Covalent Modifier Discovery Using Hydrogen/Deuterium Exchange–Mass Spectrometry

Abstract

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 γ (PPARγ) 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 PPARγ ligand-binding domain (PPARγ−LBD) with synergistic activation. Furthermore, iodoacetic acid was identified as a novel covalent modifier that stabilizes the PPARγ−LBD.