Wang Xiaoyun, Sun Jianxian, Ahmad Shabbir, Yang Diwen, Li Fengling, Chan U Hang, Zeng Hong, Simoben Conrad V, Houliston Scott, Dong Aiping, Bolotokova Albina, Gibson Elisa, Kutera Maria, Ghiabi Pegah, Kondratov Ivan, Matviyuk Tetiana, Chuprina Alexander, Mavridi Danai, Lenz Christopher, Joerger Andreas C, Brown Benjamin D, Heath Richard B, Yue Wyatt W, Robbie Lucy K, Beyett Tyler S, Müller Susanne, Knapp Stefan, Harding Rachel, Schapira Matthieu, Brown Peter J, Santhakumar Vijayaratnam, Ackloo Suzanne, Arrowsmith Cheryl H, Edwards Aled M, Peng Hui, Halabelian Levon
Department of Chemistry, University of Toronto, Toronto, ON, Canada.
Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
bioRxiv. 2025 Jan 22:2025.01.17.633682. doi: 10.1101/2025.01.17.633682.
We report an enantioselective protein affinity selection mass spectrometry screening approach (EAS-MS) that enables the detection of weak binders, informs about selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screened 31 human proteins against a designed library of 8,210 chiral compounds. 16 binders to 12 targets, including many proteins predicted to be "challenging to ligand", were discovered and confirmed in orthogonal assays. 7 binders to 6 targets bound in an enantioselective manner, with s ranging from 3 to 20 μM. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) were selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanism for enantioselective selectivity was readily explained. EAS-MS can be used to identify and characterize selective and weakly-binding ligands for novel protein targets with unprecedented throughput and sensitivity.
我们报道了一种对映选择性蛋白质亲和选择质谱筛选方法（EAS-MS），该方法能够检测弱结合剂，提供选择性信息，并生成结合的正交确认。在用对照蛋白进行方法开发后，我们针对一个设计的包含8210种手性化合物的文库筛选了31种人类蛋白质。发现了16种与12个靶标的结合剂，包括许多预计“对配体具有挑战性”的蛋白质，并在正交试验中得到了确认。7种与6个靶标的结合剂以对映选择性方式结合，解离常数（KD）范围为3至20 μM。选择了针对四个靶标（DDB1、WDR91、WDR55和HAT1）的结合剂，使用X射线晶体学进行深入表征。在所有四种情况下，对映选择性的机制都很容易解释。EAS-MS可用于以前所未有的通量和灵敏度识别和表征新型蛋白质靶标的选择性和弱结合配体。
