Kinome-wide Cell-Based Assays

 描述(申请人提供)：人类基因组编码518个蛋白激酶，催化客户蛋白的磷酸化。由蛋白质介导的信号转导几乎影响细胞生理的方方面面，从细胞周期和CLL分裂的协调到细胞凋亡。不足为奇的是，激酶的解除调控与许多疾病有关，包括糖尿病、炎症、心血管疾病、肿瘤细胞增殖和转移，使它们成为药物开发的靶点。在过去的十年中，针对蛋白激酶的小分子药物的发现取得了巨大的成功，超过25种药物被批准用于肿瘤和最近的类风湿性关节炎，还有更多的药物正在筹备中，用于不同的适应症。然而，小分子靶向的激酶中的ATP结合活性部位的相似性继续使选择性成为药物发现和开发中的一个重要问题，因为混杂的药物可能会产生不良反应，特别是对于长期治疗。在这个第二阶段的应用中，我们将开发一种基于分裂荧光素酶的发光分析方法，用于在细胞环境中对抑制剂进行全基因组筛选和图谱分析。目前，有几种动态组规模的体外生化分析用于筛选和鉴定激酶抑制剂，但这些分析没有提供任何关于分子穿越细胞膜或在细胞中与其期望的靶点结合的能力的信息。此外，大多数生化分析使用催化激活域，从而限制了潜在的更具选择性的变构抑制剂的鉴定，这些变构抑制剂结合AT结合位点的远端。我们假设，处于初级阶段的基于KINOME的宽细胞分析的发展将满足关键的需求。本文提出的基于细胞的动态组分析将允许直接监测化合物对靶蛋白的影响，不仅可以确定细胞通透性和细胞毒性，而且还可以在存在其他ATP结合蛋白的情况下建立细胞环境中的有效性。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，从而导致早期发现故障，从而带来更多的成功机会和新的更安全的临床候选对象。

 DESCRIPTION (provided by applicant): The human genome encodes 518 protein kinases that catalyze the phosphorylation of client proteins. Signal transduction mediated by protein impacts virtually all aspects of cellular physiology, from the coordination of the cell cycle and cll division to apoptosis. Not surprisingly, the deregulation of kinases is implicated in many diseases including diabetes, inflammation, cardiovascular diseases, tumor cell proliferation and metastasis, making them a target for drug development. Small molecule drug discovery targeting protein kinases has seen enormous success in the past decade with over 25 drugs approved for oncology and most recently rheumatoid arthritis with many more in the pipeline for diverse indications. However, the similarity of the ATP binding active site in kinases targeted by small molecules continues to make selectivity a significant concern in drug discovery and development, since a promiscuous drug is expected to give rise to undesired adverse-effects, especially for long term therapy. In this Phase II application, we will develop a split-luciferase based luminescent assay for kinome-wide screening and profiling of inhibitors in a cellular setting. Currently, there are several kinome scale in vitro biochemical assays that are used for screening and identification of kinase inhibitors, but these assays do not provide any information on the ability of a molecule to cross cell membranes or engage its desired target in a cell. In addition, most biochemical assays use the catalytic kinase domain, thereby limiting the identification of potentially more selective allosteric inhibitors that bind sites distal to the AT-binding site. We hypothesize that the development of kinome wide cell-based assays, which are in its infancy, will meet a critical need. The cell-based kinome assays proposed herein will allow for directly monitoring the effect of compounds on a target kinase to not only to ascertain cell-permeability and cellular toxicity, but also to establish efficacy in the cellular milieu in the presence of other ATP binding proteins. Our goal is to render 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 in many more opportunities for success and new and safer clinical candidates.