Identifying endogenous peptide ligands for orphan G protein-coupled receptors to explore their roles in CNS disorders

鉴定孤儿 G 蛋白偶联受体的内源性肽配体，探索其在中枢神经系统疾病中的作用

• 批准号: 10701897
• 负责人: Lakshmi A Devi
• 金额: $ 21.13万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10701897
• 关键词: Adenylate Cyclase; Agonist; Alzheimer' s Disease; Arrestins; Behavior; Binding; Binding Sites; Biological; Biological Assay; Brain; CNR1 gene; Calcium; Cells; Central Nervous System; Central Nervous System Diseases; Cholera Toxin; Collaborations; Conserved Sequence; Detection; Development; Disease; Enzymes; FDA approved; G-Protein-Coupled Receptors; Gases; Genome; Glutaminase; Grant; Human; Individual; Knowledge; Knowledge Management Center; Laboratories; Libraries; Ligand Binding; Ligands; Measures; Mediating; Mental Depression; Messenger RNA; Mood Disorders; Mus; Names; Neuropeptide Receptor; Neuropeptides; Obesity; Orphan; Pathology; Pathway interactions; Peptide Receptor; Peptides; Pertussis Toxin; Pharmaceutical Preparations; Phosphotransferases; Physiological; Physiology; Play; Publishing; Quantitative Reverse Transcriptase PCR; Reagent; Receptor Activation; Research Personnel; Resources; Rewards; Role; Running; Screening Result; Signal Transduction; Signaling Molecule; System; Testing; Time; Tissues; United States National Institutes of Health; Visit; addiction; antagonist; design; drug candidate; drug development; drug resource; extracellular; gain of function; high throughput screening; insight; knock-down; loss of function; neuroblastoma cell; new therapeutic target; novel; overexpression; peptide hormone; receptor; receptor binding; recruit; screening; small hairpin RNA; validation studies

Project summary G protein-coupled receptors (GPCRs) play many important roles in physiology and pathology. GPCRs are the largest target of FDA approved drugs. Over 100 GPCRs are considered orphans because their endogenous ligands have not yet been identified, which is necessary for understanding the physiological role of the GPCR as well as their contribution to CNS disorders. A large fraction of GPCRs are activated by neuropeptides and/or peptide hormones, and our hypothesis is that many of the orphan receptors are activated by peptides, presumably novel peptides, because other investigators have screened established bioactive peptides against orphan receptors. A large number of novel peptides have been identified by peptidomic studies, and functions for these are not known and hence they are considered ‘orphan peptides’ – these are very likely to function as bioactive peptides based on their tissue and cellular distribution, localization to the regulatory secretory pathway, and high sequence conservation across species. Our laboratory has successfully deorphanized six different orphan peptides in that we identified receptors that are activated by them. In this proposal, we will use a gain-of- function and a loss of function strategy to identify and validate a larger list of orphan ‘neuropeptide-receptor’ pairs. Our aims are: To identify ‘orphan’ receptors activated by ‘orphan’ neuropeptides using a gain-of- function strategy (Aim 1), and to validate orphan ‘receptor-neuropeptide’ pairs using secondary screens including a loss-of-function strategy (Aim 2). In Aim 1 we will use the PRESTO-Tango GPCR kit to screen orphan GPCRs with orphan peptides. The assay involves overexpression of the GPCRs in the kit in HTLA cells and measuring peptide-mediated arrestin recruitment. We will screen, 83 validated orphan GPCRs available in the PRESTO-Tango system with each of the 42 highly curated orphan peptides that fit many of the criteria for being a bioactive neuropeptide. We have found that several of these peptides cause a rapid and robust increase in intracellular Ca2+ levels in Neuro 2A neuroblastoma cells and therefore these peptides are excellent candidates to test against a library of orphan receptors. In Aim 2 we will validate the ‘orphan-neuropeptide’ pairs using secondary screens as well as with shRNA-mediated knockdown of the target receptor. The identification of ligands for orphan receptors will provide essential insights into the physiological roles of the receptors as well as facilitate structural studies investigating receptor-ligand binding sites that are fundamental to the development of new therapeutics targeting the receptors.

项目总结 G蛋白偶联受体(GPCRs)在生理和病理中发挥着重要作用。GPCR是 FDA批准的药物的最大目标。超过100个GPCR被认为是孤儿，因为他们的内源 配体尚未确定，这对于理解gpr的生理作用是必要的。 以及它们对中枢神经系统疾病的贡献。很大一部分GPCRs是由神经肽和/或 多肽荷尔蒙，我们的假设是许多孤儿受体被多肽激活， 推测是新的多肽，因为其他研究人员已经筛选了已建立的生物活性多肽来对抗 孤儿受体。多肽学研究发现了大量新的多肽，并对其功能进行了研究。 因为这些都是未知的，因此它们被认为是“孤儿多肽”--这些很可能起到 基于其组织和细胞分布的生物活性多肽，对调节分泌途径的定位， 以及跨物种的高度序列保守性。我们的实验室已经成功地使六种不同的 孤儿多肽，因为我们确定了由它们激活的受体。在这项提案中，我们将使用收益- 识别和验证更多孤儿‘神经肽受体’的功能和功能丧失策略 成对的。我们的目标是：使用一种增益率来识别由孤儿神经肽激活的孤儿受体。 功能策略(目标1)，并使用二次筛查验证孤儿‘受体-神经肽’对 包括丧失功能战略(目标2)。在目标1中，我们将使用Presto-Tango GPCR试剂盒进行筛查 具有孤儿多肽的孤儿GPCR。该检测包括在HTLA细胞中过表达试剂盒中的GPCRs 并测量多肽介导的arrestin募集。我们将筛选83个有效的孤立GPCR，可在 Presto-Tango系统包含42个高度精选的孤儿多肽，每一个都符合许多标准 是一种生物活性神经肽。我们已经发现，其中几种多肽会引起快速而强劲的增长。 神经母细胞瘤细胞内钙离子水平的变化，因此这些多肽是很好的候选者 与孤儿感受器库进行对比测试。在目标2中，我们将使用以下方法验证“孤儿-神经肽”对 二次筛选以及shRNA介导的靶受体敲除。身份的鉴定 孤儿受体的配体将为了解受体的生理作用以及 促进研究受体-配体结合部位的结构研究，这些结合部位是发展的基础 针对受体的新疗法。