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）是制药行业最优先考虑的靶点之一 大约有三分之一的获批药物针对特定的GPCR，这相当于全球销售量超过 每年1800亿美元。GPCR是人类最大的基因家族之一，具有多种生理功能， 还涉及许多疾病的病理生理学，例如癌症，尿崩症， 心脏病和许多代谢紊乱。了解GPCR的特征仍然是一个 生物学的中心主题和各种商业测定可用于表征配体结合亲和力， 该受体家族成员的激动剂和拮抗剂的鉴定。尽管它们的效用， 测定具有许多限制，这些限制妨碍了它们的广泛使用。这些包括需要昂贵的， 人工GPCR细胞系;有限的分析输出; GPCR特性的间接测量;以及 光漂白和光损伤。表面增强拉曼光谱（Sers）是一种 用于分析细胞和亚细胞中的表面分子的极其灵敏和高度特异性的手段， 域.拟议的第一阶段研究计划的重点是开发基于SERS的探针， 作为这些受体的主要类别（A类GPCR）的成员的两种GPCR的表征 家人这种方法比现有技术具有优势，包括更大的分析输出，直接 GPCR的措施，而不是下游目标，以及将这项技术应用于任何本地的能力， 细胞与其正常的细胞蛋白质互补。这项研究的长期目标是开发一种低成本的 用于表征GPCR的低成本、多功能和强大的平台，这是对那些 目前可用的分析。为了实现这一目标，我们有以下具体目标。 ·GPCR Sers探针的开发（具体目标1）。本部分的初步目标（目标1.1） 计划是鉴定合适的抗体，用于使用免疫组织化学最终构建Sers探针， GPR 84和GPR 120在表达这些受体的细胞系中的表面等离子共振。目标1.2将 使用鉴定的抗体合成、构建和表征对GPR 84特异的Sers探针， GPR120。 ·开发基于SERS的GPCR活化分析并利用现有技术进行验证 （具体目标2）。Sers探针将用于目标2.1中，以开发逐点和光谱 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。本目标中生成的数据将与现有技术进行比较 通过在相同的两种细胞系上运行市售测定（PathHunter® β-抑制蛋白测定; Eurofins （目标2.2）。