Covalent Modifier Discovery Using Hydrogen/Deuterium Exchange-Mass Spectrometry

Covalent ligands are generally filtered out of chemical libraries used for high-throughput screening, because electrophilic functional groups are considered to be pan-assay interference compounds (PAINS). Therefore, screening strategies that can distinguish true covalent ligands from PAINS are required. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for evaluating protein stability. Here, we report a covalent modifier screening approach using HDX-MS. In this study, HDX-MS was used to classify peroxisome proliferatoractivated receptor gamma (PPAR gamma) and vitamin D receptor ligands. HDX-MS could discriminate the strength of ligand-protein interactions. Our HDX-MS screening method identified LT175 and nTZDpa, which can bind concurrently to the PPAR gamma ligand-binding domain (PPAR gamma-LBD) with synergistic activation. Furthermore, iodoacetic acid was identified as a novel covalent modifier that stabilizes the PPAR gamma-LBD.