Human Protein Kinase Microarrays

描述（由申请人提供）：蛋白激酶是细胞内反应的中心调节因子，构成第二大药物靶点类别。我们已经开发了一种微孔格式，用于显示在水性介质中的固定化蛋白质，使得可以在单个显微镜载玻片上以低体积与许多不同的蛋白质、底物或抑制剂或结合剂进行单独的分离反应。在第一阶段，我们产生了大量的活性激酶，并在这些微孔中证明了20纳升体积的激酶测定。在这个第二阶段的应用中，我们建议扩展这一程序，以制备微孔阵列基本上是整个补充的人蛋白激酶和蛋白激酶催化结构域，并开发这些阵列的定量和全面的抑制测定。我们将通过测试阵列对一组已知激酶抑制剂的抑制剂特异性分析来证明阵列的实用性，并将对阵列中存在的每种激酶进行功能表征。全面的激酶抑制谱将显著提高激酶药物开发的有效性和效率。

DESCRIPTION (provided by applicant): Protein kinases are central regulators of intracellular responses and constitute the second largest drug target class. We have developed a microwell format for displaying immobilized proteins in an aqueous medium such that separate isolated reactions can be carried out with many different proteins, substrates, or inhibitors, or binding agents in low volumes on a single microscope slide. In Phase I, we produced significant numbers of active kinases and demonstrated 20 nanoliter volume kinase assays in these microwells. In this Phase II application, we propose to extend this procedure to prepare microwell arrays of essentially the entire complement of human protein kinases and protein kinase catalytic domains, and to develop these arrays for quantitative and comprehensive inhibition assays. We will demonstrate the utility of the arrays for inhibitor specificity profiling by testing the array against a panel of known kinase inhibitors, and will perform functional characterizations of each kinase present in the array. Comprehensive kinase inhibition profiling will significantly enhance the effectiveness and efficiency of kinase drug development.