Pushing the Limits of Hydrogen/Deuterium Exchange Mass Spectrometry to Study Protein:Fragment Low Affinity Interactions

Characterization of protein-ligand interactions is essential for the pre-clinical development of drug candidates and Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) has emerged as a valuable tool in this process. HDX-MS has predominantly been employed with high affinity compounds with only a few examples of its application for weaker binders such as fragments. Nevertheless, HDX-MS usage could be instrumental in Fragment-Based Drug Discovery (FBDD) programs, especially when dealing with challenging targets that cannot be studied by other higher resolution structural techniques, such as X-Ray crystallography, Cryogenic Electron Microscopy (Cryo-EM) or Nuclear Magnetic Resonance (NMR). In this work we used the drug-target protein Cyclophilin D (CypD) as model to explore how far fragments binding characterization by HDX-MS (fHDX-MS) can be pushed. We performed a systematic study on the best conditions for fHDX-MS execution and found that fragments with binding affinities in the double-digit mM range are still amenable to HDX-MS. We observed how, despite the intrinsic low resolution of HDX-MS, partially overlapping fragments binding sites can still be distinguished. This study contributes to asserting fHDX-MS as an instrumental method for FBDD and offers a methodological framework to investigate such interactions using HDX-MS.