A Virion-Display Oscillator Array and Detection Platform for Quantification of Transmembrane Protein Binding Kinetics

用于量化跨膜蛋白结合动力学的病毒粒子显示振荡器阵列和检测平台

• 批准号: 10115647
• 负责人: SHAOPENG WANG
• 金额: $ 37.74万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115647
• 关键词: Address; Adverse effects; Affinity; Antibodies; Arizona; Binding; Binding Proteins; Biological; Biosensing Techniques; Cell physiology; Cellular Membrane; Charge; Collection; Detection; Disease; Dissociation; Drug Screening; Drug Targeting; Environment; FDA approved; Family; G-Protein-Coupled Receptors; Glass; Goals; Gold; Golgi Apparatus; Herpesviridae; Herpesvirus 1; Human; Image; Imaging Techniques; Integral Membrane Protein; Kinetics; Knowledge; Label; Ligand Binding; Ligands; Light; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Proteins; Microarray Analysis; Mission; Modification; Molecular; Molecular Analysis; Molecular Conformation; Pharmaceutical Preparations; Physiological; Play; Polymers; Process; Proteins; Reader; Signal Transduction; Slide; Structure; Technology; Time; Transfection; United States National Institutes of Health; Universities; Viral; Virion; Virus; base; cancer initiation; cancer therapy; detection platform; drug candidate; drug development; electric field; experimental study; expression vector; flexibility; imaging system; nanometer; novel therapeutics; plasmonics; preservation; protein B; receptor; reconstitution; sensor; small molecule; success; tool; tumor progression; vector

ABSTRACT Transmembrane proteins, such as G-protein-coupled receptors (GPCRs), are critical for many cellular functions. They are also the most popular drug targets for various diseases, including cancer. For both understanding cellular functions and drug development, it is necessary to measure their binding activities with molecular ligands and drug candidates. However, this has been a difficult task because of two challenges. First, transmembrane proteins are difficult to extract and purify, and they often lose their native conformations after isolation from the cellular membranes. Second, even if a membrane protein is successfully isolated, it remains challenging to measure its binding to ligands, especially with small molecule ligands. Small molecules comprise ~90% of the current drugs, but their binding kinetics cannot be easily measured with the existing detection technologies. This project addresses both challenges with a virion oscillator technology. Human GPCRs are displayed on the viral envelopes of human herpes simplex virus-1 (HSV-1), which removes the need of extraction, purification, and reconstitution of the transmembrane proteins. Each virion is then tethered to a sensor chip with a flexible polymer linker to form an oscillator. By applying an alternating electric field to the chip, the virion oscillates, and the oscillation amplitude is tracked in real-time with sub-nanometer precision using a plasmonic imaging technique. Upon binding of ligands or drugs to the GPCRs on the virion envelopes, the oscillation amplitude changes, from which binding kinetics and affinity are quantified. This project combines the virion display and microarray strengths at Johns Hopkins University, and plasmonic imaging and biosensing expertise at Arizona State University. The team has been working together and completed substantial preliminary experiments to demonstrate this new detection platform. The goal of this R33 project is to transform the technology into a powerful high-throughput platform for studying membrane proteins by 1) developing virion oscillator microarray chips (with 315 non-odorant human GPCRs on a single chip), 2) developing a plasmonic imaging system for high-throughput quantification of molecular binding kinetics, and 3) validating the virion oscillator microarray technology with cancer related GPCRs. It is anticipated that the virion oscillator detection technology will become a unique tool for studying cellular functions of membrane proteins, and quantifying binding of large and small molecule drugs with any types of membrane proteins.

抽象的 跨膜蛋白（例如G蛋白偶联受体（GPCR））对于许多细胞功能至关重要。 它们也是包括癌症在内的各种疾病的最受欢迎的药物靶标。两者都理解 细胞功能和药物发展，有必要测量其与分子配体的结合活性 和候选毒品。但是，由于两个挑战，这是一项艰巨的任务。首先，跨膜 蛋白质很难提取和纯化，并且它们经常在与 细胞膜。其次，即使膜蛋白成功地隔离了，也仍然具有挑战性 测量其与配体的结合，尤其是与小分子配体的结合。小分子占约90％的 当前的药物，但是使用现有检测技术可以轻松测量其结合动力学。 该项目通过Virion振荡器技术解决了这两个挑战。人类GPCR显示在 人类疱疹病毒1（HSV-1）的病毒信封，它消除了提取，纯化的需求 并重构跨膜蛋白。然后将每个病毒座绑在具有柔性的传感器芯片上 聚合物接头形成振荡器。通过在芯片上施加交替的电场，病毒粒子振荡和 使用等离子体成像，使用子纳米精度实时跟踪振荡振幅 技术。配体或药物与病毒信封上的GPCR结合后，振荡幅度 变化，结合动力学和亲和力被量化。 该项目结合了约翰·霍普金斯大学和等离子的Virion展示和微阵列的优势 亚利桑那州立大学的成像和生物传输专业知识。团队一直在一起工作， 完成了实质性的初步实验，以证明这个新的检测平台。 R33的目标 项目是将技术转换为一个强大的高通量平台，用于研究膜蛋白 通过1）开发病毒体振荡器微阵列芯片（单个芯片上有315个非温和的人GPCR），2） 开发用于分子结合动力学的高通量定量的等离子体成像系统，3） 使用与癌症相关的GPCR验证病毒粒子振荡器微阵列技术。预计病毒座 振荡器检测技术将成为研究膜蛋白的细胞功能的独特工具， 并量化大型和小分子药物与任何类型的膜蛋白的结合。