Kinome-wide Cell-Based Assays

 描述（由申请人提供）：人类基因组编码518种催化客户蛋白磷酸化的蛋白激酶。蛋白质介导的信号转导几乎影响细胞生理学的各个方面，从细胞周期和细胞分裂的协调到细胞凋亡。毫不奇怪，激酶的失调与许多疾病有关，包括糖尿病、炎症、心血管疾病、肿瘤细胞增殖和转移，使其成为药物开发的靶点。靶向蛋白激酶的小分子药物发现在过去十年中取得了巨大的成功，超过25种药物被批准用于肿瘤学，最近被批准用于类风湿性关节炎，还有更多药物正在用于各种适应症。然而，小分子靶向的激酶中ATP结合活性位点的相似性继续使选择性成为药物发现和开发中的重要问题，因为预期混杂药物会引起不期望的副作用，特别是对于长期治疗。在这个II期申请中，我们将开发一种基于分裂荧光素酶的发光检测方法，用于在细胞环境中进行全激酶组筛选和抑制剂分析。目前，有几种用于筛选和鉴定激酶抑制剂的激酶组规模的体外生物化学测定法，但这些测定法不提供关于分子穿过细胞膜或在细胞中接合其所需靶标的能力的任何信息。此外，大多数生物化学测定使用催化激酶结构域，从而限制了对可能更具选择性的变构抑制剂的鉴定，所述变构抑制剂结合AT结合位点远端的位点。我们假设，发展的激酶组广泛的细胞为基础的测定，这是在其婴儿期，将满足一个关键的需求。本文提出的基于细胞的激酶组测定将允许直接监测化合物对靶激酶的作用，以不仅确定细胞渗透性和细胞毒性，而且在其他ATP结合蛋白存在下在细胞环境中建立功效。我们的目标是使细胞激酶谱分析既容易获得，又负担得起，以便在细胞或天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，从而获得更多的成功机会和新的更安全的临床候选人。

 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.