HTS of small molecule-protein interactions (RMI)

小分子-蛋白质相互作用 (RMI) 的 HTS

• 批准号: 7288817
• 负责人: DALE NORMAN LARSON
• 金额: $ 60.68万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7288817
• 关键词: Address; Automation; Binding; Biological; Biological Assay; Characteristics; Clinic; Collaborations; Complementary DNA; Data; Detection; Devices; Doctor of Philosophy; Engineering; Evaluation; Expression Library; Generations; Glass; Gold; Human; Human Cloning; In Situ; Institutes; Kinetics; Label; Lead; Life; Methods; Nucleic Acids; Performance; Printing; Protein Array; Protein Microchips; Proteins; Proteomics; Reagent; Research Personnel; Resolution; Sampling; Surface; System; Technology; Testing; Time; Toxic effect; Work; base; drug development; gene cloning; miniaturize; novel; photonics; protein expression; protein protein interaction; protein purification; scale up; sensor; single molecule; size; small molecule; tool

DESCRIPTION (provided by applicant): Understanding the selectivity of small molecule binding is a central question in drug development. It impinges on both determining which desirable targets might respond to the compound as well as which unintended targets might lead to toxicity. Currently these are difficult questions to answer because they require the evaluation of the small molecule's interactions with many proteins, which in turn requires methods for testing many proteins and for detecting and characterizing the interaction. Protein microarrays offer a compelling method for presenting many proteins for testing, but the methods currently available for assessing small molecule binding require modifying the small molecule with a label. This labeling frequently results in changes to the small molecule's binding performance and can be difficult. A technology that enables researchers to address these questions in a high-throughput manner will accelerate the transition of discoveries into the clinic. In this project, we propose to bring two technologies together that will result in a high-throughput system to detect and characterize small molecule-protein interactions using a label-free system that is extremely sensitive, quantitative and provides information on binding kinetics. The two technologies are a label-free multiplexed detection system that is based on nanohole arrays and a protein microarray system, nucleic acid programmable protein array (NAPPA). The nanohole array sensor, technology is a detection method based on a novel photonics device whose key characteristics are: single molecule sensitivity, a very high level of multiplexing, label-free detection, and the generation of kinetic binding data. The NAPPA technology is a method for the miniaturized presentation of thousands of proteins based on in situ expression of protein from cDNAs printed onto a surface. NAPPA addresses the key constraints associated with other protein microarray systems, namely, protein stability and storage, elimination of the need for protein purification, and captures 1000 fold more protein than other approaches. This project also makes use of a library of expression ready cDNA clones for human proteins that is being assembled by the Harvard Institute of Proteomics. These technologies have the potential to produce a valuable tool in lead identification and optimization.

描述（由申请人提供）：了解小分子结合的选择性是药物发育中的一个核心问题。这既影响了哪些理想的目标可能对化合物的反应以及哪些意外目标可能导致毒性。目前，这些问题是难以回答的问题，因为它们需要评估小分子与许多蛋白质的相互作用，这反过来又需要测试许多蛋白质并检测和表征相互作用的方法。蛋白质微阵列提供了一种令人信服的方法，用于展示许多用于测试的蛋白质，但是目前可用于评估小分子结合的方法需要用标签修改小分子。这种标记通常会导致小分子的结合性能变化，这可能很困难。一项使研究人员能够以高通量方式解决这些问题的技术将加速发现向诊所的过渡。在这个项目中，我们建议将两种技术聚集在一起，这些技术将导致高通量系统，以使用无标记的系统来检测和表征小分子 - 蛋白质相互作用，该系统非常敏感，定量，并提供有关结合动力学的信息。 这两种技术是基于纳米荷尔阵列和蛋白质微阵列系统的无标签多路复用检测系统，核酸可编程蛋白阵列（NAPPA）。 Nanohole阵列传感器，技术是一种基于一种新型光子设备的检测方法，其关键特征是：单分子灵敏度，非常高的多重多路复用，无标记检测和动力学结合数据的产生。 NAPPA技术是一种基于印刷在表面上的cDNA的原位表达的数千种蛋白质的微型呈现的方法。 NAPPA解决了与其他蛋白质微阵列系统相关的关键约束，即蛋白质稳定性和储存，消除对蛋白质纯化的需求，并捕获比其他方法更多的蛋白质。 该项目还利用了由哈佛蛋白质组学研究所组装的人类蛋白质的表达现成的cDNA克隆库。 这些技术有可能生产出铅识别和优化的有价值的工具。