CRYSTAL STRUCTURES OF TWO THROMBIN COMPLEXES WITH NOVEL PEPTIDE MIMETIC INHIBITORS. Robert St. Charles, Peggy Vanderhoff-Hanaver, Doug Boatman*, Cy Ogdu*, A. Tulinsky, Department of Chemistry, Michigan State University, East Lansing, MI 48824, *Molecumetics, Bellevue, WA 98005

Thrombin is a trypsin-like blood serine protease specialized in catalyzing the proteolysis of fibrinogen to fibrin monomers during the final stage of the coagulation cascade. Synthetic active site inhibitors of thrombin represent potentially useful antithrombotic agents and are showing promise as alternatives to current heparin-based therapeutics.

The crystal structures of human [[alpha]]-thrombin complexed separately to two similar peptidomimetic active site inhibitors (MOL124-1 and MOL126-1) have been solved to 2.1 Å resolution and refined using restrained least squares with R-factors less than 0.18. Ternary complexes were prepared by soaking crystals of hirugen-inactivated [[alpha]]-thrombin (space group C2) in 2 - 7 mM solutions of each synthetic inhibitor over a period of 5 days. Both inhibitors resemble D-Phe-Pro-Arg chloromethylketone (PPACK) in structure and bind in a similar fashion within the catalytic site. As with other active site inhibitors of thrombin, the arginyl side chain of the P1 residue of each inhibitor is bound within the primary specificity pocket of the protein making salt-bridged hydrogen bonds with the acidic side chain of Asp 189. Moreover, this arginine residue forms an expected hemiketal with Ser 195 in both complexes. The P2 residue of each mimetic possesses a novel bicyclic structure that constrains the precleavage portion of the inhibitor in an extended, substrate-like conformation, and interacts with the S2 site much like the proline residue of thrombin-bound PPACK. An aromatic P3 residue, D-phenylalanine, occupies the aryl binding site in both complexes. In MOL124-1, this residue is acylated at the amino-terminus, and makes several stabilizing interactions with the protein. One of the two inhibitors (MOL126-1) possesses an aromatic, [[alpha]]-ketoamide-linked P1' residue that is hydrogen-bonded to His 57 and makes numerous apolar interactions within the S1' and S2' sites of thrombin. This residue, absent in MOL124-1, is responsible for enhanced inhibitory activity.