Miyamoto Suzanne, Liu Ruiwu, Hung Susan, Wang Xiaobing, Lam Kit S
Division of Hematology and Oncology, Department of Internal Medicine, University of California Davis Cancer Center, University of California Davis, Sacramento, CA 95817, USA.
Anal Biochem. 2008 Mar 1;374(1):112-20. doi: 10.1016/j.ab.2007.10.028. Epub 2007 Oct 24.
The search for small molecules that specifically recognize protein targets is a laborious process if conducted in a one protein-one compound manner. A high-throughput antibody-based screening of one bead-one compound (OBOC) combinatorial small molecule libraries is described here, whereby libraries containing thousands of different small molecule ligands are synthesized on individual TentaGel beads and simultaneously screened for protein binding to individual beads, each with a different compound. The use of OBOC libraries greatly facilitates this simultaneous screening of thousands of compounds. Now, through the use of monoclonal or affinity-purified antibodies, small molecules that bind a particular protein contained in a complex mixture of biological molecules have been identified. This method identified small molecule ligands that bound beta-actin present in cytoplasmic cell extracts of Ramos B-lymphoma cells. These small molecule ligands were resynthesized in immobilized and soluble forms and tested for binding of beta-actin present in Ramos B-cell extracts and for activity against Ramos lymphoma cells. This high-throughput screening immunoassay method has great promise for improving our ability to find relevant, bioactive small molecules that target a specific native protein in a complex protein mixture without purification of the protein.
如果以一种蛋白质对一种化合物的方式寻找特异性识别蛋白质靶点的小分子，这将是一个费力的过程。本文描述了一种基于抗体的高通量单珠单化合物（OBOC）组合小分子文库筛选方法，即包含数千种不同小分子配体的文库在单个TentaGel珠上合成，并同时筛选蛋白质与单个珠子的结合情况，每个珠子带有不同的化合物。OBOC文库的使用极大地促进了对数千种化合物的同时筛选。现在，通过使用单克隆抗体或亲和纯化抗体，已经鉴定出了能与生物分子复杂混合物中特定蛋白质结合的小分子。该方法鉴定出了能与拉莫斯B淋巴瘤细胞胞质提取物中存在的β-肌动蛋白结合的小分子配体。这些小分子配体以固定化和可溶形式重新合成，并测试其与拉莫斯B细胞提取物中β-肌动蛋白的结合情况以及对拉莫斯淋巴瘤细胞的活性。这种高通量筛选免疫测定方法对于提高我们在不纯化蛋白质的情况下，在复杂蛋白质混合物中找到靶向特定天然蛋白质的相关生物活性小分子的能力具有很大的前景。
