Enhanced ADP Immunoassay for Multimode Kinase Detection

说明(申请人提供)：由于其在调节其他生物分子功能方面的中心作用，激酶是筛选最多的一类酶，被作为治疗干预的目标。开发选择性激酶抑制剂的策略正在迅速演变，这就产生了对在高通量筛查活动中测量激酶活性的更灵活方法的需求。具体地说，迫切需要能够适应生理范围内ATP浓度的通用检测方法。在单独的SBIR资助下，BellBrook实验室开发了一种名为Transcreener(R)的新的激酶酶分析方法，并将其商业化，该方法依赖于ADP的选择性免疫检测，从而能够检测任何激酶，而不受受体底物的影响。该检测方法已被制药行业广泛采用，并获得了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。尽管该方法具有技术优势和商业上的成功，但它的显著缺点包括需要调整抗体浓度以适应不同的ADP水平，以及由于该检测的竞争性，对ADP的反应是负面的信号。为了克服这些限制，我们将与加州大学戴维斯分校的Bruce Hammock博士合作开发能够与ADP抗体免疫复合体特异结合的噬菌体携带的多肽。在三价免疫复合体中使用这些噬菌体多肽有望提高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，由此产生的非竞争性免疫分析，称为噬菌体抗免疫复合体(PHAIA)，将产生与均相(例如，tr-fret)或固相(例如，ELISA)形式的产物直接成比例的信号，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，它们的功能障碍与许多类型的疾病有关，特别是癌症和炎症相关疾病。这项提案寻求改进工具，以确定可用作治疗与激酶相关疾病的药物的选择性激酶抑制剂。

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.