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