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，是第二大药物靶标类别。我们开发了一种微孔格式，用于在水介质中显示固定的蛋白质，这样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、抑制剂或结合剂进行分离的反应。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II期申请中，我们建议扩展这一过程，以制备基本完整的人类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。我们将通过对一组已知的激酶抑制剂测试阵列来展示阵列对抑制剂特异性分析的效用，并将对阵列中存在的每个激酶进行功能表征。全面的激酶抑制谱分析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.