A New Versatile, SERS-based GPCR assay

一种新型多功能、基于 SERS 的 GPCR 检测

• 批准号: 10324451
• 负责人: Wei Zhang
• 金额: $ 25.96万
• 依托单位: VICOLINE MEDICAL, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324451
• 关键词: ARNT gene; Affinity; Agonist; Antibodies; Aptamer Technology; Area; Binding; Biological Assay; Biology; Cell Line; Cell Surface Receptors; Cell Survival; Cell model; Cell surface; Cells; Characteristics; Complement; Computer software; Data; Data Analyses; Data Collection; Dependence; Detection; Development; Devices; Diabetes Insipidus; Disadvantaged; Disease; Dose; Drug Industry; Drug Targeting; Emerging Technologies; Endocrine System Diseases; Engineering; Family; Family member; Feasibility Studies; Florida; Fluorescence; Functional disorder; G-Protein-Coupled Receptors; GPR84 gene; GTP-Binding Protein alpha Subunits, Gs; Gene Family; Goals; Heart Diseases; Human; Immunochemistry; Immunohistochemistry; Investigation; Kinetics; Life; Ligand Binding; Ligands; Malignant Neoplasms; Measurement; Measures; Medical; Membrane Proteins; Metabolic Diseases; Mission; Modeling; Molecular; Monoclonal Antibodies; Oligonucleotides; Output; Pathology; Peptides; Performance; Pharmacologic Substance; Phase; Photobleaching; Physiological; Physiology; Plasma; Play; Population; Probability; Process; Promega; Property; Proteins; Publications; Raman Spectrum Analysis; Readiness; Receptor Activation; Research; Role; Running; Sales; Selection Criteria; Sensitivity and Specificity; Surface; System; Technology; Time; Universities; Validation; base; beta-arrestin; cell type; commercialization; cost; data acquisition; detection method; drug development; druggable target; enhancing factor; experience; extracellular; instrument; luminescence; member; novel; overexpression; receptor; receptor function; response; success; tool

PROJECT SUMMARY G protein-coupled receptors (GPCRs) represent one of the highest priority targets for the pharmaceutical industry and roughly one-third of all approved drugs target on specific GPCRs, which corresponds to global sales of over USD 180 billion annually. As one of the largest gene families in humans with varied physiology functions, GPCRs have also been implicated in the pathophysiology of numerous diseases such as cancer, diabetes insipidus, heart disease, and numerous metabolic disorders. Understanding the characteristics of GPCRs remains a central theme in biology and various commercial assays are available to characterize ligand binding affinity and the identification of agonists and antagonists for members of this receptor family. Despite their utility, these assays have a number of limitations that preclude their widespread use. These include the need for expensive, artificial GPCR cell lines; limited analytical output; indirect measures of GPCR properties; and the potential for photobleaching and photodamage during data collection. Surface-enhanced Raman spectroscopy (SERS) is an extremely sensitive and highly specific means to analyze surface molecules in the cellular and subcellular domains. The proposed Phase I research plan focuses on the development of SERS-based probes for the characterization of two GPCRs that are members of the predominant class of these receptors, the Class A GPCR family. This approach has noted advantages over existing technology including greater analytical output, direct measures of the GPCR rather than downstream targets, and the ability to apply this technology to any native cells with their normal complement of cellular proteins. The long-term goal of this research is to develop a low cost, versatile and robust platform for the characterization of GPCRs that is a significant advancement over those assays currently available. To meet this goal, we have the following specific objectives. • Development of GPCR SERS probes (Specific Aim 1). The initial goal (Obj. 1.1) in this part of the proposed plan is to identify suitable antibodies for eventual construction of SERS probes using immunohistochemistry and surface plasma resonance for GPR84 and GPR120 in cell lines expressing these receptors. Objective 1.2 will use the identified antibodies to synthesize, construct and characterize SERS probes specific for GPR84 and GPR120. • Development of the SERS-based GPCR activation assay and validation with existing technology (Specific Aim 2). The SERS probes will be used in Objective 2.1 to develop the point-by-point and spectral mapping modules to provide the kinetic analyses, dose-dependence, relative expression area, and activation probabilities for these two GPCRs. Data generated in this objective will be compared to the exiting technology by running commercially available assays (PathHunter® ß-arrestin assays; Eurofins) on the same two cell lines (Objective 2.2).

项目总结 G蛋白偶联受体(GPCRs)是制药业最优先考虑的靶点之一 所有批准的药物中，大约有三分之一针对特定的GPCR，这相当于全球销售额超过 每年1800亿美元。作为人类最大的具有多种生理功能的基因家族之一，GPCRs 也与许多疾病的病理生理学有关，如癌症、尿崩症、 心脏病，以及众多的代谢紊乱。理解GPCRs的特点仍然是一个 生物学中的中心主题和各种商业分析可用于表征配体结合亲和力和 该受体家族成员的激动剂和拮抗剂的鉴定。尽管它们很实用，但这些 化验方法有许多限制，使其无法广泛使用。这些问题包括需要昂贵的、 人工gpr细胞系；有限的分析输出；gpcr特性的间接测量；以及 数据采集过程中的光漂白和光损伤。表面增强拉曼光谱(SERS)是一种 分析细胞和亚细胞中表面分子的极其灵敏和高度特异的手段 域名。拟议的第一阶段研究计划侧重于开发基于表面增强拉曼光谱的探测器，用于 属于这些受体的主要类别的两个GPCR的特性，A类GPCRs 一家人。这种方法已经注意到了比现有技术的优势，包括更大的分析输出，直接 衡量GPCR而不是下游目标，以及将这项技术应用于任何原生设备的能力 具有正常细胞蛋白质补充的细胞。这项研究的长期目标是开发一种低成本 成本低廉、功能齐全且功能强大的GPCR表征平台，这是对 目前可用的化验方法。为实现这一目标，我们有以下具体目标。 ·发展GPCRSERS探针(具体目标1)。最初的目标(Obj.1.1)在建议的 计划是识别合适的抗体，用于最终使用免疫组织化学和 表达GPR84和GPR120受体的细胞系的表面等离子体共振。目标1.2将 利用已鉴定的抗体合成、构建和鉴定针对GPR84和GPR84的SERS探针 GPR120。 ·开发基于SERS的GPCR激活分析并利用现有技术进行验证 (具体目标2)。SERS探测器将在目标2.1中使用，以开发逐点和光谱 用于提供动力学分析、剂量依赖性、相对表达区域和激活的映射模块 这两个GPCR的概率。在此目标中产生的数据将与现有技术进行比较 通过在相同的两个细胞系上进行商业上可用的检测(PathHunter®？-arrestin检测；Eurofins) (目标2.2)。