X-Ray Crystal Structures of Thrombin in Complex with D-Phe-Pro-Arg and with Small Benzamidine- and Arginine-Based “Non-Peptidic” Inhibitors

  • Wolfram Bode
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 340)


Thrombin plays a key role in thrombosis and haemostasis. Amongst other functions, thrombin effects thrombus formation through conversion of fibrinogen into fibrin and induction of platelet aggregation. Thrombin has therefore been implicated in various disease processes such as myocardial infarction, stroke or pulmonary embolism. In such cases, the quick administration of selective thrombin inhibitors might offer an attractive means of antithrombotic therapy1. Apart from a few natural protein inhibitors such as antithrombin III and hirudin, a large number of synthetic inhibitors have been found’. Some of these inhibitors (in particular MQPA = MD805, see Figure 1) are already in clinical trials. The recent availability of experimental thrombin structures10,11 allows rationalization of these screening results with respect to substitution effects, conformation and shape19,23. In conjunction with interactive graphics and new computational methods, such experimental structures now offer the unique possibility of tailoring existing drugs, or of designing new compounds and elaborating them into drugs.


Chloromethyl Ketone Bovine Thrombin Specificity Pocket Connolly Surface Piperidine Carboxylic 
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  1. 1.
    J.W. Fenton II, F.A. Ofosu, D.G. Moon, and J.M. Maraganore, Thrombin structure and function: why thrombin is the primary target for antithrombotics, Blood Coag. Fibrinol. 2: 69 (1991).CrossRefGoogle Scholar
  2. 2.
    S. Bajusz, E. Barabas, P. Tolnay, E. Szell, and D. Bagdy, Inhibition of thrombin and trypsin by tripeptide aldehydes, Int. J. Peptide Prot.Res. 123: 217 (1978).Google Scholar
  3. 3.
    C. Kettner, and E. Shaw, Inactivation of trypsin-like enzymes with peptides of arginine chloromethyl ketone, Method Enzymol. 80: 826 (1981).CrossRefGoogle Scholar
  4. 4.
    J. Stürzebecher, F. Markwardt, B. Voigt, G. Wagner, and P. Walsmann, Cyclic amides of Na-arylsulfonylaminoacylated 4-amidinophenyl-alanine - tight binding of thrombin, Thromb. Res. 29: 635 (1983).Google Scholar
  5. 5.
    J. Stürzebecher, P. Walsmann, B. Voigt, and G. Wagner, Inhibition of bovine and human thrombins by derivatives of benzamidine, Thromb. Res. 36: 457 (1984).Google Scholar
  6. 6.
    R. Kikumoto, Y. Tamao, T. Tezuka, S. Tonomura, H. Hara, K. Ninomiya, A. Hijikata, and S. Okamoto, Selective inhibition of thrombin by (2R,4R)-4-ethyl-11 N2–3-methyl-1, 2, 3,4-tetrahydro-8-quinolinyl)sulphonyl)-L-arginyl] -2-piperidinecarboxylic acid, Biochemistry 23: 85 (1984).Google Scholar
  7. 7.
    K. Cho, T. Tanaka, R.R. Cook, W. Kisiel, K. Fujikawa, K. Kurachi, and J.C. Powers, Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates, Biochemistry 23: 644 (1984).PubMedCrossRefGoogle Scholar
  8. 8.
    C.M. Kam, W. Fujikawa, and J.C. Powers, Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants, Biochemistry 27: 2547 (1988).PubMedCrossRefGoogle Scholar
  9. 9.
    C. Kettner, L. Mersinger, and R. Knabb, The selective inhibition of thrombin by peptides of boroarginine, J. Biol. Chem. 265: 18289 (1991).Google Scholar
  10. 10.
    W. Bode, I. Mayr, U. Baumann, R. Huber, S.R. Stone, and J. Hofsteenge, The refined 1.9 A crystal structure of human a-thrombin: Interaction with D-Phe-ProArg chloromethyl-ketone and significance of the Tyr-Pro-Pro-Trp insertion segment, EMBO J. 8: 3467 (1989).PubMedGoogle Scholar
  11. 11.
    W. Bode, D. Turk, and A. Xarshikov, The refined 1.9 A crystal structure of DPhe-Pro-Arg chloromethylketone inhibited human a-thrombin. Structure analysis, overall structure, electrostatic properties, detailed active-site geometry, structure-function relationships, Protein Sci. 426 (1992).Google Scholar
  12. 12.
    W. Bode, M. and Stubbs, The spatial structure of thrombin as a guide to its multiple sites of interaction, Sem. Thromb. Hemost. 426: (1992).Google Scholar
  13. 13.
    T.J. Rydel, A. Tulinsky, W. Bode, R. and Huber, R.fined structure of the hirudinthrombin complex, J. Mol. Biol. 221: 583 (1991).Google Scholar
  14. 14.
    M.G. Grütter, U.P. Priestle, J. Rahuel, H. Grossenbacher, W. Bode, J. Hofsteenge, and Stone SR, Crystal structure of the thrombin-hirudin complex: A novel mode of serine protease inhibition, EMBO J. 9: 2361 (1990).PubMedGoogle Scholar
  15. 15.
    E. Skrzykczak-Jankun, V.E. Carperos, K.G. Ravichandran, A. Tulinsky, M. Westbrook, and J.M. Maraganore, Structure of the hirugen and hirulog 1 complexes of a-thrombin, J. Mol. Biol. 221: 1379 (1991).Google Scholar
  16. 16.
    M. Stubbs, H. Oschkinat, I. Mayr, R. Huber, H. Angliker, S.R. Stone, and W. Bode, The interaction of thrombin with fibrinogen - a structural basis for its specificity, Eur. J. Biochem. 206: 187 (1992).PubMedCrossRefGoogle Scholar
  17. 17.
    M. Stubbs, and W. Bode, A model for the specificity of fibrinogen cleavage by thrombin, Semin. Thromb. Hemost. In press (1992).Google Scholar
  18. 18.
    P.D. Martin, W. Robertson, D. Turk, R. Huber, W. Bode, and B.F.P. Edwards, The structure of residues 7–16 of the Aa-chain of human fibrinogen bound to bovine thrombin at 2.3 A resolution, J. Biol. Chem. 267: 7911 (1992).PubMedGoogle Scholar
  19. 19.
    W. Bode, D. Turk, and J. Stürzebecher, Geometry of binding of the benzamidineand arginine-based inhibitors NAPAP and MQPA to human a-thrombin. X-ray crystallographic determination of the NAPAP-trypsin complex and modelling of NAPAP-thrombin and MQPA-thrombin, Eur. J. Biochem. 193: 175 (1990).PubMedCrossRefGoogle Scholar
  20. 20.
    D. Turk, J. Stürzebecher, and W. Bode, Geometry of binding of a-tosylated piperidides of m-amidino, p-amidino-and p-guanidino phenylalanine to thrombin and trypsin. X-ray crystal structures of their trypsin complexes and modelling of their thrombin complexes, FEBS. Lett. 287: 133 (1991).Google Scholar
  21. 21.
    T. Matsuzaki, C. Sasaki, C. Okumura. and H. Umeyama, X-ray analysis of a thrombin inhibitor-trypsin complex, J. Biochem. (Tokyo) 105: 949 (1989).Google Scholar
  22. 22.
    M.M. Chow, E.F. Meyer Jr, W. Bode, C-M. Kam, R. Radhakrishnan, J. Vijayalakshmi, and J.C. Powers, X-ray crystal structure of the complex of trypsin inhibited by 4-chloro-3-ethoxy-7-guanidinocoumarin: A proposed model of the thrombin-inhibitor complex, J. Am. Chem. Soc. 112: 7783 (1990).CrossRefGoogle Scholar
  23. 23.
    H. Brandstetter, D. Turk, W. Hoeffken, D. Grosse, J. Stürzebecher, P.D. Martin, B.F.P. Edwards, and W. Bode, The 2.2 A resolution X-ray crystal structure of the complex of trypsin inhibited by 4-chloro-3-ethoxy-7-guanidinocoumarin: A proposed model of the thrombin-inhibitor complex, Mol. Biol. 226: 1985 (1992).Google Scholar
  24. 24.
    W. Bode, H. Brandstetter, D. Turk, M. Bauer, and J. Stürzebecher, Crystallographic determination of thrombin complexes with small synthetic inhibitors as a starting point for the receptor-based design of antithrombotics, Semin. Thromb. Hemost. In press (1992).Google Scholar
  25. 25.
    W. Bode, and A. Karshikov, The electrostatic properties of thrombin: Importance for structural stabilization and for ligand binding, Semin. Thromb. Hemost. In press (1992).Google Scholar
  26. 26.
    W. Bode, J. Walter, R. Huber, H.R. Wenzel, and H. Tschesche, The refined 2.2 A (0.22mm) X-ray crystal structure of the ternary complex formed by bovine trypsinogen, valine-valine, and the Arg15 analogue of bovine pancreatic trypsin inhibitor, Eur. J. Biochem. 144: 185 (1984).PubMedCrossRefGoogle Scholar
  27. 27.
    W. Bode, H.J. Greyling, R. Huber, J. Otlewski, and T. Wilusz, The refined 2.0 A X-ray crystal structure of the complex formed between bovine 13-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash inhibitors with the carboxypeptidase A inhibitor from potatoes, FEBS. Lett. 242: 285 (1989).Google Scholar
  28. 28.
    L.J. Berliner, and Y.Y.L. Shen, Physical evidence for an apolar binding site near the catalytic center of human a-thrombin, Biochemistry 16: 4622 (1977).PubMedCrossRefGoogle Scholar
  29. 29.
    W. Bode, and P. Schwager, The refined crystal structure of bovine ß-trypsin at 1.8 A resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH7, J. Mol. Biol. 98: 693 (1975).PubMedCrossRefGoogle Scholar
  30. 30.
    T. Matsuzaki, C. Sasaki, and H. Umeyama, A predicted tertiary structure of a thrombin inhibitor-trypsin complex explains the mechanisms of the selective inhibition of thrombin, factor Xa, plasmin and trypsin, J. Biochem. 103: 537 (1988).PubMedGoogle Scholar

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© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Wolfram Bode
    • 1
  1. 1.Max-Planck-Institut für BiochemieMartinsriedGermany

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