High-throughput Fluorescent Protein Kinase Assays

描述(申请人提供)：蛋白激酶催化多肽和蛋白质中丝氨酸(Ser)、苏氨酸(Thr)和酪氨酸(Tyr)残基的磷酸化，是真核生物中最大的超家族酶，在人类基因组中估计有500个基因。因为大多数细胞内的过程都是由蛋白质磷酸化来调节的，所以蛋白质激酶的失调会导致多种疾病状态。因此，蛋白激酶是药物发现和开发的重要靶点。以蛋白激酶为靶点的药物发现需要快速筛选化合物的能力，以确定其特定抑制选定的蛋白激酶的能力。为了便于高通量筛选激酶活性，我们将使用犹他大学开发的专有荧光技术开发微板蛋白激酶分析。首先，我们将制备两组荧光标记的合成肽。其中一套将用于检测蛋白质丝氨酸/苏氨酸蛋白激酶，另一套将用于检测蛋白质酪氨酸激酶。其次，我们将优化每个蛋白激酶检测系统，以用于标准的96孔和384孔荧光微孔板阅读器。根据外部评估人员的反馈，我们将优化和开发这些蛋白激酶分析试剂盒的商业版本，出售给研究社区和参与药物发现和开发的公司。

DESCRIPTION (provided by applicant): Protein kinases catalyze the phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues in peptides and proteins, and comprise the largest superfamily of enzymes in eukaryotes with an estimated 500 genes in the human genome. Because most intracellular processes are regulated by protein phosphorylation, dysregulation of protein kinases can lead to a variety of disease states. Protein kinases are therefore important targets for drug discovery and development. Protein kinase-targeted drug discovery requires the ability to rapidly screen compounds for their ability to specifically inhibit selected protein kinases. To facilitate the high-throughput screening of kinase activities we will develop microplate protein kinase assays using a proprietary fluorescence technology developed at the University of Utah. First, we will prepare two sets of fluorescently labeled synthetic peptides. One set will be used to assay protein Ser/Thr kinases, the other for protein Tyr kinases. Second, we will optimize each protein kinase assay system for use in standard 96-well and 384-well fluorescence microplate readers. Based on feedback from outside evaluators, we will optimize and develop commercial versions of these protein kinase assay kits to be sold to the research community and companies involved in drug discovery and development.