Targeting Epac synergistic component in cAMP signaling

描述（由申请人提供）：camp依赖信号调节多种生理反应，其解除参与许多病理条件。因此，它代表了多年来药物发现的主要焦点；然而，由于担心潜在的相关副作用，这种信号通路的普遍性也给药物开发带来了负担。因此，该领域迫切需要开发新的特异性靶向方法。目前的建议建立在ERM成员Radixin与cAMP效应物（即Epac1和PKA）的新复合物的鉴定以及下游底物Rap1作为集成节点的作用的基础上。cAMP效应物通过epac1介导的激活和PKA依赖的Rap1磷酸化协同作用，促进cAMP依赖的细胞增殖。在机械上，这两种效应器在一个新的亚膜腔室中共定位，形成一个以Radixin为支架单元的三元配合物。破坏这种区隔化的操作可以消除camp介导的增殖。有趣的是，组成活性Rap1的表达，但仅以其磷酸化形式（G12V-S179D）解除了这种抑制，从而证实了Rap1作为cAMP效应通路的信号整合器单元的作用。本提案的总体假设是，协同作用的Radixin-Epac1-Rap1组分代表了药物干预的有效靶点，为实现更高的特异性提供了新的理论依据。我们已经描述了一个完整的试点筛选程序，以表征Epac1-Radixin相互作用的小分子抑制剂，作为评估这一假设的一部分。我们将在三个方面完成这项任务：1)开发和优化hts兼容的荧光偏振检测方法，以监测Epac1-Radixin的相互作用；2)利用收集到的生物活性化合物，在qHTS格式中验证该偏振法；3)实施一组正交、二次和后续试验，以评估初次阳性命中的意义。初步研究验证了这一概念，并为其可行性提供了原理证明。我们认为，我们的研究的成功完成将为鉴定新的选择性药物提供有效的分析方法，这些药物可作为药理学探针，用于解决机制方面的问题，并研究Radixin-Epac1在cAMP信号传导中的功能作用，最终可能导致未来针对cAMP依赖性超增性疾病和其他epac1介导的疾病的新药理学策略。我们的研究计划符合PAR-13-364的所有规范，题为“开发用于探针和治疗前发现的高通量筛选检测方法”，即开发针对新的特定生物靶点（即Epac1-Radixin相互作用）的检测方法，并与已建立的HTS中心（即Dr . Inglese， NCATS， NIH）建立合作。在当前提案中描述的试点活动完成后，qHTS和验证分析将通过快速通道进入机制提交给分子文库计划（MLP）中的NIH分子文库探针生产中心网络（MPLCN）。

DESCRIPTION (provided by applicant): cAMP-dependent signaling regulates multiple physiological responses and its deregulation is involved in many pathological conditions. As such, it represented for many years a major focus for drug discovery; however, the ubiquitous nature of this signaling pathway also presented a burden for drug development due to fear of potential associated side effects. Thus, the development of new approaches for specific targeting is highly needed in the field. The current proposal builds upon the identification of a new complex involving the ERM member Radixin with cAMP effectors, i.e. Epac1 and PKA, and the role of the downstream substrate Rap1 as an integration node. cAMP effectors act synergistically, via Epac1-mediated activation and PKA- dependent phosphorylation of Rap1, to promote cAMP-dependent cell proliferation. Mechanistically, both effectors co-localize in a new sub-membrane compartment, forming a ternary complex with Radixin as the scaffolding unit. Maneuvers that disrupt this compartmentalization abrogate cAMP-mediated proliferation. Interestingly, expression of constitutively active Rap1 but only in its phosphorylated form (G12V-S179D) rescues this inhibition, thus confirming the role of Rap1 as a signal integrator unit of cAMP effector pathways. The overall hypothesis of this proposal is that the synergistic Radixin-Epac1-Rap1 component represents a valid target for pharmacological intervention providing a new rationale towards achieving higher specificity. We have delineated a full pilot screen program to characterize small molecule inhibitors of Epac1-Radixin interaction as part of the assessment of this hypothesis. We will accomplish this task in three integrated aims: 1) To develop and optimize HTS-compatible fluorescence polarization assays to monitor Epac1-Radixin interaction; 2) To validate this polarization assay in qHTS format utilizing a collection o bioactive compounds, and 3) To implement a set of orthogonal, secondary and follow-up assays to assess the significance of primary positive hits. Preliminary studies validate the concept and provide proof-of-principle for its feasibility. We submit that the successful completion of our studies will provide validated assays for the identification of new, selective drugs useful as pharmacological probes for addressing mechanistic aspects and investigating the functional role of Radixin-Epac1 in cAMP signaling that could eventually lead to future new pharmacologic strategies in cAMP-dependent hyperproliferative and other Epac1-mediated disorders. Our research proposal fulfills all of the specifications of PAR-13-364, entitled "Development of Assays for High- Throughput Screening for Use in Probe and Pre-therapeutic Discovery" namely, developing assays for a new specific biological target (i.e. Epac1-Radixin interaction) and establishing collaboration with established HTS centers (i.e. Dr Inglese, NCATS, NIH). Upon completion of the pilot campaign delineated in the current proposal, the qHTS and validation assays will be submitted via a Fast Track entry mechanism to the NIH Molecular Libraries Probe Production Centers Network (MPLCN) in the Molecular Libraries Program (MLP).