THROMBIN COMPLEXES WITH THIAZOLE-BASED INHIBITORS: USEFUL PROBES OF THE S1' BINDING SITE. John H. Matthews*, Raman Krishnan*, Michael J. Costanzo#, Bruce E. Maryanoff#, A. Tulinsky*, * Department of Chemistry, Michigan State University, East Lansing, MI 48824, #The R. W. Johnson Pharmaceutical Research Institute, Spring House, PA 19477

The serine protease thrombin plays a central role in blood clotting with the most prominent function being the conversion of fibrinogen to fibrin in the later stages of the coagulation cascade. A large number of inhibitor-thrombin complexes have been studied by X-ray crystallography. Most of these inhibitors bind to one or the other of the S1-S3 subsites of the active site or the fibrinogen recognition exosite. Less is known of the binding at the S' subsites that involve substrate residues downstream from the point of cleavage. We report here the results of S1'-binding thiazole-containing groups and the implications for the future design of inhibitors. The potent thrombin inhibitor RWJ-50353 is a tripeptide with a D-Phe-Pro-Arg motif (PPACK) with a benzothiazole group. The other inhibitor, RWJ-50215, is related to DAPA, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide, an early member of a class of inhibitors based on the chemical nature of thrombin binding sites and contains a 2-ketothiazole group.

The RWJ-50353-hirugen-thrombin structure was refined to an R value of 0.168 in the (7.0-2.3) Å resolution range with 125 water molecules, while the RWJ-50215 complex converged at an R value of 0.155 in the (7.0-1.8) Å resolution range with 161 water molecules.

Binding in the S1-S3 subsites is similar to the parent compounds PPACK (RWJ-50353) and DAPA (RWJ-50215). The benzothiazole in RWJ-50353 and the thiazole in RWJ-50215 bind at the S1' site of thrombin. There they are surrounded by His57, Tyr60A, Trp60D, Lys60F of thrombin, and in the case of RWJ-50215, also by the piperidine ring of the inhibitor. In RWJ-50353, the N1 atom of the benzothiazole forms a hydrogen bond with His57NE2 (2.7 Å) and the indole ring of Trp60D stacks edge onto the face of the benzothiazole ring. The sidechain of Lys60F is displaced from its normal position by the bulky benzothiazole group. Both N1 and O1 of the 2-ketothiazole of RWJ-50215 form hydrogen bonds with the sidechain of Lys60F.