权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Novel tools for identifying GPCR compounds modulating specific sub-cellular ERK a

用于识别调节特定亚细胞 ERK a 的 GPCR 化合物的新工具

基本信息

批准号：
8589496
负责人：
Haifeng Eishingdrelo
金额：
$ 27.87万
依托单位：
BIOINVENU CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8589496
关键词：
Address Agonist Animal Model Antibodies Antipsychotic Agents Arrestins Behavior Biological Assay Bipolar Disorder Brain Cell Line Cell Nucleus Cells Clinic Custom Cytoplasm Cytoplasmic Protein Data Dopamine Dopamine D2 Receptor Drug Industry Drug Kinetics Etorphine Fees G Protein-Coupled Receptor Genes GPCR Signaling Pathway GTP-Binding Proteins Importins Inhibitory Concentration 50 Lead Learning Letters Libraries Licensing Ligands Link Lithium Location Long-Term Potentiation MAPK1 gene Major Depressive Disorder Marketing Measures Mediating Memory Mental Health Mental disorders Methods Mitogen-Activated Protein Kinases Modeling Mood stabilizers Moods Morphine NIH Program Announcements National Institute of Mental Health Nuclear Nuclear Protein Opioid Receptor Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Phase Phosphorylation Physiological Play Property Protein Isoforms Reporter Reporting Research Research Proposals Role Schizophrenia Services Signal Pathway Signal Transduction Signal Transduction Pathway Synaptic plasticity System Technology To specify arrestin 1 assay development base drug candidate drug discovery drug efficacy experience in vivo interest novel preference programs protein activation protein protein interaction public health relevance receptor response screening tool transcription factor valproate

项目摘要

DESCRIPTION (provided by applicant): We plan to develop novel tools for assessing GPCR ligands modulating G-protein-dependent and ?-arrestin- dependent ERK signaling pathways. Many GPCRs display a phenomenon in which different ligands can differentially activate different signaling pathways via the same receptor. It has become clear that single signaling pathway approaches for identifying drug candidates are not adequately suited to detect the full repertoire of compounds that may have other signaling pathway activities beyond the target receptor. One has to examine multiple signaling pathways to link pathway activities to physiologic functions. ERK signaling cascades are important components of GPCR signaling pathways. Both G-protein and ? -arrestin mediated signaling pathways can lead to ERK activation. G-protein activation of ERKs results in the translocation of active ERK to the nucleus, where it can phosphorylate and activate various transcription factors. In contrast, when ERK is activated via an arrestin-dependent mechanism, active ERKs remain largely in the cytoplasm, where they can phosphorylate non-nuclear substrates. Subcellular locations of phosphorylated ERKs determine downstream signal transduction cascades. Current ERK activation assays are all antibody-based methods. The antibody-based assays measure the phosphorylation status of ERKs using cell lysates, but lack the ability to distinguish G-protein-dependent or arrestin-dependent ERK activation. Critical information on subcellular location and distribution of activated ERKs, which determine specific signal transduction cascades, is missing. In addition, some of antibody based assay methods are not HTS friendly. Currently, pathway- specific ERK activation assays with the HTS capability do not exist. We propose to develop these novel tools by applying newly developed cell-based protein-protein interaction LinkLight assay technology. The assays can measure specific subcellular ERK activation signaling pathways. ERK signaling pathways are implicated in LTP, memory, learning, mood stabilization etc. Therefore, developing these tools for identifying compounds modulating pathway-specific ERK activation should facilitate new GPCR drug discovery efforts for treating mental disorders. We propose four aims for the project. Aim 1. Develop G-protein-dependent ERK2 activation pathway assay. We plan to utilize phosphorylated ERK2 interaction with imp7 as signal readout for assay development. We plan to validate the assay by using the m-opioid receptor (MOR) and its ligand morphine for nuclear location of the ERK2 activation pathway, since morphine is known to activate G-protein-dependent ERK pathway. Aim 2. Develop arrestin-dependent ERK2 activation pathway assay. We plan to utilize phosphorylated ERK2 interaction with receptor-associated ? -arrestins as signal readout for the assay development. We plan to validate the assay by using the MOR and its ligand etorphine for arrestin-dependent cytoplasmic location of the ERK2 activation pathway, since etophine is known to activate arrestin-dependent ERK pathway. Aim 3. Generate and characterize D2R ERK pathway-selective activation assay cells. Dopamine D2 receptors (D2R) include a long (D2L) and a short (D2S) isoforms. We plan to generate stable D2L G-protein- dependent and D2S arrestin-dependent ERK activation assay cells. It is known that D2S activates ERK signaling by an arrestin-dependent pathway and D2L activates ERK signaling by an arrestin-independent pathway. The cells will be used in aim 4 for assessing D2R compounds on ERK pathway activities and for assessing HTS feasibility. Aim 4. Assessing D2R compounds on specific ERK pathway activities and assessing HTS feasibility by conducting a small pilot screening. Brain-derived dopamine D2 receptor-regulated behaviors have been associated to ERK activation. However, it is largely unknown D2R compounds on ERK activation especially specific ERK activation cascades. We plan to profile D2R compounds (listed in ref. 44) to look for their ERK pathway-selectivity preferences. Dr. Sam Kongsamut, our consultant for the project, having over 20 years experience in antipsychotic drug discovery programs in pharmaceutical industry will correlate the specific ERK pathway assay potencies with potencies of in vivo studies (animal models and clinic data). Pathway-selective compounds could have functional selectivity. We also plan to demonstrate assay feasibility for HTS. Dr. Wei Zheng's group in NCATS will conduct the pilot screenings using the LOPAC library. Based on the results of the aim 4, we will plan next phase research proposal (phase II) and collaborate with partners for developing new leads for treatment of mental health disorders. Once developed, these cells would be the unique products on the market. We plan to market specific GPCR ERK LinkLight assay cells, provide custom-services for compound profiling and screening, and license ERK pLuc reporter host cells with an annual fee. The end users can use these host cells to develop their interested GPCR ERK LinkLight assays.

描述（由申请人提供）：我们计划开发用于评估GPCR配体调节G蛋白依赖性和？抑制蛋白依赖性ERK信号通路。许多GPCR显示出一种现象，其中不同的配体可以通过相同的受体差异地激活不同的信号传导途径。已经清楚的是，用于鉴定候选药物的单一信号传导途径方法不足以适合于检测可能具有靶受体以外的其他信号传导途径活性的化合物的全部库。人们必须检查多个信号通路，以将通路活动与生理功能联系起来。ERK信号通路是GPCR信号通路的重要组成部分。G蛋白和？β-arrestin介导的信号通路可导致ERK激活。ERK的G蛋白活化导致活性ERK易位至细胞核，在那里它可以磷酸化并激活各种转录因子。相反，当ERK通过抑制蛋白依赖性机制被激活时，活性ERK主要保留在细胞质中，在那里它们可以磷酸化非核底物。磷酸化ERK的亚细胞位置决定下游信号转导级联。目前的ERK活化测定都是基于抗体的方法。基于抗体的测定使用细胞裂解物测量ERK的磷酸化状态，但缺乏区分G蛋白依赖性或抑制蛋白依赖性ERK活化的能力。确定特定信号转导级联的活化ERK的亚细胞位置和分布的关键信息缺失。此外，一些基于抗体的测定方法不是HTS友好的。目前，还不存在具有HTS能力的途径特异性ERK激活测定。我们建议通过应用新开发的基于细胞的蛋白质-蛋白质相互作用LinkLight测定技术来开发这些新工具。该测定可测量特定的亚细胞ERK活化信号传导途径。ERK信号通路与LTP、记忆、学习、情绪稳定等有关。因此，开发这些用于鉴定调节途径特异性ERK激活的化合物的工具应有助于用于治疗精神障碍的新GPCR药物发现工作。我们为该项目提出了四个目标。目标1.开发G蛋白依赖性ERK 2活化途径测定。我们计划利用磷酸化ERK 2与imp 7的相互作用作为检测开发的信号读出。我们计划通过使用m-阿片受体（莫尔）及其配体吗啡对ERK 2激活途径的核定位来验证该测定，因为已知吗啡激活G-蛋白依赖性ERK途径。目标2.开发抑制蛋白依赖性ERK 2活化途径测定。我们计划利用磷酸化ERK 2与受体相关的相互作用？- 抑制蛋白作为测定开发的信号读出。我们计划通过使用莫尔及其配体埃托啡对ERK 2激活途径的抑制蛋白依赖性细胞质定位来验证该测定，因为已知埃托啡激活抑制蛋白依赖性ERK途径。目标3.生成并表征D2 R ERK通路选择性活化测定细胞。多巴胺D2受体（D2 R）包括长（D2 L）和短（D2 S）亚型。我们计划产生稳定的D2 L G蛋白依赖性和D2 S抑制蛋白依赖性ERK激活测定细胞。已知D2 S通过抑制蛋白依赖性途径激活ERK信号传导，D2 L通过抑制蛋白非依赖性途径激活ERK信号传导。这些细胞将用于目标4中，以评估D2 R化合物对ERK通路活性的影响，并评估HTS的可行性。目标4。评估D2 R化合物对特定ERK通路活性的影响，并通过进行小型试点筛选评估HTS可行性。脑源性多巴胺D2受体调节的行为与ERK激活相关。然而，它在很大程度上是未知的D2 R化合物对ERK激活，特别是特定的ERK激活级联。我们计划分析D2 R化合物（在参考文献44中列出），以寻找它们的ERK通路选择性偏好。我们的项目顾问Sam Kongsamut博士在制药行业的抗精神病药物发现项目中拥有超过20年的经验，他将把特定ERK通路测定效力与体内研究（动物模型和临床数据）的效力相关联。途径选择性化合物可能具有功能选择性。我们还计划证明HTS的测定可行性。NCATS的Wei Zheng博士的小组将使用LOPAC库进行试点筛选。根据目标4的结果，我们将计划下一阶段的研究计划（第二阶段），并与合作伙伴合作开发治疗精神健康障碍的新线索。一旦开发出来，这些细胞将成为市场上独一无二的产品。我们计划销售特定的GPCR ERK LinkLight检测细胞，提供化合物分析和筛选的定制服务，并以年费许可ERK pLuc报告宿主细胞。最终用户可以使用这些宿主细胞开发他们感兴趣的GPCR ERK LinkLight检测试剂盒。