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
基本信息
- 批准号:10115647
- 负责人:
- 金额:$ 37.74万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressAdverse effectsAffinityAntibodiesArizonaBindingBinding ProteinsBiologicalBiosensing TechniquesCell physiologyCellular MembraneChargeCollectionDetectionDiseaseDissociationDrug ScreeningDrug TargetingEnvironmentFDA approvedFamilyG-Protein-Coupled ReceptorsGlassGoalsGoldGolgi ApparatusHerpesviridaeHerpesvirus 1HumanImageImaging TechniquesIntegral Membrane ProteinKineticsKnowledgeLabelLigand BindingLigandsLightMalignant NeoplasmsMeasurementMeasuresMembraneMembrane ProteinsMicroarray AnalysisMissionModificationMolecularMolecular AnalysisMolecular ConformationPharmaceutical PreparationsPhysiologicalPlayPolymersProcessProteinsReaderSignal TransductionSlideStructureTechnologyTimeTransfectionUnited States National Institutes of HealthUniversitiesViralVirionVirusbasecancer initiationcancer therapydetection platformdrug candidatedrug developmentelectric fieldexperimental studyexpression vectorflexibilityimaging systemnanometernovel therapeuticsplasmonicspreservationprotein Breceptorreconstitutionsensorsmall moleculesuccesstooltumor progressionvector
项目摘要
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.
摘要
跨膜蛋白,如G蛋白偶联受体(GPCR),是许多细胞功能的关键。
它们也是包括癌症在内的各种疾病最受欢迎的药物靶标。为了理解
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
和候选药物。然而,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,并且它们在从蛋白质中分离出来后经常失去它们的天然构象。
细胞膜其次,即使成功分离出膜蛋白,
测量其与配体的结合,特别是与小分子配体的结合。小分子约占90%,
目前的药物,但它们的结合动力学不能很容易地测量与现有的检测技术。
这个项目解决了这两个挑战与病毒体振荡器技术。人类GPCR显示在
人单纯疱疹病毒-1(HSV-1)的病毒包膜,这消除了提取,纯化,
和跨膜蛋白的重构。每个病毒体然后被拴在一个传感器芯片上,
聚合物连接体以形成振荡器。通过对芯片施加交变电场,病毒粒子振荡,
使用等离子体成像以亚纳米精度实时跟踪振荡幅度
法当配体或药物与病毒体包膜上的GPCR结合时,振荡幅度
变化,从中定量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展示和微阵列优势,以及等离子体激元技术。
亚利桑那州立大学的成像和生物传感专业知识。团队一直在一起工作,
完成了大量的初步实验,以证明这一新的检测平台。R33的目标是
一个项目是将这项技术转化为一个强大的高通量平台,用于研究膜蛋白
通过1)开发病毒粒子振荡器微阵列芯片(在单个芯片上具有315个无气味的人类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,以及3)
用癌症相关的GPCR验证病毒体振荡器微阵列技术。预计病毒粒子
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,
以及定量大分子和小分子药物与任何类型的膜蛋白的结合。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('SHAOPENG WANG', 18)}}的其他基金
Optical imaging of size, charge, mobility and binding of single proteins
单个蛋白质的大小、电荷、迁移率和结合的光学成像
- 批准号:
10521663 - 财政年份:2022
- 资助金额:
$ 37.74万 - 项目类别:
Optical imaging of size, charge, mobility and binding of single proteins
单个蛋白质的大小、电荷、迁移率和结合的光学成像
- 批准号:
10687006 - 财政年份:2022
- 资助金额:
$ 37.74万 - 项目类别:
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:
10357577 - 财政年份:2020
- 资助金额:
$ 37.74万 - 项目类别:
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:
9889569 - 财政年份:2020
- 资助金额:
$ 37.74万 - 项目类别:
Point-of-care antimicrobial susceptibility testing based on simultaneous tracking of multi-phenotypic features of single bacterial cells
基于同时跟踪单个细菌细胞的多表型特征的护理点抗菌药物敏感性测试
- 批准号:
10426291 - 财政年份:2018
- 资助金额:
$ 37.74万 - 项目类别:
Point-of-care antimicrobial susceptibility testing based on simultaneous tracking of multi-phenotypic features of single bacterial cells
基于同时跟踪单个细菌细胞的多表型特征的护理点抗菌药物敏感性测试
- 批准号:
10188407 - 财政年份:2018
- 资助金额:
$ 37.74万 - 项目类别:
Quantitative label-free imaging of membrane protein interaction kinetics on cells
细胞膜蛋白相互作用动力学的定量无标记成像
- 批准号:
8882482 - 财政年份:2014
- 资助金额:
$ 37.74万 - 项目类别:
Quantitative label-free imaging of membrane protein interaction kinetics on cells
细胞膜蛋白相互作用动力学的定量无标记成像
- 批准号:
9086372 - 财政年份:2014
- 资助金额:
$ 37.74万 - 项目类别:
Quantitative label-free imaging of electrical activities in cells
细胞电活动的定量无标记成像
- 批准号:
10242180 - 财政年份:2014
- 资助金额:
$ 37.74万 - 项目类别:
Quantitative label-free imaging of electrical activities in cells
细胞电活动的定量无标记成像
- 批准号:
10001533 - 财政年份:2014
- 资助金额:
$ 37.74万 - 项目类别:
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