The Broad Institute of Harvard and MIT, Howard Hughes Medical Institute, and the Department of Chemistry and Chemical Biology, Harvard University , 75 Ames Street, Cambridge, Massachusetts 02142, United States.
J Am Chem Soc. 2017 Aug 2;139(30):10192-10195. doi: 10.1021/jacs.7b04880. Epub 2017 Jul 20.
We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.
我们之前报道了使用未纯化蛋白质进行相互作用测定（IDUP）的方法，该方法可从细胞裂解物中 DNA 连接的小分子和未纯化的蛋白质靶标的混合物中选择性扩增编码配体：靶对的 DNA 序列。在这项研究中，我们将 IDUP 应用于 DNA 编码生物活性化合物文库和 DNA 标记的人类激酶文库，以在单个溶液相文库 × 文库实验中从 32096 种可能的组合中鉴定配体：蛋白质结合伙伴。结果再现了已知的小分子：蛋白质相互作用，还表明依他尼酸是 MAP2K6 激酶的新型配体和抑制剂。依他尼酸通过烷基化一个非保守的半胱氨酸残基部分抑制 MAP2K6。这项工作验证了 IDUP 发现与生物医学相关蛋白质的配体的能力。
