Human Protein Kinase Microarrays

描述(申请人提供)：蛋白激酶是细胞内反应的中心调节器，并构成第二大药物靶标类别。我们已经开发了一种微孔格式，用于在水介质中展示固定的蛋白质，以便可以在单个显微镜载玻片上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，我们生产了大量的活性激酶，并在这些微孔中展示了20纳升体积的激酶分析。在这个第二阶段的应用中，我们建议将这一程序扩展到制备基本上完整的人蛋白激酶和蛋白激酶催化结构域的微孔阵列，并开发这些阵列用于定量和全面的抑制分析。我们将通过对照一组已知的激酶抑制剂测试阵列来证明阵列用于抑制剂特异性分析的实用性，并将对阵列中存在的每一种激酶进行功能表征。全面的激酶抑制图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.