Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

"Getting it right" refers to the careful modeling of all elements in the living system, i.e. biological macromolecules, ligands and water molecules. In addition, careful attention should be paid to the protonation state of ionizable functional groups on the ligands and residues at the active site. Computational technology based on the empirical HINT program is described to: (1) calculate free energy scores for ligand binding; (2) include the implicit and explicit effects of water in and around the ligand binding site; and (3) incorporate the effects of global and local pH in molecular models. This last point argues for the simultaneous consideration of a number of molecular models, each with different protonation profiles. Data from recent studies of protein-ligand systems (trypsin, thrombin, neuraminidase, HIV-1 protease and others) are used to illustrate the concepts in the paper. Also discussed are experimental factors related to accurate free energy predictions with this and other computational technologies.
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PMID:Getting it right: modeling of pH, solvent and "nearly" everything else in virtual screening of biological targets. 1518 7

The ability to find novel bioactive scaffolds in compound similarity-based virtual screening experiments has been studied comparing Tanimoto-based, ranking-based, voting, and consensus scoring protocols. Ligand sets for seven well-known drug targets (CDK2, COX2, estrogen receptor, neuraminidase, HIV-1 protease, p38 MAP kinase, thrombin) have been assembled such that each ligand represents its own unique chemotype, thus ensuring that each similarity recognition event between ligands constitutes a scaffold hopping event. In a series of virtual screening studies involving 9969 MDDR compounds as negative controls it has been found that atom pair descriptors and 3D pharmacophore fingerprints combined with ranking, voting, and consensus scoring strategies perform well in finding novel bioactive scaffolds. In addition, often superior performance has been observed for similarity-based virtual screening compared to structure-based methods. This finding suggests that information about a target obtained from known bioactive ligands is as valuable as knowledge of the target structures for identifying novel bioactive scaffolds through virtual screening.
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PMID:Scaffold hopping through virtual screening using 2D and 3D similarity descriptors: ranking, voting, and consensus scoring. 1650 72

The common structural feature of LK direct thrombin inhibitors is a strong basic group attached to the azaphenylalanine scaffold, which is important for the appropriate interaction at the thrombin active site. Our previous results have shown that this basic group could be responsible for a reduction of tracheal air flow and a fall of mean arterial pressure in anaesthetized rats, an undesired effect of direct thrombin inhibitors which correlated with their ability to release histamine from mast cells. In the present study, we investigated the mechanism of LK direct thrombin inhibitors-induced histamine release from rat peritoneal mast cells. We demonstrated that thrombin inhibitors with basic character (LK-732, LK-639 and LK-6063) provoked release of histamine from mast cells, while less basic analogs (LK-658, LK-633 and LK-6062) had no effect. Histamine released by LK-732 and LK-639 was suppressed by removal of sialic acid residues by neuraminidase and by pertussis toxin, an inhibitor of G(i) protein activity. Additional demonstration that G proteins are the targets of LK-732 and LK-639 was provided by the increase of GTPgammaS binding rate to G proteins in rat brain cortical membranes. Our results indicate that basic direct thrombin inhibitors LK-732 and LK-639 provoke release of histamine from mast cells by direct activation of G(i) proteins through the similar biochemical pathway as basic secretagogues.
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PMID:Histamine release, an undesired effect of thrombin inhibitors with basic character, is mediated through direct activation of G(i) proteins. 1665 Apr 5

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.
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PMID:BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility. 1743 97

A new GRID-based method for scaffold hopping (SHOP) is presented. In a fully automatic manner, scaffolds were identified in a database based on three types of 3D-descriptors. SHOP's ability to recover scaffolds was assessed and validated by searching a database spiked with fragments of known ligands of three different protein targets relevant for drug discovery using a rational approach based on statistical experimental design. Five out of eight and seven out of eight thrombin scaffolds and all seven HIV protease scaffolds were recovered within the top 10 and 31 out of 31 neuraminidase scaffolds were in the 31 top-ranked scaffolds. SHOP also identified new scaffolds with substantially different chemotypes from the queries. Docking analysis indicated that the new scaffolds would have similar binding modes to those of the respective query scaffolds observed in X-ray structures. The databases contained scaffolds from published combinatorial libraries to ensure that identified scaffolds could be feasibly synthesized.
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PMID:SHOP: scaffold HOPping by GRID-based similarity searches. 1748 78

Since the development of the first docking algorithm in the early 1980s a variety of different docking approaches and tools has been created in order to solve the docking problem. Subsequent studies have shown that the docking performance of most tools strongly depends on the considered target. Thus it is hard to choose the best algorithm in the situation at hand. The docking tools FlexX and AutoDock are among the most popular programs for docking flexible ligands into target proteins. Their analysis, comparison, and combination are the topics of this study. In contrast to standard consensus scoring techniques which integrate different scoring algorithms usually only by their rank, we focus on a more general approach. Our new combined docking workflow-AutoxX-unifies the interaction models of AutoDock and FlexX rather than combining the scores afterward which allows interpretability of the results. The performance of FlexX, AutoDock, and the combined algorithm AutoxX was evaluated on the basis of a test set of 204 structures from the Protein Data Bank (PDB). AutoDock and FlexX show a highly diverse redocking accuracy at the different complexes which assures again the usefulness of taking several docking algorithms into account. With the combined docking the number of complexes reproduced below an rmsd of 2.5 A could be raised by 10. AutoxX had a strong positive effect on several targets. The highest performance increase could be found when redocking 20 protein-ligand complexes of alpha-thrombin, plasmepsin, neuraminidase, and d-xylose isomerase. A decrease was found for gamma-chymotrypsin. The results show that--applied to the right target-AutoxX can improve the docking performance compared to AutoDock and FlexX alone.
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PMID:Alternative to consensus scoring--a new approach toward the qualitative combination of docking algorithms. 1749 29

In this study, we propose a drug design approach which includes docking, molecular fingerprints based cluster analysis, and 'induced' descriptors based receptor-dependent 3D-QSAR. The method was shown to be very useful for screening and modeling structurally diverse data sets of pharmacological interest. Different from other receptor-dependent 3D-QSAR, no ambiguous alignments are required for the construction of the models, and the computational cost is relatively lower. Moreover, 'induced' descriptors were shown to be very powerful in "capturing" ligand-receptor intermolecular interactions. The methodology was validated for eight data sets sampled from the literature and from public databases: human sex hormone-binding globulin, human corticosteroid-binding globulin, anthrax lethal factor, HIV-1 reverse transcriptase, neuraminidase A, thrombin, trypsin, and Pneumocystis carinii dihydrofolate reductase data sets. The resulting models were interpretable; the constructed QSAR equations have high statistical significance and predictive strength; and the drug design solutions were shown to be useful for guiding ligand modification for the development of new inhibitors for a broad range of molecular targets.
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PMID:Using molecular docking, 3D-QSAR, and cluster analysis for screening structurally diverse data sets of pharmacological interest. 1881 24

With the ongoing progress in protein crystallography and NMR, structure-based drug design is adopting increasing importance in the search for new drugs. Modeling starts from the 3D structure of a target protein in order to construct molecules which are complementary to a binding site, in their geometry as well as in the pattern of their physicochemical properties around the molecules. The rational design process is accompanied by 3D structure determinations of different ligand-protein complexes. Most often, significantly improved binding affinities of the ligands are observed after several cycles of 3D structure determinations, the design of compounds with appropriate structural modifications, synthesis, and testing of the new drug candidates. As an alternative, pharmacophore models are derived from the 3D structures of active analogs. A risk with lead structure optimization by structure-based design is the neglect of other important biological properties, such as bioavailability and metabolic stability. Recent applications of structure-based design, as well as success stories in the search for new, potent and selective HIV protease inhibitors, thrombin inhibitors, neuraminidase inhibitors and integrin receptor antagonists, are reviewed.
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PMID:Structure-based design of enzyme inhibitors and receptor ligands. 1964 84

The stalk of influenza neuraminidase (NA) has been a target of cleavage by various proteases, resulting in the release of catalytically active globular heads from virus particles. However, despite successful cases in a number of influenza subtypes, this strategy could not be applied to all influenza viruses due to high variation of the NA stalk. In the present study, reverse genetics was employed to construct non-pathogenic recombinant influenza A viruses, termed rgH1N1(LVPR) and rgH1N1(LVPR-GS), that harbor the NA of H5N1 virus engineered to contain a specific thrombin cleavage site at the stalk region. By using thrombin to cleave NA at its stalk, a productive extraction of NA globular heads could be obtained from purified rgH1N1(LVPR). Furthermore, it was found that the NA of rgH1N1(LVPR-GS) could be cleaved by endogenous thrombin present in embryonated chicken eggs, resulting in the release of NA globular heads into allantoic fluids. These data highlight the use of thrombin cleavage as an effective strategy for extraction of active NA heads directly from live viral particles not only of H5N1 but, theoretically, of any subtype of influenza A viruses.
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PMID:Extraction of catalytically active neuraminidase of H5N1 influenza virus using thrombin proteolytic cleavage. 1976 41

Influenza A is a major cause of mortality. Knowledge on coagulation activation in influenza infection is limited. The factor V Leiden (FVL) mutation is possibly subject to positive selection pressure. It is unknown whether this mutation impacts on the outcome of severe influenza. In the present study, the effect of lethal influenza on pulmonary and systemic coagulation activation and whether or not FVL mutation alters coagulation activation in and the course of lethal influenza, was determined. Wild-type mice, and mice heterozygous or homozygous for FVL were infected intranasally with a lethal dose of H1N1 (haemagglutinin 1 and neuraminidase 1) influenza A. Mice were sacrificed after 48 or 96 h for determination of coagulation activation, histopathology, pulmonary inflammatory parameters and viral load, or were observed in a survival study. Extensive local and systemic coagulation activation during lethal influenza was demonstrated by increased lung and plasma levels of thrombin-antithrombin complexes and fibrin degradation products, and by pulmonary fibrin deposition. FVL mutation did not influence the procoagulant response, lung histopathology or survival. FVL mice demonstrated elevated viral loads 48 h after infection. In conclusion, coagulation is activated locally and systemically during lethal murine influenza A infection. The FVL mutation does not influence coagulation activation, lung inflammation or survival in lethal influenza A.
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PMID:Factor V Leiden mutation does not affect coagulopathy or outcome in lethal H1N1 influenza. 2041 39


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