Multiplex cell-based platform for kinase drug discovery

描述(由申请人提供)：酪氨酸激酶抑制剂是过去10年中最成功的癌症治疗药物之一。然而，剂量限制毒性和耐药性的迅速出现表明，进入临床的化合物可以得到重大改善。很明显，非靶向活动会导致心血管缺陷、免疫系统抑制和肝脏毒性。避免这些问题的一种方法是产生高度特异性的抑制剂，以避免偏离目标的副作用。然而，目标是乱交 在许多情况下对临床疗效负责，抑制多个多余的或补偿的激酶是临床效果所必需的。因此，今天的激酶药物开发面临的一个主要挑战是如何优化第二代抑制剂来阻断选定的激酶，同时避免广谱活性和相关的毒性。不幸的是，能够有效选择多靶点抑制剂的平台目前还不存在，因为人们通常把重点放在开发针对单一激酶的有效和选择性抑制剂上。虽然体外生化分析的动态组范围的面板可用于选择性测量，但这些分析在药物发现的早期大规模执行的成本和时间都是令人望而却步的，并且不能充分复制该激酶的复杂的细胞环境。在我们的第一阶段应用中，我们开发了一种新的改编的现有BaF3分析平台，以直接测量转化的激酶下游的信号，极大地减少了通常在激酶抑制剂筛选中观察到的假阳性。这一改进是基于酪氨酸激酶细胞的分析的根本性飞跃，使该平台能够用于即使是最混杂的抑制剂，并在药物开发的早期阶段，包括二次筛选和铅识别。为了能够针对多个激酶进行化合物优化，新的检测方法使用Primity的CellCode技术进行了多路复用，从而可以在384孔板的单个孔中分析9个转化了激酶的细胞株。对300种激酶抑制剂的筛选揭示了能够同时筛选靶标活性和选择性的能力。在这个第二阶段的应用中，我们建议将这一平台扩展到酪氨酸激动组的所有90个已知成员。这些研究的成功完成将创造第一个全面的、以细胞为基础的选择性激酶分析平台 测量、探针开发和下一代多靶点抑制剂的选择。

DESCRIPTION (provided by applicant): Tyrosine kinase inhibitors have been some of the most successful cancer treatments of the last 10 years. However, dose limiting toxicities and the rapid emergence of resistance suggest that significant improvements can be made to the compounds entering the clinic. It is clear that off-target activity can result in cardiovascular defects, immune system suppression, and liver toxicity. One way to avoid these problems is to generate highly specific inhibitors that avoid off-target side effects. However, target promiscuity is in many cases responsible for clinical efficacy, with inhibition of multiple redundant or compensatory kinases necessary for clinical effect. Thus, a major challenge facing kinase drug discovery today is how to optimize second generation inhibitors to block selected kinases, while avoiding broad spectrum activity and the associated toxicities. Unfortunately, platforms that would enable the efficient selection of multi-targeted inhibitors are currently not available as th focus has routinely been on the development of potent and selective inhibitors to a single kinase. Although kinome-wide panels of in vitro biochemical assays are available for selectivity measurements, these assays are cost and time prohibitive to perform on a large scale early in drug discovery and do not adequately replicate the complex cellular environment of the kinase. In our Phase I application we developed a novel adaptation of the existing BaF3 assay platform to measure signaling directly downstream of the transformed kinase, dramatically reducing the false positives normally observed with kinase inhibitor screening. This improvement is a fundamental leap forward in tyrosine kinase cell-based assays that allows this platform to be used with even the most promiscuous inhibitors and at the earliest phases of drug development, including secondary screening and lead identification. In order to enable compound optimization against multiple kinases, the new assay was multiplexed using Primity's CellCode technology such that nine kinase-transformed cell lines could be analyzed in a single well of a 384 well plate. A screen of 300 kinase inhibitors revealed the power of being able to screen for target activity and selectivity simultaneously. In this Phase II application, we propose the extension of this platform to all 90 known members of the tyrosine kinome. The successful completion of these studies will create the first comprehensive, cell-based kinase assay platform for selectivity measurements, probe development, and the selection of next generation multi- targeted inhibitors.