Preparation, characterization, and the crystal structure of the inhibitor ZK-807834 (CI-1031) complexed with factor Xa

Factor Xa plays a critical role in the formation of blood clots. This serin e protease catalyzes the conversion of prothrombin to thrombin, the first j oint step that links the intrinsic and extrinsic coagulation pathways. Ther e is considerable interest in the development of factor Xa inhibitors for t he intervention in thrombic diseases. This paper presents the structure of the inhibitor ZK-807834, also known as CI-1031, bound to factor Xa and prov ides the details of the protein purification and crystallization. Results f rom mass spectrometry indicate that the factor Xa underwent autolysis durin g crystallization and the first EGF-like domain was cleaved from the protei n. The crystal structure of the complex shows that the amidine of ZK-807834 forms a salt bridge with Asp189 in the S1 pocket and the basic imidazoline fits snugly into the S4 site. The central pyridine ring provides a fairly rigid linker between these groups. This rigidity helps minimize entropic lo sses during binding. In addition, the structure reveals new interactions th at were not found in the previous factor Xa/inhibitor complexes. ZK-807834 forms a strong hydrogen bond between an ionized 2-hydroxy group and Ser195 of factor Xa. There is also an aromatic ring-stacking interaction between t he inhibitor and Trp215 in the S4 pocket. These interactions contribute to both the potency of this compound (K-I = 0.11 nM) and the >2500-fold select ivity against homologous serine proteases such as trypsin.