Identification of AcAP5 as a novel factor Xa inhibitor with both direct and allosteric inhibition.

Ancylostoma caninum anticoagulant peptide 5 (AcAP5) is a potent inhibitor for coagulation factor Xa (FXa). Previous studies show that AcAP5 binds to FXa at the active site, and/or the exosite. The active site-binding contributes to direct blocking of FXa catalytic activity, but the effect of exosite-binding and the underlying mechanism remain unknown. To investigate whether and how the exosite-binding affects FXa function, we prepared several AcAP5 mutants with modifications to the active site-binding or exosite-binding region. Their FXa-inhibiting and anticoagulant activities were examined both in vitro and in rabbit plasma, and the interactions with FXa were analyzed using in silico molecular modeling, docking, and molecular dynamics simulation. Mutants abolishing either active site- or exosite-binding resulted in a dramatic decrease in their anti-FXa and anticoagulant activities. Elongation of AcAP5 exosite-binding region also impaired the FXa-inhibiting activity. Computational analysis demonstrated that the conformation of FXa becomes more rigid due to exosite-binding with AcAP5, which consequently affects its catalytic activity. Our results suggest that both active site- and exosite-binding contribute to the FXa inhibitory activity of AcAP5.

Poor comparability of coagulation screening test with specific measurement in patients receiving direct oral anticoagulants: results from a multicenter/multiplatform study.

Essentials Prothrombin and partial thromboplastin time (PT/PTT) measure direct oral anticoagulants (DOACs). PT, PTT and specific tests for DOACs were performed on patients treated for atrial fibrillation. Normal PT/PTT don't exclude DOAC activity and their prolongation doesn't confirm DOAC action. The use of PT or PTT to evaluate DOAC activity could cause dangerous misinterpretations. SUMMARY: Background Prothrombin time (PT) and activated partial thromboplastin time (APTT) have been proposed to measure the effect of oral anti-activated factor X (FXa) or anti-activated FII drugs, respectively. Aims To evaluate the relationships and responsiveness of PT and APTT versus direct oral anticoagulant (DOAC) concentrations measured with specific coagulation tests performed with different platforms in four Italian anticoagulation clinics. Methods Six hundred and thirty-five patients with atrial fibrillation participated in the study: 240 were receiving dabigatran, 264 were receiving rivaroxaban, and 131 were receiving apixaban. Blood was taken at trough and peak within the first month (15-25 days) of treatment. PT, APTT, diluted thrombin time (dTT) calibrated for dabigatran and anti-FXa calibrated for rivaroxaban or apixaban were determined. Results For dabigatran, the correlation between APTT and dTT ranged from r = 0.80 to r = 0.62. For rivaroxaban, the correlation between the anti-FXa assay and PT ranged from r = 0.91 to r = 0.73. For apixaban, the correlation between the anti-FXa assay and PT was lower than for the two other drugs (r = 0.81 to r = 0.54). Despite the above significant correlations, the responsiveness of PT or APTT was relatively poor. A discrepancy between global testing and DOAC plasma concentrations was shown in a considerable proportion of patients, depending on the platform and drug, with values ranging from 6% to 62%. Conclusions Overall, poor responsiveness of the screening tests to DOAC concentrations was observed. PT and APTT normal values cannot exclude DOAC anticoagulant activity, and PT or APTT prolongation is not always associated with DOAC anticoagulant effect as determined with specific tests.

Rivaroxaban limits complement activation compared with warfarin in antiphospholipid syndrome patients with venous thromboembolism.

Essentials Complement activation has a pathogenic role in thrombotic antiphospholipid syndrome (APS). Coagulation proteases such as factor Xa can activate complement proteins. Complement activation markers were elevated in anticoagulated thrombotic APS patients. Complement activation decreased in APS patients switching from warfarin to rivaroxaban. SUMMARY: Background Complement activation may play a major role in the pathogenesis of thrombotic antiphospholipid syndrome (APS). Coagulation proteases such as factor Xa can activate complement proteins. Aims To establish whether rivaroxaban, a direct factor Xa inhibitor, limits complement activation compared with warfarin in APS patients with previous venous thromboembolism (VTE). Methods A total of 111 APS patients with previous VTE, on warfarin target INR 2.5, had blood samples taken at baseline and at day 42 after randomization in the RAPS (Rivaroxaban in Antiphospholipid Syndrome) trial. Fifty-six patients remained on warfarin and 55 switched to rivaroxaban. Fifty-five normal controls (NC) were also studied. Markers of complement activation (C3a, C5a, terminal complement complex [SC5b-9] and Bb fragment) were assessed. Results APS patients had significantly higher complement activation markers compared with NC at both time-points irrespective of the anticoagulant. There were no differences between the two patient groups at baseline, or patients remaining on warfarin at day 42. In 55 patients randomized to rivaroxaban, C3a, C5a and SC5b-9 were lower at day 42 (median (ng mL-1 ) [confidence interval] 64 [29-125] vs. 83 [35-147], 9 [2-15] vs. 12 [4-18] and 171 [56-245] vs. 201 [66-350], respectively) but levels of Bb fragment were unchanged. There were no correlations between rivaroxaban levels and complement activation markers. Conclusions APS patients with previous VTE on warfarin exhibit increased complement activation, which is likely to occur via the classical pathway and is decreased by rivaroxaban administration. Rivaroxaban may therefore potentially provide an additional benefit to its anticoagulant effect in this patient group by limiting complement activation.

Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity-Based Probes of Matriptase-2.

Matriptase-2, a type II transmembrane serine protease, plays a key role in human iron homeostasis. Inhibition of matriptase-2 is considered as an attractive strategy for the treatment of iron-overload diseases, such as hemochromatosis and ß-thalassemia. In the present study, synthetic routes to nine dipeptidomimetic inactivators were developed. Five active compounds (41-45) were identified and characterized kinetically as irreversible inhibitors of matriptase-2. In addition to a phosphonate warhead, these dipeptides possess two benzguanidine moieties as arginine mimetics to provide affinity for matriptase-2 by binding to the S1 and S3/S4 subpockets, respectively. This binding mode was strongly supported by covalent docking analysis. Compounds 41-45 were obtained as mixtures of two diastereomers and were therefore separated into the single epimers. Compound 45 A, with S configuration at the N-terminal amino acid and R configuration at the phosphonate carbon atom, was the most potent matriptase-2 inactivator with a rate constant of inactivation of 2790 m(-1) s(-1) and abolished the activity of membrane-bound matriptase-2 on the surface of intact cells. Based on the chemotyp of phosphono bisbenzguanidines, the design and synthesis of a fluorescent probe (51 A) by insertion of a coumarin label is described. The in-gel fluorescence detection of matriptase-2 was demonstrated by applying 51 A as the first activity-based probe for this enzyme.

Docking and Scoring with Target-Specific Pose Classifier Succeeds in Native-Like Pose Identification But Not Binding Affinity Prediction in the CSAR 2014 Benchmark Exercise.

The CSAR 2014 exercise provided an important benchmark for testing current approaches for pose identification and ligand ranking using three X-ray characterized proteins: Factor Xa (FXa), Spleen Tyrosine Kinase (SYK), and tRNA Methyltransferase (TRMD). In Phase 1 of the exercise, we employed Glide and MedusaDock docking software, both individually and in combination, with the special target-specific pose classifier trained to discriminate native-like from decoy poses. All approaches succeeded in the accurate detection of native and native-like poses. We then used Glide SP and MedusaScore scoring functions individually and in combination with the pose-scoring approach to predict relative binding affinities of the congeneric series of ligands in Phase 2 of the exercise. Similar to other participants in the CSAR 2014 exercise, we found that our models showed modest prediction accuracy. Quantitative structure-activity relationship (QSAR) models developed for the FXa ligands using available bioactivity data from ChEMBL showed relatively low prediction accuracy for the CSAR 2014 ligands of the same target. Interestingly, QSAR models built with CSAR data only yielded Spearman correlation coefficients as high as ρ = 0.69 for FXa and ρ = 0.79 for SYK based on 5-fold cross-validation. Virtual screening of the DUD library using the FXa structure was successful in discriminating between active compounds and decoys in spite of poor ranking accuracy of the underlying scoring functions. Our results suggest that two of the three common tasks associated with molecular docking, i.e., native-like pose identification and virtual screening, but not binding affinity prediction, could be accomplished successfully for the CSAR 2014 challenge data set.

Thymoquinone Modulates Blood Coagulation in Vitro via Its Effects on Inflammatory and Coagulation Pathways.

Thymoquinone (THQ) is a major component of black seeds. Given that both THQ and black seeds exhibit anti-cancer and anti-inflammatory activities, we hypothesized that THQ will affect cancer-associated thrombosis (CAT), which is primarily triggered by tissue factor (TF) and inflammation. The effect of both black seed-extracted and purchased ("pure") THQ on normal blood coagulation was tested with in vitro thromboelastography (TEG) and activated partial thromboplastin time (aPTT) coagulation assays. The effect of pure THQ on CAT was tested with aPTT assay using pancreatic cancer cell lines that are either positive or negative for TF, and with TEG assay using lipopolysaccharide as an inflammatory trigger. Additionally, the direct effect of THQ on the inactivation of factors IIa and Xa was assessed. Since TNF-α facilitates crosstalk between inflammation and thrombosis by triggering the NF-κB pathway, we tested THQ's ability to interfere with this communication with a luciferase assay. Both extracted and pure THQ had minimal effects on normal blood coagulation. Pure THQ reversed CAT initiated by both TF and inflammation to basal levels (p < 0.001). Mechanistically, while THQ had minimal to no effect on factor IIa and Xa inactivation, it strongly reduced the effects of TNF-α on NF-κB elements (p < 0.001). THQ has a minimal effect on basal coagulation and can reverse CAT in vitro, possibly by interfering with the crosstalk between inflammation and coagulation. This study suggests the utility of THQ as a preventative anticoagulant and/or as a supplement to existing chemotherapies and anticoagulant therapies.

Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease.

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.

Anticoagulation by factor Xa inhibitors.

BACKGROUND: Therapeutic agents that regulate blood coagulation are critical to the management of thrombotic disorders, with the selective targeting of factor (F) Xa emerging as a promising approach. OBJECTIVE: To assess anticoagulant strategies targeting FXa. METHODS: A deterministic computational model of tissue factor (Tf)-initiated thrombin generation and two empirical experimental systems (a synthetic coagulation proteome reconstruction using purified proteins and a whole blood model) were used to evaluate clinically relevant examples of the two available types of FXa-directed anticoagulants [an antithrombin (AT)-dependent agent, fondaparinux, and an AT-independent inhibitor, Rivaroxaban] in experimental regimens relevant to long-term (suppression of new Tf-initiated events) and acute (suppression of ongoing coagulation processes) clinical applications. RESULTS: Computational representations of each anticoagulant's efficacy in suppressing thrombin generation over a range of anticoagulant concentrations in both anticoagulation regimens were validated by results from corresponding empirical reconstructions and were consistent with those recommended for long-term and acute clinical applications, respectively. All three model systems suggested that Rivaroxaban would prove more effective in the suppression of an ongoing coagulation process than fondaparinux, reflecting its much higher reactivity toward the prothrombinase complex. CONCLUSION: The success of fondaparinux in acute settings in vivo is not explained solely by its properties as an FXa inhibitor. We have reported that FIXa contributes to the long-term capacity of clot-associated catalysts to restart a coagulation process, suggesting that the enhanced anti-FIXa activity of fondaparinux-AT may be critical to its success in acute settings in vivo.

Design of novel aminopyrrolidine factor Xa inhibitors from a screening hit.

Starting from a hit identified by focused screening, 3-aminopyrrolidine factor Xa inhibitors were designed. The binding mode as determined by X-ray structural analysis as well as the pharmacokinetic behaviour of selected compounds is discussed.

Expression of human peroxisome proliferator-activated receptors ligand binding domain-maltose binding protein fusion protein in Escherichia coli: a convenient and reliable method for preparing receptor for screening ligands.

Human peroxisome proliferator-activated receptors (hPPARs) are ligand-activated transcription factors and are the target for the treatment of many diseases. Screening of their ligands is mainly based on assays of ligand binding to the ligand binding domain (LBD) of hPPARs.However, such assays are difficult because of the preparation of hPPARs LBD. In order to yield functional hPPARs LBD for screening ligands, hPPARs LBD was fused with maltose-binding protein(MBP) using the pMAL-p2x expression system through the gene engineering technique. The radioligand binding assay showed that MBP did not affect ligand binding with hPPARs LBD in the fusion proteins, which means that MBP-hPPARs LBD can be used instead of hPPARs LBD in ligand screening work. The results show that the new strategy using MBP as a fusion tag for preparing hPPARs LBD for screening ligands is a convenient and reliable method. It may be used to easily obtain the other nuclear receptors.

Rivaroxaban, an oral direct factor Xa inhibitor.

Rivaroxaban is a small molecule, direct Factor Xa inhibitor and may be a potentially attractive alternative to vitamin K antagonists. Rivaroxaban is being investigated for the prevention and treatment of venous and arterial thrombosis. A broad search of Medline, clinicaltrials.gov and the annual proceedings of the American Society of Hematology and the International Society on Thrombosis and Hemostasis was conducted. This review addresses the findings of this systematic search, including the need for new oral anticoagulants, the development and pharmacology of rivaroxaban, and the results of completed as well as ongoing trials with rivaroxaban. At present, the safety and efficacy of rivaroxaban for the prophylaxis and treatment of venous thromboembolism has been evaluated in Phase II and Phase III trials involving over 24,000 patients. Additionally, rivaroxaban is being evaluated for the treatment of pulmonary embolism, secondary prevention after acute coronary syndromes and the prevention of stroke and non-central nervous system embolism in patients with non-valvular atrial fibrillation. The drug may have its greatest impact in providing a much-needed and attractive alternative to warfarin. Further data (especially large Phase III trials) are required.

Fast and efficient in silico 3D screening: toward maximum computational efficiency of pharmacophore-based and shape-based approaches.

In continuation of our recent studies on the quality of conformational models generated with CATALYST and OMEGA we present a large-scale survey focusing on the impact of conformational model quality and several screening parameters on pharmacophore-based and shape-based virtual high throughput screening (vHTS). Therefore, we collected known active compounds of CDK2, p38 MAPK, PPAR-gamma, and factor Xa and built a set of druglike decoys using ilib:diverse. Subsequently, we generated 3D structures using CORINA and also calculated conformational models for all compounds using CAESAR, CATALYST FAST, and OMEGA. A widespread set of 103 structure-based pharmacophore models was developed with LigandScout for virtual screening with CATALYST. The performance of both database search modes (FAST and BEST flexible database search) as well as the fit value calculation procedures (FAST and BEST fit) available in CATALYST were analyzed in terms of their ability to discriminate between active and inactive compounds and in terms of efficiency. Moreover, these results are put in direct comparison to the performance of the shape-based virtual screening platform ROCS. Our results prove that high enrichment rates are not necessarily in conflict with efficient vHTS settings: In most of the experiments, we obtained the highest yield of actives in the hit list when parameter sets for the fastest search algorithm were used.

pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+.

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.

A novel search engine for virtual screening of very large databases.

Virtual screening of large chemical databases using the structure of the receptor can be computationally very demanding. We present a novel strategy that combines exhaustive similarity searches directly in SMILES format with the docking of flexible ligands, whose 3D structure is generated on the fly from the SMILES representation. Our strategy makes use of the recently developed LINGO tools to extract implicit chemical information from SMILES strings and integrates LINGO similarities into a pseudo-evolutionary algorithm. The algorithm represents a combination of a fast target-independent similarity method with a slower but information richer target-focused method. A virtual search of FactorXa ligands provided 62% of the potential hits after docking only 6.5% of a database of nearly 1 million molecules. The set of solutions showed good diversity, indicating that the method shows good scaffold hopping capabilities.

Preparation, crystallization and X-ray diffraction analysis to 1.5 A resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation.

Cysteine dioxygenase (CDO; EC 1.13.11.20) is an approximately 23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O2, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 A resolution and belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 57.55, c = 123.06 A, alpha = beta = gamma = 90 degrees. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

Relative proximity and orientation of helices 4 and 8 of the GLUT1 glucose transporter.

A structure has been proposed for glucose transporter-1 (GLUT1) based upon homology modeling that is consistent with the results of numerous mutagenesis studies (Mueckler, M., and Makepeace, C. (2004) J. Biol. Chem. 279, 10494-10499). To further test and refine this model, the relative orientation and proximity of transmembrane helices 4 and 8 were analyzed by chemical crosslinking of di-cysteine mutants created in a reporter GLUT1 construct. All six native cysteine residues of GLUT1 were changed to either glycine or serine residues by site-directed mutagenesis, resulting in a functional Glut1 construct with Cys mutated to Gly/Ser (C-less). The GLUT1 reporter molecule was engineered from C-less GLUT1 by creating a unique cleavage site for factor Xa protease within the central cytoplasmic loop and by eliminating the site of N-linked glycosylation. Fourteen functional di-cysteine mutants were then created from the C-less reporter construct, each mutant containing a single cysteine residue in helix 4 and one cysteine residue in helix 8. These mutants were expressed in Xenopus oocytes, and the sensitivity of each mutant to intramolecular crosslinking by two homo-bifunctional, thiol-specific crosslinking reagents, bismaleimidehexane and 1,4-phenylenedimaleimide, was ascertained by protease cleavage followed by immunoblot analysis. Four pairs of cysteine residues, Cys(148)/Cys(328), Cys(145)/Cys(328), Cys(148)/Cys(325), and Cys(145)/Cys(325), were observed to be in close enough proximity to be susceptible to crosslinking by one or both reagents. All five of the cysteine residues susceptible to crosslinking are predicted to lie on the same face of helix 4 or 8 and to reside close to the cytoplasmic face of the membrane. These data indicate that the cytoplasmic ends of helices 4 and 8 lie within 6-16 A of one another and that the two helices twist or tilt such that they are further than 16 A apart toward the center and the exoplasmic side of the membrane. An updated model for the clustering of the transmembrane helices of GLUT1 is presented based on these data.

Exploring the active site of human factor Xa protein by NMR screening of small molecule probes.

A collection of small molecules (MW < 350 Da) was screened for binding to human factor Xa using saturation transfer difference NMR spectroscopy to detect binding. The NMR screening experiments identified four hits. Binding isotherms constructed from NMR linewidth data showed that the binding affinities of the hits were all in the 30-210 microM range. Competition binding experiments showed that three of the ligands were displaced by a known microM inhibitor of factor Xa. The success of the method for identifying new ligands and the relevance of this information to the design of new factor Xa inhibitors are discussed.