Enhanced ADP Immunoassay for Multimode Kinase Detection

描述（由申请人提供）：激酶是最密集筛选的一类用于治疗干预的酶，因为它们在调节其他生物分子的功能中起核心作用。开发选择性激酶抑制剂的策略正在迅速发展，从而产生了对在高通量筛选活动中测量激酶活性的更灵活方法的需求。具体而言，迫切需要能够适应跨越生理范围的ATP浓度的通用测定。在单独的SBIR资助下，贝尔布鲁克Labs开发并商业化了一种称为Transcreener（R）的新型激酶酶测定方法，该方法依赖于ADP的选择性免疫检测，因此能够检测任何激酶，而不管受体底物。该检测方法已被制药行业广泛采用，并获得领先的药物发现服务提供商的许可，为BellBook提供了重要的收入来源。尽管其技术优势和商业成功，该方法的显著缺点包括需要调节抗体浓度以适应不同的ADP水平，以及由于测定的竞争性质而响应ADP的负信号。为了克服这些局限性，我们将与加州大学戴维斯分校的布鲁斯汉莫克博士合作，开发特异性结合ADP抗体免疫复合物的噬菌体携带的肽。在三价免疫复合物中使用这些噬菌体-肽预计将增加ADP检测相对于ATP的选择性，从而克服Transcreener平台的主要限制。此外，所得到的非竞争性免疫测定，称为噬菌体抗免疫复合物（PHAIA），将产生与均质（例如，TR-FRET）或固相（例如，ELISA）形式，从而极大地扩展了该方法的灵活性和适用性。公共卫生相关性：激酶是人类最大的蛋白质家族之一，其功能障碍与多种疾病有关，尤其是癌症、炎症相关疾病。该提案旨在产生用于鉴定可用作治疗激酶相关疾病的药物的选择性激酶抑制剂的改进工具。

DESCRIPTION (provided by applicant): Kinases are the most intensively screened class of enzymes being targeted for therapeutic intervention because of their central role in regulating the function of other biomolecules. Strategies for developing selective kinase inhibitors are evolving rapidly, creating a demand for more flexible methods for measuring kinase activity in high throughput screening campaigns. Specifically, generic assays that can accommodate ATP concentrations spanning the physiological range are badly needed. Under separate SBIR funding, BellBrook Labs developed and commercialized a novel kinase enzyme assay method called Transcreener(R) that relies on selective immunodetection of ADP, thus enables detection of any kinase regardless of the acceptor substrate. The assay has been widely adopted by pharmaceutical industry, and licensed by leading drug discovery service providers, providing a significant revenue stream to BellBook. Despite its technical advantages and commercial success, significant drawbacks of the method include the need to adjust antibody concentration to accommodate different ADP levels, and a negative signal in response to ADP because of the competitive nature of the assay. To overcome these limitations, we will collaborate with Dr. Bruce Hammock at University of California, Davis to develop phage-borne peptides that bind specifically to the ADP-antibody immune complex. The use of these phage-peptides in a trivalent immune complex is expected to increase the selectivity for ADP detection vs. ATP, thereby overcoming the major limitation with the Transcreener platform. In addition, the resulting noncompetitive immunoassay, called a Phage Anti-Immune Complex (PHAIA), will generate a signal that is directly proportional to product formation in either homogenous (e.g., TR-FRET), or solid phase (e.g., ELISA) formats, thus greatly extending the flexibility and applicability of the method. PUBLIC HEALTH RELEVANCE: Kinases are one of the largest families of proteins in humans, and their malfunction is involved in many types of diseases, especially cancers, inflammation-related diseases. This proposal seeks to produce improved tools for identifying selective kinase inhibitors that can be used as drugs to treat kinase-related diseases.