Xiaoyun Wang; Jianxian Sun; Shabbir Ahmad; Diwen Yang; Fengling Li; U. Hang Chan; Hong Zeng; Conrad V. Simoben; Scott Houliston; Aiping Dong; Albina Bolotokova; Elisa Gibson; Maria Kutera; Pegah Ghiabi; Ivan Kondratov; Tetiana Matviyuk; Alexander Chuprina; Danai Mavridi; Christopher Lenz; Andreas C. Joerger; Benjamin D. Brown; Richard B. Heath; Wyatt W. Yue; Lucy K. Robbie; Tyler S. Beyett; Susanne Müller; Stefan Knapp; Rachel Harding; Matthieu Schapira; Peter J. Brown; Vijayaratnam Santhakumar; Suzanne Ackloo; Cheryl H. Arrowsmith; Aled M. Edwards; Hui Peng; Levon Halabelian
bioRxiv, 2025
https://doi.org/10.1101/2025.01.17.633682

Abstract

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 KDs 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.