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

提案摘要 G蛋白偶联受体（GPCR）是一种跨膜信号蛋白，在细胞内大量存在 在整个身体。GPCR负责调节包括疼痛在内的许多生理过程 调制和药物成瘾。μ-阿片受体（莫尔）是最重要的GPCR之一，因为它 作为治疗性镇痛药的主要靶点与阿片类药物流行有关。高通量筛选 (HTS)用于检测GPCR活性的方法对于发现替代的、非成瘾性的、 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，我提出了第一个在溶液高温超导体的设计 一种用于检测GPCR活性的生物发光测定法，其将大大简化GPCR配体的过程 筛选新的检测方法具有构象特异性结合剂，包括纳米抗体（Nb）和 肽模拟物，其将在溶液中和活细胞中与GPCR的活化构象结合 cultures.裂解生物发光酶纳米荧光素酶（裂解NanoLuc）的组分将连接到纳米荧光素酶上。 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 重组并产生可定量的生物发光信号。我已经 验证了使用β 2-肾上腺素能受体（β 2 AR）的这种溶液中生物发光测定的可行性及其 构象特异性结合剂、G蛋白模拟物纳米抗体和新型肽模拟物。对试剂盒进行 应用其构象特异性纳米抗体结合剂Nb 39筛选莫尔激动剂和拮抗剂 Nb44。此外，我建议采用更广泛的应用，在溶液中的生物发光测定， G蛋白肽模拟物。该平台将用于筛选多种药物的激动剂和拮抗剂， GPCR，扩大筛查能力，以包括与药物滥用有关的其他GPCR。最后这 分析将提供一种新的方法来验证从膜分离的GPCR的结构完整性， 纳米盘。总的来说，这里提出的筛选试验可能具有深远的治疗影响， 从发现非成瘾性止痛药或激动剂来抑制阿片类药物诱导的成瘾。

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.