Kinome-wide cell-based assays

描述(申请人提供)：蛋白激酶是人类细胞信号转导的关键媒介，几乎影响细胞生理学的方方面面，从细胞周期和细胞分裂的协调到细胞凋亡。目前已知的信号通路失控与多种人类疾病有关，包括糖尿病、炎症、心血管疾病、肿瘤细胞增殖和转移，使其成为药物开发的靶点。在结构上，激酶在活性部位(ATP结合域)共享非常相似的结构，这使得选择性成为药物发现和开发中的一个问题。因此，发展快速而经济的方法，针对整个细胞中的一大组激酶来分析候选药物，不仅有助于预测潜在的长期毒物，而且有助于了解以激酶为靶点的多药理学，并有助于为旧化合物确定新的靶点。在这个第一阶段的应用中，我们将开发一种基于分裂荧光素酶的发光分析方法，用于细胞环境中的激酶图谱分析。这些以细胞为基础的分析将满足直接监测化合物对靶蛋白激酶的影响的迫切需要，不仅可以确定细胞通透性和细胞毒性，而且还可以在存在接头和调节蛋白的情况下确定细胞替代物的有效性。我们将测试该方法在不同群体中的通用性，并评估我们的方法用于高通量筛选的可行性，该方法使用了一组80种已知的激酶抑制剂。我们的目标是使细胞激酶谱分析既容易获得又负担得起，这样在细胞或自然环境中的化合物谱分析可以更早地完成，从而导致早期识别失败，从而产生更多的成功机会。

DESCRIPTION (provided by applicant): Protein kinases serve as critical mediators of signal transduction in human cells and impact virtually all aspects of cellular physiology, from the coordination of the cell cycle and cell division to apoptosis. The deregulation of kinase mediated signaling is now known to be involved in a variety of human diseases that include, diabetes, inflammation, cardiovascular diseases, tumor cell proliferation and metastasis making them a target for drug development. Structurally, kinases share a very similar architecture at the active-site (ATP-binding domain), making selectivity an issue in drug discovery and development. Thus, the development of rapid and economic methods for profiling drug candidates against a large panel of kinases in whole cells will not only aid in anticipating potential long term toxicit, but also help in understanding kinase targeted polypharmacology and aid in identifying new targets for old compounds. In this Phase I application, we will develop a split-luciferase based luminescent assay for kinase profiling in a cellular setting. These cell-based assays will meet a critical need for directly monitoring the effect of compounds on a target kinase not only to ascertain cell-permeability and cellular toxicity, but also to establish efficacy in the cellular mlieu in the presence of adaptor and regulatory proteins. We will test the generality of the approach for kinases across various groups and evaluate the feasibility of our assay for high throughput screening using a panel of 80 known kinase inhibitors. Our goal is to make cellular kinase profiling assays both easily available and affordable, so that compound profiling in a cellular or native context can be done earlier and thereby lead to early identification of failures, resulting n many more opportunities for success.