Structural basis for thrombin activation of a protease-activated receptor: inhibition of intramolecular liganding

Protease-activated G protein-coupled receptors (PAR1-4) are tethered-ligand receptors that are activated by proteolytic cleavage of the extracellular domain (exodomain) of the receptor. PAR1, the prototypic member of the PAR family, is the high-affinity thrombin receptor of platelets and vascular endothelium and plays a critical role in blood coagulation, thrombosis, and inflammation. Here, we describe the solution structure of the thrombin-cleaved exodomain of PAR1. The side chains of a hydrophobic hirudin-like (Hir) sequence and adjacent anionic motif project into solution. Docking of the exodomain Hir sequence to exosite I of thrombin reveals that the tethered ligand in the cleaved exodomain bends away from thrombin, leaving its active site available to another large macromolecular substrate. The N-terminal ligand is longer than anticipated and forms an intramolecular complex with a region located in the C terminus of the exodomain. Mutational analysis confirmed that this C-terminal region is a ligand binding site for both intra- and intermolecular ligands. A lipidated-ligand binding site peptide was found to be an effective inhibitor of thrombin-induced platelet aggregation.     

PEG在线定点修饰水蛭素及其修饰位点的理论预测与分析

比较了液相和固相修饰水蛭素的实验结果, 并在实验的基础上对水蛭素以及水蛭素/凝血酶复合物进行分子动力学模拟, 分析了容易被修饰的水蛭素赖氨酸残基的位点. 结果表明, 在液相修饰的时候, 水蛭素在水溶液中可被修饰的赖氨酸残基除Lys47外, 都容易被修饰. 而在固相修饰的时候, 水蛭素仅Lys35和Lys27容易被修饰.     

Effects of shear rate on propagation of blood clotting determined using microfluidics and numerical simulations

This paper describes microfluidic experiments with human blood plasma and numerical simulations to determine the role of fluid flow in the regulation of propagation of blood clotting. We demonstrate that propagation of clotting can be regulated by different mechanisms depending on the volume-to-surface ratio of a channel. In small channels, propagation of clotting can be prevented by surface-bound inhibitors of clotting present on vessel walls. In large channels, where surface-bound inhibitors are ineffective, propagation of clotting can be prevented by a shear rate above a threshold value, in agreement with predictions of a simple reaction-diffusion mechanism. We also demonstrate that propagation of clotting in a channel with a large volume-to-surface ratio and a shear rate below a threshold shear rate can be slowed by decreasing the production of thrombin, an activator of clotting. These in vitro results make two predictions, which should be experimentally tested in vivo. First, propagation of clotting from superficial veins to deep veins may be regulated by shear rate, which might explain the correlation between superficial thrombosis and the development of deep vein thrombosis (DVT). Second, nontoxic thrombin inhibitors with high binding affinities could be locally administered to prevent recurrent thrombosis after a clot has been removed. In addition, these results demonstrate the utility of simplified mechanisms and microfluidics for generating and testing predictions about the dynamics of complex biochemical networks.     

Universal Approach Towards r‐Hirudin Derivatives with High Anti‐Thrombin Activity Based on Chemical Differentiation of Primary Amino Groups

Chemical modification of recombinant hirudin (r‐hirudin) is necessary whenever surface‐confinement to a biomaterial or biotinylation for subsequent conjugation with carriers is intended. Here, we report a modification strategy that permits chemical discrimination between r‐hirudin's amino groups and preserves its thrombin inhibitor activity. By reaction with Msc‐ONSu, protective groups were successively introduced in r‐hirudin yielding four derivatives (Msc)_x‐hirudin (1 ≤ x ≤ 4) and pure fractions were isolated by ion exchange chromatography. Structure–function relationships were studied for all derivatives and revealed a decrease in activity of more than 90% as compared to unprotected r‐hirudin. MALDI‐TOF MS was used to determine the locations of the Msc groups. Furthermore, evidence was provided that r‐hirudin's N‐terminal amino group is highly important for its anti‐thrombin activity. Selective modification of the lysine residues which maintained the free N‐terminal amino group preserved the anti‐thrombin activity of r‐hirudin even after biotinylation and subsequent linkage to streptavidin or confinement to a polymer surface.     

Rational design of hirulog-type inhibitors of thrombin

Summary The two crystal structures of thrombin complexed with its most potent natural inhibitor hirudin and with the active-site inhibitor d-Phe-Pro-Arg-CH₂Cl [Rydel, T.J. et al., J. Mol. Biol., 221 (1991) 583; Bode, W. et al., EMBO J., 8 (1989) 3467] were used as a basis to design a new inhibitor, combining the high specificity of the polypeptide hirudin with the simpler chemistry of an organic compound. In the new inhibitor, the C-terminal amino acid residues 53–65 of hirudin are linked by a spacer peptide of four glycines to the active-site inhibitor NAPAP (N-(2-naphthyl-sulfonyl-glycyl)-dl-p-amidinophenylalanyl-piperidine). Energy minimization techniques served as a tool to determine the preferred configuration at the amidinophenylalanine and the modified piperidine moiety of the inhibitor. The predictions are supported by the interaction energies determined for d- and l-NAPAP in complex with thrombin, which are in good agreement with experimentally determined dissociation constants. The conformational flexibility of the linker peptide in the new inhibitors was investigated with molecular dynamics techniques. A correlation between the P1 position and the interactions of the linker peptide with the protein is suggested. Modifications of the linker peptide are proposed based on the distribution of its main-chain torsion angles in order to enhance its binding to thrombin.     

Prediction of enzyme binding: human thrombin inhibition study by quantum chemical and artificial intelligence methods based on X-ray structures

Thrombin is a serine protease which plays important roles in the human body, the key one being the control of thrombus formation. The inhibition of thrombin has become a target for new antithrombotics. The aim of our work was to (i) construct a model which would enable us to predict Ki values for the binding of an inhibitor into the active site of thrombin based on a database of known X-ray structures of inhibitor-enzyme complexes and (ii) to identify the structural and electrostatic characteristics of inhibitor molecules crucially important to their effective binding. To retain as much of the 3D structural information of the bound inhibitor as possible, we implemented the quantum mechanical/molecular mechanical (QM/MM) procedure for calculating the molecular electrostatic potential (MEP) at the van der Waals surfaces of atoms in the protein's active site. The inhibitor was treated quantum mechanically, while the rest of the complex was treated by classical means. The obtained MEP values served as inputs into the counter-propagation artificial neural network (CP-ANN), and a genetic algorithm was subsequently used to search for the combination of atoms that predominantly influences the binding. The constructed CP-ANN model yielded Ki values predictions with a correlation coefficient of 0.96, with Ki values extended over 7 orders of magnitude. Our approach also shows the relative importance of the various amino acid residues present in the active site of the enzyme for inhibitor binding. The list of residues selected by our automatic procedure is in good correlation with the current consensus regarding the importance of certain crucial residues in thrombin's active site.     

Light regulation of aptamer activity: an anti-thrombin aptamer with caged thymidine nucleobases

"Caged" derivatives of a 15 nucleotide ssDNA anti-thrombin aptamer have been synthesized in which thymidine nucleotides are modified with photolabile protecting groups. One caged thymidine in a key location is enough to completely mask the aptamer's function in respect to their affinity for thrombin and their inhibition of the blood clotting cascade. With light (366 nm) the caging group can be removed, yielding the unmodified active aptamer.     

An Immucillin‐Based Transition‐State‐Analogous Inhibitor of tRNA–Guanine Transglycosylase (TGT)

Shigellosis is one of the most severe diarrheal diseases worldwide without any efficient treatment so far. The enzyme tRNA–guanine transglycosylase (TGT) has been identified as a promising target for small‐molecule drug design. Herein, we report a transition‐state analogue, a small, immucillin‐derived inhibitor, as a new lead structure with a novel mode of action. The complex inhibitor synthesis was accomplished in 18 steps with an overall yield of 3 %. A co‐crystal structure of the inhibitor bound to Z. mobilis TGT confirmed the predicted conformation of the immucillin derivative in the enzyme active site.     

Label free binding assay with spectroscopic detection for pharmaceutical screening

Many therapeutic drugs exert their effects by interaction with well defined molecular targets. Increasing knowledge in molecular biology allows identification of more and more molecular key compounds and in consequence a molecular approach to disease and therapy. As binding of drugs to their target compounds is a key event, binding assays with an appropriate target molecule are useful means for primary screening of novel substances. We have investigated the potential of thin film interference spectroscopy (RIFS) as a label free detection method for pharmaceutical screening in a binding inhibition assay. To meet the throughput requirements in pharmaceutical screening a parallel detection system based on imaging spectroscopy was constructed. Thrombin/thrombin inhibitor interaction was investigated as a model system. The thin film transducer was covalently modified with a thrombin inhibitor. Specific binding of thrombin and binding inhibition by inhibitor compounds could be observed. A test cycle of less than 10 min could be reached. The parallel setup allows the simultaneous detection of 96 binding curves and can reach a throughput of more than 10⁶ samples per year.     

PHOTOCOAGULATION OF HUMAN PLASMA: ACYL SERINE PROTEINASE PHOTOCHEMISTRY

Human alpha-thrombin or bovine Factor Xa was acylated at the active site serine hydroxyl with alpha-methyl-2-hydroxy-4-diethylaminocinnamic acid. These modified serine proteinase enzymes showed no plasma coagulation biological activity in the absence of light. Photolysis of the acyl serine proteinase enzymes in plasma for 1-35 s with monochromatic 366 nm light isolated from a high pressure mercury arc results in coagulation of the plasma. For example, photolysis of 3 NIH U of the acyl human alpha-thrombin for 5 s in human plasma results in a clot in 23 s. For comparison, 1 NIH U of unmodified human alpha-thrombin gave a clot in 21 s under the conditions of the assay but without photolysis. Appropriate controls showed that the coagulation is the result of the formation of active thrombin due to photodeacylation of the enzymes. The photoinduced clotting time measured is dependent on acyl thrombin concentration and photolysis time. Thus higher concentrations of acyl thrombin and longer photolysis times give a shorter clotting time. A kinetic scheme based upon Lineweaver-Burke analysis of the clotting process is developed.     

A model for the binding of low molecular weight inhibitors to the active site of thrombin

This paper describes the construction, validation and application of an active site model of the serine protease thrombin. Initial use was made of medium resolution X-ray crystallographic structures of thrombin complexed with low molecular weight, non-specific inhibitors to create a computationally useable active site shell of the enzyme. Molecular mechanics methods were then applied to dock known ligands into the active site region in order to derive a model that would accurately predict binding conformations. Validation of the modelling process was achieved by comparison of the predicted enzyme-bound conformations with their known, crystallographic binding conformations. The resultant model was used extensively for predictive purposes prior to obtaining confirmatory crystal data relating to a ligand possessing a novel and unexpected binding component complexed to thrombin. The data served both to confirm the accuracy of the binding site model and to provide information for the further refinement of the model.     

The binding site for an inhibitor of squalene:hopene cyclase determined using photoaffinity labeling and molecular modeling.

BACKGROUND: The squalene:hopene cyclases (SHCs) are bacterial enzymes that convert squalene into hopanoids, a function analogous to the action of oxidosqualene cyclases (OSCs) in eukaryotic steroid and triterpenoid biosynthesis. We have identified the binding site for a selective, potent, photoactivatable inhibitor of an SHC. RESULTS: SHC from Alicyclobacillus acidocaldarius was specifically labeled by [3H]Ro48-8071, a benzophenone-containing hypocholesteremic drug. Edman degradation of a peptide fragment of covalently modified SHC confirmed that Ala44 was specifically modified. Molecular modeling, using X-ray-derived protein coordinates and a single point constraint for the inhibitor, suggested several geometries by which Ro48-8071 could occupy the active site. CONCLUSIONS: A covalent complex of a potent inhibitor with a squalene cyclase has been characterized. The amino acid modification and molecular modeling suggest that Ro48-8071 binds at the junction between the central cavity and substrate entry channel, therefore inhibiting access of the substrate to the active site.     

Design, synthesis and biological evaluation of thrombin inhibitors based on a pyridine scaffold

A series of 2,4-disubstituted pyridine derivatives has been designed, synthesised and evaluated as thrombin inhibitors. A Grignard exchange reaction was used to introduce various benzoyl substituents in position 4 of the pyridine ring, where they serve as P3 residues in binding to thrombin. In position 2 of the pyridine ring, a para-amidinobenzylamine moiety was incorporated as P1 residue by an SNAr reaction using ammonia as nucleophile followed by a reductive amination. A crystal structure obtained for one of the compounds in the active site of thrombin revealed that the basic amidine group of the inhibitor was anchored to Asp 189 at the bottom of the S1 pocket. A comparison with melagatran, bound in the active site of thrombin, revealed a good shape match but lack of hydrogen bonding possibilities in the S2-S3 region for the thrombin inhibitors reported in this study.     

Binding conformation of 2-oxoamide inhibitors to group IVA cytosolic phospholipase A2 determined by molecular docking combined with molecular dynamics

The group IVA cytosolic phospholipase A(2) (GIVA cPLA(2)) plays a central role in inflammation. Long chain 2-oxoamides constitute a class of potent GIVA cPLA(2) inhibitors that exhibit potent in vivo anti-inflammatory and analgesic activity. We have now gained insight into the binding of 2-oxoamide inhibitors in the GIVA cPLA(2) active site through a combination of molecular docking calculations and molecular dynamics simulations. Recently, the location of the 2-oxoamide inhibitor AX007 within the active site of the GIVA cPLA(2) was determined using a combination of deuterium exchange mass spectrometry followed by molecular dynamics simulations. After the optimization of the AX007-GIVA cPLA(2) complex using the docking algorithm Surflex-Dock, a series of additional 2-oxoamide inhibitors have been docked in the enzyme active site. The calculated binding affinity presents a good statistical correlation with the experimental inhibitory activity (r(2) = 0.76, N = 11). A molecular dynamics simulation of the docking complex of the most active compound has revealed persistent interactions of the inhibitor with the enzyme active site and proves the stability of the docking complex and the validity of the binding suggested by the docking calculations. The combination of molecular docking calculations and molecular dynamics simulations is useful in defining the binding of small-molecule inhibitors and provides a valuable tool for the design of new compounds with improved inhibitory activity against GIVA cPLA(2).     

Label free binding assay with spectroscopic detection for pharmaceutical screening

Many therapeutic drugs exert their effects by interaction with well defined molecular targets. Increasing knowledge in molecular biology allows identification of more and more molecular key compounds and in consequence a molecular approach to disease and therapy. As binding of drugs to their target compounds is a key event, binding assays with an appropriate target molecule are useful means for primary screening of novel substances. We have investigated the potential of thin film interference spectroscopy (RIFS) as a label free detection method for pharmaceutical screening in a binding inhibition assay. To meet the throughput requirements in pharmaceutical screening a parallel detection system based on imaging spectroscopy was constructed. Thrombin/thrombin inhibitor interaction was investigated as a model system. The thin film transducer was covalently modified with a thrombin inhibitor. Specific binding of thrombin and binding inhibition by inhibitor compounds could be observed. A test cycle of less than 10 min could be reached. The parallel setup allows the simultaneous detection of 96 binding curves and can reach a throughput of more than 10⁶ samples per year. Received: 26 November 1996 / Revised: 17 February 1997 / Accepted: 22 February 1997     

A novel chitosan-based sponge coated with self-assembled thrombin/tannic acid multilayer films as a hemostatic dressing

In order to prepare a novel hemostatic dressing for uncontrolled hemorrhage, a porous chitosan sponge was coated with self-assembled(thrombin/tannic acid)n films, which were based on hydrogen bonding interactions between thrombin and tannic acid at physiologic p H. According to the whole blood clotting test, the coated chitosan sponges showed a significantly high rate of blood clotting due to the addition of thrombin. On the other hand, the storable half-life of immobilized thrombin is extended to 66.9 days at room temperature, which is 8.5 times longer than unfixed thrombin. It is because of the immobilization effect of, not only the porous structure of chitosan sponge but also the interactions between thrombin and tannic acid. In addition, the tannic acid has similar antibacterial effect to chitosan. Therefore, it is an excellent combination of chitosan, thrombin and tannic acid. Besides, all of materials in this research have been approved by the United States Food and Drug Administration(FDA). So the chitosan-based sponge is a promising candidate dressing for uncontrolled hemorrhage due to its storable, bio-safe and highly effective hemostatic properties.     

Total Synthesis of Homogeneous Variants of Hirudin P6: A Post‐Translationally Modified Anti‐Thrombotic Leech‐Derived Protein

Hirudin P6 is a leech‐derived anti‐thrombotic protein which possesses two post‐translational modifications, O‐glycosylation and tyrosine sulfation. In this study we report the ligation‐based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.     

L-乳酸脱氢酶抑制剂抑制成因的探讨

应用HF/3 21G研究了抑制剂H2N－CO－COO－对L 乳酸脱氢酶的抑制成因.结果表明,酶被抑制的主要原因有:(1)抑制剂与底物的稳定构象态在结构上极为相似,导致酶不能有效识别底物;(2)模型抑制剂各原子所带净电荷的优势使抑制剂更易与酶活性中心结合;(3)抑制剂通过对酶的诱导契合作用使酶活性中心的空间被缩小;(4)对活性中心有关结构的分析表明,底物的甲基分子片以及酶的氨基酸残基Gln 102,对催化反应能否顺利进行,影响极大.     

Molecular dynamics simulation of papain-E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine) complex

To investigate the possible binding mode of E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine), a potent cysteine protease inhibitor, to papain active site, molecular dynamics simulations were applied to two complex forms: R- and S- configurational forms of E-64 C2 atom for the covalent bond formation with the papain Cys-25 SH group. The tertiary structures of the papain-E-64 complexes were built by visual interactive modelling and the energy minimization technique, and were subjected to the dynamics simulations of 10 ps. Although no significant difference was observed between the potential energies of energy-minimized R- and S-complex forms, the molecular dynamics simulations suggested that the hydrogen bonding mode of the former form is more advantageous than that of the latter one. Comparing with the hydrogen bonds observed in the papain-E-64 complex crystal, it could be concluded that the present molecular dynamics simulation reflects well the three-dimensional structure concerning the interaction of E-64 with the papain active site. The conformational characteristics of E-64 and its possible interaction mode with papain were also discussed.     

Geissoschizine methyl ether, a corynanthean-type indole alkaloid from Uncaria rhynchophylla as a potential acetylcholinesterase inhibitor

Geissoschizine methyl ether (1), a newly discovered strong acetylcholinesterase (AChE) inhibitor, along with six weakly active alkaloids, vallesiachotamine (2), hisuteine (3), hirsutine (4), isorhynchophylline (5), cisocorynoxeine (6) and corynoxeine (7) have been isolated from Uncaria rhynchophylla. Geissoschizine methyl ether (1) inhibited 50% of AChE activity at concentrations of 3.7?±?0.3?μg?mL(-1) while the IC(50) value of physostigmine as a standard was 0.013?±?0.002?μg?mL(-1). The mode of AChE inhibition by 1 was reversible and non-competitive. In addition, molecular modelling was performed to explore the binding mode of inhibitor 1 at the active site of AChE.