供）：II型糖尿病的简单特征是高血糖症，但却是一种病因复杂的疾病状态。除了脂肪和骨骼肌的葡萄糖摄取减少外，慢性肝脏葡萄糖生成也有助于这种疾病的表现，并且是拟议研究解决的中心健康相关问题。胰高血糖素通过激活cAMP-信号级联来启动促凋亡程序，cAMP-信号级联部分地由CREB共激活因子TORC 2的核转位介导。事实上，TORC 2的再分布是葡萄糖稳态中的关键调节步骤，并且能够阻断TORC 2的核输入或促进其核输出的小分子可能调节肝脏葡萄糖产生。该项目的目标是开发和验证一种TORC 2亚细胞定位检测方法，该方法适用于大型化学文库的高通量筛选，其长期目标是鉴定靶向TORC 2再分布和调节肝细胞再生的小分子。为此，还将同时开发一系列确认性筛查，以区分在初步筛查活动期间产生假阳性结果的小分子。最后，还将开发一种基于细胞的胚胎发生试验，以验证来自我们的初级和二级筛选的小分子命中的生理相关性。总的来说，在拟议的研究中开发的测定将提供高通量筛选活动所需的工具集，以实现鉴定小分子化学探针的长期目标，这将有助于我们理解异常的血管生成，并可能导致用于治疗II型糖尿病的新型治疗分子。公共卫生相关性：以高血糖为特征的II型糖尿病已经达到流行病的程度，仅在美国就有大约2000万人受到影响。促成这种疾病的表现，肝脏的慢性葡萄糖产生是糖尿病的一个组成部分，由cAMP信号通路介导。通过寻找能够阻断这种信号通路的小分子，我们将发现化学探针，这将有助于我们了解这种疾病，并可能作为治疗糖尿病患者的新型治疗分子。

DESCRIPTION (provided by applicant): Type II diabetes is simply characterized by hyperglycemia but is a disease state with a complex etiology. In addition to decreased glucose uptake by adipose and skeletal muscle, chronic hepatic glucose production contributes to the manifestation of this disease and is the central health related problem addressed by the proposed studies. Glucagon initiates the gluconeogenic program by activating the cAMP-signaling cascade that is, in part, mediated by the nuclear translocation of the CREB coactivator, TORC2. Indeed, the redistribution of TORC2 is a key regulatory step in glucose homeostasis and small molecules that are capable of blocking nuclear import of TORC2, or that facilitate its nuclear export, are likely to regulate hepatic glucose production. The goal of this project is to develop and validate a TORC2 subcellular localization assay that is amenable to high-throughput screening of large chemical libraries, with a long-term goal of identifying small molecules that target TORC2 redistribution and regulate hepatic gluconeogenesis. To this end, a portfolio of confirmatory screens will also be developed in parallel, which will discriminate small molecules producing false- positive results during the primary screening campaign. Finally, a cell-based gluconeogenesis assay will also be developed to validate the physiological relevance of small molecules hits coming from our primary and secondary screens. Collectively, the assays developed in the proposed studies will provide the toolsets necessary for a high-throughput screening campaign, to fulfill the long-term goal of identifying small molecule chemical probes that will aid in our understanding of aberrant gluconeogenesis, and that may lead to novel therapeutic molecules for the treatment of type II diabetes. PUBLIC HEALTH RELEVANCE: Characterized by high blood glucose, type II diabetes has reached pandemic proportions with approximately 20 million individuals affected in the United States alone. Contributing to the manifestation of this disease, chronic glucose production by the liver is one component of diabetes that is mediated by the cAMP-signaling pathway. By search for small molecules capable of blocking this signaling pathway we will uncover chemical probes that will facilitate our understanding this disease and will possibly function as novel therapeutic molecules for the treatment of individuals suffering from diabetes.