Generic Fluorescent HTS Assay for Kinases and ATPases

描述（由申请人提供）：人类基因组中有超过500种蛋白激酶和数百种其他酶利用ATP作为磷酸盐供体或能量来源。蛋白激酶是第二密集筛选的靶标类，与所有主要治疗领域都有联系，最明显的是癌症。脂质激酶、碳水化合物激酶和三磷酸腺苷酶也出现了与癌症和抗感染有关的重要疾病联系。尽管它们具有很高的药物相关性，但由于缺乏灵活的高通量筛选（HTS）测定方法，新的ATP依赖性酶靶点快速进入药物发现受到阻碍。这个问题延伸到选择性分析和导联优化的二次筛选工作中。为了满足这一需求，BellBrook实验室开发了一种新的基于荧光的ADP免疫检测方法，ADP是所有激酶和atp酶反应的不变产物。在第一阶段，我们开发了一种单克隆抗体和荧光示踪剂，使用荧光偏振作为读数，能够以比ATP高100倍的选择性检测ADP。我们在2005年11月将ADP检测平台商业化，成为Transcreener激酶检测试剂盒，自那时起，它已经产生了接近25万美元的收入。在第二阶段，我们建议1)通过开发更具选择性的ADP抗体来提高Transcreener Kinase Assay的性能，2)通过开发类似的GDP免疫检测方法，将平台扩展到其他目标家族，包括gpcr， 3)通过将该检测格式为第二种常用的荧光检测模式（TR-FRET）来增加HTS的市场渗透率，4)开发生物反应器杂杂瘤培养方法，以扩大单克隆抗体的生产并减少动物使用。5)利用Transcreener激酶试验的独特功能来测试a)使用生理蛋白底物而不是广泛使用的肽受体进行激酶筛选的重要性；b)激酶抑制剂与其他类型的atp依赖性酶的脱靶效应的潜力。通过为制药公司提供实施更多生理激酶筛选方法的工具和策略，我们希望加速发现癌症和其他使人衰弱的疾病的改进疗法。人类基因组中编码了500多种不同的蛋白激酶蛋白，它们引起了制药公司的强烈兴趣，因为它们与许多类型的癌症、炎症性疾病（如关节炎）和其他使人衰弱的疾病有关。为了加速发现这些疾病的改进疗法，我们正在开发一种能够找到药物分子的筛选方法，这些药物分子将纠正功能失调的激酶蛋白的异常行为。

DESCRIPTION (provided by applicant): There are over 500 protein kinases in the human genome and several hundred other enzymes that utilize ATP as a phosphate donor or energy source. Protein kinases are the second most intensively screened target class, with links to all of the major therapeutic areas, most notably cancer. Important disease links for cancer and anti-infectives also are emerging for lipid kinases, carbohydrate kinases and ATPases. Despite their high pharmaceutical relevance, the rapid movement of new ATP- dependent enzyme targets into drug discovery is being hampered by a lack of flexible high throughput screening (HTS) assay methods. This problem extends into secondary screening efforts for selectivity profiling and lead optimization as well. To address this need, BellBrook Labs has developed a novel fluorescence-based immunodetection method for ADP, the invariant product of all kinase and ATPase reactions. In Phase I, we developed a monoclonal antibody and fluorescent tracer that enable detection of ADP with 100-fold selectivity over ATP using fluorescence polarization as a readout. We commercialized the ADP detection platform as the Transcreener Kinase Assay kit in November, 2005, and it has since generated revenue approaching $250,000. In Phase II we propose to 1) enhance Transcreener Kinase Assay performance by developing a more selective ADP antibody, 2) extend the platform to additional target families, including GPCRs, by developing a similar immunodetection method for GDP, 3) increase HTS market penetration by formatting the assay for a second commonly used fluorescent detection mode, TR-FRET, 4) develop bioreactor hybridoma culture methods to scale up monoclonal antibody production and reduce animal usage, 5) use the unique capabilities of the Transcreener Kinase Assay to test a) the importance of using physiological protein substrates rather than widely used peptide acceptors for kinase screening and b) the potential for off target effects of kinase inhibitors with other types of ATP-dependent enzymes. By providing pharma with the tools and strategies to implement more physiological kinase screening methods, we hope to accelerate the discovery of improved therapies for cancer and other debilitating diseases. There are over 500 distinct protein kinase proteins encoded in the human genome and they have attracted intense interest from pharmaceutical companies because of their links with numerous types of cancer, inflammatory disorders such as arthritis, and other debilitating diseases. To accelerate the discovery of improved therapies for these diseases, we are developing an enabling screening method for finding drug molecules that will correct the aberrant behavior of malfunctioning kinase proteins.