GPCR activation-based in vitro luminescent assays for high-throughput drug screening and extracted GPCR characterization

基于 GPCR 激活的体外发光测定用于高通量药物筛选和提取的 GPCR 表征

基本信息

批准号：
10462863
负责人：
Ruby May Miller
金额：
$ 3.94万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
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：
2022-05-30 至 2025-05-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10462863
关键词：
Address Adrenergic Receptor Agonist Amino Acids Analgesics Binding Binding Proteins Biological Assay Bioluminescence Cell Culture Techniques Cell Density Cells Coupled Development Drug Addiction Drug Screening Drug Targeting Drug abuse Engineering Ensure Enzymes G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Goals Hour Human body Hybrids In Vitro Ligands Luminescent Measurements Membrane Methods Molecular Conformation Neurons Opioid Opioid Receptor Opioid agonist Pain Pathway interactions Peptides Pharmaceutical Preparations Physiological Processes Process Protein Conformation Proteins Protocols documentation Reagent Receptor Activation Risk Signal Transduction Signaling Protein Suby&apos s G solution Testing Therapeutic United States Food and Drug Administration Work addiction antagonist base design experimental study high throughput analysis high throughput screening high-throughput drug screening in vivo mu opioid receptors nanobodies nanodisk nanoluciferase novel novel strategies novel therapeutics opioid epidemic peptidomimetics protein expression receptor reconstitution respiratory scale up screening side effect targeted treatment trafficking

项目摘要

PROPOSAL SUMMARY G-protein Coupled Receptors (GPCR) are transmembrane signaling proteins found abundantly throughout the body. GPCRs are responsible for regulating many physiological processes including pain modulation and drug addiction. The mu-opioid receptor (MOR) is one of the most significant GPCRs since it is implicated in the opioid epidemic as the primary target for therapeutic analgesics. High-throughput screening (HTS) methods for detecting GPCR activity are highly desirable for discovering alternative, nonaddictive agonists. All the existing screening platforms are based on maintaining live-cell cultures due to the lack of methods for characterizing GPCR activity in-solution. Here I propose the design of the first in-solution HTS bioluminescent assay for detecting GPCR activity which will greatly streamline the process of GPCR ligand screening. The new assay features conformation specific binders, including nanobodies (Nb) and peptidomimetics, which will bind to the activated conformation of the GPCR, both in-solution and in live-cell cultures. Components of a split bioluminescent enzyme, Nanoluciferase (split NanoLuc), will be attached to the GPCR and the conformation specific binder. When the agonist-dependent interaction occurs, the NanoLuc will reconstitute and produce a quantifiable bioluminescent signal in the presence of furimazine. I have already validated the feasibility of this in-solution bioluminescent assay using beta2-adrenergic receptor (β2AR) and its conformation-specific binders, a G-protein mimic Nanobody and a novel peptidomimetic. The assay will be applied for screening MOR agonists and antagonists using its conformation-specific nanobody binders, Nb39 and Nb44. Additionally, I propose to adapt the in-solution bioluminescent assay for broader applications using G-protein peptidomimetics. The platform will be used to screen agonists and antagonists for a wide variety of GPCRs, extending the screening capabilities to include additional GPCRs implicated in drug abuse. Lastly, this assay will provide a new approach to validate the structural integrity of GPCRs isolated from membranes using nanodiscs. Overall, the screening assay proposed here could have far-reaching therapeutic impacts resulting from the discovery of non-addictive pain medications or agonists to suppress opioid-induced addiction.

提案摘要 G蛋白偶联受体（GPCR）是跨膜信号蛋白，绝对发现整个身体。 GPCR负责控制许多身体过程，包括疼痛调节和吸毒成瘾。 MU-阿片受体（MOR）是最重要的GPCR之一，因为它是在阿片类药物流行中实施，作为热镇痛药的主要靶标。高通量筛选（HTS）检测GPCR活性的方法非常需要发现替代性，非属性激动剂。由于缺乏表征GPCR活性内解决的方法。在这里，我提出了第一个解决HTS的设计用于检测GPCR活性的生物发光测定，这将大大简化GPCR配体的过程筛选。新测定具有构象特定的粘合剂，包括纳米体（NB）和肽仪，将结合GPCR的活化构象，无论文化。分裂生物发光酶的成分，纳米酸酯酶（分裂纳米酸）将附着在 GPCR和会议特定的活页夹。当发生激动剂依赖性相互作用时，纳米核将在呋喃嗪存在下，重建并产生可量化的生物发光信号。我已经使用beta2-肾上腺素受体（β2AR）及其ITS验证了该实质生物发光测定的可行性构象特异性粘合剂，G蛋白模拟纳米肌和一种新颖的辣椒瘤。测定将是使用其构象特异性纳米粘结剂NB39申请筛选激动剂和拮抗剂和NB44。此外，我建议适应使用的生物发光测定法 G蛋白辣妹。该平台将用于筛选激动剂和对手 GPCR，扩展了筛查功能，包括在药物滥用中实施的其他GPCR。最后，这个测定将提供一种新的方法来验证使用膜分离的GPCR的结构完整性纳米盘。总体而言，这里提出的筛选测定可能会产生深远的治疗影响从发现非成瘾性止痛药或激动剂到抑制Ooid引起的成瘾。