Abstract
MAPK-activated protein kinase 2 (MAPKAPK2, MK2) is a downstream substrate of p38 MAPK that regulates inflammatory signaling and cytokine synthesis, making it an attractive therapeutic target. Although several crystal structures of MAPKAPK2 with ATP-competitive inhibitors have supported drug discovery, structural elucidation of selective non-ATP-competitive inhibitors has remained challenging. MK-25 is a non-ATP-competitive furan-2-carboxamide MAPKAPK2 inhibitor developed at Merck to overcome the selectivity and cellular-efficacy limitations of ATP-competitive chemotypes. Here, we show through isothermal titration calorimetry that MK-25 binds the activated MAPKAPK2, consistent with state-dependent allosteric binding. To determine the binding mode of MK-25, we solved a 2.8 A resolution crystal structure of an activated MAPKAPK2 construct in ternary complex with the ATP-site inhibitor PF-3644022 and a bromo-analogue of MK-25. The ligand occupies a pocket in the C-lobe overlapping the protein-substrate recognition region and formed through rearrangements of αD and αG helices and the activation segment. These interactions are consistent with the structure-activity relationships reported for the furan-2-carboxamide chemotype and identify a ligandable allosteric pocket that may facilitate future structure-guided design of selective, non-ATP-competitive MAPKAPK2 inhibitors.