Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Jun;5(6):1174–1183. doi: 10.1002/pro.5560050620

Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

J Féthière 1, Y Tsuda 1, R Coulombe 1, Y Konishi 1, M Cygler 1
PMCID: PMC2143430  PMID: 8762149

Abstract

The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banner D. W., Hadváry P. Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors. J Biol Chem. 1991 Oct 25;266(30):20085–20093. [PubMed] [Google Scholar]
  2. Bode W., Mayr I., Baumann U., Huber R., Stone S. R., Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467–3475. doi: 10.1002/j.1460-2075.1989.tb08511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bode W., Turk D., Karshikov A. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci. 1992 Apr;1(4):426–471. doi: 10.1002/pro.5560010402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brandstetter H., Turk D., Hoeffken H. W., Grosse D., Stürzebecher J., Martin P. D., Edwards B. F., Bode W. Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics. J Mol Biol. 1992 Aug 20;226(4):1085–1099. doi: 10.1016/0022-2836(92)91054-s. [DOI] [PubMed] [Google Scholar]
  5. Cairns J. A., Fuster V., Kennedy J. W. Coronary thrombolysis. Chest. 1992 Oct;102(4 Suppl):482S–507S. doi: 10.1378/chest.102.4_supplement.482s. [DOI] [PubMed] [Google Scholar]
  6. Coller B. S. Platelets and thrombolytic therapy. N Engl J Med. 1990 Jan 4;322(1):33–42. doi: 10.1056/NEJM199001043220107. [DOI] [PubMed] [Google Scholar]
  7. DiMaio J., Gibbs B., Lefebvre J., Konishi Y., Munn D., Yue S. Y., Hornberger W. Synthesis of a homologous series of ketomethylene arginyl pseudodipeptides and application to low molecular weight hirudin-like thrombin inhibitors. J Med Chem. 1992 Sep 4;35(18):3331–3341. doi: 10.1021/jm00096a004. [DOI] [PubMed] [Google Scholar]
  8. DiMaio J., Ni F., Gibbs B., Konishi Y. A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond. FEBS Lett. 1991 Apr 22;282(1):47–52. doi: 10.1016/0014-5793(91)80441-5. [DOI] [PubMed] [Google Scholar]
  9. Fenton J. W., 2nd Thrombin specificity. Ann N Y Acad Sci. 1981;370:468–495. doi: 10.1111/j.1749-6632.1981.tb29757.x. [DOI] [PubMed] [Google Scholar]
  10. Grütter M. G., Priestle J. P., Rahuel J., Grossenbacher H., Bode W., Hofsteenge J., Stone S. R. Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition. EMBO J. 1990 Aug;9(8):2361–2365. doi: 10.1002/j.1460-2075.1990.tb07410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hauptmann J., Markwardt F. Pharmacologic aspects of the development of selective synthetic thrombin inhibitors as anticoagulants. Semin Thromb Hemost. 1992;18(2):200–217. doi: 10.1055/s-2007-1002426. [DOI] [PubMed] [Google Scholar]
  12. Janin J., Wodak S. Conformation of amino acid side-chains in proteins. J Mol Biol. 1978 Nov 5;125(3):357–386. doi: 10.1016/0022-2836(78)90408-4. [DOI] [PubMed] [Google Scholar]
  13. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  14. Kikumoto R., Tamao Y., Ohkubo K., Tezuka T., Tonomura S., Okamoto S., Funahara Y., Hijikata A. Thrombin inhibitors. 2. Amide derivatives of N alpha-substituted L-arginine. J Med Chem. 1980 Aug;23(8):830–836. doi: 10.1021/jm00182a004. [DOI] [PubMed] [Google Scholar]
  15. Kikumoto R., Tamao Y., Ohkubo K., Tezuka T., Tonomura S., Okamoto S., Hijikata A. Thrombin inhibitors. 3. Carboxyl-containing amide derivatives of N alpha-substituted L-arginine. J Med Chem. 1980 Dec;23(12):1293–1299. doi: 10.1021/jm00186a003. [DOI] [PubMed] [Google Scholar]
  16. Krstenansky J. L., Mao S. J. Antithrombin properties of C-terminus of hirudin using synthetic unsulfated N alpha-acetyl-hirudin45-65. FEBS Lett. 1987 Jan 19;211(1):10–16. doi: 10.1016/0014-5793(87)81264-4. [DOI] [PubMed] [Google Scholar]
  17. Lefkovits J., Topol E. J. Direct thrombin inhibitors in cardiovascular medicine. Circulation. 1994 Sep;90(3):1522–1536. doi: 10.1161/01.cir.90.3.1522. [DOI] [PubMed] [Google Scholar]
  18. Mao S. J., Yates M. T., Owen T. J., Krstenansky J. L. Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity. Biochemistry. 1988 Oct 18;27(21):8170–8173. doi: 10.1021/bi00421a027. [DOI] [PubMed] [Google Scholar]
  19. Markwardt F., Nowak G., Stürzebecher J., Griessbach U., Walsmann P., Vogel G. Pharmacokinetics and anticoagulant effect of hirudin in man. Thromb Haemost. 1984 Oct 31;52(2):160–163. [PubMed] [Google Scholar]
  20. Okamoto S., Hijikata A., Kikumoto R., Tonomura S., Hara H., Ninomiya K., Maruyama A., Sugano M., Tamao Y. Potent inhibition of thrombin by the newly synthesized arginine derivative No. 805. The importance of stereo-structure of its hydrophobic carboxamide portion. Biochem Biophys Res Commun. 1981 Jul 30;101(2):440–446. doi: 10.1016/0006-291x(81)91279-1. [DOI] [PubMed] [Google Scholar]
  21. Okamoto S., Kinjo K., Hijikata A., Kikumoto R., Tamao Y., Ohkubo K., Tonomura S. Thrombin inhibitors. 1. Ester derivatives of N alpha-(arylsulfonyl)-L-arginine. J Med Chem. 1980 Aug;23(8):827–830. doi: 10.1021/jm00182a003. [DOI] [PubMed] [Google Scholar]
  22. Qiu X., Padmanabhan K. P., Carperos V. E., Tulinsky A., Kline T., Maraganore J. M., Fenton J. W., 2nd Structure of the hirulog 3-thrombin complex and nature of the S' subsites of substrates and inhibitors. Biochemistry. 1992 Dec 1;31(47):11689–11697. doi: 10.1021/bi00162a004. [DOI] [PubMed] [Google Scholar]
  23. Rydel T. J., Ravichandran K. G., Tulinsky A., Bode W., Huber R., Roitsch C., Fenton J. W., 2nd The structure of a complex of recombinant hirudin and human alpha-thrombin. Science. 1990 Jul 20;249(4966):277–280. doi: 10.1126/science.2374926. [DOI] [PubMed] [Google Scholar]
  24. Rydel T. J., Tulinsky A., Bode W., Huber R. Refined structure of the hirudin-thrombin complex. J Mol Biol. 1991 Sep 20;221(2):583–601. doi: 10.1016/0022-2836(91)80074-5. [DOI] [PubMed] [Google Scholar]
  25. Sawyer R. T. Thrombolytics and anti-coagulants from leeches. Biotechnology (N Y) 1991 Jun;9(6):513-5, 518. doi: 10.1038/nbt0691-513. [DOI] [PubMed] [Google Scholar]
  26. Singh J., Thornton J. M., Snarey M., Campbell S. F. The geometries of interacting arginine-carboxyls in proteins. FEBS Lett. 1987 Nov 16;224(1):161–171. doi: 10.1016/0014-5793(87)80441-6. [DOI] [PubMed] [Google Scholar]
  27. Skrzypczak-Jankun E., Carperos V. E., Ravichandran K. G., Tulinsky A., Westbrook M., Maraganore J. M. Structure of the hirugen and hirulog 1 complexes of alpha-thrombin. J Mol Biol. 1991 Oct 20;221(4):1379–1393. [PubMed] [Google Scholar]
  28. Stone S. R., Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry. 1986 Aug 12;25(16):4622–4628. doi: 10.1021/bi00364a025. [DOI] [PubMed] [Google Scholar]
  29. Tapparelli C., Metternich R., Ehrhardt C., Cook N. S. Synthetic low-molecular weight thrombin inhibitors: molecular design and pharmacological profile. Trends Pharmacol Sci. 1993 Oct;14(10):366–376. doi: 10.1016/0165-6147(93)90095-2. [DOI] [PubMed] [Google Scholar]
  30. Tsuda Y., Cygler M., Gibbs B. F., Pedyczak A., Féthière J., Yue S. Y., Konishi Y. Design of potent bivalent thrombin inhibitors based on hirudin sequence: incorporation of nonsubstrate-type active site inhibitors. Biochemistry. 1994 Dec 6;33(48):14443–14451. doi: 10.1021/bi00252a010. [DOI] [PubMed] [Google Scholar]
  31. Verstraete M., Zoldhelyi P. Novel antithrombotic drugs in development. Drugs. 1995 Jun;49(6):856–884. doi: 10.2165/00003495-199549060-00002. [DOI] [PubMed] [Google Scholar]
  32. Weber P. C., Lee S. L., Lewandowski F. A., Schadt M. C., Chang C. W., Kettner C. A. Kinetic and crystallographic studies of thrombin with Ac-(D)Phe-Pro-boroArg-OH and its lysine, amidine, homolysine, and ornithine analogs. Biochemistry. 1995 Mar 21;34(11):3750–3757. doi: 10.1021/bi00011a033. [DOI] [PubMed] [Google Scholar]
  33. Zdanov A., Wu S., DiMaio J., Konishi Y., Li Y., Wu X., Edwards B. F., Martin P. D., Cygler M. Crystal structure of the complex of human alpha-thrombin and nonhydrolyzable bifunctional inhibitors, hirutonin-2 and hirutonin-6. Proteins. 1993 Nov;17(3):252–265. doi: 10.1002/prot.340170304. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES