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. 1999 Oct;8(10):2213–2217. doi: 10.1110/ps.8.10.2213

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.

V De Filippis 1, I Russo 1, A Vindigni 1, E Di Cera 1, S Salmaso 1, A Fontana 1
PMCID: PMC2144148  PMID: 10548068

Abstract

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30-400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by beta-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 microM).

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Selected References

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  1. Ayala Y., Di Cera E. Molecular recognition by thrombin. Role of the slow-->fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components. J Mol Biol. 1994 Jan 14;235(2):733–746. doi: 10.1006/jmbi.1994.1024. [DOI] [PubMed] [Google Scholar]
  2. Betz A., Hofsteenge J., Stone S. R. Interaction of the N-terminal region of hirudin with the active-site cleft of thrombin. Biochemistry. 1992 May 19;31(19):4557–4562. doi: 10.1021/bi00134a004. [DOI] [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. Brahms S., Brahms J. Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J Mol Biol. 1980 Apr;138(2):149–178. doi: 10.1016/0022-2836(80)90282-x. [DOI] [PubMed] [Google Scholar]
  5. Callas D., Fareed J. Comparative pharmacology of site directed antithrombin agents. Implication in drug development. Thromb Haemost. 1995 Jul;74(1):473–481. [PubMed] [Google Scholar]
  6. Chang J. Y. Production, properties, and thrombin inhibitory mechanism of hirudin amino-terminal core fragments. J Biol Chem. 1990 Dec 25;265(36):22159–22166. [PubMed] [Google Scholar]
  7. Das J., Kimball S. D. Thrombin active site inhibitors. Bioorg Med Chem. 1995 Aug;3(8):999–1007. doi: 10.1016/0968-0896(95)00104-o. [DOI] [PubMed] [Google Scholar]
  8. Davie E. W., Fujikawa K., Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991 Oct 29;30(43):10363–10370. doi: 10.1021/bi00107a001. [DOI] [PubMed] [Google Scholar]
  9. De Filippis V., Quarzago D., Vindigni A., Di Cera E., Fontana A. Synthesis and characterization of more potent analogues of hirudin fragment 1-47 containing non-natural amino acids. Biochemistry. 1998 Sep 29;37(39):13507–13515. doi: 10.1021/bi980717n. [DOI] [PubMed] [Google Scholar]
  10. De Filippis V., Vindigni A., Altichieri L., Fontana A. Core domain of hirudin from the leech Hirudinaria manillensis: chemical synthesis, purification, and characterization of a Trp3 analog of fragment 1-47. Biochemistry. 1995 Jul 25;34(29):9552–9564. doi: 10.1021/bi00029a032. [DOI] [PubMed] [Google Scholar]
  11. Di Cera E. Anticoagulant thrombins. Trends Cardiovasc Med. 1998 Nov;8(8):340–350. doi: 10.1016/s1050-1738(98)00030-9. [DOI] [PubMed] [Google Scholar]
  12. Fenton J. W., 2nd, Ofosu F. A., Brezniak D. V., Hassouna H. I. Thrombin and antithrombotics. Semin Thromb Hemost. 1998;24(2):87–91. doi: 10.1055/s-2007-995828. [DOI] [PubMed] [Google Scholar]
  13. Folkers P. J., Clore G. M., Driscoll P. C., Dodt J., Köhler S., Gronenborn A. M. Solution structure of recombinant hirudin and the Lys-47----Glu mutant: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study. Biochemistry. 1989 Mar 21;28(6):2601–2617. doi: 10.1021/bi00432a038. [DOI] [PubMed] [Google Scholar]
  14. Fontana A., Zambonin M., Polverino de Laureto P., De Filippis V., Clementi A., Scaramella E. Probing the conformational state of apomyoglobin by limited proteolysis. J Mol Biol. 1997 Feb 21;266(2):223–230. doi: 10.1006/jmbi.1996.0787. [DOI] [PubMed] [Google Scholar]
  15. Haruyama H., Wüthrich K. Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance. Biochemistry. 1989 May 16;28(10):4301–4312. doi: 10.1021/bi00436a027. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Hursting M. J., Alford K. L., Becker J. C., Brooks R. L., Joffrion J. L., Knappenberger G. D., Kogan P. W., Kogan T. P., McKinney A. A., Schwarz R. P., Jr Novastan (brand of argatroban): a small-molecule, direct thrombin inhibitor. Semin Thromb Hemost. 1997;23(6):503–516. doi: 10.1055/s-2007-996128. [DOI] [PubMed] [Google Scholar]
  18. Kahn P. C. The interpretation of near-ultraviolet circular dichroism. Methods Enzymol. 1979;61:339–378. doi: 10.1016/0076-6879(79)61018-2. [DOI] [PubMed] [Google Scholar]
  19. Markwardt F. The development of hirudin as an antithrombotic drug. Thromb Res. 1994 Apr 1;74(1):1–23. doi: 10.1016/0049-3848(94)90032-9. [DOI] [PubMed] [Google Scholar]
  20. Nicastro G., Baumer L., Bolis G., Tatò M. NMR solution structure of a novel hirudin variant HM2, N-terminal 1-47 and N64-->V + G mutant. Biopolymers. 1997 Jun;41(7):731–749. doi: 10.1002/(SICI)1097-0282(199706)41:7<731::AID-BIP3>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  21. Pineo G. F., Hull R. D. Hirudin and hirudin analogues as new anticoagulant agents. Curr Opin Hematol. 1995 Sep;2(5):380–385. doi: 10.1097/00062752-199502050-00009. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Sanderson P. E., Naylor-Olsen A. M. Thrombin inhibitor design. Curr Med Chem. 1998 Aug;5(4):289–304. [PubMed] [Google Scholar]
  24. Scacheri E., Nitti G., Valsasina B., Orsini G., Visco C., Ferrera M., Sawyer R. T., Sarmientos P. Novel hirudin variants from the leech Hirudinaria manillensis. Amino acid sequence, cDNA cloning and genomic organization. Eur J Biochem. 1993 May 15;214(1):295–304. doi: 10.1111/j.1432-1033.1993.tb17924.x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Verstraete M. Direct thrombin inhibitors: appraisal of the antithrombotic/hemorrhagic balance. Thromb Haemost. 1997 Jul;78(1):357–363. [PubMed] [Google Scholar]
  27. Vindigni A., Dang Q. D., Di Cera E. Site-specific dissection of substrate recognition by thrombin. Nat Biotechnol. 1997 Sep;15(9):891–895. doi: 10.1038/nbt0997-891. [DOI] [PubMed] [Google Scholar]
  28. Vindigni A., De Filippis V., Zanotti G., Visco C., Orsini G., Fontana A. Probing the structure of hirudin from Hirudinaria manillensis by limited proteolysis. Isolation, characterization and thrombin-inhibitory properties of N-terminal fragments. Eur J Biochem. 1994 Dec 1;226(2):323–333. doi: 10.1111/j.1432-1033.1994.tb20056.x. [DOI] [PubMed] [Google Scholar]
  29. di Cera E. Site-specific analysis of mutational effects in proteins. Adv Protein Chem. 1998;51:59–119. doi: 10.1016/s0065-3233(08)60651-8. [DOI] [PubMed] [Google Scholar]

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