STEREOCHEMICAL CONSIDERATIONS IN DRUG DESIGN: THE CRYSTAL STRUCTURES OF HUMAN [[alpha]]-THROMBIN COMPLEXED WITH TWO TRIPEPTIDYL ALDEHYDE INHIBITORS AT 2.1Å RESOLUTION. E. Zhang, R. K. Arni, O. E. Levy#, A. Tulinsky, Department of Chemistry, Michigan State University, East Lansing, MI48824, USA, #Corvas International, Inc. San Diego, CA 92121, USA

Thrombin is a trypsin-like serine protease that plays a central role in thrombosis and hemostasis, inhibitors of which are potentially pharmacologically important as antithrombotics. The structures of many small molecule inhibitors complexed with thrombin reveal that the active site of thrombin has one anionic subsite (S1) and two hydrophobic subsites (S2 and S3) in addition to the catalytic site (Ser-195 and an oxyanion hole) .

The binding of transition-state analog inhibitors, such as peptidyl aldehydes to trypsin-like serine proteases may be modelled by two kinetic steps, a rapid, reversible binding of enzyme and inhibitor involving the three subsites of the active site followed by a slower reversible covalent-like interaction at the catalytic site. Two isomeric peptidyl aldehyde thrombin inhibitors containing 3-guanidylpiperidyl alaninal at the P1 position were designed and synthesized to examine the contributions of steric and dynamic features to selectivity and kinetics. The two inhibitors were identical except for the absolute configuration of the chiral C[[gamma]] atom. The inhibitors were soaked into thrombin-hirugen crystals and the structures of the complexes were determined and refined at 2.1Å resolution to R values of ~ 0.15. From the structures it was possible to assign an absolute configuration to each isomer and thus to correlate the structures/configurations with the observed inhibition kinetics. The kinetically fast inhibitor(Ki=5nM) was identified as the R configuration and the more normal slow-tight binding kinetics (Ki=0.3nM) was associated with the S configuration at the P1-C[[gamma]].

The structures of the R- and S- configurations were very similar at the S2 and S3 subsites and the catalytic site, but are different at the S1 subsite. In the Rconfiguration, the piperidyl ring is in a chair conformation with the guanidyl group forming energetically favorable interactions with Asp-189 of thrombin. In contrast, the piperidyl ring of the S-configuration is in an energetically less favorable boat conformation with its guanidyl interacting with Asp- 189. At the catalytic site, the oxygen atom of the aldehyde group of both isomers is not in the oxyanion hole. These observations suggest that the kinetically slow step is related to the interactions of the boat form in the S1 site.