Targeted Deorphanization of G Protein-Coupled Receptors

G 蛋白偶联受体的靶向去孤儿化

基本信息

批准号：
9813714
负责人：
Lakshmi A Devi
金额：
$ 16.95万
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：
2019
资助国家：
美国
起止时间：
2019-08-05 至 2020-08-04
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9813714
关键词：
Agonist Award Binding Binding Sites Biological Biological Assay Brain Brain region CRISPR/Cas technology Calcium Cell Line Cell model Cells Chinese Hamster Ovary Cell Clustered Regularly Interspaced Short Palindromic Repeats Code Communities Cyclic AMP Data Development Disease Drug Targeting Enzymes Evolution Funding Opportunities Future G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Genome Goals Grant Human Knock-out Knowledge Ligand Binding Ligands Literature Location Measures Mediating Membrane Proteins Mental Depression Methodology Methods Modeling Molecular Names Neuropeptide Receptor Neuropeptides Obesity Organism Orphan Paper Pathologic Pathway interactions Peptide Receptor Peptides Pharmacology Phospholipase C Physiological Physiology Property Protein Family Proteins Public Domains Reagent Receptor Activation Receptor Signaling Regulation Role Selection Criteria Signal Transduction Structure System Testing Time Tissues addiction base design drug candidate drug market druggable target gain of function genome editing high throughput screening insight loss of function new therapeutic target overexpression receptor receptor binding screening

项目摘要

Project summary G protein-coupled receptors (GPCRs) comprise the most abundant human membrane protein family, representing 4% of the protein-coding genome. Due to their numerous physiological and pathological roles, approximately 34% of marketed drugs target a GPCR. Although GPCRs are relevant druggable targets ~140 are still orphans i.e. their endogenous ligands unidentified. The identification of the endogenous molecules that activate the receptor is an essential step that helps define the receptor’s role in biological pathways. Here we describe studies to screen orphan GPCRs using genome editing and a high-throughput screening platform, testing each receptor against 48 distinct peptides that are predicted to be neuropeptides based on their abundance in brain, their localization to the regulatory secretory pathway, and sequence conservation. We have carefully selected 18 GPCRs that are potential neuropeptide-binding receptors based on their distribution, expression levels, and sequence conservation. We will use two complementary approaches for deorphanization: in Aim 1 we will use a loss-of-function strategy by generating GPCR knockout cell lines using CRISPR/Cas9 system; and in Aim 2 we will use a gain-of-function strategy by generating stable GPCR overexpression cell lines. A high-throughput screening assay will be used to measure the peptide ligand-induced GPCR activation- mediated changes in the intracellular calcium release in these two cellular models. Using these two independent approaches (loss- and gain- of-function) we have previously successfully deorphanized two different orphan GPCRs and hence we are confident that the proposed studies will be successfully carried out within the one- year period of this award. The results obtained from this proposal will guide future studies that explore physiological roles of the receptors as well as facilitate structural studies that seek insights into receptor-ligand binding sites, which are fundamental to the development of new therapeutics targeting the receptors.

项目摘要 G蛋白偶联受体（GPCR）包括最丰富的人膜蛋白家族，代表4％的蛋白质编码基因组。由于它们众多的身体和病理角色，大约34％的销售药物针对GPCR。尽管GPCR是相关的可药物靶标〜140 仍然是孤儿，即它们的内源配体未识别。鉴定内源分子激活受体是有助于定义受体在生物学途径中的作用的重要步骤。我们在这里描述使用基因组编辑和高通量筛选平台筛选孤儿GPCR的研究，测试每个受体对48种不同的肽，这些肽被认为是基于其神经肽的大脑中的抽象，它们的定位到调节秘密途径和序列保守。我们有精心选择的18个GPCR是根据其分布的分布，是潜在的神经肽结合受体的表达水平和序列保守。我们将使用两种完整的方法进行脱键化：在AIM 1中，我们将使用CRISPR/CAS9生成GPCR敲除细胞系来使用功能丧失策略系统;在AIM 2中，我们将通过产生稳定的GPCR过表达单元格使用功能奖励策略线。高通量筛选测定法将用于测量肽配体诱导的GPCR激活 - 在这两个细胞模型中，细胞内钙释放的介导变化。使用这两个独立我们以前已经成功地脱字了两个不同的孤儿因此，GPCR和我们有信心，拟议的研究将在一个人中成功进行该奖项的一年。从该提案中获得的结果将指导未来的研究来探索受体的生理作用，并促进了寻求对受体配体的见解的结构研究约束地点，这是针对接收者的新治疗剂的基础。