HTS of small molecule-protein interactions (RMI)

描述(由申请人提供)：了解小分子结合的选择性是药物开发中的一个中心问题。它既影响到确定哪些理想的目标可能对化合物起作用，也涉及到哪些非预期的目标可能导致毒性。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引人注目的方法来呈现许多用于测试的蛋白质，但目前可用于评估小分子结合的方法需要用标记来修饰小分子。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。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，从而产生一个高通量的系统，使用一个极其敏感、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与蛋白质的相互作用。 这两项技术分别是基于纳米孔阵列的无标记多路检测系统和蛋白质微阵列系统，即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学器件的检测方法，其关键特征是：单分子灵敏度、非常高的复用水平、无标记检测、以及生成动力学结合数据。NAPPA技术是一种基于打印到表面的cDNA原位表达蛋白质的方法，可以微型化呈现数千种蛋白质。Nappa解决了与其他蛋白质微阵列系统相关的关键限制，即蛋白质的稳定性和储存，消除了对蛋白质纯化的需要，并捕获了比其他方法多1000倍的蛋白质。该项目还利用了哈佛蛋白质组学研究所正在组装的人类蛋白质表达准备基因克隆文库。 这些技术有可能成为识别和优化铅的宝贵工具。

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.