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.

意义：G 蛋白偶联受体 (GPCR) 是一大类膜蛋白，可启动 细胞对各种细胞外信号（如神经递质或激素）的反应，通过激活 细胞内的异三聚体 G 蛋白使得 GPCR 对许多生理过程及其过程至关重要。 GPCR 不仅是最广泛的蛋白质家族，而且经常与疾病相关。 临床批准药物靶向，预计市场价值数千亿美元 今年，也是许多正在进行的新疗法运动的主题。此外，许多 GPCR 也是如此。 仍然未被充分研究和“未药物化”，这对于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方面具有生物医学重要性，但方法学 目前用于测量细胞中 GPCR 信号传导活性的方法存在局限性，阻碍了进展。 显着的限制是使用会损害读数保真度的方法，因为它们太 间接和/或扭曲信号成分的自然化学计量是另一个重要的限制。 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 一些细胞系，而不是更多的生理相关系统，如原代细胞培养物。 目标：在这里，我们建议开发能够直接测量 GPCR 信号活动的技术 以及在不同的、生理相关的细胞系统中，而不会对自然环境造成重大扰动 将制作该项目中生成的信号成分的化学计量。 其他调查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 在Aim1中，我们将建立一套广泛适用的直接检测异源三聚体G蛋白的活性。 “紧凑”载体与简单转染或病毒包装兼容以表达 G 蛋白 在目标 2 中，我们将验证小鼠转基因系的条件表达。 总的来说，这两个平台将允许直接评估内源性 G 蛋白活性。 在生理相关细胞系统中天然表达条件下的 GPCR 信号传导活性，以及 采用能够提高通量的格式，以允许并行询问可药物 GPCR-ome。 影响：我们目标的实现可能会改变对 GPCR 感兴趣的广大研究领域的方式 介导的细胞通讯涉及到测量信号活动等基本问题。 这里开发的技术将通过以下方式推动该领域的发展： (i) 能够在目前不可能的系统中直接表征 GPCR 信号传导， (ii) 在不同背景下以前所未有的保真度揭示 GPCR 信号转导机制，和/或 (iii) 以与增加的吞吐量兼容的格式促进药理学或机械询问。