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

基本信息

批准号：
10532036
负责人：
Lakshmi A Devi
金额：
$ 25.35万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
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

项目摘要

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.

项目概要 G蛋白偶联受体（GPCR）在生理学和病理学中发挥着许多重要作用。 FDA 批准药物的最大目标超过 100 种 GPCR 被视为孤儿药物，因为它们是内源性的。配体尚未确定，这对于理解 GPCR 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。大部分 GPCR 是由神经肽和/或神经肽激活的。肽激素，我们的假设是许多孤儿受体被肽激活，可能是新颖的肽，因为其他研究人员已经筛选出针对特定生物活性的肽通过肽组学研究和功能已鉴定出大量新型肽。因为这些是未知的，因此它们被认为是“孤儿肽”——它们很可能发挥作用基于其组织和细胞分布的生物活性肽，定位于调节分泌途径，我们的实验室已经成功地使六种不同的物种失去孤儿。孤儿肽，因为我们鉴定了由它们激活的受体。在本提案中，我们将使用增益。功能和功能丧失策略来识别和验证更大的孤儿“神经肽受体”列表我们的目标是：使用增益来识别由“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。孤儿 GPCR 与孤儿肽该检测涉及试剂盒中的 GPCR 在 HTLA 细胞中的过度表达。我们将筛选 83 个经过验证的孤儿 GPCR。 PRESTO-Tango 系统包含 42 种高度孤儿策划的肽，符合许多标准作为一种生物活性神经肽，我们发现其中一些肽会导致快速而强劲的增加。 Neuro 2A 神经母细胞瘤细胞的细胞内 Ca2+ 水平，因此这些肽是极好的候选者在目标 2 中，我们将使用孤儿受体库来验证“孤儿-神经肽”对。二次筛选以及 shRNA 介导的靶受体敲低的鉴定。孤儿受体的配体将为受体的生理作用以及促进结构研究，调查受体-配体结合位点，这对于开发至关重要针对受体的新疗法。