Thrombin Inhibitors With Novel Azaphenylalanine Scaffolds and a New P1 Binding Pocket Functionality: Structural Analysis of Binding

Gregor Mlinsek,¹ Rainer Friedrich² and Tom Solmajer^1,*

¹ National Institute of Chemistry, POB 660, Hajdrihova 19, 1001 Ljubljana, Slovenia
² Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany
* Corresponding author: E-mail: tom.solmajer@ki.si,
Phone: +386-01-4760-277; Fax: +386-01-4760-300

Abstract
Thrombin, a serine proteinase of the trypsin family, plays a central role in thrombosis and hemostasis. There is a high need for potent, fast, stable and selective direct thrombin inhibitors for oral use, and intensive research in this field is being carried out. Recently, we followed a rational design paradigm for discovery of two novel series of thrombin inhibitors. Firstly, we concentrated on inhibitors with an aza peptide scaffold that mimic the classical tripeptide D-Phe- Pro-Arg structure. A second focus of our work was the search for thrombin inhibitors with uncharged P1 functionalities to optimize the bioavailability and selectivity towards trypsin. Our efforts resulted in several P1 bicyclic arginine mimetics attached to the glycyl-proline amide and pyridinone acetamide scaffold. In this paper we report seven crystal structures of a series of congeneric inhibitors with the novel aza scaffold and with neutral P1 moieties in complex with thrombin. In particular, the requirements for a successful recognition of both inhibitor classes directed at the protein’s active-site pocket S1 are discussed based on detailed analysis of their electron distribution and water structure in the pocket.

Keywords: Thrombin, inhibitor, X-ray structure