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

• 批准号: 10640094
• 负责人: Ruby May Miller
• 金额: $ 1.56万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-30 至 2023-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10640094
• 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 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' s G solution; Testing; Therapeutic; United States Food and Drug Administration; Work; addiction; antagonist; design; detection assay; detection method; detection platform; 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

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) 是大量发现的跨膜信号蛋白 遍布全身。 GPCR 负责调节许多生理过程，包括疼痛 调节和药物成瘾。 mu-阿片受体 (MOR) 是最重要的 GPCR 之一，因为它 作为治疗性镇痛药的主要目标，与阿片类药物流行有关。高通量筛选 检测 GPCR 活性的 (HTS) 方法对于发现替代的、非成瘾性的方法非常有用 激动剂。所有现有的筛选平台都是基于维持活细胞培养，因为缺乏 表征溶液中 GPCR 活性的方法。在这里我提出了第一个解决方案内高温超导的设计 用于检测 GPCR 活性的生物发光测定法将大大简化 GPCR 配体的流程 筛选。新的检测方法具有构象特异性结合物，包括纳米抗体 (Nb) 和 肽模拟物，无论是在溶液中还是在活细胞中，都会与 GPCR 的激活构象结合 文化。分裂生物发光酶纳米荧光素酶（分裂 NanoLuc）的成分将附着在 GPCR 和构象特异性结合剂。当激动剂依赖性相互作用发生时，NanoLuc 将 在存在呋马嗪的情况下重建并产生可量化的生物发光信号。我已经 使用 β2 肾上腺素受体 (β2AR) 验证了这种溶液内生物发光测定的可行性及其 构象特异性结合剂、G蛋白模拟纳米抗体和新型肽模拟物。化验将是 应用于使用其构象特异性纳米抗体结合剂 Nb39 筛选 MOR 激动剂和拮抗剂 和Nb44。此外，我建议使用溶液内生物发光测定法来适应更广泛的应用 G-蛋白肽模拟物。该平台将用于筛选各种激动剂和拮抗剂 GPCR，扩展筛查能力以包括与药物滥用有关的其他 GPCR。最后，这个 该测定将提供一种新方法来验证使用膜分离的 GPCR 的结构完整性 纳米圆盘。总的来说，这里提出的筛选试验可能会产生深远的治疗影响 来自发现非成瘾性止痛药或激动剂来抑制阿片类药物引起的成瘾。

项目成果

Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.

• DOI: 10.1021/acsmeasuresciau.3c00021
• 发表时间: 2023-10-18
• 期刊: ACS MEASUREMENT SCIENCE AU
• 作者: Miller, Ruby M;Sescil, Jennifer;Sarcinella, Marina C;Bailey, Ryan C;Wang, Wenjing
• 通讯作者: Wang, Wenjing