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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月22日

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  • Catarina F. Malta,
  • Diana O. Silva,
  • Ulrich Grädler,
  • Pedro M. F. Sousa,
  • Djordje Musil,
  • Daniel Schwarz,
  • Joerg Bomke,
  • Tiago M. Bandeiras,
  • Alessio Bortoluzzi

摘要

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.