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，并且其失调涉及许多病理状况。因此，多年来它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性也给药物开发带来了负担。因此，该领域非常需要开发针对特定目标的新方法。目前的提案建立在对涉及 ERM 成员 Radixin 与 cAMP 效应子（即 Epac1 和 PKA）的新复合物的识别以及下游底物 Rap1 作为整合节点的作用的基础上。 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，促进 cAMP 依赖性细胞增殖。从机制上讲，两个效应器共定位在一个新的亚膜室中，形成以 Radixin 作为支架单元的三元复合物。破坏这种区室化的操作会消除 cAMP 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，从而证实了 Rap1 作为 cAMP 效应器途径的信号整合单元的作用。该提案的总体假设是，协同 Radixin-Epac1-Rap1 成分代表了药物干预的有效目标，为实现更高的特异性提供了新的原理。作为评估这一假设的一部分，我们已经描绘了一个完整的试点筛选计划来表征 Epac1-Radixin 相互作用的小分子抑制剂。我们将通过三个综合目标来完成这项任务：1）开发和优化 HTS 兼容的荧光偏振测定法以监测 Epac1-Radixin 相互作用； 2) 利用生物活性化合物集合以 qHTS 格式验证该极化测定，以及 3) 实施一组正交、二次和后续测定，以评估主要阳性命中的显着性。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，我们研究的成功完成将为鉴定新的选择性药物提供有效的检测方法，这些药物可用作药理学探针，以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依赖性过度增殖和其他Epac1介导的疾病的新药理学策略。我们的研究提案满足 PAR-13-364 的所有规范，题为“用于探针和治疗前发现的高通量筛选测定的开发”，即开发新的特定生物靶标（即 Epac1-Radixin 相互作用）的测定并与已建立的 HTS 中心（即 Inglese 博士、NCATS、NIH）建立合作。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至分子库计划 (MLP) 中的 NIH 分子库探针生产中心网络 (MPLCN)。