HTS of small molecule-protein interactions

小分子-蛋白质相互作用的 HTS

• 批准号: 7125563
• 负责人: DALE NORMAN LARSON
• 金额: $ 69.42万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125563
• 关键词: NIH Roadmap Initiative tag; biotechnology; complementary DNA; genetic library; high throughput technology; intermolecular interaction; nanotechnology; peptide library; protein quantitation /detection; proteins; proteomics; small molecule; technology /technique development

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开发的核心问题。它影响到确定哪些理想目标可能对化合物产生反应，以及哪些非预期目标可能导致毒性。目前，这些问题很难回答，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以及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来呈现许多蛋白质进行测试，但目前可用于评估小分子结合的方法需要用标签修饰小分子。这种标记经常会导致小分子结合性能的变化，并且可能很困难。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将发现转化为临床。在这个项目中，我们建议将两种技术结合在一起，形成一个高通量系统，使用极其灵敏、定量并提供结合动力学信息的无标记系统来检测和表征小分子-蛋白质相互作用。 这两项技术是基于纳米孔阵列的无标记多重检测系统和蛋白质微阵列系统、核酸可编程蛋白质阵列（NAPPA）。纳米孔阵列传感器技术是一种基于新型光子器件的检测方法，其主要特点是：单分子灵敏度、极高水平的多重性、无标记检测以及动力学结合数据的生成。 NAPPA 技术是一种基于从打印到表面的 cDNA 中原位表达蛋白质来小型化呈现数千种蛋白质的方法。 NAPPA 解决了与其他蛋白质微阵列系统相关的关键限制，即蛋白质稳定性和储存、消除了蛋白质纯化的需要，并且捕获的蛋白质比其他方法多 1000 倍。 该项目还利用了哈佛大学蛋白质组学研究所正在组装的人类蛋白质表达准备 cDNA 克隆库。 这些技术有潜力成为先导化合物识别和优化的宝贵工具。