Pushing the limits of hydrogen/deuterium exchange mass spectrometry to study protein:fragment low affinity interactions

Abstract

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. In this work, the drug-target protein Cyclophilin D (CypD) was used as a model to explore the boundaries of fragments binding characterization by HDX-MS (fHDX-MS). We performed a systematic study on the optimal conditions for fHDX-MS execution and found that fragments with binding affinities in the double-digit mM range are still amenable to fHDX-MS. We observed that, despite the intrinsic low resolution of HDX-MS, fragments binding sites that partially overlap can still be distinguished. Overall, this study shows that fHDX-MS can be a useful method for FBDD.