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

描述(由申请人提供)：我们计划开发新的工具来评估调节G蛋白依赖和β-arrestin依赖的ERK信号通路的GPCR配体。许多GPCRs表现出一种现象，即不同的配体可以通过相同的受体不同地激活不同的信号通路。很明显，用于识别候选药物的单一信号通路方法不足以检测除了靶受体之外可能具有其他信号通路活性的全部化合物。人们必须研究多条信号通路来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介导的信号通路均可导致ERK激活。G蛋白激活ERKs导致活性ERK转位到细胞核， 在那里它可以磷酸化并激活各种转录因子。相反，当ERK通过arrestin依赖的机制被激活时，活性的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的亚细胞位置决定了下游的信号转导级联。目前ERK活性检测都是以抗体为基础的方法。基于抗体的检测方法使用细胞裂解物来检测ERKs的磷酸化状态，但缺乏区分G蛋白依赖或arrestin依赖的ERK激活的能力。关于激活的ERKs的亚细胞位置和分布的关键信息，决定了特定的信号转导级联，目前还没有。此外，一些基于抗体的检测方法对HTS不友好。目前，还不存在具有HTS功能的通路特异性ERK激活分析。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分析技术来开发这些新工具。这些检测方法可以测量特定的亚细胞ERK激活信号通路。ERK信号通路涉及LTP、记忆、学习、情绪稳定等。因此，开发这些工具来识别调节通路特异性ERK激活的化合物将有助于GPCR药物治疗精神障碍的新努力。我们为这个项目提出了四个目标。目的1.建立G蛋白依赖的ERK2激活通路分析方法。我们计划利用磷酸化的ERK2与Imp7的相互作用作为检测发展的信号读出。我们计划通过使用m-阿片受体(MOR)及其配体吗啡来验证ERK2激活途径的核定位，因为已知吗啡激活G蛋白依赖的ERK途径。目的2.建立依赖Arrestin的ERK2激活途径分析方法。我们计划利用磷酸化的ERK2与受体相关的？-抑制素相互作用作为检测发展的信号读出。我们计划通过使用MOR及其配体埃托啡来验证该方法，以确定依赖于arrestin的ERK2激活途径的细胞质定位，因为已知依托泊芬激活依赖于arrestin的ERK途径。目的3.建立和鉴定D2R ERK通路选择性激活实验细胞。多巴胺D2受体(D2R)包括长的(D2L)和短的(D2S)亚型。我们计划建立稳定的依赖于D2L G蛋白和依赖于D2S arrestin的ERK激活实验细胞。已知D2S通过arrestin依赖途径激活ERK信号，D2L通过arrestin非依赖途径激活ERK信号。这些电池将用于AIM 4，用于评估D2R化合物对ERK途径的活性，并用于评估HTS的可行性。目的4.通过一项小型中试筛选，评估D2R化合物对特定ERK通路活性的影响，并评估HTS的可行性。脑源性多巴胺D2受体调节的行为与ERK的激活有关。然而，D2R类化合物对ERK的激活，尤其是对ERK的特异性激活级联作用，目前还很少见报道。我们计划对D2R化合物进行分析(见参考文献44)寻找他们的ERK途径选择性偏好。我们该项目的顾问Sam Kongsamut博士在制药行业的抗精神病药物发现计划方面拥有20多年的经验，他将把特定的ERK途径分析效力与活体研究(动物模型和临床数据)的效力联系起来。路径选择性化合物可能具有功能选择性。我们还计划论证HTS的化验可行性。魏征博士在NCATS的团队将使用Lopac文库进行试点筛选。根据目标4的结果，我们将规划下一阶段的研究方案(第二阶段)，并与合作伙伴合作，为精神健康障碍的治疗开发新的线索。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GPCRERK LinkLight分析细胞，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来开发他们感兴趣的GPCRERK LinkLight分析。

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.