High throughput assay for novel pharmacological probes targeting cAMP signaling

描述(由申请人提供)：cAMP介导的细胞信号在生理和病理条件下调节无数重要的生物过程，包括糖尿病、心力衰竭和癌症。在真核细胞中，cAMP的作用是通过两种细胞内cAMP受体传递的，即经典的蛋白激酶A/cAMP依赖的蛋白激酶(PKA/CAPK)和新近发现的由cAMP/cAMP调节的鸟嘌呤核苷酸交换因子(EPAC/cAMP-Global)直接激活的交换蛋白。与PKA一样，EPAC含有cAMP结合域，这是一个进化上保守的结构基序，可以作为分子开关来检测细胞内第二信使cAMP水平。EPAC的发现为研究cAMP介导的信号转导开辟了一个新的维度，因为PKA和EPAC在所有组织中都普遍表达。细胞内cAMP水平的升高将导致PKA和EPAC的激活。因此，cAMP的净细胞效应不仅取决于PKA或EPAC，而且依赖于EPAC和PKA的动态表达及其在特定组织中的特定亚细胞分布。目前，EPAC的生理功能尚不清楚。该研究领域的主要挑战之一是缺乏EPAC特异性拮抗剂来剖析EPAC在整个cAMP介导的信号转导中所发挥的特定生理功能。这项建议的目的是发展一种高通量筛选(HTS)方法，以发现针对EPAC而不是PKA的新型分子探针。这些EPAC特异的探针将为研究EPAC的生物学功能和cAMP信号的分子机制以及促进对EPAC/cAMP信号相关疾病机制的理解提供有力的药理学工具。 公共卫生相关性：我们建议的研究目标是建立一种高通量筛选试验，以发现新的靶向特异性药物探针，这些探针可用于阐明重要的第二信使cAMP介导的信号转导机制。这一研究项目的医学和药理学意义深远。针对特定cAMP信号成分的新的药理探针可能导致识别与cAMP信号相关的疾病的基于机制的治疗策略。

DESCRIPTION (provided by applicant): cAMP-mediated cell signaling regulates a myriad of important biological processes under both physiological and pathological conditions, including diabetes, heart failure, and cancer. In eukaryotic cells, the effects of cAMP are transduced by two intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (Epac/cAMP-GEF). Like PKA, Epac contains a cAMP-binding domain, an evolutionally conserved structural motif that acts as a molecular switch for sensing intracellular second messenger cAMP levels. The discovery of Epac has opened a new dimension in studying the cAMP-mediated signaling as both PKA and Epac are ubiquitously expressed in all tissues. An increase in intracellular cAMP levels will lead to the activation of both PKA and Epac. Therefore, the net cellular effects of cAMP are not just dictated by PKA or Epac alone, but dependent upon the dynamic expression of Epac and PKA and their specific subcellular distribution in a particular tissue. Currently, the physiological functions of Epac are not clear. One of the major challenges within the research field is the lack of Epac-specific antagonists to dissect the specific physiological functions that Epac play in the overall cAMP-mediated signaling. The objective of this proposal is to develop a High Throughput Screening (HTS) assay for the discovery of novel molecular probes that are specific for Epac but not PKA. These Epac-specific probes will be powerful pharmacological tools for investigating the biological functions of Epac and molecular mechanism of cAMP signaling as well as, for promoting an understanding of disease mechanisms related to Epac/cAMP signaling. PUBLIC HEALTH RELEVANCE: The goal of our proposed research is to develop a High Throughput Screening assay for the discovery of novel target-specific pharmacological probes that can be used for elucidating the mechanism of signal transduction mediated by an important second messenger, cAMP. The medical and pharmacological implications of this research program are far-reaching. Novel pharmacological probes targeting specific cAMP signaling components can potentially lead to the identification of mechanism-based therapeutic strategies for diseases associated with cAMP signaling.