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.

建议书摘要 G蛋白偶联受体是一种广泛存在的跨膜信号蛋白。 遍及全身。GPCRs负责调节许多生理过程，包括疼痛 调制和毒瘾。Mu-阿片受体(MOR)是最重要的GPCRs之一，因为它是 与阿片类药物流行有关，是治疗止痛药的主要目标。高通量筛选 (HTS)检测GPCR活性的方法对于发现替代的、非成瘾的是非常可取的 激动剂。现有的所有筛选平台都是基于维持活细胞培养的，因为缺乏 溶液中gpr活性的表征方法。在这里，我提出了第一个解决方案内HTS的设计 生物发光法检测gpr活性将大大简化gpr配基的制备过程 放映。新的分析方法具有构象特异性结合，包括纳米体(Nb)和 模拟多肽，它将结合在溶液中和活细胞中的gpr的激活构象上 文化。一种分裂的生物发光酶纳米水解素酶(Split NanoLuc)的组件将被连接到 GPCR和构象特异性结合蛋白。当激动剂依赖的相互作用发生时，NanoLuc将 在呋喃西林存在的情况下重建并产生可量化的生物发光信号。我已经去过了 用β2肾上腺素能受体(β2AR)及其受体验证了这种溶液生物发光分析的可行性 构象特异性结合蛋白、模拟G蛋白的纳米体和一种新型的多肽模拟物。化验结果将是 应用其构象特异的纳米体结合剂Nb39筛选MOR激动剂和拮抗剂 和NB44。此外，我建议调整溶液中的生物发光分析，使其适用于更广泛的应用 G蛋白多肽仿生学。该平台将用于筛选各种激动剂和拮抗剂。 GPCRs，将筛查能力扩大到包括与药物滥用有关的其他GPCRs。最后，这一点 检测将提供一种新的方法来验证从膜中分离的GPCRs的结构完整性 纳米盘。总体而言，这里提出的筛查试验可能会产生深远的治疗影响 从发现非成瘾止痛药或激动剂来抑制阿片类药物诱导的成瘾。

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