The crystal structures of human alpha-thrombin complexed with active site-directed diamino benzo[b]thiophene derivatives: A binding mode for a structurally novel class of inhibitors

The crystal structures of four active site-directed thrombin inhibitors, 1- 4, in a complex with human alpha-thrombin have been determined and refined at up to 2.0 Angstrom resolution using X-ray crystallography. These compoun ds belong to a structurally novel family of inhibitors based on a 2,3-disub stituted benzo[b]thiophene structure. Compared to traditional active-site d irected inhibitors, the X-ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleu s of the inhibitor appears to bind in the Si specificity pocket. At the sam e time, the basic amine of the C-3 side chain of the inhibitor interacts wi th the mostly hydrophobic proximal, S-2, and distal, S-3, binding sites. Th e second, basic amine side chain at C-2 was found to point away from the ac tive site, occupying a location between the S-1 and S-1' sites. Together, t he aromatic rings of the C-2 and C-3 side chains sandwich the indole ring o f Trp60D contained in the thrombin S-2 insertion loop defined by the sequen ce "Tyr-Pro-Pro-Trp." [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323-373).] In contr ast to the binding mode of more classical thrombin inhibitors (D-Phe-Pro-Ar g-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin acti ve site. A detailed analysis of the three-dimensional structures not only p rovides a clearer understanding of the interaction of these agents with thr ombin, but forms a foundation for rational structure-based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900-fold more potent than the screening hit 1.