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）：cAMP依赖性信号传导调节多种生理反应，其失调涉及许多病理状况。因此，它多年来一直是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存在也给药物开发带来了负担。因此，该领域迫切需要开发特异性靶向的新方法。目前的建议是建立在一个新的复杂的识别，涉及ERM成员Radixin与cAMP效应，即Epac 1和PKA，和下游底物Rap 1作为整合节点的作用。cAMP效应子通过Epac 1介导的激活和Rap 1的PKA依赖性磷酸化协同作用，以促进cAMP依赖性细胞增殖。从机制上讲，两种效应物共定位在一个新的亚膜区室，形成一个三元复合物与Radixin作为支架单元。破坏这种区室化的操作消除cAMP介导的增殖。有趣的是，组成型活性Rap 1的表达，但仅在其磷酸化形式（G12 V-S179 D）拯救这种抑制，从而证实了Rap 1作为cAMP效应途径的信号整合单位的作用。该提议的总体假设是协同作用的Radixin-Epac 1-Rap 1组分代表了药理学干预的有效靶标，为实现更高的特异性提供了新的理论基础。我们已经描绘了一个完整的试点筛选程序，以表征小分子抑制剂的Epac 1-Radixin相互作用的评估这一假设的一部分。我们将在三个综合目标中完成该任务：1）开发和优化HTS相容的荧光偏振测定以监测Epac 1-Radixin相互作用; 2）利用生物活性化合物的集合以qHTS形式验证该偏振测定;和3）实施一组正交、次级和后续测定以评估初级阳性命中的显著性。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提供有效的检测方法，用于鉴定新的，选择性的药物，可作为药理学探针，用于解决机制方面和研究Radixin-Epac 1在cAMP信号传导中的功能作用，最终可能导致未来新的药理学策略在cAMP依赖性过度增殖和其他Epac 1介导的疾病。我们的研究提案符合PAR-13-364的所有规范，标题为“开发用于探针和治疗前发现的高通量筛选测定法”，即开发新的特异性生物靶点（即Epac 1-Radixin相互作用）的测定法，并与已建立的HTS中心（即Inglese博士，NCATS，NIH）建立合作。完成当前提案中描述的试点活动后，qHTS和验证试验将通过快速通道进入机制提交至分子库计划（MLP）中的NIH分子库探针生产中心网络（MPLCN）。