Preparation of pyrrolidine and isoxazolidine benzamidines as potent inhibitors of coagulation factor Xa.

The serine protease factor Xa is a critical enzyme in the blood coagulation cascade. Recently, the inhibition of factor Xa has begun to emerge as an attractive strategy for the discovery of novel antithrombotic agents. Here we describe pyrrolidine and isoxazolidine benzamidines as novel and potent inhibitors of factor Xa.     

Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.     

Halothiophene benzimidazoles as P1 surrogates of inhibitors of blood coagulation factor Xa

Neutral weak halothiophene benzimidazole inhibitors of the serine protease factor Xa were identified via screening of a compound library. The X-ray crystal structure of representative 3a bound to human fXa confirmed the S1 binding mode. Starting from 3a a series of halothiophene benzimidazoles was synthesized and investigated for their factor Xa inhibitory activity. This led to potent and selective achiral inhibitors against fXa such as compounds 9k and 9w.     

Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation

Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca(2+) signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor VIIa, complexed with tissue factor, evoked a greatly delayed, oscillatory Ca(2+) response, which relied on its catalytic activity and resembled that of SLIGRL, a peptide specifically activating the protease-activated receptor 2 (PAR2). Thrombin even at low concentrations evoked a rapid, mostly non-oscillating Ca(2+) response through activation of PAR1, which reinforced the factor Xa response. The additive Ca(2+) signals persisted, when factor X and prothrombin were activated in situ, or in the presence of plasma that was triggered to coagulate with tissue factor. Further, thrombin reinforced the factor Xa-induced production of IL-8, but not of IL-6. Both interleukins were produced in the presence of coagulating plasma. In conclusion, under coagulant conditions, factor Xa and thrombin appear to contribute in different and additive ways to the Ca(2+)-mobilizing and proinflammatory reactions of endothelial cells. These data provide first evidence that these serine proteases trigger distinct signaling modules in endothelium that is activated by plasma coagulation.     

Preparation, characterization, and the crystal structure of the inhibitor ZK-807834 (CI-1031) complexed with factor Xa

Factor Xa plays a critical role in the formation of blood clots. This serine protease catalyzes the conversion of prothrombin to thrombin, the first joint step that links the intrinsic and extrinsic coagulation pathways. There is considerable interest in the development of factor Xa inhibitors for the intervention in thrombic diseases. This paper presents the structure of the inhibitor ZK-807834, also known as CI-1031, bound to factor Xa and provides the details of the protein purification and crystallization. Results from mass spectrometry indicate that the factor Xa underwent autolysis during crystallization and the first EGF-like domain was cleaved from the protein. The crystal structure of the complex shows that the amidine of ZK-807834 forms a salt bridge with Asp189 in the S1 pocket and the basic imidazoline fits snugly into the S4 site. The central pyridine ring provides a fairly rigid linker between these groups. This rigidity helps minimize entropic losses during binding. In addition, the structure reveals new interactions that were not found in the previous factor Xa/inhibitor complexes. ZK-807834 forms a strong hydrogen bond between an ionized 2-hydroxy group and Ser195 of factor Xa. There is also an aromatic ring-stacking interaction between the inhibitor and Trp215 in the S4 pocket. These interactions contribute to both the potency of this compound (K(I) = 0.11 nM) and the >2500-fold selectivity against homologous serine proteases such as trypsin.     

Probing the molecular basis of factor Xa specificity by mutagenesis of the serpin, antithrombin.

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa approximately 3-4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (approximately 2-3x10(2) M(-1)s(-1)). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300-500-fold. A similar 10(4)-10(5)-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4-P4' residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4-P4' binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.     

The design of phenylglycine containing benzamidine carboxamides as potent and selective inhibitors of factor Xa

Factor Xa, a critical serine protease in the blood coagulation cascade, has become a target for inhibition as a strategy for the invention of novel anti-thrombotic agents. Here we describe the development of phenylglycine containing benzamidine carboxamides as novel, potent and selective inhibitors of factor Xa.     

Increase of anti-metastatic efficacy by selectivity- but not affinity-optimization of synthetic serine protease inhibitors

Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model. This in vivo screening revealed efficacy of urokinase inhibitors but no correlation between urokinase selectivity or affinity and anti-metastatic effect. In contrast, factor Xa-selective inhibitors were more potent, demonstrating factor Xa or a factor Xa-like serine protease likely to be more determinant in this model. Factor Xa selectivity, but not affinity, significantly improved anti-metastatic efficacy. For example, factor Xa inhibitors CJ-504 and CJ-510 exert similar affinity for factor Xa (K(i)=14 nM versus 8.8 nM) but CJ-504 was 70-fold more selective for factor Xa. This correlated with higher anti-metastatic efficacy (58.8% with CJ-504; 28.2% with CJ-510). Our results show that among the protease inhibitors employed that have affinities in the nanomolar range, the strategy of selectivity-optimization is superior to further improvement of affinity to significantly enhance anti-metastatic efficacy. This appreciation may be important for the future rational design of new anti-proteolytic agents for cancer therapy.     

Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity

In order to investigate issues of selectivity and specificity in protein-ligand interactions, we have undertaken the reconstruction of the binding pocket of human factor Xa in the structurally related rat trypsin by site-directed mutagenesis. Three sequential regions (the "99"-, the "175"- and the "190"- loops) were selected as representing the major structural differences between the ligand binding sites of the two enzymes. Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were expressed in yeast, and analysed for their interaction with factor Xa and trypsin inhibitors. For most of the inhibitors studied, progressive loop replacement at the trypsin surface resulted in inhibitory profiles akin to factor Xa. Crystals of the variants were obtained in the presence of benzamidine (3), and could be soaked with the highly specific factor Xa inhibitor (1). Binding of the latter to X99rT results in a series of structural adaptations to the ligand, including the establishment of an "aromatic box" characteristic of factor Xa. In X(99/175/190)rT, introduction of the 175-loop results in a surprising re-orientation of the "intermediate helix", otherwise common to trypsin and factor Xa. The re-orientation is accompanied by an isomerisation of the Cys168-Cys182 disulphide bond, and burial of the critical Phe174 side-chain. In the presence of (1), a major re-organisation of the binding site takes place to yield a geometry identical to that of factor Xa. In all, binding of (1) to trypsin and its variants results in significant structural rearrangements, inducing a binding surface strongly reminiscent of factor Xa, against which the inhibitor was optimised. The structural data reveal a plasticity of the intermediate helix, which has been implicated in the functional cofactor dependency of many trypsin-like serine proteinases. This approach of grafting loops onto scaffolds of known related structures may serve to bridge the gap between structural genomics and drug design.     

Nonbenzamidine compounds as selective factor Xa inhibitors

Nonbenzamidine compounds (imidazole, pyridine, pyrimidine, and thiazole derivatives) as selective serine protease factor Xa inhibitors are discussed.     

Three-dimensional solution structure of <Emphasis Type="Italic">Tropidechis carinatus</Emphasis> venom extract trocarin: a structural homologue of Xa and prothrombin activator

Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we report a predicted complete three-dimensional all-atom structural model of trocarin equilibrated in explicit water using 4 ns of molecular dynamics simulation. The tertiary structure was modeled using the structure of human blood coagulation factor Xa. The conformational and structural features of trocarin are then compared with the X-ray crystal and solution simulation structures of human factor Xa. The modeled structure of trocarin has four individual domains (Gla, EGF1, EGF2 and SP) connected along the long axis with similar secondary structural elements to Xa. The simulations suggest that sodium ion binding in the serine protease domain is impaired in trocarin as compared to Xa. In contrast to Xa, for which the sodium ion forms an octahedral coordination network that brings two loop regions connecting four anti-parallel beta-sheets together, we do not find a similar pattern of network in trocarin. We observe that the difference in the binding pattern of sodium ion leads to a approximately 2-A "shrinkage" of the beta2 strand (B2), in comparison to human Xa, as marked by a shorter distance between 189Asp373 (S1-site residue) and 195Ser379 (active-site residue) in the B2 strand. We propose that these differences may be linked to the experimentally observed lower amidolytic activity of trocarin as compared to Xa.     

Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin.

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.     

Receptors of the PAR Family as a Link between Blood Coagulation and Inflammation

Blood coagulation plays a key role among numerous mediating systems that are activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation.Activation of PAR-1 by thrombin and of PAR-2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. Certain other receptors (EPR-1, thrombomodulin, etc.), which can modulate responses of the cells activated by proteinases through PAR receptors, are also involved in the association of coagulation and inflammation together with the receptors of the PAR family. The presence of PAR receptors on mast cells is responsible for their reactivity to thrombin and factor Xa and defines their contribution to the association of inflammation and blood clotting processes.     

Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.     

The gamma-carboxyglutamic acid and epidermal growth factor-like domains of factor X. Effect of isolated domains on prothrombin activation and endothelial cell binding of factor X

Factor Xa is the enzymatically active constituent of the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin. We have isolated fragments, from tryptic digests of factor X, that consists of the gamma-carboxyglutamic acid (Gla) region linked to one or two epidermal growth factor (EGF)-like domains. Calcium ion binding measurements indicated that these fragments have a native conformation. The factor X-GlaEGF fragments inhibit factor Xa-induced blood clotting in a manner suggesting that they compete with factor Xa for phospholipid binding sites. The same conclusion was reached when thrombin generation was studied in a system of purified components (factor Xa, factor Va, prothrombin, phospholipid, and Ca2+). There was no evidence for a strong interaction between the EGF-like domains of factor Xa and factor Va in either system. However, experiments in the purified system without phospholipid indicated a direct, albeit weak, interaction between the Gla region of factor Xa and factor Va and between the COOH-terminal EGF-like domain of factor Xa and factor Va. Using domain-specific Fab fragments, we have confirmed that the conformation of the serine protease region alters dramatically upon activation of factor X. Furthermore, we have demonstrated that the conformation of the Gla region is affected by the activation, whereas the EGF-like domains appear to be unaltered. The association constant for factor X binding to endothelial cells was two orders of magnitude lower than that for binding of factor IX to these cells. Binding of the Gla and GlaEGF fragments suggested Gla-mediated binding to phospholipid rather than binding to a specific receptor.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to human and bovine factor Xa. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: Ka = 2.1 x 10(5)M-1 (at 21.0 degrees C), delta G degree = -29.7 kJ/mol (at 21.0 degrees C), delta S degree = +161 entropy units (at 21.0 degrees C), and delta H degree = +17.6 kJ/mol (temperature-independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

A four component coupling strategy for the synthesis of D-phenylglycinamide-derived non-covalent factor Xa inhibitors

A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.     

Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419).

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and gamma-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 x 10(-10) M). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.     

The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa.

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.     

Comparative study of the active site caging of serine proteases: thrombin and factor Xa

Bovine thrombin and human factor Xa were acylated at their active site selectively with inhibitors derived from the parent compound 4-guanidinophenyl (E)-4-diethylamino-2-hydroxy-alpha-methylcinnamate hydrochloride, 1b. Peptidyl side chains were attached to the phenol ring via amide connection, which served as a recognition motif in inhibiting different serine proteases. Upon irradiation with 366 nm light, the trans-cinnamate attached to the active-site serine isomerizes to the cis isomer which then rapidly lactonizes to release the free enzyme. The peptidyl side chain sequences specific for each serine protease were revealed via constructing and screening a library of homologous compounds. This methodology may be applied to other proteases. One application based on enzyme-specific, photoactivatable inhibitors is to isolate a designated active protease from a mixture of several proteases. Thus, a cinnamate inhibitor with a biotin moiety, 1d, was synthesized. A solution of enzyme-specific, biotinylated inhibitor was added into a mixture of proteases containing a target enzyme. The target enzyme was acylated at the active site and subsequently bore a biotin tail. An avidin column was used to separate the biotinylated enzyme from the unmodified ones, by a strong binding between biotin and avidin. After a brief irradiation on the avidin column, the retained enzymes were released from the biotin tag and eluted off the column. To demonstrate the idea, thrombin and factor Xa have been separated from each other by this strategy.