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年来最成功的癌症治疗药物之一。然而，剂量限制性毒性和快速出现的耐药性表明，可以对进入临床的化合物进行显著改善。很明显，脱靶活性可导致心血管缺陷、免疫系统抑制和肝毒性。避免这些问题的一种方法是产生高度特异性的抑制剂，避免脱靶副作用。然而，目标滥交 在许多情况下负责临床功效，抑制临床效果所需的多种冗余或补偿激酶。因此，当今激酶药物发现面临的主要挑战是如何优化第二代抑制剂以阻断选定的激酶，同时避免广谱活性和相关毒性。不幸的是，能够有效选择多靶向抑制剂的平台目前不可用，因为焦点通常集中在开发针对单一激酶的有效和选择性抑制剂上。虽然激酶组范围的体外生物化学测定可用于选择性测量，但这些测定在药物发现早期大规模进行成本和时间过高，并且不能充分复制激酶的复杂细胞环境。在我们的I期申请中，我们开发了现有BaF3测定平台的新适应，以直接测量转化激酶下游的信号传导，显著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测定的根本性飞跃，允许该平台与即使是最混杂的抑制剂一起使用，并在药物开发的最早阶段使用，包括二次筛选和先导化合物鉴定。为了能够针对多种激酶进行化合物优化，使用Primont的CellCode技术对新测定进行多重化，使得可以在384孔板的单个孔中分析9种激酶转化的细胞系。对300种激酶抑制剂的筛选揭示了能够同时筛选靶活性和选择性的能力。在第二阶段的申请中，我们建议将该平台扩展到所有90个已知的酪氨酸激酶组成员。这些研究的成功完成将创造第一个全面的，基于细胞的激酶检测平台的选择性 测量、探针开发和下一代多靶向抑制剂的选择。

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.