Kinome Drug Bioassay Platform

描述（由申请人提供）：Reaction Biology Corporation（RBC）开发了一种成本极低的纳升反应微阵列，以满足高通量筛选（HTS）和化合物选择性分析的各种药物发现需求。这些微阵列可以容纳超过6000个单独的反应。每个反应比典型孔板形式中使用的反应小1000至10，000倍。激酶在癌症、炎症、糖尿病等疾病的信号转导和调节过程中起着关键作用，然而，寻找选择性激酶抑制剂仍然是一个挑战。在第一阶段，RBC使用微阵列HTS优化了超过10种酪氨酸激酶测定和5种丝氨酸/苏氨酸激酶测定。通过对LOPAC文库的筛选，已经验证了微阵列的激酶HTS。此外，在第一阶段开发的几种磷酸酶测定方法已准备好用于大规模HTS活动。在第二阶段，我们建议扩展这项技术，增加额外的30种激酶（目标1）。将在孔板和微阵列格式中使用来自LOPAC文库和商业供应商的已知化合物评价针对这些靶标的IC 50谱。在目标2中，RBC将开发一种通用的基于放射性同位素33 P的激酶测定法，以实现每10 μ g纯化激酶100，000次反应。在目标3中，HTS活动使用79，000种不同结构的化合物库针对RBC激酶和磷酸酶组，将建立一个数据库以驱动结构-活性关系（SAR）模型。通过第二阶段的资助，RBC将验证这一激酶/磷酸酶HTS平台，以服务于寻求快速分析大型文库或领先系列的客户，这些文库或领先系列针对从RBC检测集选择的众多激酶。使用少量纯化蛋白质快速实现专有客户激酶的HTS也是一个重要的机会。

DESCRIPTION (provided by applicant): Reaction Biology Corporation (RBC) has developed an extremely low cost nanoliter reaction microarray to serve various drug discovery needs for high throughput screening (HTS) and compound selectivity profiling. These microarrays can hold more than 6000 individual reactions. Each reaction is 1000 to 10,000-fold smaller than those used in typical well plate formats. Kinases play a pivotal role in signal transduction and regulation of processes in cancer, inflammation, diabetes etc. However, finding selective kinase inhibitors remains a challenge. In Phase I, RBC optimized over 10 tyrosine kinase assays and 5 serine/threonine kinase assays using microarray HTS. The microarrays have been validated for kinase HTS by screening against the LOPAC library. Additionally, several phosphatase assay methodologies developed during Phase I are ready for large scale HTS campaigns. In Phase II, we propose to expand this technology to add an additional 30 kinases (Aim 1). IC50 profiling against these targets will be evaluated in both well plate and microarray format with known compounds from the LOPAC library and commercial vendors. In Aim 2, RBC will develop a universal radioisotope 33P-based kinase assay to achieve 100,000 reactions per 10 ug of purified kinase. In Aim 3, HTS campaigns using a 79,000 compound library of diverse structures against the RBC panel of kinases and phosphatases will establish a database to drive structure-activity relationship (SAR) models. Through Phase II funding, RBC will validate this kinase/phosphatase HTS platform to serve customers seeking to rapidly profile large libraries or lead series against numerous kinases chosen from the RBC assay set. Rapid HTS implementation for proprietary customer kinases using small amounts of purified protein is also a significant opportunity.