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（GPCRs）在生理和病理学中起着重要作用。GPCR是 FDA批准药物的最大目标。超过100个GPCR被认为是孤儿，因为它们的内源性 配体尚未被鉴定，这对于理解GPCR的生理作用是必要的 以及它们对CNS疾病的贡献。大部分GPCR被神经肽和/或神经肽受体激活。 肽激素，我们的假设是许多孤儿受体被肽激活， 可能是新的肽，因为其他研究人员已经筛选了建立的生物活性肽， 孤儿受体通过肽组学研究和功能鉴定了大量新型肽 因为这些肽是未知的，因此它们被认为是“孤儿肽”-这些肽很可能作为 生物活性肽基于它们的组织和细胞分布，定位于调节分泌途径， 和物种间的高度序列保守性。我们的实验室已经成功地将六种不同的 孤儿肽，因为我们鉴定了被它们激活的受体。在本建议中，我们将使用增益- 功能和功能丧失策略，以鉴定和验证孤儿“神经肽受体”的更大列表 对.我们的目标是：通过使用增益的方法来识别由“孤儿”神经肽激活的“孤儿”受体， 功能策略（目标1），并使用二次筛选验证孤儿“受体-神经肽”对 包括功能丧失策略（目标2）。在目标1中，我们将使用PRESTO-Tango GPCR试剂盒进行筛选 具有孤儿肽的孤儿GPCR。该测定涉及试剂盒中GPCR在HTLA细胞中的过表达 和测量肽介导的抑制蛋白募集。我们将筛选83个经验证的孤儿GPCR， PRESTO-Tango系统具有42种高度精选的孤儿肽，符合许多标准， 是一种生物活性神经肽。我们已经发现，这些肽中的几种引起快速和强烈的增加， 在Neuro 2A神经母细胞瘤细胞中的细胞内Ca 2+水平，因此这些肽是极好的候选物 来测试孤儿受体库在目标2中，我们将使用 二次筛选以及shRNA介导的靶受体敲低。的识别 孤儿受体的配体将提供对受体的生理作用以及 促进结构研究，研究受体-配体结合位点，这对发展 针对受体的新疗法。