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A computational algorithm was used to design automatically novel thrombin inhibitors that are available from a single-step chemical reaction. The compounds do not contain amide bonds, are achiral and have a molecular weight below 400. Of the 10 compounds that were synthesized, five bind to thrombin with a Ki in the nanomolar range. Subsequent X-ray structure determination of the thrombin-inhibitor complex for the best compound (Ki = 95 nM) confirms the predicted binding mode. The novel algorithm is applicable to a broad range of chemical reactions.

The tissue factor:factor VIIa (TF:F.VIIa) complex is the physiological initiator of blood coagulation and plays a role both in normal hemostasis and in various thrombotic disorders. The TF:F.VIIa crystal structure presented here shows the two molecules in association as a complex and provides a ready structural explantation for most of the complementary observations on the complex obtained by other techniques. This is only one F.VII structure of a series along the activation pathway going from zymogen F.VII, through F.VIIa alone, towards that in the ternary complex with a macromolecular substrate such as F.IX or F.X. To fully understand the activation of F.VIIa by TF will require further structural work, but in the meantime this structure may be used in the search for more effective and more specific anticoagulants.

Background: The serine protease thrombin is central in the processes of hemostasis and thrombosis. To be useful, thrombin inhibitors should combine potency towards thrombin with selectivity towards other related enzymes such as trypsin. We previously reported the structure-based design of thrombin inhibitors with rigid, bicyclic core structures. These compounds were highly active towards thrombin, but showed only modest selectivity., Results: Here, we describe the rational design of selective thrombin inhibitors starting from the X-ray crystal structure of the complex between the previously generated lead molecule and thrombin. The lead molecule bound with a Ki value of 90nM and a selectivity of 7.8 for thrombin over trypsin. Our design led to inhibitors with improved activity and greatly enhanced selectivity. The binding mode for two of the new inhibitors was determined by X-ray crystallography of their complexes with thrombin. The results confirmed the structures predicted by molecular modeling and, together with the binding assays, provided profound insight into molecular recognition phenomena at the thrombin active site., Conclusions: A novel class of nonpeptidic, selective thrombin inhibitors has resulted from structure-based design and subsequent improvement of the initial lead molecule. These compounds, which are preorganized for binding to thrombin through a rigid, bicyclic or tricyclic central core, could aid in the development of new antithrombotic drugs. Correlative binding and X-ray structural studies within a series of related, highly preorganized inhibitors, which all prefer similar modes of association to thrombin, generate detailed information on the strength of individual intermolecular bonding interactions and their contribution to the overall free energy of complexation.

Blood coagulation is initiated when tissue factor binds to coagulation factor VIIa to give an enzymatically active complex which then activates factors IX and X, leading to thrombin generation and clot formation. We have determined the crystal structure at 2.0-A degrees resolution of active-site-inhibited factor VIIa complexed with the cleaved extracellular domain of tissue factor. In the complex, factor VIIa adopts an extended conformation. This structure provides a basis for understanding many molecular aspects of the initiation of coagulation.

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 x 0.12 x 0.6 mm and showed diffraction to a resolution of 3 A.

Thrombin, a serine protease, plays a central role in the initiation and propagation of thrombotic events. An extensive search for new thrombin inhibitors was performed, using an unconventional approach. Screening of small basic molecules for binding in the recognition pocket of thrombin led to the discovery of (aminoiminomethyl)piperidine (amidinopiperidine) as a weak, but intrinsically selective, thrombin inhibitor. Elaboration of this molecule provided compounds which inhibit thrombin with Ki's in the range of 20-50 nM and with selectivities of 1000-4000 against trypsin. These inhibitor compounds show a new and unexpected binding mode to thrombin. Modification of the central building block and then of one of the hydrophobic substituents led to the discovery of a new family of thrombin inhibitors which has reverted to the former binding mode to thrombin. This last class of compounds shows inhibitory activities in the picomolar range, low toxicity, and a short plasma half life which favors its use for an intravenous application. From this series of thrombin inhibitors, 19f(Ro 46-6240) was selected for clinical development as an antithrombotic agent for intravenous administration.

The X-ray crystal structure of the complex of the extracellular domain of the human 55 kd tumor necrosis factor (TNF) receptor with human TNF beta has been determined at 2.85 A resolution. The complex has three receptor molecules bound symmetrically to one TNF beta trimer. The receptor fragment, a very elongated end to end assembly of four similar folding domains, binds in the groove between two adjacent TNF beta subunits. The structure of the complex defines the orientation of the ligand with respect to the cell membrane and provides a model for TNF receptor activation. The novel fold of the TNF receptor structure is likely to be representative of the nerve growth factor (NGF)/TNF receptor family as a whole.

A complex of tumour necrosis factor-beta with the soluble extracellular domain of the human 55 kDa TNF receptor has been crystallized. Crystals of the complex were grown using polyethylene glycol 4000 as the precipitating agent in the presence of beta-octyl glucoside. The receptor-ligand complex crystallizes in a cubic space group and diffracts to 2.85 A.

The mode of binding of four active-site directed inhibitors to human thrombin has been determined by x-ray crystallographic analysis. The inhibitors studied are benzamidine, PPACK, NAPAP, and MD-805, of which the last three are compounds evolved specifically to inhibit thrombin. Crystal structures were determined in the presence of both the inhibitor and the undecapeptide [des-amino Asp55]hirudin(55-65) which binds distant from the active site. Despite having significantly different chemical structures, NAPAP and MD-805 bind to thrombin in a very similar "inhibitor binding mode" which is not that expected by direct analogy with the binding of substrate. Both inhibitors bind to thrombin in a similar way as to trypsin, but thrombin has an extra loop, the "Tyr-Pro-Pro-Trp loop," not present in trypsin, which gives further binding interactions and is seen to move somewhat to accommodate binding of the different inhibitors. The fact that NAPAP and MD-805 require different stereochemistry for potent inhibition is demonstrated, and its structural basis clarified. The wealth of data on analogs and variants of these lead compounds is shown to be compatible with this inhibitor binding mode.

Structural details of the Rop protein from plasmid ColE1 are presented, with a description of the X-ray crystal structure determination and refinement at a nominal resolution of 1.7 A. The 63 amino acid protein is a dimer. Each monomer consists almost entirely of two alpha helices, the whole molecule forming a highly regular four-alpha-helix bundle. This may be approximated by a four-stranded rope with a radius of 7.0 A, a left-handed helical twist and a pitch of 172.5 A. The packing constraints for this novel type of coiled-coil structure are given. The protein acts in the control of plasmid replication via regulation of an RNA-RNA interaction in a manner not yet understood in atomic detail.

We show that a protein of 63 amino acids is the product of the rop gene, a gene which negatively regulates the copy number of plasmids of the ColE1 family. Rop protein purified to homogeneity inhibits ColE1 plasmid replication in a bacterial extract. Furthermore, we show that Rop inhibition requires RNA I. In a purified in vitro system that can support primer transcription and processing, Rop affects primer formation in two ways: first, it elicits transcription termination oat nucleotide 220, and second, it increases the ability of RNA I to inhibit RNAase H processing of the primer. The analysis of these data and the comparison with the results obtained in vivo with transcription fusion experiments allow us to propose a tentative model of the molecular mechanism underlying Rop-RNA I inhibition.

Crystals of the green, copper-containing enzyme nitrite reductase from Alcaligenes faecalis S-6 have been obtained. They are monoclinic, space group P2(1) with a = 78.0 A, b = 92.9 A, c = 78.5 A and beta = 111.8 degrees. The crystals diffract to better than 2 A and are suitable for high-resolution X-ray crystallography.

Preliminary crystallographic data are given for Rop, a protein involved in the control of replication of plasmids of the ColE1 family.

The crystal structure of L-prolyl-L-leucyl-alpha-aminoisobutyryl-alpha- aminoiso-butyryl-alpha-L-glutamyl-L-valinol (L-Pro-L-Leu-Aib-Aib-L-Glu-L-Valol), the C-terminal hexapeptide fragment of trichotoxin, has been determined by X-ray crystallography. The hexapeptide forms a right-handed 3(10)-helix consisting of two 10-atom hydrogen-bonded beta-turns of type III and one beta-turn of type I. Two of the intramolecular hydrogen-bonds are particularly weak, thus suggesting that the title compound may adopt non-helical conformations in solution, as observed by circular dichroism. In the crystal the molecules are hydrogen-bonded head-to-tail, forming infinitely long helical columns.

The three-dimensional structure of pseudoazurin, a single copper-containing protein from Alcaligenes faecalis strain S-6, has been determined at 2.9 A resolution by X-ray crystallography. The sequences of two other pseudoazurins from Pseudomonas AM1 and Achromobacter cycloclastes may also be accommodated in this structure. The structure, an eight-stranded beta-barrel, resembles closely those of plastocyanin and azurin. It possesses two extra alpha-helices at the C-terminus, whereas azurins have an alpha-helical flap in the middle of their sequences.