New Molecular Probes for Protein Kinases

描述(申请人提供)：可逆的蛋白质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的表型反应(如生长、分化、黏附和运动)的主要方式。蛋白激酶对细胞内磷酸化事件的时空调控对于细胞的正常功能至关重要。事实上，蛋白激酶活性的错误调节是许多疾病的原因或后果，包括癌症、糖尿病和慢性炎症。因此，蛋白激酶是制药业开发新疗法最热门的靶点之一。尽管人们对确定激酶的细胞功能和开发以激酶为靶点的药物很感兴趣，但这个不同的酶家族中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仍然缺乏对复杂生物混合物中的蛋白激酶进行全球分析的技术。这项研究的总体目标是开发一套通用的分子工具，允许在细胞裂解物和活细胞中分析蛋白激酶的功能状态。这项拟议研究的具体目标是：(1)开发一组活性位点定向的、光亲和的探针，允许对激酶超家族的全球图谱进行分析。(2)开发一套通用的分析针对特定目标1开发的探针的分析方法，并使用这些分析方法来确定几种临床相关的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构象或非活性构象。如果成功，这些工具将提供一种通用的方法，用于在生理相关的环境中确定激酶抑制剂的细胞靶标，并识别人类疾病中的异常激酶活性。 与公共卫生相关：蛋白激酶活性的错误调节是许多疾病的原因或后果，包括癌症、糖尿病和慢性炎症，这使得这种不同的酶家族的成员成为开发新疗法的目标。我们提出的研究的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。这些试剂应该可以让我们更好地了解激酶的功能，并有助于发现治疗发展的新靶点。

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.