Structural Studies of the uPA-Nafamostat Complexes Reveal a Covalent Inhibitory Mechanism of Nafamostat as the Basis for its Serine Protease Inhibition

Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases produced during the coagulation cascade and inflammation. Previous studies showed that NM was a highly safe drug for the treatment of different cancers, but the detailed inhibition mechanism of NM is not clear. In this study, we determined a series of crystal structures of NM in complex with a serine protease (uPA). These structures reveal that NM was cleaved by uPA and a hydrolyzed product (GBA) remained covalently linked to Ser195 of uPA, and the other hydrolyzed product (6A2N) releases from uPA. Strikingly, in the inactive uPA (uPA-S195A):NM structure, intact NM use the 6A2N side to bind to the specific pocket. MD simulations and binding free energy calculations show the conformation of NM in the uPA-S195A:NM complex is more stable than that in the uPA:NM complex. Moreover, the imidazole group of His57 flips further away from Ser195 and disrupts the stable canonical catalytic triad conformation. These results not only reveal the inhibitory mechanism of NM as an efficient serine protease inhibitor but also might provide the structural basis for a better understanding of the catalytic mechanism of serine proteases.