Optimisation of the P2 pharmacophore in a series of thrombin inhibitors: Ion-dipole interactions with lysine 60G

Bioorganic and Medicinal Chemistry Letters

Volumn 9, Issue 9, 1999, Pages 1317-1322

  Ambler, John ^a   Brown, Lyndon ^a   Cockcroft, Xiao Ling ^a   Grütter, Markus ^a   Hayler, Judy ^a   Janus, Diana ^a   Jones, Darryl ^a   Kane, Peter ^a   Menear, Keith ^a   Priestle, John ^a   Smith, Garrick ^a   Talbot, Mark ^a   Walker V, Clive ^a   Wathey, Bernard ^a  

^a Novartis Horsham Research Center   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

ARGATROBAN; PIPERIDINE DERIVATIVE; THROMBIN INHIBITOR;

ANTICOAGULATION; ARTICLE; CHEMICAL MODIFICATION; CRYSTAL STRUCTURE; DRUG ACTIVITY; DRUG POTENCY; DRUG STRUCTURE; ENZYME INHIBITOR COMPLEX; PHARMACOPHORE; STRUCTURE ANALYSIS;

CRYSTALLOGRAPHY, X-RAY; HUMANS; KINETICS; MODELS, MOLECULAR; POLYGLUTAMIC ACID; POLYLYSINE; THROMBIN;

EID: 0033519252     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0960-894X(99)00172-9     Document Type: Article

References (8)

1. Turpie, Alexander G. G.; Weitz, Jeffrey I.; Hirsh, Jack. Thromb. Haemostasis (1995), 74(1), 565-571.
2. Fenton, J. W., II; Ofosu, F. A.; Moon, D. G.; Maraganore, J. M. Blood Coagulation Fibrinolysis (1991), 2(1), 69-75.
3. Kikumoto, Ryoji; Tamao, Yoshikuni; Ohkubo, Kazuo; Tezuka, Tohru; Tonomura, Shinji; Okamoto, Shosuke; Hijikata, Akiko. J. Med. Chem. (1980), 23(12), 1293-1299.
4. For convenience pharmacophorcs residing in the S9 pocket, also known as the distal or aryl pocket, of thrombin are referred to as P3.
5. Studies relating to the discovery of is in press. Mark C. Allen, John Ambler, Derek E. Brundish, Alice Bull, Vera Donovan, Joseph D. Fullerton, Sheila M. Garman, Judy F. Hayler, Geoffrey Howarth, William Hoyle, Diana Janus, Peter D Kane*, Mark McDonnell, Garrick P. Smith, Mark D. Talbot, Robert Wakeford, Clive V. Walker. J. Med. Chem. in press
6. The C-terminal fragment of hirudin (residues 54-65, sulfated Tyr-63), a natural inhibitor of thrombin from the European medicinal leech Hirudo medicinalis, was used to prepare well-diffracting crystals of thrombin reproducibly. A 1:1 molar ratio of thrombin:hirudin was set up against buffered sodium chloride in hanging drop experiments. Large, well-formed crystals grew within two weeks. Compounds 13 and 18 were soaked into the pre-formed crystals of the thrombin-hirudin fragment complex by adding a small amount of substance directly to a drop containing a thrombin crystal. Diffraction data were collected on a FAST area detector (Nonius) with a rotating anode generator providing graphite-monochromated CuΚα X-radiation. Crystals of the inhibitor-thrombin complexes were shown to belong to space group C2 with unit cell parameters a=71.9Å, b=72.1Å, c=72.9Å,β=101.0° (with compound 13) and a=71.5Å, b=72.2Å, c=73.0Å, β=101.0° (with compound 18), with one molecule of the complex (thrombin, C-terminal fragment of hirudin and inhibitor) in the asymmetric unit. Diffraction data to 2.0Å resolution could be collected for the complex with compound 13, while the crystal complexed with compound 18 diffracted to only 2.5Å. The structure of the complex with compound 13 was solved using molecular replacement employing thrombin alone as the trial structure. The structure was partially refined before trying to build in the inhibitor in order to improve the quality of the electron density for the inhibitor. An Fo-Fc difference Fourier showed very clear density for the inhibitor in the active site and the chemical structure of compound 13. Further refinement, including the addition of solvent molecules, was carried out until no improvement in the structure could be realised. The structure of the complex with compound 18 was solved by using the structure of the complex with compound 13, after removing the inhibitor and solvent molecules, since the crystal forms are isomorphous. Again there was electron density in the active site for the inhibitor which fit the chemical structure of compound 18. Although the quality of the density was inferior to that for the previous structure, the inhibitor could be unambiguously fitted into the electron density and refined. The solid surface of thrombin is generated by Quanta 79.
7. Howard, Judith A. K.; Hoy, Vanessa J.; O'Hagan, David; Smith, Garry T. Tetrahedron (1996), 52(38), 12613-12622.
8. Abraham, Michael H.; Duce, Philip P.; Prior, David V.; Barratt, Derek G.; Morris, Jeffrey J.; Taylor, Peter J. J. Chem. Soc., Perkin Trans. 2 (1989), 10, 1355-75.