High throughput assay for novel pharmacological probes targeting cAMP signaling

描述（由申请人提供）：cAMP 介导的细胞信号传导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 的作用由两种细胞内 cAMP 受体转导，即经典的蛋白激酶 A/cAMP 依赖性蛋白激酶 (PKA/cAPK) 和最近发现的由 cAMP/cAMP 调节的鸟嘌呤核苷酸交换因子直接激活的交换蛋白 (Epac/cAMP-GEF)。与 PKA 一样，Epac 包含一个 cAMP 结合域，这是一种进化上保守的结构基序，可作为检测细胞内第二信使 cAMP 水平的分子开关。 Epac 的发现为研究 cAMP 介导的信号传导开辟了新的维度，因为 PKA 和 Epac 在所有组织中都普遍表达。细胞内 cAMP 水平的增加将导致 PKA 和 Epac 的激活。因此，cAMP 的净细胞效应不仅由 PKA 或 Epac 单独决定，还取决于 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 介导的信号传导中发挥的特定生理功能。该提案的目的是开发一种高通量筛选 (HTS) 检测方法，以发现针对 Epac 但不针对 PKA 的新型分子探针。这些 Epac 特异性探针将成为强大的药理学工具，用于研究 Epac 的生物学功能和 cAMP 信号传导的分子机制，以及促进对 Epac/cAMP 信号传导相关疾病机制的理解。 公共健康相关性：我们提出的研究的目标是开发一种高通量筛选方法，以发现新型靶标特异性药理学探针，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。该研究项目的医学和药理学意义是深远的。针对特定 cAMP 信号传导成分的新型药理学探针可能有助于识别与 cAMP 信号传导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.