Crystal structures of two potent nonamidine inhibitors bound to factor Xa

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.     

Design,synthesis, and structure-activity relationships of unsubstituted piperazinone-based transition state factor Xa inhibitors

A series of novel transition state factor Xa inhibitors containing a variety of lactam ring systems as central templates was synthesized in an expedient manner and allowed for a great deal of structural variability. Among them, the piperazinone-based inhibitors were found to be not only active against factor Xa but also selective over thrombin. Optimization of the P4 moiety yielded several potent compounds with IC(50) below 1 nM against factor Xa.     

Aminobenzisoxazoles with biaryl P4 moieties as potent, selective, and orally bioavailable factor Xa inhibitors

We have previously reported on a series of aminobenzisoxazoles as potent, selective, and orally bioavailable factor Xa inhibitors, which culminated in the discovery of razaxaban. Herein, we describe another approach to improve factor Xa inhibitory potency and pharmacokinetic profile by incorporating basic and water soluble functionalities on the terminal ring of the P4 biaryl group found in our earlier Xa inhibitors. This approach resulted in a series of potent, selective, and orally bioavailable factor Xa inhibitors.     

Sequential receptor cascade for coagulation proteins on monocytes. Constitutive biosynthesis and functional prothrombinase activity of a membrane form of factor V/Va

Cells of monocytic differentiation can promote proteolytic activation of factor X following binding to the adhesive receptor Mac-1. We now show that the product, factor Xa, binds to a second receptor on these cells in a Ca2+-dependent reaction. Functionally, this results in the capacity to convert prothrombin to thrombin. The factor Xa receptor was identified by monoclonal antibody (7G12) reactive with plasma factor V/Va, but selected for reactivity with THP-1 cells. It reacted with 71.2 +/- 10.1% of monocytes, bound 153,600 +/- 33,500 sites/THP-1 cell, blocked binding of 125I-factor Xa, inhibited formation of thrombin, and immunoprecipitated 125I-factor Xa chemically cross-linked to its receptor on THP-1 cells. Following surface iodination or intrinsic labeling of THP-1 cells, antibody 7G12 immunoprecipitated a 74-kDa molecular species, similar to plasma factor Va light chain. Thus, monocytes and monocyte-like cells synthesize and express a factor V/Va-like receptor for factor Xa and organize a functional prothrombinase complex. The simultaneous membrane coexpression of a factor X receptor (Mac-1) and a factor Xa receptor as demonstrated by two-color flow cytofluorometric analysis of monocytes or THP-1 cells is consistent with a sequential receptor cascade for coordinated molecular assembly of coagulation proteins on specialized cells.     

Co-crystal structure and inhibition of factor Xa by PD0313052 identifies structurally stabilized active site residues of factor Xa and prothrombinase

The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.     

Orally active zwitterionic factor Xa inhibitors with long duration of action

We have optimized 2-aminomethylphenylamine derivative as a factor Xa inhibitor. Several polar functional groups were introduced in the central phenyl ring, and we focused on zwitter ionic compound showing continuous inhibitory activity in oral administration test. In vitro and oral activities were improved by optimization of S1 and S4 ligands. Incorporating the interaction with S1-β pocket enhanced in vitro factor Xa inhibitory activity to less than 1 nM. Many zwitter ionic compounds showed long duration of action and potent inhibitory activity and high AUC values in oral administration tests to monkeys.     

Neutral inhibitors of the serine protease factor Xa.

A neutral inhibitor of the serine protease factor Xa was identified via a high-throughput screen of a commercial library. The initial lead 1 demonstrated reversible and competitive inhibition kinetics for factor Xa and possessed a high degree of selectivity versus other related serine proteases. Initial modeling efforts and the generation of a series of analogues of 1 are described.     

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.     

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.     

Inhibition of ADP-induced platelet aggregation by reduced factor Xa

Treatment of blood coagulation factor Xa with insolubilized hexyl-agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes. Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.     

Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.     

Novel factor Xa inhibitors based on a 2-carboxyindole scaffold: SAR of P4 substituents in combination with a neutral P1 ligand

A series of novel, highly potent 2-carboxyindole-based factor Xa inhibitors is described. Structural requirements for P4 ligands in combination with a neutral biaryl P1 ligand were investigated with the 2-carboxyindole scaffold. A diverse set of P4 substituents was identified, which, in conjunction with a biaryl P1 ligand, gave highly potent factor Xa inhibitors, which were also selective versus other proteases and efficacious in various antithrombotic secondary assays.     

Solid-phase optimisation of achiral amidinobenzyl indoles as potent and selective factor Xa inhibitors

Starting from the achiral and potent factor Xa inhibitor 1, a new and flexible solid-phase optimisation strategy is described to reduce its cationic character. By replacing one positively charged side chain by a lipophilic substituent, a novel series of highly potent and selective achiral factor Xa inhibitors was discovered. The identified lipophilic replacements in the S4 pocket might be valuable for other approaches towards fXa inhibitors.     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

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.     

Substituted acrylamides as factor Xa inhibitors: improving bioavailability by P1 modification

To overcome the low bioavailability of our substituted acrylamide P1 benzamidine factor Xa inhibitors reported previously, neutral and less basic groups were used to replace the benzamidine. As a result, a series of P1 aminoisoquinoline substituted acrylamide Xa inhibitors was identified to be potent, selective, and orally bioavailable. Modification of P4 moiety of these compounds further improved their pharmacokinetic properties.     

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR1). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR1-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR1-specific agonists and inhibitors were used to demonstrate that PAR1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR1 and not PAR2. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Human umbilical vein endothelial cells express high affinity receptors for factor Xa

The binding of [¹²⁵I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7°C, [¹²⁵I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 ± 0.8 nM and a binding site density of 57,460 ± 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 0.15 × 10⁶ M^-1 s^-1 and 4.0 × 10^-4 s^-1, respectively. [¹²⁵I]-factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin-III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor-1 (EPR-1), a well-known receptor of factor Xa on various cell types. [¹²⁵I]-factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide-antithrombin-III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC-bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR-1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall. J. Cell. Physiol. 172:36–43, 1997. © 1997 Wiley-Liss, Inc.     

The tissue factor region that interacts with factor Xa in the activation of factor VII

Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.