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？ - arrestin-依赖性ERK信号传导途径的新颖工具。许多GPCR表现出一种现象，其中不同的配体可以通过同一受体差异地激活不同的信号通路。很明显，识别候选药物的单个信号传导途径方法不足以检测可能在靶受体以外的其他信号通路活动的化合物中的完整曲目。必须检查多个信号通路将途径活动与生理功能联系起来。 ERK信号级联是GPCR信号通路的重要组成部分。 G蛋白和？ -arrestin介导的信号通路可以导致ERK激活。 ERK的G蛋白激活导致活性ERK转移到核，它可以磷酸化并激活各种转录因子。相反，当通过抑制蛋白依赖性机制激活ERK时，活性ERK在很大程度上保留在细胞质中，它们可以在其中磷酸化非核底物。磷酸化ERK的亚细胞位置决定了下游信号转导级联。当前的ERK激活测定是所有基于抗体的方法。基于抗体的测定方法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激活的能力。缺少有关确定特定信号转导级联的激活ERK的亚细胞位置和分布的关键信息。此外，某些基于抗体的测定方法不友好。当前，具有HTS功能的途径特定于ERK激活测定法。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 Assay技术来开发这些新工具。这些测定可以测量特定的亚细胞ERK激活信号通路。 ERK信号通路与LTP，记忆，学习，情绪稳定相关。因此，开发这些工具以识别调节途径特异性ERK激活的化合物，应促进新的GPCR药物发现治疗精神疾病的工作。我们提出了该项目的四个目标。 AIM 1。开发依赖G蛋白的ERK2激活途径测定法。我们计划利用与IMP7磷酸化的ERK2相互作用作为测定开发的信号读数。我们计划通过使用M-阿片受体（MOR）及其配体吗啡来验证ERK2激活途径的核位置，因为已知吗啡可激活G蛋白依赖性ERK途径，因此对ERK2活化途径的核位置进行验证。目标2。开发依赖于抑制蛋白的ERK2激活途径测定法。我们计划利用与受体相关的磷酸化ERK2相互作用？ -Arrestins作为测定开发的信号读数。我们计划通过使用MOR及其配体伊托啡派来验证测定法，以依赖ERK2激活途径的抑制蛋白依赖性细胞质位置，因为已知etpophine可以激活依赖性ERK途径。 AIM 3。生成并表征D2R ERK途径选择性激活测定单元。多巴胺D2受体（D2R）包括长（D2L）和短（D2S）同工型。我们计划生成稳定的D2L G蛋白依赖性和D2S依赖性ERK激活测定细胞。众所周知，D2S通过抑制蛋白依赖性途径激活ERK信号传导，而D2L通过抑制蛋白独立的途径激活ERK信号传导。这些细胞将用于AIM 4用于评估ERK途径活动中的D2R化合物并评估HTS可行性。 AIM 4。评估D2R化合物在特定的ERK途径活动上，并通过进行小型试点筛选来评估HTS的可行性。脑衍生的多巴胺D2受体调节的行为与ERK激活有关。然而，它在ERK激活上尤其是特定的ERK激活级联反应上是未知的D2R化合物。我们计划介绍D2R化合物（参考文献44中列出），以寻找其ERK途径选择性偏好。我们的项目顾问Sam Kongsamut博士在制药行业具有20多年的抗精神病药物发现计划经验，将使特定的ERK途径测定能力与体内研究（动物模型和临床数据）相关联。途径选择性化合物可以具有功能选择性。我们还计划证明HTS的测定可行性。 Wei Zheng博士在NCATS中的小组将使用LOPAC图书馆进行飞行员筛选。根据目标4的结果，我们将计划下一阶段研究建议（第二阶段），并与合作伙伴合作开发新的潜在客户以治疗精神疾病。一旦开发，这些细胞将是市场上独特的产品。我们计划销售特定的GPCR ERK Linklight分析单元格，为复合分析和筛选提供定制服务，并以年费为ERK PLUC记者宿主细胞。最终用户可以使用这些主机单元开发其感兴趣的GPCR ERK Linklight分析。