Generic Fluorescent HTS Assay for Kinases and ATPases

描述(申请人提供)：人类基因组中有500多种蛋白激酶，数百种其他酶利用三磷酸腺苷作为磷酸供体或能量来源。蛋白激酶是第二密集筛选的靶类，与所有主要治疗领域有联系，最明显的是癌症。与癌症和抗感染药物有关的重要疾病还包括脂肪酶、碳水化合物酶和三磷酸腺苷。尽管它们与药物有很高的相关性，但由于缺乏灵活的高通量筛选(HTS)分析方法，新的ATP依赖酶靶标的快速移动到药物发现中正受到阻碍。这个问题延伸到了选择性分析和铅优化的二次筛选工作中。为了满足这一需求，贝尔布鲁克实验室开发了一种新的基于荧光的免疫检测方法，用于检测ADP，ADP是所有激酶和ATPase反应的不变产物。在第一阶段，我们开发了一种单抗和荧光示踪剂，可以使用荧光偏振作为读数来检测ADP，其选择性是ATP的100倍。我们于2005年11月将ADP检测平台商业化，成为Transcreener Kinase分析试剂盒，自那以来已产生近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分析的性能，2)通过为GDP开发类似的免疫检测方法来将平台扩展到包括GPCRs在内的其他靶家族，3)通过将该检测方法格式化为第二种常用的荧光检测模式--TRRET来增加HTS的市场渗透率，4)开发生物反应器杂交瘤培养方法以扩大单抗的生产并减少动物的使用，5)使用Transcreener Kinase Assay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多肽受体进行KK筛选的重要性，以及b)KK抑制剂与其他类型的ATP依赖酶一起产生脱靶效应的可能性。通过为制药公司提供工具和策略来实施更多的生理性激酶筛查方法，我们希望加快发现癌症和其他衰弱疾病的改进治疗方法。人类基因组中有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，因为它们与多种类型的癌症、炎症性疾病(如关节炎)和其他令人衰弱的疾病有关。为了加速发现这些疾病的改进治疗方法，我们正在开发一种使人能够筛选的方法，以寻找能够纠正功能失调的激酶蛋白的异常行为的药物分子。

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.