Targeting Epac synergistic component in cAMP signaling

靶向 cAMP 信号传导中的 Epac 协同成分

• 批准号: 9115657
• 负责人: DANIEL L ALTSCHULER
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115657
• 关键词: Address; Adverse effects; Biological; Biological Assay; Biology; Cell Proliferation; Cells; Collaborations; Collection; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Development; Disease; Evaluation; Fluorescence Polarization; Fright; Future; Generations; Goals; Health; Hormones; In Vitro; Intervention; Lead; Libraries; Mediating; Membrane; Molecular Bank; Monitor; Nature; Nuclear Translocation; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Physiological; Physiology; Production; Reproducibility; Research Proposals; Robotics; Role; Scaffolding Protein; Second Messenger Systems; Signal Pathway; Signal Transduction; Specificity; Statistical Data Interpretation; Therapeutic; Triage; United States National Institutes of Health; Validation; assay development; base; cell type; cytotoxicity; drug development; drug discovery; follow-up; high throughput screening; inhibitor/antagonist; member; miniaturize; novel strategies; programs; public health relevance; radixin protein; response; scaffold; screening; second messenger; small molecule inhibitor; synergism

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).

描述（由申请人提供）：依赖CAMP的信号传导调节多种生理反应及其放松管制在许多病理条件下涉及。因此，它代表了多年来的主要重点。但是，由于担心潜在的相关副作用，该信号通路的普遍性也给药物发育带来了负担。因此，在现场非常需要开发针对特定靶向的新方法。当前的提案建立在鉴定涉及ERM成员radixin的新综合体中，即cAMP效应子，即ePAC1和PKA，以及下游基板RAP1作为集成节点的作用。 cAMP效应子通过EPAC1介导的激活和RAP1的PKA依赖性磷酸化协同作用，以促进依赖CAMP的细胞增殖。从机械上讲，两个效应子在新的亚膜室中共定位，形成了与radixin作为脚手架单元的三元复合物。破坏这种隔室化的操纵消除了营地介导的增殖。有趣的是，组成型活性RAP1的表达，但仅以其磷酸化形式（G12V-S179D）挽救了这种抑制作用，从而证实了RAP1作为cAMP效应途径的信号积分单位的作用。该提案的总体假设是协同的radixin-epac1-rap1成分代表了药理学干预的有效目标，从而为实现更高的特异性提供了新的理由。我们已经描述了一个完整的试点屏幕程序，以表征EPAC1-脱脂蛋白相互作用的小分子抑制剂，作为评估该假设的一部分。我们将以三个综合目的完成此任务：1）开发和优化与HTS兼容的荧光极化测定法以监测EPAC1-RADIXIN相互作用； 2）使用收集O生物活性化合物验证QHTS格式的这种极化测定，以及3）实施一组正交，次级和后续测定，以评估原发性正命中的重要性。初步研究验证了该概念并为其可行性提供原则证明。我们认为，我们的研究成功完成将为鉴定新的选择性药物提供验证的测定法，可作为药物学探针来解决机理方面，并研究Radixin-EPAC1在CAMP信号中的功能作用，从而最终会导致未来的新药理策略在CAMP依赖性超增殖性和EPAC1介导的疾病中。 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).在当前提案中划定的试点运动完成后，QHT和验证测定将通过快速轨道进入机制提交给NIH分子图书馆探测生产中心网络（MPLCN）在Molecular Libraries计划（MLP）中。