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.

项目摘要 G蛋白偶联受体（GPCR）在生理和病理学中起许多重要的作用。 GPCR是 FDA批准的药物的最大目标。超过100 GPCR被认为是孤儿，因为它们的内源性 尚未确定配体，这对于理解GPCR的身体作用是必不可少的 以及他们对中枢神经系统疾病的贡献。大部分GPCR被神经肽激活和/或 肽马酮，我们的假设是许多孤儿受体被佩蒂德激活， 大概是新颖的宠物，因为其他研究人员已经筛选了既定的生物活性宠物 孤儿受体。通过肽症研究已经鉴定出大量新型肽，功能 因为这些尚不清楚，因此它们被认为是“孤儿辣椒” - 这些很可能充当 生物活性胡椒基于其组织和细胞分布，定位到调节秘密途径， 和跨物种的高序列保守。我们的实验室已成功地脱字了六个不同的 孤儿辣椒是我们确定了被它们激活的受体。在此提案中，我们将使用 功能和功能策略丧失，以识别和验证更大的孤儿列表“神经肽受体” 成对。我们的目标是：识别使用“孤儿”神经肽激活的“孤儿”受体 功能策略（AIM 1），并使用次级筛选来验证孤儿的受体 - 神经肽”对 包括功能丧失策略（AIM 2）。在AIM 1中，我们将使用Presto-Tango GPCR套件进行屏幕 带有孤儿肽的孤儿GPCR。该测定涉及HTLA细胞中GPCR的过表达 并测量肽介导的阻止素的募集。我们将筛选83个经过验证的孤儿GPCR Presto-tango系统具有42个高度策划的孤儿辣椒中的每一个，它们适合许多标准 是生物活性神经肽。我们发现其中几个辣椒会导致快速而强大的增加 在神经2A神经母细胞瘤细胞中细胞内Ca2+水平中，这些宠物是极好的候选者 测试针对孤儿受体库。在AIM 2中，我们将使用使用“孤儿神经肽”对验证 次级筛选以及shRNA介导的目标接收器的敲低。识别 孤儿受体的配体将为受体的生理作用提供基本见解 促进结构研究研究受体 - 配体结合位点，这是对发展的基础 针对接收者的新疗法。