Synthese und Charakterisierung von Inhibitoren der hämagglutininspaltenden Proteasen Furin und HAT

In der vorliegenden Arbeit wurden Inhibitoren der hämagglutininspaltenden Proteasen Furin und HAT synthetisiert und charakterisiert. Während die Proproteinkonvertase Furin das Hämagglutinin (HA) hochpathogener Influenzaviren vom Subtyp H5 und H7 aktiviert, bewirkt die trypsinartige Serinprotease HAT...

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1. Verfasser: Sielaff, Frank
Beteiligte: Steinmetzer, Torsten (Prof. Dr. ) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Veröffentlicht: Philipps-Universität Marburg 2011
Pharmazeutische Chemie
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1. Lin, C. F., Yang, J. S., Chang, C. Y., Kuo, S. C., Lee, M. R., Huang, L. J., 2005. Synthesis and anticancer activity of benzyloxybenzaldehyde derivatives against HL-60 cells. Bioorg Med Chem 13, 1537-1544.


3. Coughlin, S. R., 2005. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost 3, 1800-1814.

4. Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195-201.

5. Hansen, I. A., Fassnacht, M., Hahner, S., Hammer, F., Schammann, M., Meyer, S. R., Bicknell, A. B., Allolio, B., 2004. The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease. Endocrinology 145, 1898-1905.

6. Chokki, M., Yamamura, S., Eguchi, H., Masegi, T., Horiuchi, H., Tanabe, H., Kamimura, T., Yasuoka, S., 2004. Human airway trypsin-like protease increases mucin gene expression in airway epithelial cells. Am J Respir Cell Mol Biol 30, 470-478.

7. Roebroek, A. J., Umans, L., Pauli, I. G., Robertson, E. J., van Leuven, F., Van de Ven, W. J., Constam, D. B., 1998. Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin. Development 125, 4863-4876.

8. Rockwell, N. C., Krysan, D. J., Komiyama, T., Fuller, R. S., 2002. Precursor processing by kex2/furin proteases. Chem Rev 102, 4525-4548.

9. Powers, J. C., Asgian, J. L., Ekici, O. D., James, K. E., 2002. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem Rev 102, 4639-4750.

10. Wysocka, M., Spichalska, B., Lesner, A., Jaros, M., Brzozowski, K., Legowska, A., Rolka, K., 2010. Substrate specificity and inhibitory study of human airway trypsin-like protease. Bioorg Med Chem 18, 5504-5509.

11. Shiraishi, T., Kadono, S., Haramura, M., Kodama, H., Ono, Y., Iikura, H., Esaki, T., Koga, T., Hattori, K., Watanabe, Y., Sakamoto, A., Yoshihashi, K., Kitazawa, T., Esaki, K., Ohta, M., Sato, H., Kozono, T., 2008. Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination. Bioorg Med Chem Lett 18, 4533-4537.

12. Becker, G. L., Hardes, K., Steinmetzer, T., 2011. New substrate analogue furin inhibitors derived from 4-amidinobenzylamide. Bioorg Med Chem Lett 21, 4695-4697.

13. Baum, B., Mohamed, M., Zayed, M., Gerlach, C., Heine, A., Hangauer, D., Klebe, G., 2009. More than a simple lipophilic contact: a detailed thermodynamic analysis of nonbasic residues in the s1 pocket of thrombin. J Mol Biol 390, 56-69.

14. Hatta, M., Kawaoka, Y., 2002. The continued pandemic threat posed by avian influenza viruses in Hong Kong. Trends Microbiol 10, 340-344.

15. Garten, W., Klenk, H. D., 1999. Understanding influenza virus pathogenicity. Trends Microbiol 7, 99-100.

16. Liebeschuetz, J. W., Jones, S. D., Morgan, P. J., Murray, C. W., Rimmer, A. D., Roscoe, J. M., Waszkowycz, B., Welsh, P. M., Wylie, W. A., Young, S. C., Martin, H., Mahler, J., Brady, L., Wilkinson, K., 2002. PRO_SELECT: combining structure-based drug design and array-based chemistry for rapid lead discovery. 2. The development of a series of highly potent and selective factor Xa inhibitors. J Med Chem 45, 1221-1232.

17. Tucker, T. J., Lumma, W. C., Mulichak, A. M., Chen, Z., Naylor-Olsen, A. M., Lewis, S. D., Lucas, R., Freidinger, R. M., Kuo, L. C., 1997. Design of highly potent noncovalent thrombin inhibitors that utilize a novel lipophilic binding pocket in the thrombin active site. J Med Chem 40, 830-832.

18. Müller, E. J., Caldelari, R., Posthaus, H., 2004. Role of subtilisin-like convertases in cadherin processing or the conundrum to stall cadherin function by convertase inhibitors in cancer therapy. J Mol Histol 35, 263-275.

19. Podsiadlo, P., Komiyama, T., Fuller, R. S., Blum, O., 2004. Furin inhibition by compounds of copper and zinc. J Biol Chem 279, 36219-36227.

20. Schwede, T., Kopp, J., Guex, N., Peitsch, M. C., 2003. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31, 3381-3385.

21. Rawlings, N. D., Barrett, A. J., Bateman, A., 2010. MEROPS: the peptidase database. Nucleic Acids Res 38, D227-233.

22. Okumura, Y., Takahashi, E., Yano, M., Ohuchi, M., Daidoji, T., Nakaya, T., Böttcher, E., Garten, W., Klenk, H. D., Kido, H., 2010. Novel type II transmembrane serine proteases, MSPL and TMPRSS13, Proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication. J Virol 84, 5089-5096.

23. Zambon, M. C., 1999. Epidemiology and pathogenesis of influenza. J Antimicrob Chemother 44 Suppl B, 3-9.

24. Garten, W., Klenk, H. D., 2008. Cleavage activation of the influenza virus hemagglutinin and its role in pathogenesis. In: Klenk, H. D., Matrosovich, M., Stech, J. (Eds.), Avian Influenza., vol. 27. Karger, Basel, pp. 156-167.

25. Gong, J., Fang, H., Li, M., Liu, Y., Yang, K., Liu, Y., Xu, W., 2009. Potential Targets and Their Relevant Inhibitors in Anti-influenza Fields. Current Medicinal Chemistry 16, 3716-3739.

26. Basak, A., Chen, A., Scamuffa, N., Mohottalage, D., Basak, S., Khatib, A. M., 2010. Blockade of furin activity and furin-induced tumor cells malignant phenotypes by the chemically synthesized human furin prodomain. Curr Med Chem 17, 2214-2221.

27. Tian, S., Huang, Q., Fang, Y., Wu, J., 2011. FurinDB: A Database of 20-Residue Furin Cleavage Site Motifs, Substrates and Their Associated Drugs. Int J Mol Sci 12, 1060- 1065.

28. Gabriel, G., Klingel, K., Otte, A., Thiele, S., Hudjetz, B., Arman-Kalcek, G., Sauter, M., Shmidt, T., Rother, F., Baumgarte, S., Keiner, B., Hartmann, E., Bader, M., Brownlee, G. G., Fodor, E., Klenk, H. D., 2011. Differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus. Nat Commun 2, 156.

29. Shiryaev, S. A., Remacle, A. G., Ratnikov, B. I., Nelson, N. A., Savinov, A. Y., Wei, G., Bottini, M., Rega, M. F., Parent, A., Desjardins, R., Fugere, M., Day, R., Sabet, M., Pellecchia, M., Liddington, R. C., Smith, J. W., Mustelin, T., Guiney, D. G., Lebl, M., Strongin, A. Y., 2007. Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens. J Biol Chem 282, 20847-20853.

30. Györgydeák, Z., Holzer, W., Mereiter, K., 1999. Guanylhydrazones of (hetero)aryl methyl ketones: Structure and reaction with acetic anhydride. Monatshefte Fur Chemie 130, 899-913.

31. Michaelis, M., Doerr, H. W., Cinatl, J., Jr., 2009. Of chickens and men: avian influenza in humans. Curr Mol Med 9, 131-151.

32. Stürzebecher, J., Vieweg, H., Steinmetzer, T., Schweinitz, A., Stubbs, M. T., Renatus, M., Wikström, P., 1999. 3-Amidinophenylalanine-based inhibitors of urokinase. Bioorg Med Chem Lett 9, 3147-3152.

33. Bennett, B. D., Denis, P., Haniu, M., Teplow, D. B., Kahn, S., Louis, J. C., Citron, M., Vassar, R., 2000. A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's beta-secretase. J Biol Chem 275, 37712-37717.

34. Jean, F., Stella, K., Thomas, L., Liu, G., Xiang, Y., Reason, A. J., Thomas, G., 1998. alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent. Proc Natl Acad Sci U S A 95, 7293-7298.

35. Soll, R. M., Lu, T., Tomczuk, B., Illig, C. R., Fedde, C., Eisennagel, S., Bone, R., Murphy, L., Spurlino, J., Salemme, F. R., 2000. Amidinohydrazones as guanidine bioisosteres: application to a new class of potent, selective and orally bioavailable, non- amide-based small-molecule thrombin inhibitors. Bioorg Med Chem Lett 10, 1-4.

36. Baum, T., Eckfeld, D. K., Metz, N., Dinish, J. L., Rowles, G., Van Pelt, R., Shropshire, A. T., Fernandez, S. P., Gluckman, M. I., Bruce, W. F., 1969. 2,6-Dichlorobenzylidene amino guanidine acetate (Wy-8678). A new hypotensive agent. Experientia 25, 1066- 1067.

37. Saupe, S. M., Steinmetzer, T., 2011. A new strategy for the development of highly potent and selective plasmin inhibitors. J Med Chem in press.

38. Kar, A., Argade, N., 2002. A Simple Key for Benzylic Mono-and gem-Dibromination of Primary Aromatic Amine Derivatives Using Molecular Bromine. Synthesis, 221-224.

39. Judkins, B. D., Allen, D. G., Cook, T. A., Evans, B., Sardharwala, T. E., 1996. A versatile synthesis of amidines from nitriles via amidoximes. Synthetic Communications 26, 4351-4367.

40. Ruggli, P., Gassenmeier, E., 1939. Über Di-und Tri-acetylbenzol und p-Phenylen-di- glyoxal. Helvetica Chimica Acta 22, 496-511.

41. Bicknell, A. B., Lomthaisong, K., Woods, R. J., Hutchinson, E. G., Bennett, H. P., Gladwell, R. T., Lowry, P. J., 2001. Characterization of a serine protease that cleaves pro-gamma-melanotropin at the adrenal to stimulate growth. Cell 105, 903-912.

42. Cloning and characterization of the cDNA for human airway trypsin-like protease.

43. Kyrieleis, O. J., Huber, R., Ong, E., Oehler, R., Hunter, M., Madison, E. L., Jacob, U., 2007. Crystal structure of the catalytic domain of DESC1, a new member of the type II transmembrane serine proteinase family. FEBS J 274, 2148-2160.

44. Fugere, M., Day, R., 2005. Cutting back on pro-protein convertases: the latest approaches to pharmacological inhibition. Trends Pharmacol Sci 26, 294-301.

45. Tucker, T. J., Brady, S. F., Lumma, W. C., Lewis, S. D., Gardell, S. J., Naylor-Olsen, A. M., Yan, Y., Sisko, J. T., Stauffer, K. J., Lucas, B. J., Lynch, J. J., Cook, J. J., Stranieri, M. T., Holahan, M. A., Lyle, E. A., Baskin, E. P., Chen, I. W., Dancheck, K. B., Krueger, J. A., Cooper, C. M., Vacca, J. P., 1998. Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position. J Med Chem 41, 3210-3219.

46. Lumma, W. C., Jr., Witherup, K. M., Tucker, T. J., Brady, S. F., Sisko, J. T., Naylor- Olsen, A. M., Lewis, S. D., Lucas, B. J., Vacca, J. P., 1998. Design of novel, potent, noncovalent inhibitors of thrombin with nonbasic P-1 substructures: rapid structure- activity studies by solid-phase synthesis. J Med Chem 41, 1011-1013.

47. Sielaff F., Böttcher-Friebertshäuser E., Meyer D., Saupe S.M., Volk I.M., Garten W., Stein- metzer T.; Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT. BMCL, 21(16), 4860-4864.

48. Zündorf, I., Dingermann, T., 2011. Die neuen (drohenden?) Grippe-Pamdemien. Pharm Unserer Zeit 40, 120-125.

49. Gustafsson, D., Elg, M., Lenfors, S., Borjesson, I., Teger-Nilsson, A. C., 1996. Effects of inogatran, a new low-molecular-weight thrombin inhibitor, in rat models of venous and arterial thrombosis, thrombolysis and bleeding time. Blood Coagul Fibrinolysis 7, 69-79.

50. Komiyama, T., Fuller, R. S., 2000. Engineered eglin c variants inhibit yeast and human proprotein processing proteases, Kex2 and furin. Biochemistry 39, 15156-15165.

51. Dixon, S. L., Jurs, P. C., 1993. Estimation of pKa for Organic Oxyacids Using Calculated Atomic Charges. J. comp. chem. 14, 1460-1467.

52. Ludwig, S., Stitz, L., Planz, O., Van, H., Fitch, W. M., Scholtissek, C., 1995. European swine virus as a possible source for the next influenza pandemic? Virology 212, 555- 561.

53. Hahner, S., Fassnacht, M., Hammer, F., Schammann, M., Weismann, D., Hansen, I. A., Allolio, B., 2005. Evidence against a role of human airway trypsin-like protease--the human analogue of the growth-promoting rat adrenal secretory protease-in adrenal tumourigenesis. Eur J Endocrinol 152, 143-153.

54. Dönnecke, D., Schweinitz, A., Stürzebecher, A., Steinmetzer, P., Schuster, M., Stürzebecher, U., Nicklisch, S., Stürzebecher, J., Steinmetzer, T., 2007. From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3. Bioorg Med Chem Lett 17, 3322-3329.

55. Thomas, G., 2002. Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol 3, 753-766.

56. Garred, O., van Deurs, B., Sandvig, K., 1995. Furin-induced cleavage and activation of Shiga toxin. J Biol Chem 270, 10817-10821.

57. Pfleiderer, M., 2011. Grippe-Impfstoffe. Pharm Unserer Zeit 40, 126-134.

58. Shearer, C. M., DeAngelis, N. J., 1979. Guanabenz degradation products and stability assay. J Pharm Sci 68, 1010-1012.

59. " Haemagglutinin cleaving proteases-potential targets for influenza treatment " ; DPhG Jahrestagung, Braunschweig; Oktober 2010.

60. Sisay, M. T., Steinmetzer, T., Stirnberg, M., Maurer, E., Hammami, M., Bajorath, J., Gütschow, M., 2010. Identification of the first low-molecular-weight inhibitors of matriptase-2. J Med Chem 53, 5523-5535.

61. Osterhaus, A. D., Rimmelzwaan, G. F., Martina, B. E., Bestebroer, T. M., Fouchier, R. A., 2000. Influenza B virus in seals. Science 288, 1051-1053.

62. Klenk, H. D., 2011. Influenzaviren auf dem Weg vom Tier zum Menschen. Pharm Unserer Zeit 40, 104.

63. Kacprzak, M. M., Peinado, J. R., Than, M. E., Appel, J., Henrich, S., Lipkind, G., Houghten, R. A., Bode, W., Lindberg, I., 2004. Inhibition of furin by polyarginine- containing peptides: nanomolar inhibition by nona-D-arginine. J Biol Chem 279, 36788-36794.

64. Hallenberger, S., Bosch, V., Angliker, H., Shaw, E., Klenk, H. D., Garten, W., 1992. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature 360, 358-361.

65. Basak, A., Cooper, S., Roberge, A. G., Banik, U. K., Chretien, M., Seidah, N. G., 1999. Inhibition of proprotein convertases-1, -7 and furin by diterpines of Andrographis paniculata and their succinoyl esters. Biochem J 338 ( Pt 1), 107-113.

66. Garten, W., Stieneke, A., Shaw, E., Wikström, P., Klenk, H. D., 1989. Inhibition of proteolytic activation of influenza virus hemagglutinin by specific peptidyl chloroalkyl ketones. Virology 172, 25-31.

67. Dahlen, J. R., Jean, F., Thomas, G., Foster, D. C., Kisiel, W., 1998. Inhibition of soluble recombinant furin by human proteinase inhibitor 8. J Biol Chem 273, 1851-1854.

68. Bajusz, S., Barabas, E., Tolnay, P., Szell, E., Bagdy, D., 1978. Inhibition of thrombin and trypsin by tripeptide aldehydes. Int J Pept Protein Res 12, 217-221.

69. Posterbeiträge: " Inhibitors of haemagglutinin cleaving proteases – Potential drugs for the treatment of influeza " ; Frontiers in Medicinal Chemistry, Münster; März 2010. " Development of substrate-analogue plasmin inhibitors " ; 10 th German Peptide Symposium, Berlin; März 2011.

70. Basak, A., 2005. Inhibitors of proprotein convertases. J Mol Med 83, 844-855.

71. Clark, F. H., Cahoon, N. M., 1987. Ionization Constants by Curve Fitting: Determination of Partition and Distribution Coefficients of Acids and Bases and Their Ions. J. Pharm. Sci. 76, 611-620.

72. Lila, C., Gloanec, P., Cadet, L., Herve, Y., Fournier, J., Leborgne, F., Verbeuren, T. J., De Nanteuil, G., 1998. Large scale preparation of protected 4-aminomethyl- benzamidine. Application to the synthesis of the thrombin inhibitor, melagatran. Synthetic Communications 28, 4419-4429.

73. Takahashi, M., Sano, T., Yamaoka, K., Kamimura, T., Umemoto, N., Nishitani, H., Yasuoka, S., 2001. Localization of human airway trypsin-like protease in the airway: an immunohistochemical study. Histochem Cell Biol 115, 181-187.

74. Klebe, G., Schlitzer, M., 2011. M2-Inhibitoren und Neuraminidase-Inhibitoren. Pharm Unserer Zeit 40, 144-150.

75. Uhland, K., 2006. Matriptase and its putative role in cancer. Cell Mol Life Sci 63, 2968- 2978.

76. King, J. F., Lam, J. Y. L., Skonieczny, S., 1992. Mechanisms of Hydrolysis and Related Necleophilic Displacement Reactions of Alkanesulfonyl Chlorides: pH Dependence and the Mechanism of Hydration of Sulfenes J. Am. Chem. Soc. 114, 1743-1749.

77. Antalis, T. M., Bugge, T. H., Wu, Q., 2011. Membrane-anchored serine proteases in health and disease. Prog Mol Biol Transl Sci 99, 1-50.

78. Klenk, H. D., Garten, W., Matrosovich, M., 2011. Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic. Bioessays 33, 180-188.

79. Parry, M. A., Zhang, X. C., Bode, I., 2000. Molecular mechanisms of plasminogen activation: bacterial cofactors provide clues. Trends Biochem Sci 25, 53-59.

80. Baraldi, P. G., Preti, D., Tabrizi, M. A., Fruttarolo, F., Saponaro, G., Baraldi, S., Romagnoli, R., Moorman, A. R., Gessi, S., Varani, K., Borea, P. A., 2007. N(6)- [(hetero)aryl/(cyclo)alkyl-carbamoyl-methoxy-phenyl]-(2-chloro)-5'-N-ethylcarbox- amido-adenosines: the first example of adenosine-related structures with potent agonist activity at the human A(2B) adenosine receptor. Bioorg Med Chem 15, 2514-2527.

81. Markwardt, F., Wagner, G., Stürzebecher, J., Walsmann, P., 1980. N-alpha- arylsulfonyl-omega-(4-amidinophenyl)-alpha-aminoalkylcarboxylic acid amides -novel selective inhibitors of thrombin. Thromb Res 17, 425-431.

82. Reece, P. A., 2007. Neuraminidase inhibitor resistance in influenza viruses. J Med Virol 79, 1577-1586.

83. Schweinitz, A., Stürzebecher, A., Stürzebecher, U., Schuster, O., Stürzebecher, J., Steinmetzer, T., 2006. New substrate analogue inhibitors of factor Xa containing 4-amidinobenzylamide as P1 residue: part 1. Med Chem 2, 349-361.

84. Egbertson, M. S., Chang, C. T. C., Duggan, M. E., Gould, R. J., Halczenko, W., Hartman, G. D., Laswell, W. L., Lynch, J. J., Lynch, R. J., Manno, P. D., Naylor, A. M., Prugh, J. D., Ramjit, D. R., Sitko, G. R., Smith, R. S., Turchi, L. M., Zhang, G. X., 1994. Nonpeptide Fibrinogen Receptor Antagonists .2. Optimization of a Tyrosine Template as a Mimic for Arg-Gly-Asp. Journal of Medicinal Chemistry 37, 2537-2551.

85. Steinmetzer, T., Nowak, G., 2002. Oligo or Polyalkylene Glycol-coupled Inhibitors. WIPO, Patent No. WO 2002/059065.

86. Schechter, I., Berger, A., 1967. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27, 157-162.

87. Holzer, W., Györgydeák, Z., 1992. On the Structure of Guanylhydrazones Derived from Aromatic-Aldehydes. Monatshefte Fur Chemie 123, 1163-1173.

88. Stürzebecher, A., 2005. Optimierung synthetischer Inhibitoren des Gerinnungsfaktors Xa vom 4-Amidinobenzylamid-Typ. Dissertation, Fachbereich Pharmazie, Philipps Universität Marburg.

89. Herold, S., 2011. Pathogenese, Klinik und Therapie der Virusgrippe. Pharmazie in unserer Zeit 40, 115-119.

90. Csizmadia, F., Tsantili-Kakoulidou, A., Panderi, I., Darvas, F., 1997. Prediction of Distribution Coefficient from Structure. 1. Estimation Method. J. Pharm. Sci. 86, 865- 871.

91. Stüber, W., Koschinsky, R., Reers, M., Hoffmann, D., Czech, J., Dickneite, G., 1995. Preparation and Evaluation of PEG-Bound Thrombin Inhibitors Based on 4-Amidinophenylalanine. Peptide Research 8, 78-85.

92. Beliveau, F., Desilets, A., Leduc, R., 2009. Probing the substrate specificities of matriptase, matriptase-2, hepsin and DESC1 with internally quenched fluorescent peptides. FEBS J 276, 2213-2226.

93. Dubois, C. M., Laprise, M. H., Blanchette, F., Gentry, L. E., Leduc, R., 1995. Processing of transforming growth factor beta 1 precursor by human furin convertase.

94. Laskowski, M., Jr., Kato, I., 1980. Protein inhibitors of proteinases. Annu Rev Biochem 49, 593-626.

95. Yasuoka, S., Ohnishi, T., Kawano, S., Tsuchihashi, S., Ogawara, M., Masuda, K., Yamaoka, K., Takahashi, M., Sano, T., 1997. Purification, characterization, and localization of a novel trypsin-like protease found in the human airway. Am J Respir Cell Mol Biol 16, 300-308.

96. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63.

97. Kim, S., Hwang, S. Y., Kim, Y. K., Yun, M., Oh, Y. S., 1997. Rational design of selective Thrombin inhibitors. Bioorg Med Chem Lett 7, 769-774.

98. Zhang, Y., Kida, Y., Kuwano, K., Misumi, Y., Ikehara, Y., Arai, S., 2001. Role of furin in delivery of a CTL epitope of an anthrax toxin-fusion protein. Microbiol Immunol 45, 119-125.

99. Steinmetzer, T., Schweinitz, A., Stürzebecher, A., Dönnecke, D., Uhland, K., Schuster, O., Steinmetzer, P., Müller, F., Friedrich, R., Than, M. E., Bode, W., Stürzebecher, J., 2006. Secondary amides of sulfonylated 3-amidinophenylalanine. New potent and selective inhibitors of matriptase. J Med Chem 49, 4116-4126.

100. Loewenberg, M., Verhaar, A., van den Blink, B., ten Kate, F., van Deventer, S., Peppelenbosch, M., Hommes, D., 2005. Specific inhibition of c-Raf activity by semapimod induces clinical remission in severe Crohn's disease. J Immunol 175, 2293- 2300.

101. Berry, C. N., Lassalle, G., Lunven, C., Altenburger, J. M., Guilbert, F., Lale, A., Herault, J. P., Lecoffre, C., Pfersdorff, C., Herbert, J. M., O'Connor, S. E., 2002. SSR182289A, a novel, orally active thrombin inhibitor: in vitro profile and ex vivo anticoagulant activity. J Pharmacol Exp Ther 303, 1189-1198.

102. Sielaff F., Than M.E., Bevec D., Lindberg I. Steinmetzer T.; New furin inhibitors based on weakly basic amidinohydrazones. BMCL 21(2), 836-840.

103. Steinmetzer, T., 2011. Strategien für die Entwicklung neuartiger Grippemittel. Pharm Unserer Zeit 40, 95.

104. Stürzebecher, J., Prasa, D., Wikström, P., Vieweg, H., 1995. Structure-activity relationships of inhibitors derived from 3-amidinophenylalanine. J Enzyme Inhib 9, 87- 99.

105. Structure-based design of novel potent nonpeptide thrombin inhibitors. J Med Chem 45, 1757-1766.

106. Guex, N., Peitsch, M. C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714-2723.

107. Schwarz, G., Alberts, H., Kricheldorf, H. R., 1981. Syntheses and Reactions of (Trimethylsiloxy)benzoyl Chlorides. Liebigs Ann. Chem. 1981, 1257-1270.

108. Stürzebecher, J., Prasa, D., Hauptmann, J., Vieweg, H., Wikström, P., 1997. Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine. J Med Chem 40, 3091-3099.

109. Angliker, H., 1995. Synthesis of tight binding inhibitors and their action on the proprotein-processing enzyme furin. J Med Chem 38, 4014-4018.

110. Turk, B., 2006. Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 5, 785-799.

111. Seidah, N. G., Mayer, G., Zaid, A., Rousselet, E., Nassoury, N., Poirier, S., Essalmani, R., Prat, A., 2008. The activation and physiological functions of the proprotein convertases. Int J Biochem Cell Biol 40, 1111-1125.

112. Ascenzi, P., Bocedi, A., Bolognesi, M., Spallarossa, A., Coletta, M., De Cristofaro, R., Menegatti, E., 2003. The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein. Curr Protein Pept Sci 4, 231-251.

113. Roger, R., Neilson, D. G., 1961. The Chemistry of Imidates. Chemical Reviews 61, 179-211.

114. Jones, S. D., Liebeschuetz, J. W., Morgan, P. J., Murray, C. W., Rimmer, A. D., Roscoe, J. M., Waszkowycz, B., Welsh, P. M., Wylie, W. A., Young, S. C., Martin, H., Mahler, J., Brady, L., Wilkinson, K., 2001. The design of phenylglycine containing benzamidine carboxamides as potent and selective inhibitors of factor Xa. Bioorg Med Chem Lett 11, 733-736.

115. Kozyraki, R., Kristiansen, M., Silahtaroglu, A., Hansen, C., Jacobsen, C., Tommerup, N., Verroust, P. J., Moestrup, S. K., 1998. The human intrinsic factor-vitamin B12 receptor, cubilin: molecular characterization and chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region. Blood 91, 3593-3600.

116. Levitin, F., Stern, O., Weiss, M., Gil-Henn, C., Ziv, R., Prokocimer, Z., Smorodinsky, N. I., Rubinstein, D. B., Wreschner, D. H., 2005. The MUC1 SEA module is a self- cleaving domain. J Biol Chem 280, 33374-33386.

117. Steiner, D. F., 1998. The proprotein convertases. Curr Opin Chem Biol 2, 31-39.

118. Ulloa, L., 2005. The vagus nerve and the nicotinic anti-inflammatory pathway. Nat Rev Drug Discov 4, 673-684.

119. Bergner, A., Bauer, M., Brandstetter, H., Stürzebecher, J., Bode, W., 1995. The X-ray crystal structure of thrombin in complex with N alpha-2-naphthylsulfonyl-L-3-amidino- phenylalanyl-4-methylpiperidide: the beneficial effect of filling out an empty cavity.

120. Bignon, E., 2002. Triazole Derivatives and Pharmaceutical Compositions Comprising them. WIPO, Patent No. WO 02/34743 A1.

121. Ulrich, P., Cerami, A., 1984. Trypanocidal 1,3-arylene diketone bis(guanylhydrazone)s.

122. Schäfer, W., Stroh, A., Berghöfer, S., Seiler, J., Vey, M., Kruse, M. L., Kern, H. F., Klenk, H. D., Garten, W., 1995. Two independent targeting signals in the cytoplasmatic domain determine trans-golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J. 14, 2424-2435.

123. Hooper, J. D., Clements, J. A., Quigley, J. P., Antalis, T. M., 2001. Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes. J Biol Chem 276, 857-860.

124. Holzgrabe, U., 2011. Wie sicher war die Oseltamivir-Versorgung in Zeiten der Grippe- Pandemie? Pharm Unserer Zeit 40, 151-154.

125. Stürzebecher, A., Dönnecke, D., Schweinitz, A., Schuster, O., Steinmetzer, P., Stürzebecher, U., Kotthaus, J., Clement, B., Stürzebecher, J., Steinmetzer, T., 2007. Highly potent and selective substrate analogue factor Xa inhibitors containing D-homophenylalanine analogues as P3 residue: part 2. ChemMedChem 2, 1043-1053.

126. Bassi, D. E., Fu, J., Lopez de Cicco, R., Klein-Szanto, A. J., 2005. Proprotein convertases: "master switches" in the regulation of tumor growth and progression. Mol Carcinog 44, 151-161.

127. Zambon, M. C., 2001. The pathogenesis of influenza in humans. Rev Med Virol 11, 227-241.

128. Hellstern, P., Stürzebecher, U., Wuchold, B., Haubelt, H., Seyfert, U. T., Bauer, M., Vogt, A., Stürzebecher, J., 2007. Preservation of in vitro function of platelets stored in the presence of a synthetic dual inhibitor of factor Xa and thrombin. J Thromb Haemost 5, 2119-2126.

129. Henrich, S., Cameron, A., Bourenkov, G. P., Kiefersauer, R., Huber, R., Lindberg, I., Bode, W., Than, M. E., 2003. The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat Struct Biol 10, 520-526.

130. Bernatowicz, M. S., Wu, Y., Matsueda, G. R., 1992. 1H-Pyrazole-1-carboxamidine hydrochloride an attractive reagent for guanylation of amines and its application to peptide synthesis. The Journal of Organic Chemistry 57, 2497-2502.

131. Matrosovich, M., Stech, J., Klenk, H. D., 2009. Influenza receptors, polymerase and host range. Rev Sci Tech 28, 203-217.

132. Skehel, J. J., Wiley, D. C., 2000. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531-569.

133. Steinmetzer, T., Stürzebecher, J., 2004. Progress in the development of synthetic thrombin inhibitors as new orally active anticoagulants. Curr Med Chem 11, 2297-2321.

134. Lu, W., Zhang, W., Molloy, S. S., Thomas, G., Ryan, K., Chiang, Y., Anderson, S., Laskowski, M., Jr., 1993. Arg15-Lys17-Arg18 turkey ovomucoid third domain inhibits human furin. J Biol Chem 268, 14583-14585.

135. Takahashi, S., Nakagawa, T., Banno, T., Watanabe, T., Murakami, K., Nakayama, K., 1995. Localization of furin to the trans-golgi network and recycling from the cell surface involves Ser and Tyr residues within the cytoplasmic domain. J Biol Chem 270, 28397-28401.

136. Schweinitz, A., Steinmetzer, T., Banke, I. J., Arlt, M. J., Stürzebecher, A., Schuster, O., Geissler, A., Giersiefen, H., Zeslawska, E., Jacob, U., Krüger, A., Stürzebecher, J., 2004. Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents. J Biol Chem 279, 33613-33622.

137. Gustafsson, D., Bylund, R., Antonsson, T., Nilsson, I., Nystrom, J. E., Eriksson, U., Bredberg, U., Teger-Nilsson, A. C., 2004. A new oral anticoagulant: the 50-year challenge. Nat Rev Drug Discov 3, 649-659.

138. Seidah, N. G., Benjannet, S., Pareek, S., Savaria, D., Hamelin, J., Goulet, B., Laliberte, J., Lazure, C., Chretien, M., Murphy, R. A., 1996. Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases. Biochem J 314 (Pt 3), 951-960.

139. Plaimauer, B., Mohr, G., Wernhart, W., Himmelspach, M., Dorner, F., Schlokat, U., 2001. 'Shed' furin: mapping of the cleavage determinants and identification of its C- terminus. Biochem J 354, 689-695.

140. Dixon, M., 1953. The determination of enzyme inhibitor constants. Biochem J 55, 170- 171.

141. List, K., Bugge, T. H., Szabo, R., 2006. Matriptase: potent proteolysis on the cell surface. Mol Med 12, 1-7.

142. Kadono, S., Sakamoto, A., Kikuchi, Y., Oh-Eda, M., Yabuta, N., Koga, T., Hattori, K., Shiraishi, T., Haramura, M., Kodama, H., Ono, Y., Esaki, T., Sato, H., Watanabe, Y., Itoh, S., Ohta, M., Kozono, T., 2005. Structure of human factor VIIa/tissue factor in complex with a peptide-mimetic inhibitor: high selectivity against thrombin by introducing two charged groups in P2 and P4. Acta Crystallogr Sect F Struct Biol Cryst Commun 61, 169-173.

143. Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S., Seidah, N. G., Israel, A., 1998. The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc Natl Acad Sci U S A 95, 8108-8112.

144. Scamuffa, N., Siegfried, G., Bontemps, Y., Ma, L., Basak, A., Cherel, G., Calvo, F., Seidah, N. G., Khatib, A. M., 2008. Selective inhibition of proprotein convertases represses the metastatic potential of human colorectal tumor cells. J Clin Invest 118, 352-363.

145. Komiyama, T., Coppola, J. M., Larsen, M. J., van Dort, M. E., Ross, B. D., Day, R., Rehemtulla, A., Fuller, R. S., 2009. Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules. J Biol Chem 284, 15729- 15738.

146. Morens, D. M., Taubenberger, J. K., Fauci, A. S., 2009. The persistent legacy of the 1918 influenza virus. N Engl J Med 361, 225-229.

147. Petrera, N. S., Stafford, A. R., Leslie, B. A., Kretz, C. A., Fredenburgh, J. C., Weitz, J. I., 2009. Long range communication between exosites 1 and 2 modulates thrombin function. J Biol Chem 284, 25620-25629.

148. Basak, A., Khatib, A. M., Mohottalage, D., Basak, S., Kolajova, M., Bag, S. S., 2009. A novel enediynyl peptide inhibitor of furin that blocks processing of proPDGF-A, B and proVEGF-C. PLoS One 4, e7700.

149. Fuchs, S. M., Raines, R. T., 2006. Internalization of cationic peptides: the road less (or more?) traveled. Cell Mol Life Sci 63, 1819-1822.

150. Becker, G. L., Sielaff, F., Than, M. E., Lindberg, I., Routhier, S., Day, R., Lu, Y., Garten, W., Steinmetzer, T., 2010. Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics. J Med Chem 53, 1067- 1075.

151. Taubenberger, J. K., Morens, D. M., 2010. Influenza: the once and future pandemic. Public Health Rep 125 Suppl 3, 16-26.

152. Böttcher-Friebertshäuser E., Freuer C., Sielaff F., Schmidt S., Eickmann M., Uhlendorff J., Steinmetzer T., Klenk H. D., Garten W.; Cleavage of influenza virus hemagglutinin by airway proteases TMPRSS2 and HAT differs in subcellular localization and susceptibility to protease inhibitors. J Virol. 84(11), 5605-5614.

153. Gamblin, S. J., Skehel, J. J., 2010. Influenza hemagglutinin and neuraminidase membrane glycoproteins. J Biol Chem 285, 28403-28409.

154. Stieneke-Gröber, A., Vey, M., Angliker, H., Shaw, E., Thomas, G., Roberts, C., Klenk, H. D., Garten, W., 1992. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J 11, 2407-2414.

155. Bassi, D. E., Lopez De Cicco, R., Mahloogi, H., Zucker, S., Thomas, G., Klein-Szanto, A. J., 2001. Furin inhibition results in absent or decreased invasiveness and tumorigenicity of human cancer cells. Proc Natl Acad Sci U S A 98, 10326-10331.

156. Klenk, H. D., Rott, R., Orlich, M., Blödorn, J., 1975. Activation of influenza A viruses by trypsin treatment. Virology 68, 426-439.

157. Garten, W., Hallenberger, S., Ortmann, D., Schäfer, W., Vey, M., Angliker, H., Shaw, E., Klenk, H. D., 1994. Processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones. Biochimie 76, 217-225.

158. Klenk, H. D., Garten, W., 1994. Host cell proteases controlling virus pathogenicity. Trends Microbiol 2, 39-43.

159. Frerot, E., Coste, J., Pantaloni, A., Dufour, M.-N., Jouin, P., 1991. PyBop and PyBroP: Two reagents for the difficult coupling of the -dialkyl amino acid, Aib. Tetrahedron 47, 259-270.

160. Künzel, S., Schweinitz, A., Reissmann, S., Stürzebecher, J., Steinmetzer, T., 2002. 4-amidinobenzylamine-based inhibitors of urokinase. Bioorg Med Chem Lett 12, 645- 648.

161. Molloy, S. S., Anderson, E. D., Jean, F., Thomas, G., 1999. Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9, 28-35.

162. Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H. D., 2006. New low-viscosity overlay medium for viral plaque assays. Virol J 3, 63.

163. Roebroek, A. J., Taylor, N. A., Louagie, E., Pauli, I., Smeijers, L., Snellinx, A., Lauwers, A., Van de Ven, W. J., Hartmann, D., Creemers, J. W., 2004. Limited redundancy of the proprotein convertase furin in mouse liver. J Biol Chem 279, 53442- 53450.

164. Specht, S., Sarite, S. R., Hauber, I., Hauber, J., Goerbig, U. F., Meier, C., Bevec, D., Hoerauf, A., Kaiser, A., 2008. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase. Parasitol Res 102, 1177-1184.

165. Grützmacher, H.-F., Mehdizadeh, A., Mülverstedt, A., 1994. A convenient Synthesis of Macrocyclic[]Paracyclophanes. Chemische Berichte 127, 1163-1166.