A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening
A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening
复制标题
DOI：
10.1177/108705702237674
复制
发表时间：
2002-10-01
影响因子：
--
通讯作者：
Fesik, SW
中科院分区：
文献类型：
--
作者：
Hajduk, PJ;Betz, SF;Fesik, SW
描述了一种用于开发针对未知功能的靶点的高通量筛选测定的策略，所述靶点涉及使用核磁共振（NMR）光谱。使用这种方法，结合到蛋白质目标的分子从基于NMR的底物、辅因子和已知结合到许多蛋白质和酶的其他化合物的文库的筛选中鉴定。一旦发现了配体，就合成配体的荧光或放射性标记的类似物，其可用于高通量筛选。该方法是说明在开发一个高通量筛选测定HI-0033，一个保守的蛋白质流感嗜血杆菌，其功能是目前未知的。通过NMR发现腺苷与HI-0033结合，并且快速鉴定出在亚微摩尔范围内与HI-0033结合的荧光类似物。使用这些荧光化合物，荧光偏振分析，是适合于高通量筛选，并获得详细的结构-活性关系的铅优化。
A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.