New Molecular Probes for Protein Kinases

描述（由申请人提供）：可逆蛋白磷酸化级联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）的主要方式。蛋白激酶对细胞内磷酸化事件的适当空间和时间控制对于正常细胞功能是必不可少的。事实上，蛋白激酶活性的错误调节是许多疾病的原因或结果，包括癌症、糖尿病和慢性炎症。出于这个原因，蛋白激酶是制药工业开发新疗法的最高度追求的靶标之一。尽管在激酶细胞功能的测定和激酶靶向药物的开发方面有着广泛的兴趣，但这种多样化的酶家族的大多数成员仍然缺乏特征。这一缺点的一个主要原因是，仍然缺乏技术的蛋白激酶在复杂的生物混合物的全球分析。这项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进行分析。本研究的具体目标是：（1）开发一组活性位点导向的光亲和探针，用于激酶超家族的全局分析。(2)开发一套通用检测方法，用于分析特定目标1中开发的探针，并使用这些检测方法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，选择性地结合蛋白激酶的活性或非活性构象。如果成功，这些工具将提供一个通用的方法，用于确定激酶抑制剂在生理相关环境中的细胞靶点，并用于识别人类疾病中的异常激酶活性。 公共卫生相关性：蛋白激酶活性的错误调节是许多疾病（包括癌症、糖尿病和慢性炎症）的原因或结果，这使得该多样化酶家族的成员成为开发新疗法的靶点。我们提出的研究目标是开发新的化学工具，用于细胞裂解物和活细胞中蛋白激酶的分子分析。这些试剂应该使我们能够更好地了解激酶的功能，并有助于发现新的治疗靶点。

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.