MOLECULAR RECOGNITION AT THE THROMBIN ACTIVE-SITE - STRUCTURE-BASED DESIGN AND SYNTHESIS OF POTENT AND SELECTIVE THROMBIN INHIBITORS AND THE X-RAY CRYSTAL-STRUCTURES OF 2 THROMBIN-INHIBITOR COMPLEXES

Background: The serine protease thrombin is central in the processes o f hemostasis and thrombosis, To be useful, thrombin inhibitors should combine potency towards thrombin with selectivity towards other relate d enzymes such as trypsin. We previously reported the structure-based design of thrombin inhibitors with rigid, bicyclic core structures. Th ese compounds were highly active towards thrombin, but showed only mod est selectivity. Results: Here, we describe the rational design of sel ective thrombin inhibitors starting from the X-ray crystal structure o f the complex between the previously generated lead molecule and throm bin. The lead molecule bound with a Ki value of 90 nM and a selectivit y of 7.8 for thrombin over trypsin. Our design led to inhibitors with improved activity and greatly enhanced selectivity. The binding mode f or two of the new inhibitors was determined by X-ray crystallography o f their complexes with thrombin. The results confirmed the structures predicted by molecular modeling and, together with the binding assays, provided profound insight into molecular recognition phenomena at the thrombin active site. Conclusions: A novel Glass of nonpeptidic, sele ctive thrombin inhibitors has resulted from structure-based design and subsequent improvement of the initial lead molecule, These compounds, which are preorganized for binding to thrombin through a rigid, bicyc lic or tricyclic central core, could aid in the development of new ant ithrombotic drugs. Correlative binding and X-ray structural studies wi thin a series of related, highly preorganized inhibitors, which all pr efer similar modes of association to thrombin, generate detailed infor mation on the strength of individual intermolecular bonding interactio ns and their contribution to the overall free energy of complexation.