New Molecular Probes for Protein Kinases

描述（由申请人提供）：可逆蛋白磷酸化级联反应是细胞处理细胞外信息并将这些信号转化为复杂表型反应（如生长、分化、粘附和运动）的主要手段。适当的空间和时间控制蛋白激酶的细胞内磷酸化事件是必要的正常细胞功能。事实上，蛋白激酶活性的错误调控是许多疾病的原因或后果，包括癌症、糖尿病和慢性炎症。由于这个原因，蛋白激酶是制药工业开发新疗法最高度追求的目标之一。尽管人们对激酶细胞功能的测定和激酶靶向药物的开发广泛感兴趣，但这一多样化酶家族的大多数成员仍然缺乏特征。造成这一缺陷的一个主要原因是，在复杂的生物混合物中，仍然缺乏对蛋白激酶进行全局分析的技术。本提案研究的总体目标是开发一套通用的分子工具，使蛋白激酶的功能状态能够在细胞裂解物和活细胞中被描述。提出的研究的具体目标是：(1)开发一组活性位点导向的光亲和探针，允许激酶超家族的全局分析。(2)开发一套通用的检测方法来分析在Specific Aim 1中开发的探针，并使用这些检测方法来确定几种临床相关激酶抑制剂的选择性。(3)开发一套通用的分子探针，可以选择性地结合蛋白激酶的活性或非活性构象。如果成功，这些工具将为确定生理相关环境中激酶抑制剂的细胞靶点和识别人类疾病中的异常激酶活性提供一种通用方法。

DESCRIPTION (provided by applicant): Reversible protein phosphorylation cascades are the dominant means by which cells process extracellular information and convert these signals into complex phenotypic responses such as growth, differentiation, adhesion and motility. The proper spatial and temporal control of intracellular phosphorylation events by protein kinases is essential for normal cellular function. Indeed, mis-regulation of protein kinase activity is a cause or consequence of a number of diseases, including cancer, diabetes and chronic inflammation. For this reason, protein kinases are one of the most highly pursued targets for the development of new therapeutics by the pharmaceutical industry. Despite widespread interest in the determination of kinase cellular function and in the development of kinase-targeted pharmacological agents, most members of this diverse enzyme family remain poorly characterized. A major reason for this shortcoming is that there remains a paucity of techniques for the global analysis of protein kinases in complex biological mixtures. The overall goal of the research in this proposal is to develop a general set of molecular tools that will allow the functional state of protein kinases to be profiled in cell lysates and living cells. The Specific Aims of the proposed research are: (1) To develop a panel of active site-directed, photo-affinity probes that allow global profiling of the kinase superfamily. (2) To develop a general set of assays for the analysis of probes developed in Specific Aim 1 and to use these assays to determine the selectivity of several clinically relevant kinase inhibitors. (3) To develop a general set of molecular probes that selectively bind to either the active or inactive conformation of protein kinases. If successful, these tools will provide a general method for determining the cellular targets of kinase inhibitors in physiologically relevant environments and for identifying aberrant kinase activities in human disease. PUBLIC HEALTH RELEVANCE: The mis-regulation of protein kinase activity is a cause or consequence of a number of diseases, including cancer, diabetes and chronic inflammation, which has made members of this diverse enzyme family targets for the development of new therapeutics. The goal of our proposed research is to develop new chemical tools for the molecular analysis of protein kinases in cellular lysates and in living cells. These reagents should allow us to gain a greater understanding of kinase function and aid in the discovery of new targets for the development of therapeutics.