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).

项目摘要 G蛋白偶联受体（GPCR）代表了制药行业的最高优先级目标之一 大约在特定GPCR的所有批准药物目标中，大约三分之一 每年1800亿美元。 GPCR是人类具有不同生理功能的人类最大的基因家族之一 还与许多疾病的病理生理学有关，例如癌症，糖尿病，糖尿病， 心脏病和许多代谢疾病。了解GPCR的特征仍然是 生物学和各种商业测定中的中心主题可以表征配体结合亲和力和 为该接收者家族成员的激动剂和拮抗剂的鉴定。尽管有实用性，这些 测定具有许多限制，以排除其宽度的使用。其中包括需要昂贵的 人造GPCR细胞系；有限的分析输出； GPCR特性的间接度量；以及潜力 数据收集过程中的光漂白和光损伤。表面增强的拉曼光谱法（SERS）是 非常敏感且高度特异性的手段，用于分析细胞和亚细胞中的表面分子 域。拟议的I期研究计划着重于基于SERS的问题的发展 两个GPCR的表征是这些受体的主要类别的成员，A类GPCR 家庭。这种方法指出了比现有技术的优势，包括更大的分析输出，直接 GPCR而不是下游目标的度量，以及将该技术应用于任何本地的能力 细胞及其正常完成细胞蛋白。这项研究的长期目标是发展低 成本，多功能和强大的平台用于表征GPCR，这是对这些的重大进步 当前可用的测定。为了实现这一目标，我们有以下特定目标。 •开发GPCR问题问题（特定目标1）。提议的这一部分中的最初目标（OBJ。1.1） 计划是使用免疫组织化学和 表达这些受体的细胞系中GPR84和GPR120的表面血浆共振。目标1.2将 使用确定的抗体合成，构建和表征针对GPR84和的SERS问题 GPR120。 •使用现有技术开发基于SERS的GPCR激活测定法和验证 （特定目标2）。 SERS问题将在目标2.1中用于开发逐点和频谱 映射模块以提供动力学分析，剂量依赖性，相对表达区域和激活 这两个GPCR的概率。该目标中产生的数据将与退出技术进行比较 通过在同一两个单元线上运行市售的测定（Pathhunter®ß-arrestin分析； Eurofins） （目标2.2）。