Application of a MALDI mass spectrometry assay to identify covalent fragments targeting the methyl-lysine reader protein MPP8.

In recent decades, the development of targeted covalent inhibitors (TCIs) has significantly advanced, as evidenced by the increasing number of FDA approved drugs with covalent mechanisms of action. This study focuses on the application of a robust and efficient screening platform to identify covalent fragments that modify the methyl-lysine reader protein M-phase phosphoprotein 8 (MPP8). Using a commercially available covalent fragment library with various electrophile-based warheads, we screened for fragments that covalently label MPP8 using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Follow-up validation and prioritization studies involving evaluation of the reactivity of hit fragments with glutathione led to the identification of two novel acrylamide-containing fragments that covalently label MPP8 at cysteine 99, which is adjacent to the methyl-lysine binding pocket. These results emphasize the value of efficient screening approaches in discovering novel covalent fragments for challenging targets and demonstrate the potential for developing covalent antagonists targeting MPP8.