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

摘要 跨膜蛋白，如G蛋白偶联受体(GPCRs)，对许多细胞功能至关重要。 它们也是治疗包括癌症在内的各种疾病的最受欢迎的药物靶点。为了双方的理解 细胞功能和药物开发，有必要测量它们与分子配体的结合活性 以及毒品候选人。然而，由于两个挑战，这一直是一项艰巨的任务。第一，跨膜 蛋白质很难提取和纯化，而且在从蛋白质中分离出来后往往会失去天然构象。 细胞膜。其次，即使成功分离出一种膜蛋白，它仍然具有挑战性 测量其与配体的结合，特别是与小分子配体的结合。小分子占~90% 目前的药物，但它们的结合动力学不容易用现有的检测技术测量。 该项目利用病毒粒子振荡器技术解决了这两个挑战。人类GPCR显示在 人单纯疱疹病毒1型(HSV-1)的病毒包膜，它不需要提取，纯化， 和跨膜蛋白的重组。然后，每个病毒粒子都被绑在一个传感器芯片上， 形成振荡器的聚合物连接物。通过向芯片施加交变电场，病毒粒子振荡，并且 使用等离子体成像以亚纳米精度实时跟踪振荡幅度 技术。当配体或药物与病毒粒子包膜上的GPCRs结合时，振荡幅度 变化，由此量化结合动力学和亲和力。 该项目结合了约翰·霍普金斯大学的病毒粒子显示器和微阵列的优势，以及等离子 亚利桑那州立大学的成像和生物传感专业知识。该团队一直在共同努力， 完成了大量的初步实验，以演示这一新的检测平台。这款R33的目标是 项目是将该技术转化为研究膜蛋白的强大的高通量平台 通过1)开发病毒粒子振荡器微阵列芯片(单个芯片上有315个无味人类GPCR)，2) 开发高通量定量分子结合动力学的等离子体成像系统，以及3) 病毒粒子振荡器微阵列技术与癌症相关GPCR的验证。据预计，病毒粒子 振荡器检测技术将成为研究膜蛋白细胞功能的独特工具， 以及定量大小分子药物与任何类型的膜蛋白的结合。

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.