Versatile and high-fidelity optical biosensor platforms for GPCR signaling

用于 GPCR 信号传导的多功能高保真光学生物传感器平台

• 批准号: 10679863
• 负责人: Mikel Garcia-Marcos
• 金额: $ 35.48万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679863
• 关键词: Acceleration; Achievement; Area; Asthma; Astrocytes; Biological; Biological Assay; Bioluminescence; Biosensor; Breeding; Cardiovascular Diseases; Cell Communication; Cell Line; Cell Surface Receptors; Cell model; Cells; Clinical; Cytoplasm; Detection; Disease; Dissociation; Drug usage; Energy Transfer; Event; Experimental Models; Family; Future; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Proteins; Gene Delivery; Goals; Guanosine Triphosphate; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Hormones; Human Genome; Hypersensitivity; In Vitro; Malignant Neoplasms; Marketing; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Methodology; Molecular; Mus; Names; Neurons; Neurotransmitters; Opioid Analgesics; Optics; Pain management; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Physiological; Physiological Processes; Physiology; Primary Cell Cultures; Process; Protein Family; Proteins; Reporting; Reproducibility; Research; Research Personnel; Resources; Signal Transduction; System; Technology; Transfection; Transgenic Mice; Translations; Viral Packaging; cell type; design; dimer; drug discovery; druggable target; expectation; extracellular; improved; in vivo; interest; neuropsychiatric disorder; novel; novel therapeutics; optical sensor; pharmacologic; prototype; receptor; response; sensor; stoichiometry; success; technology development; tool; vector; virtual

SIGNIFICANCE: G protein-coupled receptors (GPCRs) are a large family of membrane proteins that initiate cellular responses to a wide range of extracellular signals, like neurotransmitters or hormones, by activating heterotrimeric G proteins inside cells. This makes GPCRs critical for many physiological processes and their dysregulation is frequently associated with disease. GPCRs are not only the family of proteins most widely targeted by clinically approved drugs, with an estimated market value of hundreds of billions of dollars per year, but also the subject of many ongoing campaigns towards novel therapeutics. Moreover, many GPCRs still remain understudied and “undrugged”, which has great untapped potential for future drug discovery. Despite the biomedical importance of these receptors in physiology and pharmacology, the methodologies currently used to measure GPCR signaling activity in cells have limitations that hinder progress. One significant limitation is the use of approaches that compromise the fidelity of the readout because they are too indirect and/or distort the natural stoichiometry of signaling components. Another significant limitation is that many of the approaches are only feasible in cells in which gene delivery is easy, limiting their implementation to a few cell lines instead of more physiologically relevant systems like primary cell cultures. GOAL: Here, we propose to develop technologies that will allow to measure GPCR signaling activity directly and in diverse, physiologically-relevant cell systems without introducing major perturbations on the natural stoichiometry of signaling components. The tools and resources generated in this project will be made available to other investigators without restrictions. SYNOPSIS OF AIMS: We will develop two complementary assay platforms based on optical biosensors that directly detect the activity of heterotrimeric G proteins. In Aim1, we will establish a suite of broadly applicable “compact” vectors compatible with simple transfection or with viral packaging for the expression of G protein activity biosensors. In Aim 2, we will validate mouse transgenic lines for the conditional expression of biosensors of endogenous G protein activity. Collectively, these two platforms will allow the direct assessment of GPCR signaling activity under native expression conditions, in physiologically relevant cellular systems, and in formats that enable increased throughput to permit the parallel interrogation of the druggable GPCR-ome. IMPACT: The achievement of our goal could transform how the large field of research interested in GPCR- mediated cell communication approaches something as fundamental as measuring signaling activity. The technologies to be developed here would advance the field by: (i) enabling the direct characterization of GPCR signaling in systems in which it is currently not possible, (ii) revealing mechanisms of GPCR signaling with unprecedented fidelity in diverse contexts, and/or (iii) facilitating pharmacological or mechanistic interrogation in formats compatible with increased throughput.

G蛋白偶联受体（GPCRs）是一个大家族的膜蛋白， 细胞对广泛的细胞外信号的反应，如神经递质或激素，通过激活 细胞内的异源三聚体G蛋白这使得GPCR对于许多生理过程及其生物学特性至关重要。 调节失调通常与疾病有关。GPCR不仅是最广泛的蛋白质家族， 临床批准的药物的目标，估计市场价值数千亿美元， 这一年，而且也是许多正在进行的新疗法运动的主题。此外，许多GPCR 仍然是未充分研究和“未开发的”，这对未来的药物发现具有巨大的未开发潜力。 尽管这些受体在生理学和药理学中具有生物医学重要性， 目前用于测量细胞中GPCR信号传导活性的方法具有阻碍进展的局限性。一 显著的限制是使用损害读出保真度的方法 间接和/或扭曲信号组分的天然化学计量。另一个重要的限制是， 许多方法仅在基因传递容易的细胞中可行，限制了它们的实施 而不是像原代细胞培养这样的生理学上更相关的系统。 目标：在这里，我们建议开发能够直接测量GPCR信号活性的技术 并且在不同的生理相关细胞系统中，而不会对自然细胞系统引入重大干扰。 信号传导组分的化学计量。在这个项目中产生的工具和资源将 其他研究人员可以不受限制地使用。 目标概要：我们将开发两个基于光学生物传感器的互补检测平台， 直接检测异源三聚体G蛋白的活性。在目标1中，我们将建立一套广泛适用的 与简单转染或与用于表达G蛋白的病毒包装相容的“紧凑”载体 活性生物传感器在目标2中，我们将验证小鼠转基因系的条件表达， 内源性G蛋白活性的生物传感器。总的来说，这两个平台将允许直接评估 在天然表达条件下，在生理学相关的细胞系统中的GPCR信号传导活性，以及 以能够增加吞吐量的形式，以允许可药物化的GPCR组的并行询问。 影响：我们目标的实现可能会改变对GPCR感兴趣的大型研究领域- 介导的细胞通讯接近测量信号传导活性的基本方法。的 在此开发的技术将通过以下方式推动该领域的发展： (i)使得能够在目前不可能的系统中直接表征GPCR信号， (ii)在不同的背景下以前所未有的保真度揭示GPCR信号传导机制，和/或 (iii)以与增加的通量相容的形式促进药理学或机械学询问。