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Sample records for factor xa inhibitor

  1. Phenyltriazolinones as potent factor Xa inhibitors.

    PubMed

    Quan, Mimi L; Pinto, Donald J P; Rossi, Karen A; Sheriff, Steven; Alexander, Richard S; Amparo, Eugene; Kish, Kevin; Knabb, Robert M; Luettgen, Joseph M; Morin, Paul; Smallwood, Angela; Woerner, Francis J; Wexler, Ruth R

    2010-02-15

    We have discovered that phenyltriazolinone is a novel and potent P1 moiety for coagulation factor Xa. X-ray structures of the inhibitors with a phenyltriazolinone in the P1 position revealed that the side chain of Asp189 has reoriented resulting in a novel S1 binding pocket which is larger in size to accommodate the phenyltriazolinone P1 substrate. PMID:20100660

  2. Apixaban, an oral, direct inhibitor of activated Factor Xa.

    PubMed

    Shantsila, Eduard; Lip, Gregory Y H

    2008-09-01

    Apixaban is an oral, direct Factor Xa inhibitor that is being developed by Bristol-Myers Squibb Co and Pfizer Inc. Apixaban is currently undergoing phase III clinical trials for cerebrovascular ischemia, deep vein thrombosis and lung embolism, and phase II clinical trials for coronary artery disease. PMID:18729009

  3. Apixaban, an oral direct Factor Xa inhibitor: awaiting the verdict.

    PubMed

    Carreiro, Jennifer; Ansell, Jack

    2008-12-01

    For the last half-century, despite its many limitations warfarin has been the mainstay of treatment for patients with venous and arterial thromboembolic disease. During the past decade, a number of new oral anticoagulant agents have been developed that may offer an alternative to warfarin. Emerging data suggest that Factor Xa may be a target for inhibition. Apixaban is one such agent. It is a potent, selective, reversible, and orally bioavailable FXa inhibitor that demonstrates antithrombotic efficacy, with a favorable pharmacokinetic profile. At present, the safety and efficacy of apixaban for the prophylaxis and treatment of venous thromboembolism is being evaluated in Phase II and Phase III trials involving nearly 25,000 patients. Trials are also underway involving over 20,000 patients for secondary prevention after acute coronary syndromes and the prevention of stroke in patients with non-valvular atrial fibrillation. This review article discusses the discovery, pharmacokinetics, attributes, and current clinical trials of this emerging drug. PMID:19012508

  4. Factor Xa inhibitors--new anticoagulants for secondary haemostasis.

    PubMed

    Perzborn, E

    2009-08-01

    Oral factor Xa (FXa) inhibitors are a promising alternative to current anticoagulants. This paper reviews the latest developments of oral direct FXa inhibitors and focuses on those which have been approved for the prevention of venous thromboembolism (VTE) after total hip or knee replacement or are in advanced development and have passed phase II (proof of principle) testing. The most advanced drugs are apixaban, betrixaban, edoxaban, eribaxaban, rivaroxaban, LY517717, TAK-442, and YM150. Rivaroxaban (Xareltoâ) is the first direct FXa inhibitor which has recently been approved for the prevention of VTE in adult patients after elective hip or knee replacement in several countries, including the European Union and Canada. Rivaroxaban has a flat dose-dependent anticoagulant response with a wide therapeutic window and low potential for drug-drug and drug-food interactions. Rivaroxaban can be given in fixed doses without coagulation monitoring. This review describes the pharmacodynamic and pharmacokinetic profiles and the results of clinical trials with FXa inhibitors in the prevention and treatment of thromboembolic disorders. PMID:19644596

  5. Reversal Strategies for Intracranial Hemorrhages in Patients Taking Oral Factor Xa Inhibitors.

    PubMed

    Karli, Betsy; Bartel, Billie; Pavelko, Rachel

    2015-07-01

    Factor Xa (fXa) inhibitors are becoming more common in clinical practice due to a variety of reasons. Unfortunately, limited data are currently available on the safe and efficacious reversal of these agents. This series presents 3 patient cases of intracranial hemorrhage and illustrates the observed effect of different methodologies undertaken in an attempt to reverse the fXa inhibitors implicated. Additionally, a brief review of the current available literature in reversal strategies is provided. The appropriate reversal for fXa inhibitors at this time is unknown. The cases described indicate that the administration of fresh frozen plasma and 4-factor prothrombin complex concentrate may provide minimal benefit in reversing the coagulation abnormalities caused by fXa inhibitors. However, in a life-threatening situation, the addition of these agents should be considered to prevent further progression of the bleed. PMID:26448667

  6. Reversal Strategies for Intracranial Hemorrhages in Patients Taking Oral Factor Xa Inhibitors

    PubMed Central

    Bartel, Billie; Pavelko, Rachel

    2015-01-01

    Factor Xa (fXa) inhibitors are becoming more common in clinical practice due to a variety of reasons. Unfortunately, limited data are currently available on the safe and efficacious reversal of these agents. This series presents 3 patient cases of intracranial hemorrhage and illustrates the observed effect of different methodologies undertaken in an attempt to reverse the fXa inhibitors implicated. Additionally, a brief review of the current available literature in reversal strategies is provided. The appropriate reversal for fXa inhibitors at this time is unknown. The cases described indicate that the administration of fresh frozen plasma and 4-factor prothrombin complex concentrate may provide minimal benefit in reversing the coagulation abnormalities caused by fXa inhibitors. However, in a life-threatening situation, the addition of these agents should be considered to prevent further progression of the bleed. PMID:26448667

  7. Current and future prospects for anticoagulant therapy: inhibitors of factor Xa and factor IIa.

    PubMed

    Harenberg, Job; Wehling, Martin

    2008-02-01

    Indirect systemic and direct oral factor Xa and direct oral factor IIa inhibitors with improved pharmacologic profiles compared with heparins and vitamin K antagonists are currently in clinical development. This overview focuses on the indirect antithrombin dependent pentasaccharide derivatives of idraparinux and on the most advanced oral direct inhibitors to factor Xa (rivaroxaban and apixaban) and IIa (dabigatran). Specifically, the results of dose-finding studies for the prevention of venous thromboembolism after elective orthopedic surgery, the results of dose-finding studies for treatment of acute venous thromboembolism including prolonged prophylaxis of recurrent events, and the designs of ongoing clinical trials are reviewed. PMID:18393142

  8. Application of Molecular Modeling to Development of New Factor Xa Inhibitors

    PubMed Central

    Sulimov, Vladimir B.; Gribkova, Irina V.; Kochugaeva, Maria P.; Katkova, Ekaterina V.; Sulimov, Alexey V.; Kutov, Danil C.; Shikhaliev, Khidmet S.; Medvedeva, Svetlana M.; Krysin, Michael Yu.; Sinauridze, Elena I.; Ataullakhanov, Fazoil I.

    2015-01-01

    In consequence of the key role of factor Xa in the clotting cascade and absence of its activity in the processes that do not affect coagulation, this protein is an attractive target for development of new blood coagulation inhibitors. Factor Xa is more effective and convenient target for creation of anticoagulants than thrombin, inhibition of which may cause some side effects. This study is aimed at finding new inhibitors of factor Xa by molecular computer modeling including docking SOL and postdocking optimization DISCORE programs. After validation of molecular modeling methods on well-known factor Xa inhibitors the virtual screening of NCI Diversity and Voronezh State University databases of ready-made low molecular weight species has been carried out. Seventeen compounds selected on the basis of modeling results have been tested experimentally in vitro. It has been found that 12 of them showed activity against factor Xa (IC50 = 1.8–40 μM). Based on analysis of the results, the new original compound was synthesized and experimentally verified. It shows activity against factor Xa with IC50 value of 0.7 μM. PMID:26484350

  9. Reversal of oral factor Xa inhibitors by prothrombin complex concentrates: a re-appraisal.

    PubMed

    Dzik, W H

    2015-06-01

    Oral factor Xa inhibitors are an attractive class of anticoagulants expected to have broad application. Rapid and reliable reversal of the anticoagulant effect is important for patients with bleeding complications or those in need of urgent reversal for procedures. While no specific reversal agent is yet available, multiple published clinical guidelines suggest that four-factor prothrombin complex concentrates (PCC) should be considered when urgent reversal is desired. This presentation updates prior reviews on this topic (Crit Care, 17, 2013, 230; Thromb Haemost, 111, 2014, 189; J Thromb Thrombolysis, 2015, 39, 395); and summarizes more recent evidence in human studies indicating that four-factor PCCs available in North America do not reverse oral factor Xa-inhibitor anticoagulants. New agents on the horizon appear to be far more promising as therapies for reversal or oral factor Xa inhibitors. PMID:26149022

  10. Identification of anthranilamide derivatives as potential factor Xa inhibitors: drug design, synthesis and biological evaluation.

    PubMed

    Xing, Junhao; Yang, Lingyun; Li, Hui; Li, Qing; Zhao, Leilei; Wang, Xinning; Zhang, Yuan; Zhou, Muxing; Zhou, Jinpei; Zhang, Huibin

    2015-05-01

    The coagulation enzyme factor Xa (fXa) plays a crucial role in the blood coagulation cascade. In this study, three-dimensional fragment based drug design (FBDD) combined with structure-based pharmacophore (SBP) model and structural consensus docking were employed to identify novel fXa inhibitors. After a multi-stage virtual screening (VS) workflow, two hit compounds 3780 and 319 having persistent high performance were identified. Then, these two hit compounds and several analogs were synthesized and screened for in-vitro inhibition of fXa. The experimental data showed that most of the designed compounds displayed significant in vitro potency against fXa. Among them, compound 9b displayed the greatest in vitro potency against fXa with the IC50 value of 23 nM and excellent selectivity versus thrombin (IC50 = 40 μM). Moreover, the prolongation of the prothrombin time (PT) was measured for compound 9b to evaluate its in vitro anticoagulant activity. As a result, compound 9b exhibited pronounced anticoagulant activity with the 2 × PT value of 8.7 μM. PMID:25839438

  11. The role of structural information in the discovery of direct thrombin and factor Xa inhibitors.

    PubMed

    Nar, Herbert

    2012-05-01

    The quest for novel medications to treat thromboembolic disorders such as venous thrombosis, pulmonary embolism and stroke received a boost when the 3D structures of two major players in the blood coagulation cascade were determined in 1989 and 1993. Structure-guided design of inhibitors of thrombin (factor IIa, fIIa) and factor Xa (fXa) eventually led to the discovery of potent, selective, efficacious, orally active and safe compounds that proved successful in clinical studies. In 2008, the direct thrombin inhibitor dabigatran etexilate developed by Boehringer Ingelheim became the first novel antithrombotic molecular entity to enter the market in 50 years. Additional compounds targeting factor Xa were subsequently granted marketing authorization or are in late-stage clinical studies. In this review, I use selected case studies to describe the discovery of novel fIIa and fXa inhibitors, with a particular emphasis on the pre-eminent role that structural information played in this process. PMID:22503439

  12. Pharmacophore identification, in silico screening, and virtual library design for inhibitors of the human factor Xa.

    PubMed

    Krovat, Eva M; Frühwirth, Karin H; Langer, Thierry

    2005-01-01

    Factor Xa inhibitors are innovative anticoagulant agents that provide a better safety/efficacy profile compared to other anticoagulative drugs. A chemical feature-based modeling approach was applied to identify crucial pharmacophore patterns from 3D crystal structures of inhibitors bound to human factor Xa (Pdb entries 1fjs, 1kns, 1eqz) using the software LIGANDSCOUT and CATALYST. The complex structures were selected regarding the criteria of high inhibitory potency (i.e. all ligands show K(i) values against factor Xa in the subnanomolar range) and good resolution (i.e. at least 2.2 A) in order to generate selective and high quality pharmacophore models. The resulting chemical-feature based hypotheses were used for virtual screening of commercial molecular databases such as the WDI database. Furthermore, a ligand-based molecular modeling approach was performed to obtain common-feature hypotheses that represent the relevant chemical interactions between 10 bioactive factor Xa inhibitors and the protein, respectively. In a next step a virtual combinatorial library was designed in order to generate new compounds with similar chemical and spatial properties as known inhibitors. The software tool ILIB DIVERSE was used for this procedure in order to provide new scaffolds of this group of anticoagulants. Finally we present the combination of these two techniques, hence virtual screening was performed with selective pharmacophore models in a focused virtual combinatorial database. De novo derived molecular scaffolds that were able to adequately satisfy the pharmacophore criteria are revealed and are promising templates for candidates for further development. PMID:15667140

  13. Synthesis of 3,4-diaminobenzoyl derivatives as factor Xa inhibitors.

    PubMed

    Yang, Jiabin; Su, Guoqiang; Ren, Yu; Chen, Yang

    2015-08-28

    The coagulation factor Xa (FXa) plays a central role in the blood coagulation cascade. Recent studies have shown that FXa is a particularly attractive target for the development of oral antithrombotic agents. In view of the excellent pharmaceutical properties of 1,2-phenylenediamine-based FXa inhibitors and the reported structure-activity relationship (SAR) analysis of FXa inhibitors, we designed and synthesized a series of 3,4-diaminobenzoyl-based FXa inhibitors. Intensive SAR studies on this new series led to the discovery of 3,4-dimethoxyl substituted compound 7b. 7b is a highly potent, selective, direct FXa inhibitor with excellent in vivo antithrombotic activity. PMID:26114810

  14. Structure-based drug design of pyrrolidine-1, 2-dicarboxamides as a novel series of orally bioavailable factor Xa inhibitors.

    PubMed

    Van Huis, Chad A; Bigge, Christopher F; Casimiro-Garcia, Agustin; Cody, Wayne L; Dudley, Danette A; Filipski, Kevin J; Heemstra, Ronald J; Kohrt, Jeffrey T; Narasimhan, Lakshmi S; Schaum, Robert P; Zhang, Erli; Bryant, John W; Haarer, Staci; Janiczek, Nancy; Leadley, Robert J; McClanahan, Thomas; Thomas Peterson, J; Welch, Kathleen M; Edmunds, Jeremy J

    2007-06-01

    A novel series of pyrrolidine-1,2-dicarboxamides was discovered as factor Xa inhibitors using structure-based drug design. This series consisted of a neutral 4-chlorophenylurea P1, a biphenylsulfonamide P4 and a D-proline scaffold (1, IC(50) = 18 nM). Optimization of the initial hit resulted in an orally bioavailable, subnanomolar inhibitor of factor Xa (13, IC(50) = 0.38 nM), which was shown to be efficacious in a canine electrolytic model of thrombosis with minimal bleeding. PMID:17581239

  15. [Successful treatment of venous thromboembolism with a Factor Xa inhibitor, edoxaban, in patients with lenalidomide-treated multiple myeloma].

    PubMed

    Kawaguchi, Masato; Uchimura, Norio; Okuda, Yuko; Konuma, Satomi; Nehashi, Yoshio

    2015-08-01

    Two multiple myeloma (MM) patients developed venous thromboembolism (VTE) while being treated with lenalidomide and low-dose dexamethasone. Aspirin is recommended for VTE prophylaxis when using lenalidomide/dexamethasone for MM patients with a standard risk of VTE. Despite aspirin administration, however, these two patients experienced VTE. Following VTE development, warfarin and then a Factor Xa inhibitor, edoxaban, were administered. The edoxaban treatment, especially, resulted in favorable and effective control of VTE. Considering these observations, Factor Xa inhibitors may in future become a preferred option for prevention and treatment of VTE when managing MM patients. PMID:26345573

  16. Oral, direct factor Xa inhibitors in development for the prevention and treatment of thromboembolic diseases.

    PubMed

    Turpie, Alexander G G

    2007-06-01

    Anticoagulants are recommended for the prevention and treatment of a wide variety of thromboembolic events. Although existing anticoagulants are effective, their use is limited by parenteral administration or the requirement for frequent monitoring and subsequent dose adjustment. Therefore, there is an urgent need for novel, oral agents with a predictable anticoagulant action. Because of its key position in the coagulation cascade and its limited roles outside of coagulation, Factor Xa has emerged as an attractive target for novel anticoagulants. As a result, the past decade has witnessed an explosion of research into small-molecule, oral, direct Factor Xa inhibitors, and several are now in clinical development. Rivaroxaban, LY517717, YM150, apixaban, PRT054021, and DU-176b, among others, have shown considerable promise; rivaroxaban is currently furthest ahead in its developmental program, having entered phase III in 3 indications. It is hoped that, before long, these anticoagulants will allow us to enter an era of convenient, oral anticoagulation, without the need for regular monitoring or dose adjustment. PMID:17379841

  17. Oral factor Xa inhibitors for thromboprophylaxis in major orthopedic surgery: a review.

    PubMed

    Imberti, Davide; Dall'Asta, Chiara; Pierfranceschi, Matteo Giorgi

    2009-12-01

    Venous thromboembolism (VTE), which includes deep vein thrombosis and pulmonary embolism, is a major cause of morbidity and mortality in patients undergoing major orthopedic surgery. Routine thromboprophylaxis has been the standard of care over the last 20 years. Currently available options for the prevention of VTE in major orthopedic surgery include low molecular weight heparins, vitamin K antagonists, and, more recently, the synthetic pentasaccharide fondaparinux. Although effective, all these drugs have several limitations and new oral antithrombotics offering predictable, effective and safe anticoagulation are strongly needed. This overview focuses on the most advanced oral direct inhibitors to factor Xa rivaroxaban, apixaban, LY517717 and YM150; specifically, the results of phase II and III studies and the designs of ongoing clinical trials in patients undergoing elective hip and knee replacement are reviewed. PMID:19696978

  18. Oral factor Xa inhibitors for venous thromboembolism prevention in major orthopedic surgery: a review.

    PubMed

    Imberti, Davide; Prisco, Domenico

    2008-01-01

    Venous thromboembolism (VTE), which includes deep vein thrombosis and pulmonary embolism, is a major cause of morbidity and mortality in patients undergoing major orthopedic surgery, and routine thromboprophylaxis has been the standard of care over the last 20 years. Currently available options for the prevention of VTE in major orthopedic surgery include low-molecular-weight heparins, vitamin K antagonists and, more recently, the synthetic pentasaccharide fondaparinux. Although effective, these drugs have several limitations, and new oral antithrombotics offering predictable, effective and safe anticoagulation are strongly needed. This overview focuses on the most advanced oral direct inhibitors of factor Xa, rivaroxaban, apixaban, LY517717, YM150 and betrixaban. Specifically, the results of phase II and III studies and the designs of ongoing clinical trials in patients undergoing elective hip and knee replacement are reviewed. PMID:19996630

  19. Effect of ketoconazole and diltiazem on the pharmacokinetics of apixaban, an oral direct factor Xa inhibitor

    PubMed Central

    Frost, Charles E; Byon, Wonkyung; Song, Yan; Wang, Jessie; Schuster, Alan E; Boyd, Rebecca A; Zhang, Donglu; Yu, Zhigang; Dias, Clapton; Shenker, Andrew; LaCreta, Frank

    2015-01-01

    Aim Apixaban is an orally active inhibitor of coagulation factor Xa and is eliminated by multiple pathways, including renal and non-renal elimination. Non-renal elimination pathways consist of metabolism by cytochrome P450 (CYP) enzymes, primarily CYP3A4, as well as direct intestinal excretion. Two single sequence studies evaluated the effect of ketoconazole (a strong dual inhibitor of CYP3A4 and P-glycoprotein [P-gp]) and diltiazem (a moderate CYP3A4 inhibitor and a P-gp inhibitor) on apixaban pharmacokinetics in healthy subjects. Method In the ketoconazole study, 18 subjects received apixaban 10 mg on days 1 and 7, and ketoconazole 400 mg once daily on days 4–9. In the diltiazem study, 18 subjects received apixaban 10 mg on days 1 and 11 and diltiazem 360 mg once daily on days 4–13. Results Apixaban maximum plasma concentration and area under the plasma concentration–time curve extrapolated to infinity increased by 62% (90% confidence interval [CI], 47, 78%) and 99% (90% CI, 81, 118%), respectively, with co-administration of ketoconazole, and by 31% (90% CI, 16, 49%) and 40% (90% CI, 23, 59%), respectively, with diltiazem. Conclusion A 2-fold and 1.4-fold increase in apixaban exposure was observed with co-administration of ketoconazole and diltiazem, respectively. PMID:25377242

  20. Structure-activity relationships of anthranilamide-based factor Xa inhibitors containing piperidinone and pyridinone P4 moieties.

    PubMed

    Corte, James R; Fang, Tianan; Pinto, Donald J P; Han, Wei; Hu, Zilun; Jiang, Xiang-Jun; Li, Yun-Long; Gauuan, Jolicia F; Hadden, Mark; Orton, Darren; Rendina, Alan R; Luettgen, Joseph M; Wong, Pancras C; He, Kan; Morin, Paul E; Chang, Chong-Hwan; Cheney, Daniel L; Knabb, Robert M; Wexler, Ruth R; Lam, Patrick Y S

    2008-05-01

    Introduction of the phenyl piperidinone and phenyl pyridinone P4 moieties in the anthranilamide scaffold led to potent, selective, and orally bioavailable inhibitors of factor Xa. Anthranilamide 28 displayed comparable efficacy to apixaban in the rabbit arteriovenous-shunt (AV) thrombosis model. PMID:18424044

  1. Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

    SciTech Connect

    Huang, Xin; Dementiev, Alexey; Olson, Steven T.; Gettins, Peter G.W.

    2012-12-13

    The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction {approx}2000-fold in the presence of phospholipid and Ca{sup 2+}. To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ{sub {Delta}GD}) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing {approx}5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only {approx}2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional {approx}1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.

  2. Contemporary developments in the discovery of selective factor Xa inhibitors: A review.

    PubMed

    Patel, Nirav R; Patel, Dushyant V; Murumkar, Prashant R; Yadav, Mange Ram

    2016-10-01

    Thrombosis is a leading cause of death in cardiovascular diseases such as myocardial infarction (MI), unstable angina and acute coronary syndrome (ACS) in the industrialized world. Venous thromboembolism is observed in about 1 million people every year in United States causing significant morbidity and mortality. Conventional antithrombotic therapy has been reported to have several disadvantages and limitations like inconvenience in oral administration, bleeding risks (heparin analogs), narrow therapeutic window and undesirable interactions with food and drugs (vitamin K antagonist-warfarin). The unmet medical demand for orally active safe anticoagulants has generated widespread interest among the medicinal chemists engaged in this field. To modulate blood coagulation, various enzymes involved in the coagulation process have received great attention as potential targets by various research groups for the development of oral anticoagulants. Among these enzymes, factor Xa (FXa) has remained the centre of attention in the last decade. Intensive research efforts have been made by various research groups for the development of small, safe and orally bioavailable FXa inhibitors. This review is an attempt to compile the research work of various researchers in the direction of development of FXa inhibitors reported since 2010 onward. PMID:27322757

  3. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    PubMed Central

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  4. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting.

    PubMed

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  5. Rivaroxaban – an oral, direct Factor Xa inhibitor – lessons from a broad clinical study programme

    PubMed Central

    Haas, Sylvia

    2009-01-01

    Anticoagulants are recommended for the prevention and treatment of venous thromboembolism (VTE), prevention of stroke in patients with atrial fibrillation (AF) and secondary prevention in patients with acute coronary syndrome (ACS). There is a clinical need for novel anticoagulants offering improvements over current standard of care, such as fixed oral dosing and no need for routine monitoring. Rivaroxaban, an oral, once-daily, direct Factor Xa inhibitor, has recently completed the RECORD phase III programme for the prevention of VTE in patients undergoing total hip or knee replacement (THR or TKR), an indication for which it is approved in Europe and Canada. It is being investigated in large-scale phase III studies for VTE treatment and prevention of stroke in patients with AF, and phase III studies will soon commence for secondary prevention in patients with ACS. Phase I studies demonstrated that no routine anticoagulation monitoring was required, while phase II studies suggested that fixed daily doses had a wide therapeutic window. The four RECORD studies consistently showed that rivaroxaban was significantly more effective than enoxaparin in the prevention of VTE after THR and TKR, with a similar safety profile. This review describes the development of this novel anticoagulant, from bench to bedside. PMID:19187276

  6. Identification of potent orally active factor Xa inhibitors based on conjugation strategy and application of predictable fragment recommender system.

    PubMed

    Ishihara, Tsukasa; Koga, Yuji; Iwatsuki, Yoshiyuki; Hirayama, Fukushi

    2015-01-15

    Anticoagulant agents have emerged as a promising class of therapeutic drugs for the treatment and prevention of arterial and venous thrombosis. We investigated a series of novel orally active factor Xa inhibitors designed using our previously reported conjugation strategy to boost oral anticoagulant effect. Structural optimization of anthranilamide derivative 3 as a lead compound with installation of phenolic hydroxyl group and extensive exploration of the P1 binding element led to the identification of 5-chloro-N-(5-chloro-2-pyridyl)-3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzamide (33, AS1468240) as a potent factor Xa inhibitor with significant oral anticoagulant activity. We also reported a newly developed Free-Wilson-like fragment recommender system based on the integration of R-group decomposition with collaborative filtering for the structural optimization process. PMID:25523211

  7. Structure-guided creation of AcAP5-derived and platelet targeted factor Xa inhibitors.

    PubMed

    Zhu, Yuanjun; Lin, Yuan; Liu, Aihua; Shui, Mengyang; Li, Ruyi; Liu, Xiaoyan; Hu, Wenhui; Wang, Yinye

    2015-06-15

    Anticoagulants and anti-platelet agents are simultaneously administrated in clinical practice (i.e. percutaneous coronary intervention), which cause significant risk of systemic bleeding. Targeted delivery of anticoagulants to the activated platelets at sites of vascular injuries may condense the site-specific anticoagulant effect and reduce the hemorrhage side effects in uninjured vessels. To this end, we prepared three ancylostoma caninum anticoagulant peptide 5 (AcAP5) variants NR1, NR2 and NR3 engineered with a platelet-binding Arg-Gly-Asp (RGD) motif and evaluated their anti-Factor Xa (FXa) and platelet-binding effects. These RGD-containing AcAP5 variants were capable of interacting with platelet receptor αIIbβ3 as shown in computational analysis. All variants, especially NR2 and NR3, retained entirely the anti-FXa function of parent AcAP5. Moreover, they prevented the formation of occlusive thrombi in rat carotid artery injury model, suggesting that they inhibit platelet aggregation in vivo. Further functional investigation of NR3 demonstrated that NR3 inhibited platelet aggregation in vitro and FXa activity in vivo, and prolonged the coagulation time, all in a dose-dependent manner. Through flow cytometry assay, we confirmed the binding of NR3 to αIIbβ3 receptor. In mouse model of carotid artery endothelium injury, NR3-treated mice showed less tail bleeding time than AcAP5-treated mice, and aspirin plus NR3 treatment exhibited moderate reduction of blood loss compared with aspirin plus AcAP5 treatment. These results indicate the feasibility to engineer a novel FXa inhibitor specifically targeting the activated platelets, which centralizes its anticoagulation efficacy in the injured vascular endothelium and reduces the risk of systemic bleeding. PMID:25887920

  8. Oral anticoagulation with Factor Xa and thrombin inhibitors: Is there an alternative to warfarin?

    PubMed

    Zikria, Jennifer; Ansell, Jack

    2009-12-01

    Vitamin K antagonists (VKA), such as warfarin, have been the only available oral anticoagulants despite their many limitations. The greatest medical need is to find a replacement for warfarin for long-term therapy, particularly for stroke prevention in atrial fibrillation (AF) patients. Emerging oral anticoagulants are free from many of warfarin's drawbacks and may offer a convenient alternative. Drugs in advanced development target factor Xa (rivaroxaban, apixaban) or thrombin (dabigatran etexilate). Recently, the RE-LY phase III study found dabigatran etexilate was an effective and convenient alternative to warfarin in stroke prevention for AF patients. Within the next two years, similar studies comparing rivaroxaban and apixaban versus warfarin in AF patients will become available. This paper reviews warfarin's limitations, discusses the pharmacokinetics of emerging anticoagulants in advanced development, and summarizes trials with an emphasis on head-to-head studies comparing novel anticoagulants to warfarin. PMID:20040270

  9. Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa.

    PubMed

    Iyer, Janaki Krishnamoorthy; Otvos, Reka A; Kool, Jeroen; Kini, R Manjunatha

    2016-02-01

    Thrombin and factor Xa (FXa) are critical enzymes of the blood coagulation cascade and are excellent targets of anticoagulant agents. Natural sources present an array of anticoagulants that can be developed as antithrombotic drugs. High-resolution, online screening techniques have been developed for the identification of drug leads from complex mixtures. In this study, we have developed and optimized a microfluidic online screening technique coupled to nano-liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are suitable for the identification of inhibitors from sample amounts as low as sub-microliter volumes. PMID:26323281

  10. Enhanced endogenous thrombolysis induced by a specific factor Xa inhibitor, DX-9065a, evaluated in a rat arterial thrombolysis model in vivo.

    PubMed

    Hashimoto, Masaru; Onobayashi, Yuko; Oiwa, Kazuhiro; Giddings, John C; Yamamoto, Junichiro

    2002-04-15

    We have previously established an animal model to investigate mechanisms of arterial thrombolysis in vivo and have demonstrated that endogenous thrombolysis, mediated by thrombin-activatable fibrinolysis inhibitor, is enhanced by administration of specific thrombin inhibitors. The aim of the present study was to evaluate the effects of a synthetic and specific factor Xa inhibitor, DX-9065a, on endogenous fibrinolysis. Mural thrombi were formed in rat mesenteric arterioles by helium-neon laser irradiation in the presence of Evans blue. Thrombolysis was continuously monitored by video microscopy and was quantified using image analysis software. Oral and intravenous administration of DX-9065a enhanced endogenous thrombolysis in vivo. The mechanisms require additional investigation using other experimental systems, but nevertheless, the present results extended our previous findings and further suggested that the enhanced fibrinolysis might be due to depressed activity thrombin-activatable fibrinolysis inhibitor. The synthetic factor Xa inhibitor could provide the basis for a useful thrombolytic agent. PMID:12182917

  11. Intron-exon organization of the human gene coding for the lipoprotein-associated coagulation inhibitor: The factor Xa dependent inhibitor of the extrinsic pathway of coagulation

    SciTech Connect

    van der Logt, C.P.E.; Reitsma, P.H.; Bertina, R.M. )

    1991-02-12

    Blood coagulation can be initiated when factor VII(a) binds to its cofactor tissue factor. This factor VIIa/tissue factor complex proteolytically activates factors IX and X, which eventually leads to the formation of a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits factor Xa directly and, in a Xa-dependent manner, also inhibits the factor VIIa/tissue factor complex. Here the authors report the cloning of the human LACI gene and the elucidation of its intron-exon organization. The LACI gene, which spans about 70 kb, consists of nine exons separated by eight introns. As has been found for other Kunitz-type protease inhibitors, the domain structure of human LACI is reflected in the intron-exon organization of the gene. The 5{prime} terminus of the LACI mRNA has been determined by primer extension and S1 nuclease mapping. The putative promoter was examined and found to contain two consensus sequences for AP-1 binding and one for NF-1 binding, but no TATA consensus promoter element.

  12. Comparative pharmacodynamics and pharmacokinetics of oral direct thrombin and factor xa inhibitors in development.

    PubMed

    Eriksson, Bengt I; Quinlan, Daniel J; Weitz, Jeffrey I

    2009-01-01

    For the past five decades, there has been little progress in the development of oral anticoagulants, with the choices being limited to the vitamin K antagonists (VKAs). The situation is changing with the development of orally active small molecules that directly target thrombin or activated factor X (FXa). The two agents in the most advanced stages of development are dabigatran etexilate and rivaroxaban, which inhibit thrombin and FXa, respectively. Both are approved in the EU and Canada for venous thromboprophylaxis in patients undergoing elective hip- or knee-replacement surgery. Other agents in the early stages of development include several FXa inhibitors (apixaban, DU 176b, LY 517717, YM 150, betrixaban, eribaxaban [PD 0348292] and TAK 442) and one thrombin inhibitor (AZD 0837). With a predictable anticoagulant response and low potential for drug-drug interactions, these new agents can be given in fixed doses without coagulation monitoring. This renders them more convenient than VKAs. While the anticoagulant effect of the new thrombin and FXa inhibitors is similar, differences in the pharmacokinetic and pharmacodynamic parameters may influence their use in clinical practice. Here, we compare the pharmacokinetic and pharmacodynamic features of these new oral agents. PMID:19071881

  13. Inhibition of coagulation activation and inflammation by a novel Factor Xa inhibitor synthesized from the earthworm Eisenia andrei.

    PubMed

    Joo, Seong Soo; Won, Tae Joon; Kim, Jong Sung; Yoo, Yeong Min; Tak, Eun Sik; Park, So-Young; Park, Hee Yong; Hwang, Kwang Woo; Park, Soon Cheol; Lee, Do Ik

    2009-02-01

    We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (p<0.0001), indicating that protease-activated receptor-2 (PAR-2) was effectively inactivated. We also found that ESP reduced the expressions of pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-8, IL-16, MCP-1, MIP-1alpha and MIP-1beta) by cultured cells treated with both ESP and FXa. Our results provide the first evidence that ESP might interrupt coagulation cascades by inhibiting FXa, and thereby may effectively control the bidirectional alternation between coagulation and inflammation. PMID:19182385

  14. Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)*

    PubMed Central

    Dockal, Michael; Hartmann, Rudolf; Fries, Markus; Thomassen, M. Christella L. G. D.; Heinzmann, Alexandra; Ehrlich, Hartmut; Rosing, Jan; Osterkamp, Frank; Polakowski, Thomas; Reineke, Ulrich; Griessner, Andreas; Brandstetter, Hans; Scheiflinger, Friedrich

    2014-01-01

    Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients. PMID:24275667

  15. Edoxaban: A Novel Factor Xa Inhibitor for the Management of Non-valvular Atrial Fibrillation and Venous Thromboembolism.

    PubMed

    Kubli, Kara A; Snead, Jessica A; Cheng-Lai, Angela

    2016-01-01

    Warfarin has been a highly prevalent agent for over 70 years; however, its use has been limited by drug-drug interactions, adverse events, and the need for frequent monitoring. To minimize these complications, several non-vitamin K oral anticoagulants have been approved, including the latest agent, edoxaban. Edoxaban is a factor Xa inhibitor approved for the prevention of stroke/systemic embolism in patients with non-valvular atrial fibrillation and for the treatment of venous thromboembolism. Edoxaban was largely studied in the Edoxaban versus Warfarin in Patients with Atrial Fibrillation (ENGAGE AF-TIMI 48) and Edoxaban versus Warfarin for the Treatment of Symptomatic Venous Thromboembolism (Hokusai-VTE) trials, both showing noninferiority when compared with warfarin. Similar to other oral anticoagulants, the most serious adverse effects of edoxaban are related to bleeding. However, there are currently no approved reversal agents. Andexanet alfa and ciraparantag are the latest agents being studied for reversal. This article provides an overview of the safety and efficacy along with the advantages and disadvantages of edoxaban. PMID:26991962

  16. Exploration of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides as potent, orally active Factor Xa inhibitors with extended duration of action.

    PubMed

    Van Huis, Chad A; Casimiro-Garcia, Agustin; Bigge, Christopher F; Cody, Wayne L; Dudley, Danette A; Filipski, Kevin J; Heemstra, Ronald J; Kohrt, Jeffrey T; Leadley, Robert J; Narasimhan, Lakshmi S; McClanahan, Thomas; Mochalkin, Igor; Pamment, Michael; Peterson, J Thomas; Sahasrabudhe, Vaishali; Schaum, Robert P; Edmunds, Jeremy J

    2009-03-15

    Aiming to improve upon previously disclosed Factor Xa inhibitors, a series of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides were explored with the intent of increasing the projected human half-life versus 5 (projected human t(1/2)=6 h). A stereospecific route to compounds containing a 4-aryl-4-hydroxypyrrolidine scaffold was developed, resulting in several compounds that demonstrated an increase in the half-life as well as an increase in the in vitro potency compared to 5. Reported herein is the discovery of 26, containing a (2R,4S)-4-hydroxy-4-(2,4-difluorophenyl)-pyrrolidine scaffold, which is a selective, orally bioavailable, efficacious Factor Xa inhibitor that appears suitable for a once-daily dosing (projected human t(1/2)=23 h). PMID:19231206

  17. Novel anticoagulants in clinical development: focus on factor Xa and direct thrombin inhibitors.

    PubMed

    Steffel, Jan; Lüscher, Thomas F

    2009-08-01

    Vitamin K antagonists are the mainstay in the prevention and treatment of thromboembolic diseases. Although effective under optimal conditions, several drawbacks are imminent to the long-term application of these drugs due to their narrow therapeutic window, interactions with other drugs as well as the need for regular monitoring and the risk of a recurrent event versus the risk of bleeding. To overcome these downsides, novel anticoagulants are being developed; in contrast to vitamin K antagonists, these novel agents specifically and selectively block central elements of the coagulation cascade. Several clinical trials have demonstrated the efficacy and safety of selective FXa inhibitors (such as fondaparinux, rivaroxaban, apixaban) and direct thrombin inhibitors (such as lepirudin, bivalirudin, dabigatran etexilate) in the treatment of typical indications for conventional vitamin K antagonists, in particular, the prevention and treatment of venous thromboembolism. This review summarizes the results and designs of recently published and ongoing clinical trials of novel anticoagulants. PMID:19561526

  18. [Surgery and invasive procedures in patients on long-term treatment with oral direct thrombin or factor Xa inhibitors].

    PubMed

    Sié, P; Samama, C-M; Godier, A; Rosencher, N; Steib, A; Llau, J-V; van der Linden, P; Pernod, G; Lecompte, T; Gouin-Thibault, I; Albaladejo, P

    2011-09-01

    Direct oral anticoagulants (DOAs), inhibitors of factor IIa or Xa, are expected to replace vitamin K antagonists in most of their indications. It is likely that patients on long-term treatment with DOAs will be exposed to elective or emergency surgery or invasive procedures. Due to the present lack of experience in such conditions, we cannot make recommendations, but only propose perioperative management for optimal safety as regards the risk of bleeding and thrombosis. DOAs may increase surgical bleeding, they have no validated antagonists, they cannot be monitored by simple, standardised laboratory assays, and their pharmacokinetics vary significantly from patient to patient. Although DOAs differ in many respects, the proposals in the perioperative setting need not be specific to each. For procedures with low risk of haemorrhage, a therapeutic window of 48 h (last administration 24h before surgery, restart 24h after) is proposed. For procedures with medium or high haemorrhagic risk, we suggest stopping DOAs 5 days before surgery to ensure complete elimination of the drug in all patients. The treatment should be resumed only when the risk of bleeding has been controlled. In patients with a high risk of thrombosis (e.g. those in atrial fibrillation with an antecedent of stroke), bridging with heparin (low molecular weight, or unfractionated if the former is contraindicated) is proposed. In emergency, the procedure should be postponed for as long as possible (minimum 1-2 half-lives) and non-specific anti-haemorrhagic agents, such as recombinant human activated factor VIIa, or prothrombin concentrates, should not be given for prophylactic reversal, due to their uncertain benefit-risk. PMID:21820844

  19. Prevention of arterial thrombosis by edoxaban, an oral factor Xa inhibitor in rats: monotherapy and in combination with antiplatelet agents.

    PubMed

    Honda, Yuko; Kamisato, Chikako; Morishima, Yoshiyuki

    2016-09-01

    In addition to platelet aggregation, coagulation activation is considered to be involved in arterial thrombosis. In this study, we determined antithrombotic effects of edoxaban, an oral factor Xa (FXa) inhibitor, as both a monotherapy and in combination with antiplatelet agents in a rat model of arterial thrombosis. We further examined its effects on a procoagulant biomarker and bleeding. Arterial thrombosis was induced by topical application of 15% ferric chloride to rat abdominal aortas. Bleeding time was measured by a tail incision method. Edoxaban, clopidogrel, and aspirin were orally administered 30min, 4h, and 2h before thrombus or bleeding induction. As a biomarker of coagulation activation, plasma thrombin-antithrombin complex (TAT) was measured. Edoxaban dose-dependently prevented arterial thrombosis in a manner comparable to clopidogrel and aspirin. The combination of edoxaban plus clopidogrel or edoxaban plus aspirin significantly potentiated the antithrombotic effects compared with these drugs alone. The combination of edoxaban and clopidogrel was more potent than clopidogrel and aspirin. Plasma TAT concentration was elevated after thrombus induction and suppressed by edoxaban and clopidogrel, but not by aspirin, suggesting P2Y12 receptor-mediated platelet procoagulant activity. Bleeding time was prolonged by the coadministration of edoxaban and clopidogrel, but not by edoxaban and aspirin. In conclusion, the present study demonstrates that the monotherapy with edoxaban and combination therapy with edoxaban plus clopidogrel or edoxaban plus aspirin are promising options for the prevention of arterial thrombosis as effective as the standard antiplatelet agents; however, a combination of edoxaban and clopidogrel increased the risk of bleeding. PMID:27288116

  20. Effect of the factor Xa inhibitor rivaroxaban on arterial thrombosis in wild-type and apolipoprotein E-deficient mice.

    PubMed

    Wagner, Nana-Maria; Dressel, Tobias; Schäfer, Katrin; Konstantinides, Stavros

    2012-11-01

    Rivaroxaban is a potent and specific direct inhibitor of coagulation factor Xa. Recent studies have highlighted its effectiveness in the prevention of venous thrombosis and embolic stroke due to atrial fibrillation. To evaluate the antithrombotic effects of rivaroxaban in an in vivo model of arterial thrombosis, photochemical vascular injury was induced in wild-type mice by intravenous rose bengal (50 mg/kg body weight [BW]) followed by illumination of the left common carotid artery using a 543 nm helium-neon laser beam. Rivaroxaban, injected concomitantly with rose bengal at doses of 1.0, 1.5, 2.0, or 3.0 mg/kg BW, dose-dependently prolonged the times to first thrombotic occlusion and stable thrombosis. Quantitative analysis of carotid flow curves revealed higher blood volumes passing through the injured artery with increasing rivaroxaban doses (P<0.01 and P<0.001 vs. vehicle for 2.0 and 3.0 mg/kg , respectively), suggesting a dose-dependent effect on vascular patency. Consistently, a significantly higher proportion of mice that received 2.0 and 3.0 mg/kg rivaroxaban exhibited patent carotid arteries at the end of the flow monitoring period compared to vehicle alone (P<0.05 and P<0.001, respectively). Histological analysis showed complete thrombotic arterial occlusion in vehicle-treated mice compared to less thrombotic material in mice injected with 3.0 mg/kg rivaroxaban (P<0.05). Rivaroxaban also prolonged the time to cessation of tail bleeding in a dose-dependent manner, starting at 1.5 mg/kg. Similar findings were obtained in apolipoprotein E-knockout mice. Rivaroxaban may exert beneficial effects by preventing arterial thrombosis and vascular occlusion after endothelial injury. PMID:22281071

  1. SAR and X-ray Structures of Enantiopure 1,2-cis-(1R,2S)-cyclopentyldiamine and Cyclohexyldiamine Derivativies as Inhibitors of Coagulation Factor Xa

    SciTech Connect

    Qiao,J.; Chang, C.; Cheney, D.; Morin, D.; Wang, P.; King, G.; Wang, S.; Rendina, T.; Luettgen, A.; et al.

    2007-01-01

    In the search of Factor Xa (FXa) inhibitors structurally different from the pyrazole-based series, we identified a viable series of enantiopure cis-(1R,2S)-cycloalkyldiamine derivatives as potent and selective inhibitors of FXa. Among them, cyclohexyldiamide 7 and cyclopentyldiamide 9 were the most potent neutral compounds, and had good anticoagulant activity comparable to the pyrazole-based analogs. Crystal structures of 7-FXa and 9-FXa illustrate binding similarities and differences between the five- and the six-membered core systems, and provide rationales for the observed SAR of P1 and linker moieties.

  2. The antithrombotic effect of synthetic low molecular weight human factor Xa inhibitor, DX-9065a, on He-Ne laser-induced thrombosis in rat mesenteric microvessels.

    PubMed

    Yamashita, T; Tsuji, T; Matsuoka, A; Giddings, J C; Yamamoto, J

    1997-01-01

    The effect of a synthetic low molecular weight factor Xa (FXa) inhibitor, DX9065a, on thrombosis in vivo were examined in a rat animal model using a Helium-Neon (He-Ne) laser method. DX-9065a administered either intravenously or orally promoted anti factor Xa activity in a dose dependent manner. Anti Xa activity was maximal immediately after intravenous injection and persisted for approximately 30 minutes. Inhibitory activity was maximal 15-30 minutes after oral administration and persisted for approximately 90 minutes. Similarly DX-9065a inhibited platelet-rich thrombosis formation in mesenteric arterioles and venules. In these instances inhibition was relatively transient after intravenous injection (10-20 minutes), but persisted for more than 3 hours after oral administration. The minimum effective doses of DX-9065a given intravenously and orally were 3.89 mg/kg and 25.9 mg/kg, respectively. The results confirmed that DX-9065a selectively modulates thrombotic mechanisms, and suggest that development of this synthetic FXa antagonist could constitute an effective intravenous and oral antithrombotic agent. PMID:8983124

  3. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  4. Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions.

    PubMed

    Kim, Tae Kwon; Tirloni, Lucas; Radulovic, Zeljko; Lewis, Lauren; Bakshi, Mariam; Hill, Creston; da Silva Vaz, Itabajara; Logullo, Carlos; Termignoni, Carlos; Mulenga, Albert

    2015-08-01

    Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin 19 that is characterised by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterisation reveals that the ubiquitously transcribed A. americanum serpin 19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) A. americanum serpin 19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, recombinant A. americanum serpin 19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of recombinant A. americanum serpin 19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14-29%. In anti-hemostatic functional assays, recombinant A. americanum serpin 19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250s, and up to 40s in the activated partial thromboplastin time assay. Given A. americanum serpin 19 high cross-tick species conservation, and specific reactivity of recombinant A. americanum serpin 19 with antibodies to A. americanum tick saliva proteins, we conclude that recombinant A. americanum serpin 19 is a potential candidate for development of a universal tick vaccine. PMID:25957161

  5. The discovery of (2R,4R)-N-(4-chlorophenyl)-N- (2-fluoro-4-(2-oxopyridin-1(2H)-yl)phenyl)-4-methoxypyrrolidine-1,2-dicarboxamide (PD 0348292), an orally efficacious factor Xa inhibitor.

    PubMed

    Kohrt, Jeffrey T; Bigge, Christopher F; Bryant, John W; Casimiro-Garcia, Agustin; Chi, Liguo; Cody, Wayne L; Dahring, Tawny; Dudley, Danette A; Filipski, Kevin J; Haarer, Staci; Heemstra, Ron; Janiczek, Nancy; Narasimhan, Lakshmi; McClanahan, Thomas; Peterson, J Thomas; Sahasrabudhe, Vaisheli; Schaum, Robert; Van Huis, Chad A; Welch, Kathleen M; Zhang, Erli; Leadley, Robert J; Edmunds, Jeremy J

    2007-08-01

    Herein, we report the discovery of novel, proline-based factor Xa inhibitors containing a neutral P1 chlorophenyl pharmacophore. Through the additional incorporation of 1-(4-amino-3-fluoro-phenyl)-1H-pyridin-2-one 22, as a P4 pharmacophore, we discovered compound 7 (PD 0348292). This compound is a selective, orally bioavailable, efficacious FXa inhibitor that is currently in phase II clinical trials for the treatment and prevention of thrombotic disorders. PMID:17683371

  6. Lufaxin, a Novel Factor Xa Inhibitor from the Salivary Gland of the sand fly Lutzomyia longipalpis, Blocks PAR2 Activation and Inhibits Inflammation and Thrombosis in Vivo

    PubMed Central

    Collin, Nicolas; Assumpção, Teresa C. F.; Mizurini, Daniella M.; Gilmore, Dana; Dutra-Oliveira, Angélica; Kotsyfakis, Michalis; Sá-Nunes, Anderson; Teixeira, Clarissa; Ribeiro, José M. C.; Monteiro, Robson Q.; Valenzuela, Jesus G.; Francischetti, Ivo M. B.

    2012-01-01

    Objective Blood-sucking arthropods salivary glands (SGs) contain a remarkable diversity of antihemostatics. The aim of this study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results Several L. longipalpis salivary proteins were expressed in HEK293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, non-competitive, and reversible inhibitor of Factor Xa (FXa). Notably, Lufaxin primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, DEGR- FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both PT and aPTT. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with a KD ~3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents PAR2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs aPTT ex vivo, implying that it works as an anticoagulant in vivo. Finally, SG of sandflies was found to inhibit FXa and to interact with the enzyme. Conclusion Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and antiinflamatory activities. It is a useful tool to understand FXa structural features and its role in pro-hemostatic and pro-inflammatory events. PMID:22796577

  7. Membrane-dependent Interaction of Factor Xa and Prothrombin with Factor Va in the Prothrombinase Complex

    PubMed Central

    Qureshi, Shabir H.; Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R.

    2009-01-01

    Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) were deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent Kd ∼60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr63-sulfated hirudin54-65 and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that fVa interaction with PCPS improves the affinity of the activation complex for the proexosite-1 of the substrate. Direct binding studies employing OG488-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla-domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa. PMID:19378973

  8. Activation of human factor V by factor Xa and thrombin

    SciTech Connect

    Monkovic, D.D.; Tracy, P.B. )

    1990-02-06

    The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polycarylamide gel electrophoresis followed by either autoradiography of {sup 125}I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of M{sub r} 220,000 and 105,000. Although thrombin cleaved the M{sub r} 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the M{sub r} 220,000 peptide. The factor Xa dependent functional assessment of {sup 125}I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the M{sub r} 220,000 peptide. The data indicate that factor Xa is as efficient an enzyme toward factor V as thrombin.

  9. Home treatment of patients with low-risk pulmonary embolism with the oral factor Xa inhibitor rivaroxaban. Rationale and design of the HoT-PE Trial.

    PubMed

    Barco, Stefano; Lankeit, Mareike; Binder, Harald; Schellong, Sebastian; Christ, Michael; Beyer-Westendorf, Jan; Duerschmied, Daniel; Bauersachs, Rupert; Empen, Klaus; Held, Matthias; Schwaiblmair, Martin; Fonseca, Cândida; Jiménez, David; Becattini, Cecilia; Quitzau, Kurt; Konstantinides, Stavros

    2016-07-01

    Pulmonary embolism (PE) is a potentially life-threatening acute cardiovascular syndrome. However, more than 95 % of patients are haemodynamically stable at presentation, and among them are patients at truly low risk who may qualify for immediate or early discharge. The Home Treatment of Pulmonary Embolism (HoT-PE) study is a prospective international multicentre single-arm phase 4 management (cohort) trial aiming to determine whether home treatment of acute low-risk PE with the oral factor Xa inhibitor rivaroxaban is feasible, effective, and safe. Patients with confirmed PE, who have no right ventricular dysfunction or free floating thrombi in the right atrium or ventricle, are eligible if they meet none of the exclusion criteria indicating haemodynamic instability, serious comorbidity or any condition mandating hospitalisation, or a familial/social environment unable to support home treatment. The first dose of rivaroxaban is given in hospital, and patients are discharged within 48 hours of presentation. Rivaroxaban is taken for at least three months. The primary outcome is symptomatic recurrent venous thromboembolism or PE-related death within three months of enrolment. Secondary outcomes include quality of life and patient satisfaction, and health care resource utilisation compared to existing data on standard-duration hospital treatment. HoT-PE is planned to analyse 1,050 enrolled patients, providing 80 % power to reject the null hypothesis that the recurrence rate of venous thromboembolism is >3 % with α≤0.05. If the hypothesis of HoT-PE is confirmed, early discharge and out-of-hospital treatment may become an attractive, potentially cost-saving option for a significant proportion of patients with acute PE. PMID:27010343

  10. Evaluation of Factor Xa-Specific Chromogenic Substrate Assays and the Determination of Pharmacokinetics of Fondaparinux.

    PubMed

    Yuri, Maiko; Tabe, Yoko; Tsuchiya, Koji; Sadatsuki, Ryo; Aoki, Jun; Horii, Takashi; Iba, Toshiaki; Ohsaka, Akimichi

    2016-07-01

    Fondaparinux (FPX), a synthesized factor Xa inhibitor, is one of the most popular anticoagulants for the prevention of postoperative venous thromboembolism (VTE). Although routine monitoring is not required, the bleeding adverse events cannot be neglected, and the measurement of anti-Xa activity is expected to be monitored. The primary purpose of this study is to evaluate the performances of 2 chromogenic assays for the detection of anti-Xa activity. Furthermore, the pharmacokinetics of FPX was examined using chromogenic assays. Anti-Xa activity was measured using 2 FPX-based chromogenic substrates (S2222 and STA-Liquid Anti-Xa). The reproducibility, detection limits, linearity, and correlations between the substrates were examined using normal plasma doped with low and high concentrations of FPX formulation. In addition, anti-Xa activity in 235 clinical samples from 164 cases treated was measured, and the pharmacokinetics of FPX was evaluated. Both of the tested substrates were capable of accurately measuring the anti-Xa activity of FPX, with a lower limit of 0.05 μg/mL and a coefficient of variation of less than 10%. The repeated administration of FPX induced a gradual but significant increase in the anti-Xa activity, which was negatively correlated with body weight and estimated glomerular filtration rate. No significant correlation between the anti-Xa activity and the occurrence of postoperative VTE or bleeding event was observed. Anti-Xa activity can be successfully determined using 2 chromogenic assays and automated biochemical analyzers. The clinical significance of anti-Xa activity monitoring should be examined in the future study. PMID:26177660

  11. Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR{sub 1} activation

    SciTech Connect

    Blanc-Brude, Olivier P. . E-mail: olivier.blanc-brude@larib.inserm.fr; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

    2005-03-10

    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 (PAR{sub 1}). 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 PAR{sub 1}-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR{sub 1}-specific agonists and inhibitors were used to demonstrate that PAR{sub 1} 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 PAR{sub 1} and not PAR{sub 2}. 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.

  12. The antithrombotic activity of EP224283, a neutralizable dual factor Xa inhibitor/glycoprotein IIbIIIa antagonist, exceeds that of the coadministered parent compounds.

    PubMed

    Hechler, Béatrice; Freund, Monique; Alame, Ghina; Leguay, Cécile; Gaertner, Sébastien; Cazenave, Jean-Pierre; Petitou, Maurice; Gachet, Christian

    2011-08-01

    EP224283 combines in a single molecule idraparinux and tirofiban, which allows obtaining a predictable and sustained antiplatelet effect through the transfer of the pharmacokinetics properties of idraparinux to the anti-αIIbβ3 antagonist. The activity can be instantaneously neutralized by injection of avidin, a specific antidote. We have tested the effects of this new profile anticoagulant in various thrombosis models. The antithrombotic effect of EP224283 was compared with those of the parent compounds used alone or in association at doses achieving low to moderate inhibition of platelet aggregation ex vivo. In a model of systemic thromboembolism independent of thrombin generation, tirofiban and EP224283 had similar effects at equimolar doses. On the other hand, EP224283 was more potent than tirofiban or idraparinux under thrombin-dependent conditions. In a ferric chloride-induced thrombosis model, EP224283 was more potent than either parent compound or their combination. Similar results were obtained after atherosclerotic plaque rupture in ApoE(-/-) mice. Thus, the dual action of EP224283 exceeds that of the parent compounds used in combination. A possible explanation is that EP224283 could concentrate antithrombin inside the thrombus by binding to αIIbβ3 through the tirofiban moiety, as shown by immunolabeling of the occluded vessel. No prolongation of the bleeding time was observed at doses achieving strong antithrombotic effects, suggesting that low to moderate αIIbβ3 inhibition combined with factor Xa inhibition minimizes the bleeding risk. The favorable antithrombotic profile of EP224283 together with its possible neutralization by avidin makes it an interesting drug candidate for the treatment and prevention of acute ischemic events. PMID:21527535

  13. A Novel Factor Xa-Inhibiting Peptide from Centipedes Venom.

    PubMed

    Kong, Yi; Shao, Yu; Chen, Hao; Ming, Xin; Wang, Jin-Bin; Li, Zhi-Yu; Wei, Ji-Fu

    2013-01-01

    Centipedes have been used as traditional medicine for thousands of years in China. Centipede venoms consist of many biochemical peptides and proteins. Factor Xa (FXa) is a serine endopeptidase that plays the key role in blood coagulation, and has been used as a new target for anti-thrombotic drug development. A novel FXa inhibitor, a natural peptide with the sequence of Thr-Asn-Gly-Tyr-Thr (TNGYT), was isolated from the venom of Scolopendra subspinipes mutilans using a combination of size-exclusion and reverse-phase chromatography. The molecular weight of the TNGYT peptide was 554.3 Da measured by electrospray ionization mass spectrometry. The amino acid sequence of TNGYT was determined by Edman degradation. TNGYT inhibited the activity of FXa in a dose-dependent manner with an IC50 value of 41.14 mg/ml. It prolonged the partial thromboplastin time and prothrombin time in both in vitro and ex vivo assays. It also significantly prolonged whole blood clotting time and bleeding time in mice. This is the first report that an FXa inhibiting peptide was isolated from centipedes venom. PMID:24273471

  14. Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells.

    PubMed Central

    Gasic, G P; Arenas, C P; Gasic, T B; Gasic, G J

    1992-01-01

    Smooth muscle cells (SMCs) in the rat carotid artery leave the quiescent state and proliferate after balloon catheter injury. The precise signals responsible for this SMC mitogenesis need to be elucidated. Although platelet-derived growth factor (PDGF), a potent SMC mitogen, is released from activated platelets, damaged endothelium, and macrophages, it cannot be solely responsible for this proliferation. In search of other SMC growth factors, we have examined several proteins of the coagulation cascade. At nanomolar concentrations, factors X, Xa, and protein S promote cultured rat aortic SMC mitosis. In contrast, factor IX is only weakly mitogenic, whereas factor VII and protein C fail to stimulate SMC division. Protein S, the most mitogenic of these coagulation cascade factors, stimulates DNA synthesis in cultured SMCs with a time course similar to that of PDGF-AA and without the delay observed for transforming growth factor beta. Antistasin and tick anticoagulant peptide, two specific factor Xa inhibitors, inhibit SMC mitogenesis due to Xa and protein S. Coagulation factors that possess mitogenic activity may contribute to intimal SMC proliferation after vascular injury as a result of angioplasty or vascular compromise during atherogenesis. Images PMID:1532256

  15. Factor Xa dimerization competes with prothrombinase complex formation on platelet-like membrane surfaces.

    PubMed

    Koklic, Tilen; Chattopadhyay, Rima; Majumder, Rinku; Lentz, Barry R

    2015-04-01

    Exposure of phosphatidylserine (PS) molecules on activated platelet membrane surface is a crucial event in blood coagulation. Binding of PS to specific sites on factor Xa (fXa) and factor Va (fVa) promotes their assembly into a complex that enhances proteolysis of prothrombin by approximately 10⁵. Recent studies demonstrate that both soluble PS and PS-containing model membranes promote formation of inactive fXa dimers at 5 mM Ca²⁺. In the present study, we show how competition between fXa dimerization and prothrombinase formation depends on Ca²⁺ and lipid membrane concentrations. We used homo-FRET measurements between fluorescein-E-G-R-chloromethylketone (CK)-Xa [fXa irreversibly inactivated by alkylation of the active site histidine residue with FEGR (FEGR-fXa)] and prothrombinase activity measurements to reveal the balance between fXa dimer formation and fXa-fVa complex formation. Changes in FEGR-fXa dimer homo-FRET with addition of fVa to model-membrane-bound FEGR-fXa unambiguously demonstrated that formation of the FEGR-fXa-fVa complex dissociated the dimer. Quantitative global analysis according to a model for protein interaction equilibria on a surface provided an estimate of a surface constant for fXa dimer dissociation (K(fXa×fXa)(d, σ)) approximately 10-fold lower than K(fXa×fVa)(d,σ) for fXa-fVa complex. Experiments performed using activated platelet-derived microparticles (MPs) showed that competition between fXa dimerization and fXa-fVa complex formation was even more prominent on MPs. In summary, at Ca²⁺ concentrations found in the maturing platelet plug (2-5 mM), fVa can compete fXa off of inactive fXa dimers to significantly amplify thrombin production, both because it releases dimer inhibition and because of its well-known cofactor activity. This suggests a hitherto unanticipated mechanism by which PS-exposing platelet membranes can regulate amplification and propagation of blood coagulation. PMID:25572019

  16. [Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors. Proposals of the Working Group on Perioperative Haemostasis (GIHP) - March 2013].

    PubMed

    Pernod, G; Albaladejo, P; Godier, A; Samama, C M; Susen, S; Gruel, Y; Blais, N; Fontana, P; Cohen, A; Llau, J V; Rosencher, N; Schved, J F; de Maistre, E; Samama, M M; Mismetti, P; Sié, P

    2013-10-01

    New direct oral anticoagulants (NOAC), inhibitors of factor IIa or Xa, are expected to be widely used for the treatment of venous thromboembolic disease, or in case of atrial fibrillation. Such anticoagulant treatments are known to be associated with haemorrhagic complications. Moreover, it is likely that such patients on long-term treatment with NOAC will be exposed to emergency surgery or invasive procedures. Due to the present lack of experience in such conditions, we cannot make recommendations, but only propose management for optimal safety as regards the risk of bleeding in such emergency conditions. In this article, only dabigatran and rivaroxaban were discussed. For emergency surgery at risk of bleeding, we propose to dose the plasmatic concentration of drug. Levels inferior or equal to 30ng/mL for both dabigatran and rivaroxaban, should enable the realization of a high bleeding risk surgery. For higher concentration, it was proposed to postpone surgery by monitoring the evolution of the drug concentration. Action is then defined by the kind of NOAC and its concentration. If the dosage of the drug is not immediately available, proposals only based on the usual tests, PT and aPTT, also are presented. However, these tests do not really assess drug concentration or bleeding risk. In case of severe haemorrhage in a critical organ, it is proposed to reduce the effect of anticoagulant therapy using a nonspecific procoagulant drug (activated prothrombin concentrate, FEIBA, 30-50U/kg, or non-activated 4-factors prothrombin concentrates 50U/kg). For any other type of severe haemorrhage, the administration of such a procoagulant drug, potentially thrombogenic in these patients, will be discussed regarding concentration of NACO and possibilities for mechanical haemostasis. PMID:23993157

  17. Surgery and invasive procedures in patients on long-term treatment with direct oral anticoagulants: thrombin or factor-Xa inhibitors. Recommendations of the Working Group on Perioperative Haemostasis and the French Study Group on Thrombosis and Haemostasis.

    PubMed

    Sié, Pierre; Samama, Charles M; Godier, Anne; Rosencher, Nadia; Steib, Annick; Llau, Juan V; Van der Linden, Philippe; Pernod, Gilles; Lecompte, Thomas; Gouin-Thibault, Isabelle; Albaladejo, Pierre

    2011-12-01

    Direct oral anticoagulants (DOAs)--inhibitors of thrombin or factor-Xa--are expected to replace vitamin K antagonists in most of their indications. Patients receiving long-term treatment with DOAs are likely to be exposed to elective or emergency surgery or invasive procedures. Owing to the present lack of experience in such conditions, we cannot make recommendations, but only propose perioperative management for optimal safety regarding the risk of bleeding and thrombosis. DOAs may increase surgical bleeding, they have no validated antagonists, they cannot be monitored by simple standardized laboratory assays and their pharmacokinetics vary significantly between patients. Although DOAs differ in many respects, the proposals in the perioperative setting need not be specific to each. For procedures with low haemorrhagic risk, a therapeutic window of 48 hours (last administration 24 hours before surgery, restart 24 hours after) is proposed. For procedures with medium or high haemorrhagic risk, we suggest stopping DOAs 5 days before surgery to ensure complete elimination in all patients. Treatment should be resumed only when the risk of bleeding has been controlled. In patients at high thrombotic risk (e.g. those in atrial fibrillation with a history of stroke), bridging with heparin (low molecular-weight heparin, or unfractionated heparin, if the former is contraindicated) is proposed. In an emergency, the procedure should be postponed for as long as possible (minimum 1-2 half-lives) and non-specific antihaemorrhagic agents, such as recombinant human activated factor VIIa or prothrombin complex concentrates should not be given for prophylactic reversal due to their uncertain benefit-risk. PMID:22152517

  18. RUBY-1: a randomized, double-blind, placebo-controlled trial of the safety and tolerability of the novel oral factor Xa inhibitor darexaban (YM150) following acute coronary syndrome

    PubMed Central

    Steg, Ph. Gabriel; Mehta, Shamir R.; Jukema, J. Wouter; Lip, Gregory Y.H.; Gibson, C. Michael; Kovar, Frantisek; Kala, Petr; Garcia-Hernandez, Alberto; Renfurm, Ronny W.; Granger, Christopher B.

    2011-01-01

    Aims To establish the safety, tolerability and most promising regimen of darexaban (YM150), a novel, oral, direct factor Xa inhibitor, for prevention of ischaemic events in acute coronary syndrome (ACS). Methods In a 26-week, multi-centre, double-blind, randomized, parallel-group study, 1279 patients with recent high-risk non-ST-segment or ST-segment elevation ACS received one of six darexaban regimens: 5 mg b.i.d., 10 mg o.d., 15 mg b.i.d., 30 mg o.d., 30 mg b.i.d., or 60 mg o.d. or placebo, on top of dual antiplatelet treatment. Primary outcome was incidence of major or clinically relevant non-major bleeding events. The main efficacy outcome was a composite of death, stroke, myocardial infarction, systemic thromboembolism, and severe recurrent ischaemia. Results Bleeding rates were numerically higher in all darexaban arms vs. placebo (pooled HR: 2.275; 95% CI: 1.13–4.60, P = 0.022). Using placebo as reference (bleeding rate 3.1%), there was a dose–response relationship (P = 0.009) for increased bleeding with increasing darexaban dose (6.2, 6.5, and 9.3% for 10, 30, and 60 mg daily, respectively), which was statistically significant for 30 mg b.i.d. (P = 0.002). There was no decrease (indeed a numerical increase in the 30 and 60 mg dose arms) in efficacy event rates with darexaban, but the study was underpowered for efficacy. Darexaban showed good tolerability without signs of liver toxicity. Conclusions Darexaban when added to dual antiplatelet therapy after ACS produces an expected dose-related two- to four-fold increase in bleeding, with no other safety concerns but no signal of efficacy. Establishing the potential of low-dose darexaban in preventing major cardiac events after ACS requires a large phase III trial. ClinicalTrials.gov Identifier: NCT00994292 PMID:21878434

  19. Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors: proposals of the working group on perioperative haemostasis (GIHP) - March 2013.

    PubMed

    Pernod, Gilles; Albaladejo, Pierre; Godier, Anne; Samama, Charles M; Susen, Sophie; Gruel, Yves; Blais, Normand; Fontana, Pierre; Cohen, Ariel; Llau, Juan V; Rosencher, Nadia; Schved, Jean-François; de Maistre, Emmanuel; Samama, Meyer M; Mismetti, Patrick; Sié, Pierre

    2013-01-01

    Direct new oral anticoagulants (NOACs) - inhibitors of thrombin or factor Xa - are intended to be used largely in the treatment of venous thromboembolic disease or the prevention of systematic embolism in atrial fibrillation, instead of vitamin K antagonists. Like any anticoagulant treatment, they are associated with spontaneous or provoked haemorrhagic risk. Furthermore, a significant proportion of treated patients are likely to be exposed to emergency surgery or invasive procedures. Given the absence of a specific antidote, the action to be taken in these situations must be defined. The lack of data means that it is only possible to issue proposals rather than recommendations, which will evolve according to accumulated experience. The proposals presented here apply to dabigatran (Pradaxa(®)) and rivaroxaban (Xarelto(®)); data for apixaban and edoxaban are still scarce. For urgent surgery with haemorrhagic risk, the drug plasma concentration should be less or equal to 30ng/mL for dabigatran and rivaroxaban should enable surgery associated with a high bleeding risk. Beyond that, if possible, the intervention should be postponed by monitoring the drug concentration. The course to follow is then defined according to the NOAC and its concentration. If the anticoagulant dosage is not immediately available, worse propositions, based on the usual tests (prothrombin time and activated partial thromboplastin time), are presented. However, these tests do not really assess drug concentration or the risk of bleeding that depends on it. In case of serious bleeding in a critical organ, the effect of anticoagulant therapy should be reduced using a non-specific procoagulant drug as a first-line approach: activated prothrombin complex concentrate (aPCC) (FEIBA(®) 30-50U/kg) or non-activated PCC (50U/kg). In addition, for any other type of severe haemorrhage, the administration of a procoagulant drug, which is potentially thrombogenic in these patients, is discussed according

  20. Evaluation of a Heparin-Calibrated Antifactor Xa Assay for Measuring the Anticoagulant Effect of Oral Direct Xa Inhibitors.

    PubMed

    Beyer, Jacob; Trujillo, Toby; Fisher, Sheila; Ko, Ann; Lind, Stuart E; Kiser, Tyree H

    2016-07-01

    The introduction of oral direct anti-Xa anticoagulants apixaban and rivaroxaban has significantly impacted the treatment and prevention of thromboembolic disease. Clinical scenarios exist in which a quantitative assessment for degree of anticoagulation due to these agents would aid management. The purpose of this work was to evaluate the chromogenic antifactor Xa assay calibrated with heparin standards at our institution for assessment of intensity of anticoagulation with rivaroxaban or apixaban in addition to its current use for unfractionated heparin or low-molecular-weight heparin. We also aimed to propose expected steady state peak and trough antifactor Xa activities for these agents based upon dosing regimens approved for nonvalvular atrial fibrillation. Antifactor Xa activity correlated very strongly with apixaban and rivaroxaban concentration in both spiked samples and treated patient plasma samples (r (2) = .99, P < .001). This correlation was observed over a broad range (20-500 ng/mL) of drug concentrations, as sample dilution with pooled normal plasma significantly extended the range of quantitative assessment. Based on drug concentrations previously published in pharmacokinetic studies, the expected steady state peak and trough antifactor Xa activity ranges for apixaban are 1.80 to 2.20 IU/mL and 0.70 to 1.10 IU/mL, respectively. For rivaroxaban, these ranges are 3.80 to 6.20 IU/mL and 0.60 to 1.00 IU/mL, respectively. In conclusion, our findings demonstrate that heparin-calibrated antifactor Xa activity correlates strongly with apixaban and rivaroxaban concentration. The dilution of samples allowed for this correlation to be extended over the majority of on-therapy drug concentrations. PMID:26842561

  1. Effects of water soluble phosphotidylserine on bovine factor Xa: functional and structural changes plus dimerization.

    PubMed

    Majumder, Rinku; Wang, Jianfang; Lentz, Barry R

    2003-02-01

    Previous work has shown that two molecules of a soluble form of phosphatidylserine, C6PS, bind to human and bovine factor X(a). Activity measurements along with the fluorescence of active-site-labeled human factor X(a) showed that two linked sites specifically regulate the active site conformation and proteolytic activity of the human enzyme. These results imply, but cannot demonstrate, a C6PS-induced factor X(a) conformational change. The purpose of this paper is to extend these observations to bovine factor X(a) and to demonstrate that they do reflect conformational changes. We report that the fluorescence of active-site-labeled bovine factor X(a) also varied with C6PS concentration in a sigmoidal manner, whereas amidolytic activity of unlabeled enzyme varied in a simple hyperbolic fashion, also as seen for human factor X(a). C6PS induced a 70-fold increase in bovine factor X(a)'s autolytic activity, consistent with the 60-fold increase in proteolytic activity reported for human factor X(a). In addition, circular dichroism spectroscopy clearly demonstrated that C6PS binding to bovine factor X(a) induces secondary structural changes. In addition, C6PS binding to the tighter of the two sites triggered structural changes that lead to Ca(2+)-dependent dimer formation, as demonstrated by changes in intrinsic fluorescence and quantitative native gel electrophoresis. Dimerization produced further change in secondary structure, either inter- or intramolecularly. These results, along with results presented previously, support a model in which C6PS binds in a roughly sequential fashion to two linked sites whose occupancy in both human and bovine factor X(a) elicits different structural and functional responses. PMID:12547804

  2. Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

    SciTech Connect

    Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian . E-mail: bzheng@hkucc.hku.hk

    2007-07-20

    The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.

  3. Measuring Anti–Factor Xa Activity to Monitor Low-Molecular-Weight Heparin in Obesity: A Critical Review

    PubMed Central

    Egan, Gregory; Ensom, Mary H H

    2015-01-01

    Background: The choice of whether to monitor anti–factor Xa (anti-Xa) activity in patients who are obese and who are receiving low-molecular-weight heparin (LMWH) therapy is controversial. To the authors’ knowledge, no systematic review of monitoring of anti-Xa activity in such patients has been published to date. Objective: To systematically ascertain the utility of monitoring anti-Xa concentrations for LMWH therapy in obese patients. Data Sources: MEDLINE (1946 to September 2014), the Cochrane Database of Systematic Reviews, Embase (1974 to September 2014), PubMed (1947 to September 2014), International Pharmaceutical Abstracts (1970 to September 2014), and Scopus were searched using the terms obesity, morbid obesity, thrombosis, venous thrombosis, embolism, venous thromboembolism, pulmonary embolism, low-molecular weight heparin, enoxaparin, dalteparin, tinzaparin, anti-factor Xa, anti-factor Xa monitoring, anti-factor Xa activity, and anti-factor Xa assay. The reference lists of retrieved articles were also reviewed. Study Selection and Data Extraction: English-language studies describing obese patients treated with LMWH or reporting anti-Xa activity were reviewed using a 9-step decision-making algorithm to determine whether monitoring of LMWH therapy by means of anti-Xa activity in obesity is warranted. Studies published in abstract form were excluded. Data Synthesis: The analysis showed that anti-Xa concentrations are not strongly associated with thrombosis or hemorrhage. In clinical studies of LMWH for thromboprophylaxis in bariatric surgery, orthopedic surgery, general surgery, and medical patients, and for treatment of venous thrombo embolism and acute coronary syndrome, anti-Xa activity can be predicted from dose of LMWH and total body weight; no difference in clinical outcome was found between obese and non-obese participants. Conclusions: Routinely determining anti-Xa concentrations in obese patients to monitor the clinical effectiveness of LMWH is

  4. Hemostatic agents of broad applicability produced by selective tuning of factor Xa zymogenicity.

    PubMed

    Ivanciu, Lacramioara; Camire, Rodney M

    2015-07-01

    There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios. PMID:25896653

  5. FRET studies with Factor X mutants provide insight into the topography of the membrane-bound Factor X/Xa

    PubMed Central

    Qureshi, Shabir H.; Yang, Likui; Yegneswaran, Subramanian; Rezaie, Alireza R.

    2007-01-01

    FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl–FPR (fluorescein–D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56±1 Å (1 Å=0.1 nm) and 63±1 Å for fXa-desEGF1 compared with 72±2 Å and 75±1 Å for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming κ2=2/3. In comparison, an L value of 95±6 Å was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys195 of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to Fl–FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface. PMID:17635109

  6. Inhibition of thrombin and factor Xa by Fucus evanescens fucoidan and its modified analogs.

    PubMed

    Lapikova, E S; Drozd, N N; Tolstenkov, A S; Makarov, V A; Zvyagintseva, T N; Shevchenko, N M; Bakunina, I U; Besednova, N N; Kuznetsova, T A

    2008-09-01

    Specimens of fucoidan extracted from Fucus evanescens were purified from protein and polyphenols, deacetylated and depolymerized by fucoidanase for evaluation of their biological activity. Deacetylation did not modify the capacity of fucoidan to inhibit thrombin and factor Xa, while purification from protein and polyphenols reduced this capacity. Depolymerization of fucoidan increased its capacity to inhibit thrombin mainly through heparin cofactor II. All the studied specimens formed complexes with protamine sulfate. PMID:19240852

  7. Phosphatidylserine-induced Factor Xa Dimerization and Binding to Factor Va Are Competing Processes in Solution

    PubMed Central

    Majumder, Rinku; Koklic, Tilen; Rezaie, Alireza R.; Lentz, Barry R.

    2013-01-01

    A soluble, short chain phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to promote FXa dimerization (Kd ~ 14 nM), and to enhance both the catalytic activity of FXa and the cofactor activity of FVa. In the presence of calcium, C6PS binds to two sites on FXa, one in the epidermal growth factor like (EGF) domain and one in the catalytic domain; the latter interaction is sensitive to Na+ binding and probably represents a protein recognition site. Here we ask whether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes. We monitored FXa activity at 5, 20 and 50 nM FXa while titrating with FVa in the presence of 400 µM C6PS and 3 or 5 mM Ca2+ to show that the apparent Kd of FVa-FXa interaction increased with increasing FXa concentration at 5 mM Ca2+, but the Kd was only slightly affected at 3 mM Ca2+. A mixture of 50 nM FXa and 50 nM FVa in the presence of 400 µM C6PS yielded both Xa homodimers and Xa ·Va heterodimers but no FXa dimers bound to FVa. A mutant FXa (R165A) that has reduced prothrombinase activity showed both reduced dimerization (Kd~147 nM) and reduced FVa binding (apparent Kd, = 58, 92 and 128 nM, respectively for 5, 20 and 50 nM R165A FXa). Native gel electrophoresis showed that the GLA-EGFNC fragment of FXa (lacking the catalytic domain) neither dimerized nor formed a complex with FVa in the presence of 400 µM C6PS and 5 mM Ca2+. Our results demonstrate that the dimerization site and FVa binding site are both located in the catalytic domain of FXa and that these sites are linked thermodynamically. PMID:23214401

  8. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa.

    PubMed

    Pinto, Donald J P; Orwat, Michael J; Koch, Stephanie; Rossi, Karen A; Alexander, Richard S; Smallwood, Angela; Wong, Pancras C; Rendina, Alan R; Luettgen, Joseph M; Knabb, Robert M; He, Kan; Xin, Baomin; Wexler, Ruth R; Lam, Patrick Y S

    2007-11-01

    Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P1 moieties that resulted in the identification of the p-methoxyphenyl P1, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P4 ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4. PMID:17914785

  9. Discovery of 1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro- 1H-pyrazolo[3,4-c]pyridine-3-carboxamide (Apixaban, BMS-562247), a Highly Potent, Selective, Efficacious, and Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa

    SciTech Connect

    Pinto, Donald J.P.; Orwat, Michael J.; Koch, Stephanie; Rossi, Karen A.; Alexander, Richard S.; Smallwood, Angela; Wong, Pancras C.; Rendina, Alan R.; Luettgen, Joseph M.; Knabb, Robert M.; He, Kan; Xin, Baomin; Wexler, Ruth R.; Lam, Patrick Y.S.

    2010-03-08

    Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P{sub 1} moieties that resulted in the identification of the p-methoxyphenyl P{sub 1}, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P{sub 4} ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.

  10. The Anti-Factor Xa Range For Low Molecular Weight Heparin Thromboprophylaxis

    PubMed Central

    Ward, Salena M.

    2015-01-01

    Low molecular weight heparins (LMWHs) are now the mainstay option in the prevention and treatment of venous thromboembolism. In some patients receiving therapeutic doses of LMWH, activity can be measured by quantifying the presence of Anti-factor Xa (AFXa) for dose adjustment. However, currently there are no guidelines for LMWH monitoring in patients on thromboprophylactic, doses, despite certain patient populations may be at risk of suboptimal dosing. This review found that while the AFXa ranges for therapeutic levels of LMWHs are relatively well defined in the literature, prophylactic ranges are much less clear, thus making it difficult to interpret current research data. From the studies published to date, we concluded that a reasonable AFXa target range for LMWH deep venous thromboses prophylaxis might be 0.2-0.5 IU/mL. PMID:26733269

  11. Correction of the coagulation defect in hemophilia using a factor Xa variant with novel engineered protease function

    PubMed Central

    Ivanciu, Lacramioara; Toso, Raffaella; Margaritis, Paris; Pavani, Giulia; Kim, Haein; Schlachterman, Alexander; Liu, Jian-Hua; Clerin, Valerie; Pittman, Debra D.; Rose-Miranda, Rosalind; Shields, Kathleen M.; Erbe, David V.; Tobin, James F.; Arruda, Valder R.; Camire, Rodney M.

    2011-01-01

    Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We describe a surprisingly useful approach to improve hemostasis in vivo using a variant of coagulation factor Xa (FXaI16L). This conformationally pliant derivative is partially inactive due to a defect in transitioning from zymogen to protease 1,2. Using mouse models of hemophilia, we show that FXaI16L has a prolonged half-life, relative to wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXaI16L is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXaI16L is more efficacious than FVIIa which is used to treat bleeding in hemophilia inhibitor patients3. Because of its underlying mechanism of action, FXaI16L may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions. PMID:22020385

  12. Cooperative Regulation of the Activity of Factor Xa within Prothrombinase by Discrete Amino Acid Regions from Factor Va Heavy Chain†

    PubMed Central

    2008-01-01

    The prothrombinase complex catalyzes the activation of prothrombin to α-thrombin. We have repetitively shown that amino acid region 695DYDY698 from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. We have also recently demonstrated that amino acid region 334DY335 is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334–335 and 695−698 as follows: factor V3K (334DY335 → KF and 695DYDY698 → KFKF), factor VKF/4A (334DY335 → KF and 695DYDY698 → AAAA), and factor V6A (334DY335 → AA and 695DYDY698 → AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va3K, factor VaK4/4A, and factor Va6A had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor VaWt) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va3K had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor VaWt, while prothrombinase assembled with saturating concentrations of factor VaKF/4A and factor Va6A had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334–335 together with amino acid region 695−698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa. PMID:18991406

  13. A novel microfluidic anti-factor Xa assay device for monitoring anticoagulant therapy at the point-of-care

    NASA Astrophysics Data System (ADS)

    Harris, Leanne F.; Rainey, Paul; Castro-López, Vanessa; O'Donnell, James S.; Killard, Anthony J.

    2013-05-01

    Millions of patients worldwide are receiving anticoagulant therapy to treat hypercoagulable diseases. While standard testing is still performed in the central laboratory, point-of-care (POC) diagnostics are being developed due to the increasing number of patients requiring long-term anticoagulation and with a need for more personalized and targeted therapy. Many POC devices on the market focus on clot measurement, a technique which is limited in terms of variability, highlighting the need for more reliable assays of anticoagulant status. The anti-Xa assay, a factor specific optical assay, was developed to measure the extent to which exogenous factor Xa (FXa) is inhibited by heparinantithrombin complexes. We have developed a novel microfluidic device and assay for monitoring the effect of heparin anticoagulant therapy at the point-of-care. The assay which was also developed in our institute is based on the anti-Xa assay principle but uses fluorescence as the method of detection. Our device is a disposable laminate microfluidic strip, fabricated from the cyclic polyolefin (COP), Zeonor®, which is extremely suitable for application to fluorescent device platforms. We present data on the execution of the anti-Xa assay in this microfluidic format, demonstrating that the assay can be used to measure heparin in human plasma samples from 0 to 0.8 U/ml, with average assay reproducibility of 8% and a rapid result obtained within 60 seconds. Results indicate that with further development, the fluorogenic anti-Xa assay and device could become a successful method for monitoring anticoagulant therapy.

  14. Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

    PubMed

    Podoplelova, Nadezhda A; Sveshnikova, Anastasia N; Kurasawa, James H; Sarafanov, Andrey G; Chambost, Herve; Vasil'ev, Sergey A; Demina, Irina A; Ataullakhanov, Fazly I; Alessi, Marie-Christine; Panteleev, Mikhail A

    2016-06-01

    Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow. PMID:26874201

  15. Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

    PubMed Central

    Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

  16. Coagulation factor Xa drives tumor cells into apoptosis through BH3-only protein Bim up-regulation

    SciTech Connect

    Borensztajn, Keren S. . E-mail: K.S.Borensztajn@amc.uva.nl; Bijlsma, Maarten F.; Groot, Angelique P.; Brueggemann, Lois W.; Versteeg, Henri H.; Reitsma, Pieter H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

    2007-07-15

    Coagulation Factor (F)Xa is a serine protease that plays a crucial role during blood coagulation by converting prothrombin into active thrombin. Recently, however, it emerged that besides this role in coagulation, FXa induces intracellular signaling leading to different cellular effects. Here, we show that coagulation factor (F)Xa drives tumor cells of epithelial origin, but not endothelial cells or monocytes, into apoptosis, whereas it even enhances fibroblast survival. FXa signals through the protease activated receptor (PAR)-1 to activate extracellular-signal regulated kinase (ERK) 1/2 and p38. This activation is associated with phosphorylation of the transcription factor CREB, and in tumor cells with up-regulation of the BH3-only pro-apoptotic protein Bim, leading to caspase-3 cleavage, the main hallmark of apoptosis. Transfection of tumor cells with dominant negative forms of CREB or siRNA for either PAR-1, Bim, ERK1 and/or p38 inhibited the pro-apoptotic effect of FXa. In fibroblasts, FXa-induced PAR-1 activation leads to down-regulation of Bim and pre-treatment with PAR-1 or Bim siRNA abolishes proliferation. We thus provide evidence that beyond its role in blood coagulation, FXa plays a key role in cellular processes in which Bim is the central player in determining cell survival.

  17. Ca2+ Switches the Effect of PS-containing Membranes on Factor Xa from Activating to Inhibiting: Implications for Initiation of Blood Coagulation

    PubMed Central

    Koklic, Tilen; Majumder, Rinku; Lentz, Barry R.

    2014-01-01

    Calcium (Ca2+) plays a pivotal role in cellular and organismal physiology. The Ca2+ ion has an intermediate protein-binding affinity, thus it can serve as an on/off switch in regulation of different biochemical processes. The serum level of ionized Ca2+ is regulated with normal ionized Ca2+ being in the range from 1.10 to 1.29 mM. Hypocalcaemia (free Ca2+ < 1.1mM) in critically ill patients is commonly accompanied by hemostatic abnormalities, ranging from isolated thrombocytopenia to complex defects such as disseminated intravascular coagulation, commonly thought to be due to insufficient functioning of anticoagulation pathways. A small amount of Factor Xa (fXa) produced by Factor VIIa and exposed tissue factor is key to initiating blood coagulation by producing enough thrombin to induce later stages of coagulation. FXa must bind to phosphatidylserine (PS)-containing membranes to produce thrombin at a physiologically significant rate. In this work, we show that overall fXa activity on PS-containing membranes is sharply regulated by a “Ca2+ switch” centered at 1.16 mM, below which fXa is active and above which fXa forms inactive dimers on PS-exposing membranes. Our data lead to a mathematical model that predicts the variation of fXa activity as a function of both calcium and membrane concentrations. Because the critical Ca2+ concentration is at the lower end of the normal plasma ionized Ca2+ concentration range, we propose a new regulatory mechanism by which local Ca2+ concentration switches fXa from an intrinsically active form to a form requiring its cofactor (fVa) to achieve significant activity. PMID:24920080

  18. Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

    PubMed Central

    Iyer, Janaki Krishnamurthy; Shih, Norrapat; Majumder, Munmi; Mattaparthi, Venkata Satish Kumar; Mukhopadhyay, Rupak; Doley, Robin

    2016-01-01

    In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. PMID:27089306

  19. Pharmacokinetics and Pharmacodynamics of Edoxaban, a Non-Vitamin K Antagonist Oral Anticoagulant that Inhibits Clotting Factor Xa.

    PubMed

    Parasrampuria, Dolly A; Truitt, Kenneth E

    2016-06-01

    Edoxaban, a once daily non-vitamin K antagonist oral anticoagulant, is a direct, selective, reversible inhibitor of factor Xa (FXa). In healthy subjects, single oral doses of edoxaban result in peak plasma concentrations within 1.0-2.0 h of administration, followed by a biphasic decline. Exposure is approximately dose proportional for once daily doses of 15-150 mg. Edoxaban is predominantly absorbed from the upper gastrointestinal tract, and oral bioavailability is approximately 62 %. Food does not affect total exposure to edoxaban. The terminal elimination half-life in healthy subjects ranges from 10 to 14 h, with minimal accumulation upon repeat once daily dosing up to doses of 120 mg. The steady-state volume of distribution is approximately 107 L, and total clearance is approximately 22 L/h; renal clearance accounts for approximately 50 % of total clearance, while metabolism and biliary secretion account for the remaining 50 %. Intrinsic factors, such as age, sex and race, do not affect edoxaban pharmacokinetics after renal function is taken into account. Oral administration of edoxaban results in rapid changes in anticoagulatory biomarkers, with peak effects on anticoagulation markers (such as anti-FXa), the prothrombin time and the activated partial thromboplastin time occurring within 1-2 h of dosing. PMID:26620048

  20. Selective inhibition of factor Xa during thrombolytic therapy markedly improves coronary artery patency in a canine model of coronary thrombosis.

    PubMed

    Nicolini, F A; Lee, P; Malycky, J L; Lefkovits, J; Kottke-Marchant, K; Plow, E F; Topol, E J

    1996-01-01

    The success of current thrombolytic strategies is undermined by ongoing thrombin activity, but it is uncertain whether prevention of thrombin generation or direct thrombin antagonism is effective in achieving more optimal thrombolysis. To address this question, 24 dogs with electrically induced coronary thrombus undergoing thrombolysis with tissue-type plasminogen activator (1 mg/kg) over 20 min, were given one of the following adjunctive regimens in a random fashion. Twelve dogs received saline, and served as the control group; a direct thrombin antagonist, hirudin, was given at a dose of 20 micrograms/kg/min for 90 min to six dogs, and a selective factor Xa inhibitor, tick anticoagulant peptide (TAP), was administered to six dogs at a dose of 30 micrograms/kg/min for 90 min. The time to reperfusion was similar in the saline and hirudin groups (34 +/- 4 vs 37 +/- 7 min; P = NS) but shorter in the TAP group (21 +/- 4 min; P < 0.05). Coronary blood flow was restored to 100% of its baseline value for 7 +/- 2 min in control dogs, and for 20 +/- 6 min in the hirudin group (P < 0.05). In the TAP group, coronary blood flow was restored to 100% of its baseline value for more than 120 min in all dogs (P < 0.01 vs others treatments). Reocclusion occurred in 89% and 50% of dogs receiving saline and hirudin, respectively (P = NS), but in none of the TAP-treated dogs (P < 0.01). Plasma fibrinopeptide A (FpA) and thrombin-antithrombin III complex (TAT) levels were determined in all dogs as indicators of thrombin activation. In the saline group, FpA and TAT during reperfusion were 19 +/- 2 ng/ml and 104 +/- 24 ng/ml respectively (P < 0.02 vs baseline) indicating high thrombin activity. In contrast, during reperfusion in hirudin-treated dogs FpA and TAT remained similar to baseline (10 +/- 3 ng/ml and 53 +/- 4 ng/ml respectively; both P < 0.05 vs saline). Reperfusion in TAP-treated dogs did not alter FpA and TAT in plasma, which remained similar to baseline (9 +/- 1 ng/ml and 39

  1. Reproducibility of the anti-Factor Xa and anti-Factor IIa assays applied to enoxaparin solution.

    PubMed

    Martinez, Céline; Savadogo, Adama; Agut, Christophe; Anger, Pascal

    2013-01-01

    Enoxaparin is a widely used subcutaneously administered antithrombotic agent comprising a complex mixture of glycosaminoglycan chains. Owing to this complexity, its antithrombotic potency cannot be defined by physicochemical methods and is therefore evaluated using an enzymatic assay of anti-Xa and anti-IIa activity. Maintaining consistent anti-Xa activity in the final medicinal product allows physicians to ensure administration of the appropriate dosage to their patients. Bioassays are usually complex and display poorer reproducibility than physicochemical tests such as HPLC assays. Here, we describe the implementation of a common robotic platform and standard release potency testing procedures for enoxaparin sodium injection (Lovenox, Sanofi, Paris, France) products at seven quality control sites within Sanofi. Qualification and analytical procedures, as well as data handling, were optimized and harmonized to improve assay reproducibility. An inter-laboratory study was performed in routine-release conditions. The coefficients of variation for repeatability and reproducibility in assessments of anti-Xa activity were 1.0% and 1.2%, respectively. The tolerance interval in reproducibility precision conditions, expressed as percentage potency, was 96.8-103.2% of the drug product target of 10,000 IU/ml, comparing favorably with the United States of America Pharmacopeia specification (90-110%). The maximum difference between assays in two different laboratories is expected to be 4.1%. The reproducibility characteristics of anti-IIa activity assessments were found to be similar. These results demonstrate the effectiveness of the standardization process established and allow for further improvements to quality control in Lovenox manufacture. This process guarantees closeness between actual and target potencies, as exemplified by the results of release assays obtained during a three-year period. PMID:23644908

  2. [Advances in antithrombotic treatment--antithrombotics with anti-Xa effect].

    PubMed

    Bátorová, A

    2009-03-01

    The use of anticoagulants in the prophylaxis and treatment of arterial and venous thrombosis has substantially expanded during the last years. Increasing knowledge about the inherited and acquired thrombophilia and the risk factors predisposing to the recurrency of thromboembolic events result in a new indications for primary and secondary thromboprophylaxis with prolonged or even life-long duration. The limitations of classical anticoagulans, heparin and vitamin K antagonists support the development of medicaments with a specific antithrombotic action. The new generation anticoagulants inhibit in a specific way either particular coagulation enzyme or hemostasis activation step. Based on the in vitro studies and extensive clinical observations the activated factor Xa (FXa) seems to be one of the most advantageous targets for a specific action of perspective antithrombotic agents. Two selective F Xa inhibitors have been approved for clinical use: fondaparinux is an indirect parenteral F Xa inhibitor, and most recently approved rivaroxaban is the first oral anti-Xa inhibitor. Other anti-Xa molecules are under development for either parenteral (idraparinux, DX-9065a) or oral use (razaxaban, apixaban, rivaroxaban, LY-51, 7717, BMS-56247 a DU-176b). PMID:19378862

  3. Contribution of Amino Acid Region 659−663 of Factor Va Heavy Chain to the Activity of Factor Xa within Prothrombinase†,‡

    PubMed Central

    2010-01-01

    Factor Va, the cofactor of prothrombinase, is composed of heavy and light chains associated noncovalently in the presence of divalent metal ions. The COOH-terminal region of the heavy chain contains acidic amino acid clusters that are important for cofactor activity. In this work, we have investigated the role of amino acid region 659−663, which contains five consecutive acidic amino acid residues, by site-directed mutagenesis. We have generated factor V molecules in which all residues were mutated to either lysine (factor V5K) or alanine (factor V5A). We have also constructed a mutant molecule with this region deleted (factor VΔ659−663). The recombinant molecules along with wild-type factor V (factor VWT) were transiently expressed in mammalian cells, purified, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to that of factor VaWT. Kinetic analyses of prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all mutant molecules for factor Xa was similar to that for factor VaWT. Gel electrophoresis analyses of plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg320 and Arg271 by prothrombinase assembled with the mutant molecules, resulting in meizothrombin lingering throughout the activation process. These results were confirmed after analysis of the cleavage of FPR-meizothrombin. Our findings provide new insights into the structural contribution of the acidic COOH-terminal region of factor Va heavy chain to factor Xa activity within prothrombinase and demonstrate that amino acid region 659−663 from the heavy chain of the cofactor contributes to the regulation of the rate of cleavage of prothrombin by prothrombinase. PMID:20722419

  4. Independent anti-angiogenic capacities of coagulation factors X and Xa.

    PubMed

    Lange, Soledad; Gonzalez, Ibeth; Pinto, Mauricio P; Arce, Maximiliano; Valenzuela, Rodrigo; Aranda, Evelyn; Elliot, Matias; Alvarez, Marjorie; Henriquez, Soledad; Velasquez, Ethel V; Orge, Felipe; Oliva, Barbara; Gonzalez, Pamela; Villalon, Manuel; Cautivo, Kelly M; Kalergis, Alexis M; Pereira, Karla; Mendoza, Camila; Saez, Claudia; Kato, Sumie; Cuello, Mauricio A; Parborell, Fernanda; Irusta, Griselda; Palma, Veronica; Allende, Miguel L; Owen, Gareth I

    2014-11-01

    Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX. PMID:24615682

  5. A new peptide (Ruviprase) purified from the venom of Daboia russelii russelii shows potent anticoagulant activity via non-enzymatic inhibition of thrombin and factor Xa.

    PubMed

    Thakur, Rupamoni; Kumar, Ashok; Bose, Biplab; Panda, Dulal; Saikia, Debashree; Chattopadhyay, Pronobesh; Mukherjee, Ashis K

    2014-10-01

    Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E(1)-V(2)-X(3)-W(4)-W(5)-W(6)-A(7)-Q(8)-L(9)-S(10)) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 μM and 0.46 μM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 μM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising. PMID:25038567

  6. Pharmacokinetics, biotransformation, and mass balance of edoxaban, a selective, direct factor Xa inhibitor, in humans.

    PubMed

    Bathala, Mohinder S; Masumoto, Hiroshi; Oguma, Toshihiro; He, Ling; Lowrie, Chris; Mendell, Jeanne

    2012-12-01

    This study determined the mass balance and pharmacokinetics of edoxaban in humans after oral administration of [¹⁴C]edoxaban. After oral administration of 60 mg (as active moiety) of [¹⁴C]edoxaban to six healthy male subjects, serial blood/plasma and urinary and fecal samples were collected for up to 168 h postdose. All samples were analyzed for total radioactivity by liquid scintillation counting and for concentrations of edoxaban and four metabolites in plasma, urine, and fecal samples by either high-performance liquid chromatography/tandem mass spectrometry method using multiple reaction modes, or a liquid chromatography radiometric method. The mean recovery of radioactivity was >97% of the administered radioactive dose, with 62.2% eliminated in feces and 35.4% in urine. Unchanged edoxaban accounted for the majority of radioactivity, with 49.1 and 23.8% of the dose as parent observed in feces and urine, respectively. Unchanged edoxaban was the most abundant species in plasma, with a mean area under the curve (AUC)(0-∞) of 1596 ng · h/ml. The next most abundant species was metabolite M4, with a mean AUC(0-∞) 147 ng · h/ml. The mass balance of edoxaban was well described, with unchanged edoxaban as the most abundant component of total radioactivity. Edoxaban is eliminated through multiple pathways, but each accounts for only a small amount of overall elimination. PMID:22936313

  7. Clinical Scenarios for Discordant Anti-Xa.

    PubMed

    Vera-Aguilera, Jesus; Yousef, Hindi; Beltran-Melgarejo, Diego; Teng, Teng Hugh; Jan, Ramos; Mok, Mary; Vera-Aguilera, Carlos; Moreno-Aguilera, Eduardo

    2016-01-01

    Anti-Xa test measures the activity of heparin against the activity of activated coagulation factor X; significant variability of anti-Xa levels in common clinical scenarios has been observed. Objective. To review the most common clinical settings in which anti-Xa results can be bias. Evidence Review. Guidelines and current literature search: we used PubMed, Medline, Embase, and MEDION, from 2000 to October 2013. Results. Anti-Xa test is widely used; however the assay underestimates heparin concentration in the presence of significant AT deficiency, pregnancy, end stage renal disease, and postthrombolysis and in patients with hyperbilirubinemia; limited published data evaluating the safety and effectiveness of anti-Xa assays for managing UH therapy is available. Conclusions and Relevance. To our knowledge this is the first paper that summarizes the most common causes in which this assay can be affected, several "day to day" clinical scenarios can modify the outcomes, and we concur that these rarely recognized scenarios can be affected by negative outcomes in the daily practice. PMID:27293440

  8. Clinical Scenarios for Discordant Anti-Xa

    PubMed Central

    Vera-Aguilera, Jesus; Yousef, Hindi; Beltran-Melgarejo, Diego; Teng, Teng Hugh; Jan, Ramos; Mok, Mary; Vera-Aguilera, Carlos; Moreno-Aguilera, Eduardo

    2016-01-01

    Anti-Xa test measures the activity of heparin against the activity of activated coagulation factor X; significant variability of anti-Xa levels in common clinical scenarios has been observed. Objective. To review the most common clinical settings in which anti-Xa results can be bias. Evidence Review. Guidelines and current literature search: we used PubMed, Medline, Embase, and MEDION, from 2000 to October 2013. Results. Anti-Xa test is widely used; however the assay underestimates heparin concentration in the presence of significant AT deficiency, pregnancy, end stage renal disease, and postthrombolysis and in patients with hyperbilirubinemia; limited published data evaluating the safety and effectiveness of anti-Xa assays for managing UH therapy is available. Conclusions and Relevance. To our knowledge this is the first paper that summarizes the most common causes in which this assay can be affected, several “day to day” clinical scenarios can modify the outcomes, and we concur that these rarely recognized scenarios can be affected by negative outcomes in the daily practice. PMID:27293440

  9. Lessons learned, DC-XA

    SciTech Connect

    Steinmeyer, D.

    1997-01-01

    The Delta Clipper-Experimental A (DC-XA) program was conceived and specifically developed to provide a low cost reusable flight vehicle testbed for demonstrating performance and operability testing of advanced technologies required for the development of next generation Reusable Launch Vehicles (RLVs). The three primary program objectives addressed were: To integrate a variety of advanced launch vehicle technology components into the DC-XA flight vehicle testbed. Demonstrate performance, operability, and supportability of Advanced Launch Technologies (ALT) components through ground and flight testing of the DC-XA. Demonstrate rapid prototyping of hardware and software in a combined government, industry cooperative effort. {copyright} {ital 1997 American Institute of Physics.}

  10. Maximize x(a - x)

    ERIC Educational Resources Information Center

    Lange, L. H.

    1974-01-01

    Five different methods for determining the maximizing condition for x(a - x) are presented. Included is the ancient Greek version and a method attributed to Fermat. None of the proofs use calculus. (LS)

  11. Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

    PubMed Central

    Thomas, Owain; Lybeck, Emanuel; Strandberg, Karin; Tynngård, Nahreen; Schött, Ulf

    2015-01-01

    Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is

  12. Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors

    SciTech Connect

    Fuchs, H.E.; Trapp, H.G.; Griffith, M.J.; Roberts, H.R.; Pizzo, S.V.

    1984-06-01

    The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the /sup 125/I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the /sup 125/I-Factor IXa was bound to ATIII by 1 min. The distribution of /sup 125/I-Factor IXa in mouse plasma was similar. The clearance of /sup 125/I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the /sup 125/I-Factor IXa was bound to ATIII. Organ distribution studies with /sup 125/I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.

  13. Improvement of low bioavailability of a novel factor Xa inhibitor through formulation of cationic additives in its oral dosage form.

    PubMed

    Fujii, Yoshimine; Kanamaru, Taro; Kikuchi, Hiroshi; Nakagami, Hiroaki; Yamashita, Shinji; Akashi, Mitsuru; Sakuma, Shinji

    2011-12-15

    A clinical trial of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naphtyl) propanoic acid (DX-9065) revealed that its oral bioavailability was only 3% when it was administered as a conventional capsule formulation. The low bioavailability of DX-9065 was likely caused by both its poor membrane permeability and its electrostatic interaction with anionic bile acids. We hypothesized that DX-9065 absorption would be enhanced when the cationic drug was free from the complex through its replacement with other cationic substances. Polystyrene nanospheres coated with cationic poly(vinylamine) and cholestyramine, which is clinically used as a cholesterol-lowering agent, dramatically prevented DX-9065 from interacting with chenodeoxycholic acid in vitro. Successive animal experiments showed that bioavailability of DX-9065 administered with these cationic substances was 2-3 times that of DX-9065 administered solely. A dry syrup formulation with one-half of a minimal cholesterol-lowering equivalent dose of cholestyramine was designed, and the clinical trial was resumed. A 1.3-fold increase in bioavailability of DX-9065 was observed when the dry syrup was administered. We successfully demonstrated that DX-9065 absorption was enhanced when the drug was administered with cationic additives; however, it appeared that the absorption-enhancing function of cholestyramine largely depended on its dose. The dose escalation is probably prerequisite for the significant improvement of DX-9065 absorption in humans. PMID:22001539

  14. Inhibitors of the Metalloproteinase Anthrax Lethal Factor.

    PubMed

    Goldberg, Allison B; Turk, Benjamin E

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LFinhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and highthroughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection. PMID:27072692

  15. Optogenetic Inhibitor of the Transcription Factor CREB.

    PubMed

    Ali, Ahmed M; Reis, Jakeb M; Xia, Yan; Rashid, Asim J; Mercaldo, Valentina; Walters, Brandon J; Brechun, Katherine E; Borisenko, Vitali; Josselyn, Sheena A; Karanicolas, John; Woolley, G Andrew

    2015-11-19

    Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events. PMID:26590638

  16. Inhibition of Hageman factor (factor XII) by popcorn inhibitor.

    PubMed

    Kambhu, S A; Ratnoff, O D; Everson, B

    1985-05-01

    A protein derived from sweet corn or popcorn inhibits the enzymatic activity of the carboxy-terminal fragment of Hageman factor (HFf) and of ellagic acid-activated Hageman factor (HF, factor XII). Not clarified is whether the inhibitor is directed at the active site of HF. Filtration of normal plasma or purified HF through columns of popcorn inhibitor bound to agarose gels demonstrated that HF was bound to these gels and could then be eluted by buffers containing 2.0 mol/L sodium chloride. The eluted HF was in the precursor form. Thus, popcorn inhibitor appeared to attach to a point on the carboxy-terminal HFf that was distinct from the enzymatically active site of this clotting factor. PMID:3989356

  17. Polyphenol Compound as a Transcription Factor Inhibitor.

    PubMed

    Park, Seyeon

    2015-11-01

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)). PMID:26529010

  18. Polyphenol Compound as a Transcription Factor Inhibitor

    PubMed Central

    Park, Seyeon

    2015-01-01

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)). PMID:26529010

  19. Substrate-Guided Design of Selective FXIIa Inhibitors Based on the Plant-Derived Momordica cochinchinensis Trypsin Inhibitor-II (MCoTI-II) Scaffold.

    PubMed

    Swedberg, Joakim E; Mahatmanto, Tunjung; Abdul Ghani, Hafiza; de Veer, Simon J; Schroeder, Christina I; Harris, Jonathan M; Craik, David J

    2016-08-11

    Thrombosis is a leading cause of morbidity and mortality associated with cardiovascular diseases. Inhibition of factor XIIa (FXIIa) provides thrombus protection without bleeding complications. Here, we defined the extended substrate specificity of FXIIa and its close homologue factor Xa and used these data, together with inhibitor-based and structure-guided methods, to engineer selective FXIIa inhibitors based on Momordica cochinchinensis trypsin inhibitor-II. PMID:27434175

  20. Novel inhibitors of Anthrax edema factor

    PubMed Central

    Chen, Deliang; Misra, Milind; Sower, Laurie; Peterson, Johnny W.; Kellogg, Glen E.; Schein, Catherine H.

    2008-01-01

    Several pathogenic bacteria produce adenylyl cyclase toxins, such as the edema factor (EF) of Bacillus anthracis. These disturb cellular metabolism by catalyzing production of excessive amounts of the regulatory molecule cAMP. Here, a structure-based method, where a 3D- pharmacophore that fit the active site of EF was constructed from fragments, was used to identify non-nucleotide inhibitors of EF. A library of small molecule fragments was docked to the EF- active site in existing crystal structures and those with the highest HINT scores were assembled into a 3D-pharmacophore. About 10,000 compounds, from over 2.7 million compounds in the ZINC database, had a similar molecular framework. These were ranked according to their docking scores, using methodology that was shown to achieve maximum accuracy (i.e., how well the docked position matched the experimentally determined site for ATP analogues in crystal structures of the complex). Finally, 19 diverse compounds with the best AutoDock binding/docking scores were assayed in a cell based assay for their ability to reduce cAMP secretion induced by EF. Four of the test compounds, from different structural groups, inhibited in the low micromolar range. One of these has a core structure common to phosphatase inhibitors previously identified by high-throughput assays of a diversity library. Thus, the fragment based pharmacophore identified a small number of diverse compounds for assay, and greatly enhanced the selection process of advanced lead compounds for combinatorial design. PMID:18620864

  1. Prognostic Factors in Cholinesterase Inhibitor Poisoning

    PubMed Central

    Sun, In O; Yoon, Hyun Ju; Lee, Kwang Young

    2015-01-01

    Background Organophosphates and carbamates are insecticides that are associated with high human mortality. The purpose of this study is to investigate the prognostic factors affecting survival in patients with cholinesterase inhibitor (CI) poisoning. Material/Methods This study included 92 patients with CI poisoning in the period from January 2005 to August 2013. We divided these patients into 2 groups (survivors vs. non-survivors), compared their clinical characteristics, and analyzed the predictors of survival. Results The mean age of the included patients was 56 years (range, 16–88). The patients included 57 (62%) men and 35 (38%) women. When we compared clinical characteristics between the survivor group (n=81, 88%) and non-survivor group (n=11, 12%), there were no differences in renal function, pancreatic enzymes, or serum cholinesterase level, except for serum bicarbonate level and APACHE II score. The serum bicarbonate level was lower in non-survivors than in survivors (12.45±2.84 vs. 18.36±4.73, P<0.01). The serum APACHE II score was higher in non-survivors than in survivors (24.36±5.22 vs. 12.07±6.67, P<0.01). The development of pneumonia during hospitalization was higher in non-survivors than in survivors (n=9, 82% vs. n=31, 38%, P<0.01). In multiple logistic regression analysis, serum bicarbonate concentration, APACHE II score, and pneumonia during hospitalization were the important prognostic factors in patients with CI poisoning. Conclusions Serum bicarbonate and APACHE II score are useful prognostic factors in patients with CI poisoning. Furthermore, pneumonia during hospitalization was also important in predicting prognosis in patients with CI poisoning. Therefore, prevention and active treatment of pneumonia is important in the management of patients with CI poisoning. PMID:26411989

  2. Small Molecule Inhibitors of Anthrax Lethal Factor Toxin

    PubMed Central

    Williams, John D.; Khan, Atiyya R.; Cardinale, Steven C.; Butler, Michelle M.; Bowlin, Terry L.; Peet, Norton P.

    2014-01-01

    This manuscript describes the preparation of new small molecule inhibitors of Bacillus anthracis lethal factor. Our starting point was the symmetrical, bis-quinolinyl compound 1 (NSC 12155). Optimization of one half of this molecule led to new LF inhibitors that were desymmetrized to afford more drug-like compounds. PMID:24290062

  3. Tumor necrosis factor inhibitors – state of knowledge

    PubMed Central

    Lis, Krzysztof; Kuzawińska, Olga

    2014-01-01

    Tumor necrosis factor (TNF) is considered a major proinflammatory cytokine, affecting various aspects of the immune reaction. All five TNF inhibitors currently available on the market (i.e., etanercept, infliximab, adalimumab, certolizumab and golimumab) are top sellers, although indicated only in autoimmune diseases, including rheumatoid arthritis, Crohn's disease and psoriasis. This article briefly discusses the background and place for TNF inhibitors in modern therapy. The main safety aspects of TNF inhibitor administration are described in particular, with special consideration of the available meta-analyses. Finally, perspectives on the next-generation TNF inhibitors and their use in the clinic are given. PMID:25624856

  4. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus

    PubMed Central

    Girish, Vallerinteavide Mavelli; Kini, R. Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki’ and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  5. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus.

    PubMed

    Girish, Vallerinteavide Mavelli; Kini, R Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki' and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  6. Tissue factor pathway inhibitor levels in patients with homocystinuria.

    PubMed

    Cella, G; Burlina, A; Sbarai, A; Motta, G; Girolami, A; Berrettini, M; Strauss, W

    2000-06-01

    Thrombotic events are a well-recognized complication of homocystinuria. However, the mechanisms involved in the atherogenic and thrombotic effects of homocyst(e)ine remain incompletely understood. The objective of this study was to determine the role of endothelial cell activation/damage as indicated by levels of thrombomodulin, tissue factor and tissue factor pathway inhibitor, and factor VII activity in patients with homocystinuria. Six patients with homocystinuria, nonresponsive to pyridoxine, treated only with trimethylglycine (betaine) were injected with a bolus of 20 IU/kg body weight of unfractionated commercial heparin to induce the release of tissue factor pathway inhibitor from the vascular endothelium. Tissue factor, thrombomodulin, and factor VII activity were measured by enzyme-linked immunosorbent assay and clotting assay before heparin administration. Tissue factor pathway inhibitor antigen and activity were measured before and 5 minutes after the bolus of heparin. Levels of homocyst(e)ine were elevated (patients: 144.2+/-19.2 micromol/L; controls: 10.2+/-0.9 micromol/L); however, levels of thrombomodulin, tissue factor, and tissue factor pathway inhibitor antigen were not statistically different from the control group. In contrast, tissue factor pathway inhibitor activity showed a significantly increased level (patients: 2.09+/-0.34 U/L; controls: 1.14+/-0.20 U/L; p<0.05) that was correlated with homocyst(e)ine. Factor VII activity was significantly decreased (patients: 64.7+/-5.1%; controls: 91.4+/-4.7%; p<0.05) and inversely correlated with homocyst(e)ine. After heparin the patients released higher amounts of tissue factor pathway inhibitor antigen and activity compared with the control group; however, the difference was not statistically significant. Although not treated with antithrombotic drugs, none of the patients had any thromboembolic complications after starting betaine. In addition to betaine treatment, the enhanced factor pathway

  7. Hypoxia inducible factor pathway inhibitors as anticancer therapeutics

    PubMed Central

    Burroughs, Sarah K; Kaluz, Stefan; Wang, Danzhu; Wang, Ke

    2013-01-01

    Hypoxia is a significant feature of solid tumor cancers. Hypoxia leads to a more malignant phenotype that is resistant to chemotherapy and radiation, is more invasive and has greater metastatic potential. Hypoxia activates the hypoxia inducible factor (HIF) pathway, which mediates the biological effects of hypoxia in tissues. The HIF complex acts as a transcription factor for many genes that increase tumor survival and proliferation. To date, many HIF pathway inhibitors indirectly affect HIF but there have been no clinically approved direct HIF inhibitors. This can be attributed to the complexity of the HIF pathway, as well as to the challenges of inhibiting protein–protein interactions. PMID:23573973

  8. Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis

    PubMed Central

    Herman, Michael P.; Sukhova, Galina K.; Kisiel, Walter; Foster, Don; Kehry, Marilyn R.; Libby, Peter; Schönbeck, Uwe

    2001-01-01

    Degradation of ECM, particularly interstitial collagen, promotes plaque instability, rendering atheroma prone to rupture. Previous studies implicated matrix metalloproteinases (MMPs) in these processes, suggesting that dysregulated MMP activity, probably due to imbalance with endogenous inhibitors, promotes complications of atherosclerosis. We report here that the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) can function as an MMP inhibitor. TFPI-2 diminished the ability of the interstitial collagenases MMP-1 and MMP-13 to degrade triple-helical collagen, the primary load-bearing molecule of the ECM within human atheroma. In addition, TFPI-2 also reduced the activity of the gelatinases MMP-2 and MMP-9. In contrast to the “classical” tissue inhibitors of MMPs (TIMPs), TFPI-2 expression in situ correlated inversely with MMP levels in human atheroma. TFPI-2 colocalized primarily with smooth muscle cells in the normal media as well as the plaque’s fibrous cap. Conversely, the macrophage-enriched shoulder region, the prototypical site of matrix degradation and plaque rupture, stained only weakly for TFPI-2 but intensely for gelatinases and interstitial collagenases. Evidently, human mononuclear phagocytes, an abundant source of MMPs within human atheroma, lost their ability to express this inhibitor during differentiation in vitro. These findings establish a new, anti-inflammatory function of TFPI-2 of potential pathophysiological significance for human diseases, including atherosclerosis. PMID:11342575

  9. A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

    PubMed

    Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

    2016-05-01

    We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. PMID:26974491

  10. Cutaneous adverse reactions specific to epidermal growth factor receptor inhibitors

    PubMed Central

    Lupu, I; Voiculescu, VM; Bacalbasa, N; Prie, BE; Cojocaru, I; Giurcaneanu, C

    2015-01-01

    Classical antineoplastic therapy is encumbered by extensively studied adverse reactions, most often of systemic nature. The emergence of new generations of anticancer treatments, including epidermal growth factor receptor inhibitors, besides improving the response to treatment and the survival rate, is accompanied by the occurrence of new specific side effects, incompletely studied. These side effects are most often cutaneous (hand foot syndrome, acneiform reactions), and in some cases are extremely severe, requiring dose reduction or drug discontinuation. The prevention of the cutaneous adverse effects and their treatment require a close collaboration between the oncologist and the dermatologist. The occurrence of some of these skin adverse effects may be a favorable prognostic factor for the response to the cancer treatment and the overall survival. Abbreviations: EGFR = epidermal growth factor receptors; EGFRI = epidermal growth factor receptors inhibitors PMID:26361513

  11. Unexpected postpartum hemorrhage due to an acquired factor VIII inhibitor.

    PubMed

    Paidas, Michael J; Hossain, Nazli

    2014-09-01

    Unexplained postpartum hemorrhage (PPH) refractory to standard hemostatic measures should trigger a heightened clinical suspicion of an acquired bleeding disorder. When hemostatic medical interventions and surgical procedures fail to control the bleeding, then significant postoperative blood loss, debilitating morbidity, loss of fertility, and death may occur. In the setting of an autoantibody inhibitor to factor VIII (FVIII), control of life-threatening PPH and avoidance of subsequent bleeding episodes depends on a timely and accurate diagnosis, prompt hemostatic treatment and eradication of FVIII inhibitors, and appropriate long-term patient care and management. Acquired postpartum hemophilia due to a FVIII inhibitor is a rare cause of PPH; however, delayed treatment can lead to increased maternal morbidity and mortality. Acquired FVIII inhibitors also pose an emerging bleeding threat to the neonate as a result of possible transplacental transfer of FVIII autoantibodies to the fetus during the last trimester of pregnancy. The purpose of this review is to increase awareness among hematologists and obstetricians/gynecologists regarding the occurrence of FVIII neutralizing autoantibodies as a cause of PPH, and emphasize the importance of collaboration between obstetrician/gynecologists and hematology specialists to optimize the diagnostic evaluation, treatment, and long-term management of women who experience PPH due to an acquired FVIII inhibitor. PMID:24338123

  12. Inhibition of expression of monocyte interleukin-1 by inhibitors of Hageman factor (factor XII).

    PubMed

    Ratnoff, O D; Voytus, J A; Toossi, Z

    1995-02-01

    In an earlier study, activated species of Hageman factor (factor XII) induced elaboration of interleukin-1 by human monocytes. These observations did not address whether Hageman factor participated in endotoxin-induced release of interleukin-1. To examine this question, the release of interleukin-1 by endotoxin-stimulated human mononuclear cells was measured in the presence of popcorn inhibitor, a specific inhibitor of Hageman factor. In the experiments herein described, popcorn inhibitor sharply decreased the release of interleukin-1 by human mononuclear cells that were incubated with endotoxin. This observation suggests that Hageman factor may play a role in the elaboration of interleukin-1 by human mononuclear cells. Conforming with this view, the addition of antiserum directed against Hageman factor inhibited the release of interleukin-1 from endotoxin-stimulated mononuclear cells. PMID:7844472

  13. Development of Trypsin-Like Serine Protease Inhibitors as Therapeutic Agents: Opportunities, Challenges, and their Unique Structure-Based Rationales.

    PubMed

    Liang, Guyan; Bowen, J Phillip

    2016-01-01

    There has been a revolution in the development of effective, small-molecule anticoagulants and antiplatelet agents. Numerous trypsin-like serine proteases have been under active pursuit as therapeutic targets. Important examples include thrombin, factor VIIa, factor Xa, and β-tryptase with indications ranging from thrombosis and inflammation to asthma and chronic obstructive pulmonary disease (COPD). Trypsin-like serine proteases exhibit a highly similar tertiary folding pattern, especially for the region near the substrate binding pocket that includes the conserved catalytic triad consisting of histidine 57, aspartic acid 102, and serine 195. A rich collection of X-ray structures for many trypsin-like serine proteases is available, which greatly facilitated the optimization of small organic inhibitors as therapeutic agents. The present review surveyed those inhibitors disclosed in peer-reviewed scientific journals and patent publications with a special focus on structural features and protein-inhibitor interactions that implicated the inhibitor optimization process. The role played by the residue 190 of trypsin-like serine proteases is critical. While many inhibitors without a basic group have progressed into the clinic for ones with alanine 190, the task for those with serine 190 remains extremely challenging, if not impossible. In addition to warfarin, heparin, and low molecular weight heparins (LMWHs), treatment options have expanded with the development and approval of the new oral anticoagulants (NOACs). The NOACs are superior to vitamin K antagonists in terms of rapid onset, pharmacokinetics, drug/food interactions, and regular coagulation monitoring; but one serious drawback is the lack of an effective antidote at this time. Apixaban (Eliquis), rivaroxaban (Xarelto), and edoxaban (Savaysa) are the new Xa inhibitors that have been recently approved by the U.S. FDA and are in current clinical practice. These drugs bind to the active site of factor Xa (fXa

  14. A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)*

    PubMed Central

    Bai, Fengwei; Asojo, Oluwatoyin A.; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A.; Leng, Lin; Koski, Raymond A.; Powell, Thomas J.; Bucala, Richard; Anthony, Karen G.

    2012-01-01

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases. PMID:22782901

  15. Potent inhibitors of anthrax lethal factor from green tea

    PubMed Central

    Dell'Aica, Isabella; Donà, Massimo; Tonello, Fiorella; Piris, Alejandro; Mock, Michèle; Montecucco, Cesare; Garbisa, Spiridione

    2004-01-01

    The anthrax lethal factor (LF) has a major role in the development of anthrax. LF is delivered by the protective antigen (PA) inside the cell, where it exerts its metalloprotease activity on the N-terminus of MAPK-kinases. PA+LF are cytotoxic to macrophages in culture and kill the Fischer 344 rat when injected intravenously. We describe here the properties of some polyphenols contained in green tea as powerful inhibitors of LF metalloproteolytic activity, and how the main catechin of green tea, (−)epigallocatechin-3-gallate, prevents the LF-induced death of macrophages and Fischer 344 rats. PMID:15031715

  16. Draft genome sequence of Enterobacter cloacae subsp. cloacae strain 08XA1, a fecal bacterium of giant pandas.

    PubMed

    Yan, Yue; Zhao, Chuan-Wu; Zhang, Yi-Zheng; Zhang, Zhi-He; Pan, Guang-Lin; Liu, Wen-Wang; Ma, Qing-Yi; Hou, Rong; Tan, Xue-Mei

    2012-12-01

    Enterobacter cloacae, a common pathogenic bacterium, is a Gram-negative bacillus. We analyzed the draft genome of Enterobacter cloacae subsp. cloacae strain 08XA1 from the feces of a giant panda in China. Genes encoding a β-lactamase and efflux pumps, as well as other factors, have been found in the genome. PMID:23209197

  17. Treatment of rheumatoid arthritis with tumour necrosis factor inhibitors

    PubMed Central

    Mewar, Devesh; Wilson, Anthony G

    2011-01-01

    Advances in our understanding of the key mediators of chronic inflammation and tissue damage characteristic of rheumatoid arthritis (RA) have resulted in the development of novel therapies primarily targeting pro-inflammatory cytokines. Inhibitors of tumour necrosis factor (TNF) are the most widely used of the biological therapies at present with five different agents currently available; four are based on monoclonal anti-TNF antibodies and a soluble TNF receptor-Fc fusion protein. Long-term use of these molecules has proven to be highly effective in the majority of patients; however, around one-third have a suboptimal response potentially leading to further cartilage and bone damage, furthermore these agents are expensive compared with conventional therapies such as methotrexate. Many recent studies have attempted to identify therapeutic response biomarkers of TNF inhibitors which could be used to improve therapeutic targeting. The presence of rheumatoid factor and anti-cyclic citullinated protein antibodies, present in around 65% of RA patients, are associated with a poorer response to anti-TNF agents. Poorer response is also associated with levels of C-reactive protein and cartilage degradation product at initiation of treatment. Intriguingly, genetic studies of variants of TNF and of genes encoding members of the Toll-like receptors, nuclear factor-kappa B and p38 mitogen-activated protein kinase signalling families have been associated with response to individual anti-TNF agents. Continued advances in technologies such as ultra high throughput sequencing and proteomics should facilitate the discovery of additional biomarkers of response to anti-TNF resulting in improved disease control and quality of life for RA patients and reduced costs for healthcare funders. PMID:21039421

  18. EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

    PubMed

    Stavik, Benedicte; Espada, Sandra; Cui, Xue Yan; Iversen, Nina; Holm, Sverre; Mowinkel, Marie-Christine; Halvorsen, Bente; Skretting, Grethe; Sandset, Per Morten

    2016-04-01

    Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders. PMID:26826018

  19. Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21.

    PubMed

    Ellur, Ranjith K; Khanna, Apurva; S, Gopala Krishnan; Bhowmick, Prolay K; Vinod, K K; Nagarajan, M; Mondal, Kalyan K; Singh, Nagendra K; Singh, Kuldeep; Prabhu, Kumble Vinod; Singh, Ashok K

    2016-01-01

    Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India's forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region. PMID:27403778

  20. Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21

    PubMed Central

    Ellur, Ranjith K.; Khanna, Apurva; S, Gopala Krishnan.; Bhowmick, Prolay K.; Vinod, K. K.; Nagarajan, M.; Mondal, Kalyan K.; Singh, Nagendra K.; Singh, Kuldeep; Prabhu, Kumble Vinod; Singh, Ashok K.

    2016-01-01

    Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India’s forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region. PMID:27403778

  1. Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains.

    PubMed

    Tian, Dongsheng; Yin, Zhongchao

    2009-01-01

    The vascular pathogen Xanthomonas oryzae pv. oryzae (Xoo) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola (Xoc) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99(A), which specifically induces the expression of the rice resistance gene Xa27, ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host-pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27, progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related (PR) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27. Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc. The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants. PMID:19161350

  2. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

    SciTech Connect

    Kato, Y.; Iwamoto, M. )

    1990-04-05

    The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate. Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, {sup 45}Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of ({sup 35}S)sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.

  3. Factor eight inhibitor bypass activity (FEIBA) in the management of bleeds in hemophilia patients with high-titer inhibitors

    PubMed Central

    Tjønnfjord, Geir E; Andre Holme, Pål

    2007-01-01

    The development of high-titer inhibitors to FVIII and less often to other coagulation factors are the most serious complication of hemophilia therapy and makes treatment of bleeds very challenging. At present, bypassing agents, such as factor eight inhibitor bypass activity (FEIBA) and activated recombinant factor VII (rFVIIa) are the only coagulation factor concentrates available for the treatment of bleeds in inhibitor patients. Both products are effective and safe, and their efficacy has been found to be comparable (approximately 80%) in a recent prospective study. A significant number of patients report a better effect of one or the other of the products, and in a minority of the patients none of the products are particularly effective. The hemostatic efficacy of bypassing agents is not considered equal to that of coagulation factor replacement in patients without inhibitors by most physicians. An improvement in hemostatic efficacy may be achieved by optimizing the dosing of by passing agents. However, the lack of standardized and validated laboratory assays reflecting the hemostatic efficacy of the bypassing agents is an obstacle to this achievement. PMID:17969383

  4. Inhibitors in childhood hemophilia A: genetic and treatment-related risk factors for development and eradication.

    PubMed

    DiMichele, Donna M

    2013-01-01

    The development of neutralizing antibodies remains a serious complication of hemophilia replacement therapy. Factor VIII inhibiting antibodies (inhibitors) occur commonly following replacement therapy in hemophilia A, creating a significant burden of clinical disease. This article will review our current understanding of risk factors and their known impact on inhibitor development in previously untreated or minimally treated children with severe and mild hemophilia A. It will also explore how the most recently elucidated immunology of inhibitor development might hold important clues to more effective inhibitor eradication and prevention in this heavily impacted patient population. PMID:23109404

  5. Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.

    PubMed

    Schmidt, Anja; Brettschneider, Kerstin; Kahle, Jörg; Orlowski, Aleksander; Becker-Peters, Karin; Stichel, Diana; Schulze, Jörg; Braner, Markus; Tampé, Robert; Schwabe, Dirk; Königs, Christoph

    2016-07-01

    Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations. PMID:27009573

  6. Concurrent acquired inhibitors to factor VIII and IX, a laboratory artifact: a case report

    PubMed Central

    Doma, Saša Anžej; Hillarp, Andreas; Pajič, Tadej; Andoljšek, Dušan; Černelč, Peter; Preldžnik Zupan, Irena

    2016-01-01

    Acquired inhibitors to coagulation factors other than factor VIII are extremely rare. We describe a case of a 59-year-old woman with abnormal bleeding, diagnosed with concurrent inhibitor antibodies to factor VIII and IX by Bethesda testing. We demonstrate that anti-FVIII antibodies of a very high titre are capable of disturbing the aPTT-based Bethesda assay, resulting in falsely-positive antibodies to factor IX. The case also illustrates the usefulness of the immunological assay (ELISA) in complementing the inhibitor diagnosis. PMID:27346976

  7. Ectopic expression of rice Xa21 overcomes developmentally controlled resistance to Xanthomonas oryzae pv. oryzae

    PubMed Central

    Park, Chang-Jin; Lee, Sang-Won; Chern, Mawsheng; Sharma, Rita; Canlas, Patrick E.; Song, Min-Young; Jeon, Jong-Seong; Ronald, Pamela C.

    2010-01-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) activates the innate immune response. The rice PRR, XA21, confers robust resistance at adult stages to most strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Seedlings are still easily infected by Xoo, causing severe yield losses. Here we report that Xa21 is induced by Xoo infection and that ectopic expression of Xa21 confers resistance at three leaf stage (three-week-old), overcoming the developmental limitation of XA21-mediated resistance. Ectopic expression of Xa21 also up-regulates a larger set of defense-related genes as compared to Xa21 driven by the native promoter. These results indicate that altered regulation of Xa21 expression is useful for developing enhanced resistance to Xoo at multiple developmental stages. PMID:21076626

  8. Inactivation of factor XII active fragment in normal plasma. Predominant role of C-1-inhibitor.

    PubMed

    de Agostini, A; Lijnen, H R; Pixley, R A; Colman, R W; Schapira, M

    1984-06-01

    To define the factors responsible for the inactivation of the active fragment derived from Factor XII (Factor XIIf ) in plasma, we studied the inactivation kinetics of Factor XIIf in various purified and plasma mixtures. We also analyzed the formation of 125I-Factor XIIf -inhibitor complexes by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In purified systems, the bimolecular rate constants for the reactions of Factor XIIf with C-1-inhibitor, alpha 2-antiplasmin, and antithrombin III were 18.5, 0.91, and 0.32 X 10(4) M-1 min-1, respectively. Furthermore, SDS-PAGE analysis revealed that 1:1 stoichiometric complexes were formed between 125I-Factor XIIf and each of these three inhibitors. In contrast, kinetic and SDS-PAGE studies indicated that Factor XIIf did not react with alpha 1-antitrypsin or alpha 2-macroglobulin. The inactivation rate constant of Factor XIIf by prekallikrein-deficient plasma was 14.4 X 10(-2) min-1, a value that was essentially identical to the value predicted from the studies in purified systems (15.5 X 10(-2) min-1). This constant was reduced to 1.8 X 10(-2) min-1 when Factor XIIf was inactivated by prekallikrein-deficient plasma that had been immunodepleted (less than 5%) of C-1-inhibitor. In addition, after inactivation in normal plasma, 74% of the active 125I-Factor XIIf was found to form a complex with C-1-inhibitor, whereas 26% of the enzyme formed complexes with alpha 2-antiplasmin and antithrombin III. Furthermore, 42% of the labeled enzyme was still complexed with C-1-inhibitor when 125I-Factor XII was inactivated in hereditary angioedema plasma that contained 32% of functional C-1-inhibitor. This study quantitatively demonstrates the dominant role of C-1-inhibitor in the inactivation of Factor XIIf in the plasma milieu. PMID:6725552

  9. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  10. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    PubMed

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols. PMID:26739399

  11. Evaluation of bisbenzamidines as inhibitors for matriptase-2.

    PubMed

    Beckmann, Anna-Madeleine; Gilberg, Erik; Gattner, Susanne; Huang, Tien L; Vanden Eynde, Jean Jacques; Mayence, Annie; Bajorath, Jürgen; Stirnberg, Marit; Gütschow, Michael

    2016-08-01

    The serine protease matriptase-2 has attracted much attention as a potential target for the treatment of iron overload diseases. In this study, a series of 27 symmetric, achiral bisbenzamidines was evaluated for inhibitory activity against human matriptase-2, against the closely related enzyme human matriptase, as well as against human thrombin, bovine factor Xa and human trypsin. The conformationally restricted piperazine derivative 19 and the oxamide-derived bisbenzamidine 1 were identified as the most potent inhibitors of this series for matriptase-2 and matriptase, respectively. PMID:27287367

  12. Factor V, tissue factor pathway inhibitor, and east Texas bleeding disorder

    PubMed Central

    Broze, George J.; Girard, Thomas J.

    2013-01-01

    In a report reading like a fascinating detective story, Vincent and colleagues crack the mysterious case of east Texas bleeding disorder. They show that affected individuals have a mutation in exon 13 of the coagulation F5 gene that causes increased expression of an alternatively spliced transcript, which encodes a previously unrecognized factor V (FV) isoform they call FV-short. This FV isoform lacks a large portion of the B domain of FV, which is normally released upon the proteolytic activation of FV by thrombin and binds tightly to the coagulation regulator tissue factor pathway inhibitor-α (TFPIα). This interaction leads to an approximately 10-fold increase in the level of TFPIα circulating in plasma and a resultant anticoagulant effect that produces a hemorrhagic diathesis. PMID:23979154

  13. Factor V, tissue factor pathway inhibitor, and east Texas bleeding disorder.

    PubMed

    Broze, George J; Girard, Thomas J

    2013-09-01

    In a report reading like a fascinating detective story, Vincent and colleagues crack the mysterious case of east Texas bleeding disorder. They show that affected individuals have a mutation in exon 13 of the coagulation F5 gene that causes increased expression of an alternatively spliced transcript, which encodes a previously unrecognized factor V (FV) isoform they call FV-short. This FV isoform lacks a large portion of the B domain of FV, which is normally released upon the proteolytic activation of FV by thrombin and binds tightly to the coagulation regulator tissue factor pathway inhibitor-α (TFPIα). This interaction leads to an approximately 10-fold increase in the level of TFPIα circulating in plasma and a resultant anticoagulant effect that produces a hemorrhagic diathesis. PMID:23979154

  14. Protease inhibitors interfere with the necessary factors of carcinogenesis.

    PubMed Central

    Troll, W

    1989-01-01

    Many tumor promoters are inflammatory agents that stimulate the formation of oxygen radicals (.O2-) and hydrogen peroxide (H2O2) in phagocytic neutrophils. The neutrophils use the oxygen radicals to kill bacteria, which are recognized by the cell membrane of phagocytic cells causing a signal to mount the oxygen response. The tumor promoter isolated from croton oil, 12-O-tetradecanoylphorbol-13-acetate (TPA), mimics the signal, causing an oxygen radical release that is intended to kill bacteria; instead, it injures cells in the host. Oxygen radicals cause single strand breaks in DNA and modify DNA bases. These damaging reactions appear to be related to tumor promotion, as three types of chemopreventive agents, retinoids, onion oil, and protease inhibitors, suppress the induction of oxygen radicals in phagocytic neutrophils and suppress tumor promotion in skin cancer in mice. Protease inhibitors also suppress breast and colon cancers in mice. Protease inhibitors capable of inhibiting chymotrypsin show a greater suppression of the oxygen effect and are better suppressors of tumor promotion. In addition, oxygen radicals may be one of the many agents that cause activation of oncogenes. Since retinoids and protease inhibitors suppress the expression of the ras oncogene in NIH 3T3 cells, NIH 3T3 cells may serve as a relatively facile model for finding and measuring chemopreventive agents that interfere with the carcinogenic process. PMID:2667986

  15. Discovery of a Highly Potent, Selective, and Orally Bioavailable Macrocyclic Inhibitor of Blood Coagulation Factor VIIa-Tissue Factor Complex.

    PubMed

    Zhang, Xiaojun; Glunz, Peter W; Johnson, James A; Jiang, Wen; Jacutin-Porte, Swanee; Ladziata, Vladimir; Zou, Yan; Phillips, Monique S; Wurtz, Nicholas R; Parkhurst, Brandon; Rendina, Alan R; Harper, Timothy M; Cheney, Daniel L; Luettgen, Joseph M; Wong, Pancras C; Seiffert, Dietmar; Wexler, Ruth R; Priestley, E Scott

    2016-08-11

    Inhibitors of the tissue factor (TF)/factor VIIa complex (TF-FVIIa) are promising novel anticoagulants which show excellent efficacy and minimal bleeding in preclinical models. Starting with an aminoisoquinoline P1-based macrocyclic inhibitor, optimization of the P' groups led to a series of highly potent and selective TF-FVIIa inhibitors which displayed poor permeability. Fluorination of the aminoisoquinoline reduced the basicity of the P1 group and significantly improved permeability. The resulting lead compound was highly potent, selective, and achieved good pharmacokinetics in dogs with oral dosing. Moreover, it demonstrated robust antithrombotic activity in a rabbit model of arterial thrombosis. PMID:27455395

  16. Adverse Reaction to Cetuximab, an Epidermal Growth Factor Receptor Inhibitor.

    PubMed

    Štulhofer Buzina, Daška; Martinac, Ivana; Ledić Drvar, Daniela; Čeović, Romana; Bilić, Ivan; Marinović, Branka

    2016-04-01

    Dear Editor, Inhibition of the epidermal growth factor receptor (EGFR) is a new strategy in treatment of a variety of solid tumors, such as colorectal carcinoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, and pancreatic cancer (1). Cetuximab is a chimeric human-murine monoclonal antibody against EGFR. Cutaneous side effects are the most common adverse reactions occurring during epidermal growth factor receptor inhibitors (EGFRI) therapy. Papulopustular rash (acne like rash) develop with 80-86% patients receiving cetuximab, while xerosis, eczema, fissures, teleangiectasiae, hyperpigmentations, and nail and hair changes occur less frequently (2). The mechanism underlying these skin changes has not been established and understood. It seems EGFRI alter cell growth and differentiation, leading to impaired stratum corneum and cell apoptosis (3-5). An abdominoperineal resection of the rectal adenocarcinoma (Dukes C) was performed on a 43-year-old female patient. Following surgery, adjuvant chemo-radiotherapy was applied. After two years, the patient suffered a metastatic relapse. Abdominal lymphadenopathy was detected on multi-slice computer tomography (MSCT) images, with an increased value of the carcinoembryonic antigen (CEA) tumor marker (maximal value 57 ng/mL). Hematological and biochemical tests were within normal limits, so first-line chemotherapy with oxaliplatin and a 5-fluorouracil (FOLFOX4) protocol was introduced. A wild type of the KRAS gene was confirmed in tumor tissue (diagnostic prerequisite for the introduction of EGFRI) and cetuximab (250 mg per m2 of body surface) was added to the treatment protocol. The patient responded well to the treatment with confirmed partial regression of the tumor formations. Three months after the patient started using cetuximab, an anti-EGFR monoclonal antibody, the patient presented with a papulopustular eruption in the seborrhoeic areas (Figure 1) and eczematoid reactions on the extremities

  17. Evaluation of aspirin metabolites as inhibitors of hypoxia-inducible factor hydroxylases.

    PubMed

    Lienard, Benoit M; Conejo-García, Ana; Stolze, Ineke; Loenarz, Christoph; Oldham, Neil J; Ratcliffe, Peter J; Schofield, Christopher J

    2008-12-21

    Known and potential aspirin metabolites were evaluated as inhibitors of oxygen-sensing hypoxia-inducible transcription factor (HIF) hydroxylases; some of the metabolites were found to stabilise HIF-alpha in cells. PMID:19048166

  18. A Spider-Derived Kunitz-Type Serine Protease Inhibitor That Acts as a Plasmin Inhibitor and an Elastase Inhibitor

    PubMed Central

    Wan, Hu; Lee, Kwang Sik; Kim, Bo Yeon; Zou, Feng Ming; Yoon, Hyung Joo; Je, Yeon Ho; Li, Jianhong; Jin, Byung Rae

    2013-01-01

    Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K+ channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (Ki 7.34 nM) and chymotrypsin (Ki 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (Ki 4.89 nM) and neutrophil elastase (Ki 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor. PMID:23308198

  19. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence.

    PubMed

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R; Kehn-Hall, Kylene; Omichinski, James G

    2015-05-12

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  20. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

    PubMed Central

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R.; Kehn-Hall, Kylene; Omichinski, James G.

    2015-01-01

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  1. Transcriptional Inhibitor of Virulence Factors in Enteropathogenic Escherichia coli

    PubMed Central

    Gauthier, Annick; Robertson, Marilyn L.; Lowden, Michael; Ibarra, J. Antonio; Puente, José Luis; Finlay, B. Brett

    2005-01-01

    The type III secretion system (TTSS) is a key virulence mechanism of many important gram-negative bacterial pathogens. The TTSS is conserved among different bacterial pathogens, and mutations and deletions to the system significantly decrease virulence, making the TTSS an important potential therapeutic target. We have developed a high-throughput assay to search for inhibitors of the TTSS. We screened a commercial library of 20,000 small molecules for their ability to inhibit type III secretion by enteropathogenic Escherichia coli (EPEC). After discarding compounds that had no effect on secretion, inhibited bacterial growth, and/or caused degradation of EPEC-secreted proteins, the search was focused on a class of compounds that, while not direct inhibitors of type III secretion, inhibit expression of TTSS-related genes and other genes involved in virulence. This class of compounds does not affect bacterial viability or motility, indicating that it is not significantly affecting the expression of essential genes and is specific to virulence-associated genes. Transcriptional fusion assays confirmed that virulence-associated promoters were more sensitive to inhibition by this class of compounds. Overall, we have identified a class of compounds that can be used as a tool to probe the mechanism(s) that regulates virulence gene expression in EPEC. PMID:16189086

  2. Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection

    PubMed Central

    Peng, Hai; Chen, Zheng; Fang, Zhiwei; Zhou, Junfei; Xia, Zhihui; Gao, Lifen; Chen, Lihong; Li, Lili; Li, Tiantian; Zhai, Wenxue; Zhang, Weixiong

    2015-01-01

    Rice bacterial blight (BB) is a devastating rice disease. The Xa21 gene confers a broad and persistent resistance against BB. We introduced Xa21 into Oryza sativa L ssp indica (rice 9311), through multi-generation backcrossing, and generated a nearly isogenic, blight-resistant 9311/Xa21 rice. Using next-generation sequencing, we profiled the transcriptomes of both varieties before and within four days after infection of bacterium Xanthomonas oryzae pv. oryzae. The identified differentially expressed (DE) genes and signaling pathways revealed insights into the functions of Xa21. Surprisingly, before infection 1,889 genes on 135 of the 316 signaling pathways were DE between the 9311/Xa21 and 9311 plants. These Xa21-mediated basal pathways included mainly those related to the basic material and energy metabolisms and many related to phytohormones such as cytokinin, suggesting that Xa21 triggered redistribution of energy, phytohormones and resources among essential cellular activities before invasion. Counter-intuitively, after infection, the DE genes between the two plants were only one third of that before the infection; other than a few stress-related pathways, the affected pathways after infection constituted a small subset of the Xa21-mediated basal pathways. These results suggested that Xa21 primed critically important genes and signaling pathways, enhancing its resistance against bacterial infection. PMID:26184504

  3. Comparison of three different anti-Xa assays in major orthopedic surgery patients treated with fondaparinux.

    PubMed

    Ikejiri, Makoto; Wada, Hideo; Yamaguchi, Toshio; Miyazaki, Shinichi; Hasegawa, Masahiro; Wakabayashi, Hiroki; Asanuma, Kunihiro; Sakaguchi, Akane; Matsumoto, Takeshi; Ohishi, Kohshi; Fujimoto, Naoki; Yamada, Norikazu; Ito, Masaaki; Katayama, Naoyuki; Sudo, Akihiro

    2016-05-01

    Anti-Xa assays are useful for monitoring the effects of selective anti-Xa drugs, such as fondaparinux, in the prophylaxis of deep vein thrombosis. In the present study, anti-Xa activity was measured using three different assays, Testzym(®) Heparin S, STA(®)-Liquid Anti-Xa and HemosIL(®) Liquid Heparin. Anti-Xa activity in each assay gradually increased from day one after administration to day eight, and still remained on day 15. Although there were significant differences in anti-Xa activity among the three assays, the activity showed significant correlation across assays. There were no significant differences in the anti-Xa activity between patients with and without DVT or between patients with and without massive bleeding on day one before and after administration, day four, day eight and day 15. Anti-Xa activity in each assay was weakly correlated with antithrombin (AT) activity. The AT activity in patients were significantly higher on days four, eight and 15 compared with day one before and after administration, suggesting that AT activity increases following the administration of fondaparinux. The three anti-Xa assay kits tested are useful for monitoring fondaparinux treatment in orthopedic surgery patients. PMID:26922193

  4. Sulfated Pentagalloylglucoside is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

    PubMed Central

    Al-Horani, Rami A.; Ponnusamy, Pooja; Mehta, Akul Y.; Gailani, David; Desai, Umesh R.

    2013-01-01

    Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced VMAX of FXIa hydrolysis of chromogenic substrate without affecting the KM suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance. PMID:23316863

  5. Microtubule inhibitors block the morphological changes induced in Drosophila blood cells by a parasitoid wasp factor.

    PubMed

    Rizki, R M; Rizki, T M

    1990-03-15

    The shape change of Drosophila melanogaster blood cells (lamellocytes) from discoidal to bipolar that is caused by a factor from the female parasitoid Leptopilina heterotoma is blocked by the tubulin inhibitors vinblastine and vincristine in vitro. The actin inhibitor, cytochalasin B, causes arborization of Drosophila lamellocytes and acts synergistically with the wasp factor to alter lamellocyte morphology. Lamellocyte aborization induced by cytochalasin B is blocked by simultaneous treatment with vinblastine. These observations indicate that the changes in lamellocyte shape induced by both the wasp factor and cytochalasin B require microtubule assembly. PMID:2311722

  6. Von Willebrand factor-containing factor VIII concentrates and inhibitors in haemophilia A. A critical literature review.

    PubMed

    Franchini, Massimo; Lippi, Giuseppe

    2010-11-01

    The development of inhibitors that neutralise the function of factor VIII (FVIII) is currently not only the most challenging complication associated with the treatment of haemophilia A but it also increases the disease-related morbidity as bleeding episodes do not respond to standard therapy. The main short-term goal of the treatment of inhibitor patients is to control bleeding episodes while the long-term one is to permanently eradicate the inhibitor by immune tolerance induction, particularly in the case of high-titer antibodies. Due to some in vitro studies and clinical observations, some investigators have suggested that FVIII concentrates containing von Willebrand factor (VWF) may be less immunogenic than high-purity or recombinant FVIII products. It has also been suggested that success rates for immune tolerance induction are higher when plasma-derived FVIII products are used. The currently available data from laboratory and clinical studies on the role of VWF in inhibitor development and eradication in haemophilia A is critically analysed in this review. As a result, we have not found definitive evidence supporting a role for product type on inhibitor incidence and inhibitor eradication in haemophilia A patients. PMID:20838738

  7. Factor VIII gene (F8) mutation and risk of inhibitor development in nonsevere hemophilia A.

    PubMed

    Eckhardt, Corien L; van Velzen, Alice S; Peters, Marjolein; Astermark, Jan; Brons, Paul P; Castaman, Giancarlo; Cnossen, Marjon H; Dors, Natasja; Escuriola-Ettingshausen, Carmen; Hamulyak, Karly; Hart, Daniel P; Hay, Charles R M; Haya, Saturnino; van Heerde, Waander L; Hermans, Cedric; Holmström, Margareta; Jimenez-Yuste, Victor; Keenan, Russell D; Klamroth, Robert; Laros-van Gorkom, Britta A P; Leebeek, Frank W G; Liesner, Ri; Mäkipernaa, Anne; Male, Christoph; Mauser-Bunschoten, Evelien; Mazzucconi, Maria G; McRae, Simon; Meijer, Karina; Mitchell, Michael; Morfini, Massimo; Nijziel, Marten; Oldenburg, Johannes; Peerlinck, Kathelijne; Petrini, Pia; Platokouki, Helena; Reitter-Pfoertner, Sylvia E; Santagostino, Elena; Schinco, Piercarla; Smiers, Frans J; Siegmund, Berthold; Tagliaferri, Annarita; Yee, Thynn T; Kamphuisen, Pieter Willem; van der Bom, Johanna G; Fijnvandraat, Karin

    2013-09-12

    Neutralizing antibodies (inhibitors) toward factor VIII form a severe complication in nonsevere hemophilia A, profoundly aggravating the bleeding pattern. Identification of high-risk patients is hampered by lack of data that take exposure days to therapeutic factor VIII concentrates into account. In the INSIGHT study, we analyzed the association between F8 mutation and inhibitor development in patients with nonsevere hemophilia A (factor VIII 2-40 IU/dL). This analysis included 1112 nonsevere hemophilia A patients from 14 centers in Europe and Australia that had genotyped at least 70% of their patients. Inhibitor risk was calculated as Kaplan-Meier incidence with cumulative number of exposure days as the time variable. During 44 800 exposure days (median, 24 exposure days per patient; interquartile range [IQR], 7-90), 59 of the 1112 patients developed an inhibitor; cumulative incidence of 5.3% (95% confidence interval [CI], 4.0-6.6) after a median of 28 exposure days (IQR, 12-71). The inhibitor risk at 50 exposure days was 6.7% (95% CI, 4.5-8.9) and at 100 exposure days the risk further increased to 13.3% (95% CI, 9.6-17.0). Among a total of 214 different F8 missense mutations 19 were associated with inhibitor development. These results emphasize the importance of F8 genotyping in nonsevere hemophilia A. PMID:23926300

  8. A sensitive, accurate assay for extrinsic pathway inhibitor (EPI) activity in rabbit plasma: Paradoxical effect of excess exogenous factor X

    SciTech Connect

    Warr, T.A.; Rao, L.V.; Rapaport, S.I. )

    1990-08-15

    A sensitive assay is described for the measurement of rabbit plasma EPI activity in experimental studies of induced hypercoagulable states in this species. It is based upon the ability of a dilution of rabbit plasma to inhibit human factor VIIa/rabbit brain tissue factor (TF) catalyzed activation of human factor IX (tritiated activation peptide release assay). Addition of {sup 3}H-factor IX to the reaction mixture is delayed for 45 minutes to allow full inhibition by EPI/factor Xa complex before the residual catalytic activity of factor VIIa/TF is measured. Although the diluted rabbit plasma test sample contains both EPI and factor X, supplemental factor X is added to the reaction mixture to assure that only EPI content of the test sample affects the assay result. However, the final concentration of factor X in the reaction mixture is critical. Too high a concentration of factor X diminishes the sensitivity of the assay. The reason for this phenomenon, which was observed with both human and rabbit factor X preparations, is unknown.

  9. Drug Survival Rates of Tumor Necrosis Factor Inhibitors in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis

    PubMed Central

    2014-01-01

    We investigated the compliance of Korean patients using tumor necrosis factor (TNF) inhibitors to treat rheumatoid arthritis (RA) and ankylosing spondylitis (AS), and identified potential predictors associated with treatment discontinuation. The study population comprised 114 RA and 310 AS patients treated with TNF inhibitors at a single tertiary center for at least 1 yr from December 2002 to November 2011. Of the 114 RA patients, 64 (56.1%) discontinued their first TNF inhibitors with a mean duration of 18.1 months. By contrast, 65 of 310 patients (21.0%) with AS discontinued their first TNF inhibitors, with a mean duration of 84 months. Although the survival rate did not differ among the three TNF inhibitors in the AS patients, the etanercept group had a lower discontinuation rate than the infliximab group in the RA patients. In addition, RA patients who received corticosteroids in combination with TNF inhibitors were more likely to discontinue their TNF inhibitors. The independent predictors of drug discontinuation in AS patients were male gender and complete ankylosis on radiographs of the sacroiliac joint. Our results provide further evidence that real-life treatment outcomes of RA and AS patients may be different from those observed in randomized clinical trials. Graphical Abstract PMID:25246737

  10. Critical Factors Determining Dimerization of Human Antizyme Inhibitor*

    PubMed Central

    Su, Kuo-Liang; Liao, Ya-Fan; Hung, Hui-Chih; Liu, Guang-Yaw

    2009-01-01

    Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer Kd, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (Kd value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a Kd value of ∼0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained. PMID:19635796

  11. Distribution of Xanthomonas oryzae pv. oryzae Strains Virulent to Xa21 in Korea.

    PubMed

    Lee, S W; Choi, S H; Han, S S; Lee, D G; Lee, B Y

    1999-10-01

    ABSTRACT Strains of Xanthomonas oryzae pv. oryzae that are virulent to rice lines carrying the Xa21 resistance gene were widely distributed in Korea. A total of 105 strains collected during 1987 to 1996 in Korea was characterized by pathogenicity tests and restriction fragment length polymorphism analysis of the XorII methyltransferase (xorIIM) and avrXa10 genes. Although the lesion lengths on rice line IRBB21, which carries Xa21, decreased as plant age increased, resistance and susceptibility of the plants to 31 strains were clearly differentiated at the seedling (14, 21, and 28 days old), maximum tillering, and flag leaf stages. The resistance or susceptibility of seedlings was correlated with bacterial populations within an inoculated leaf. There was a significant change in the population structure of X. oryzae pv. oryzae with regard to virulence to Xa21 over the last 10 years; this change in population was confirmed by genome analysis. Lineage I, which is avirulent to Xa21 and does not have a genomic xorIIM homolog, was the predominant lineage found between 1987 and 1989, while lineage II, which is virulent to Xa21 and contains the xorIIM homolog, was predominant in strains collected between 1994 and 1995. Our results demonstrate that introduction of Xa21 into commercial rice should be based on the regional structure of X. oryzae pv. oryzae populations and suggest that Xa21 will not be useful in Korea. PMID:18944737

  12. Feedback Activation of Leukemia Inhibitory Factor Receptor Limits Response to Histone Deacetylase Inhibitors in Breast Cancer.

    PubMed

    Zeng, Hanlin; Qu, Jia; Jin, Nan; Xu, Jun; Lin, Chenchu; Chen, Yi; Yang, Xinying; He, Xiang; Tang, Shuai; Lan, Xiaojing; Yang, Xiaotong; Chen, Ziqi; Huang, Min; Ding, Jian; Geng, Meiyu

    2016-09-12

    Histone deacetylase (HDAC) inhibitors have demonstrated clinical benefits in subtypes of hematological malignancies. However, the efficacy of HDAC inhibitors in solid tumors remains uncertain. This study takes breast cancer as a model to understand mechanisms accounting for limited response of HDAC inhibitors in solid tumors and to seek combination solutions. We discover that feedback activation of leukemia inhibitory factor receptor (LIFR) signaling in breast cancer limits the response to HDAC inhibition. Mechanistically, HDAC inhibition increases histone acetylation at the LIFR gene promoter, which recruits bromodomain protein BRD4, upregulates LIFR expression, and activates JAK1-STAT3 signaling. Importantly, JAK1 or BRD4 inhibition sensitizes breast cancer to HDAC inhibitors, implicating combination inhibition of HDAC with JAK1 or BRD4 as potential therapies for breast cancer. PMID:27622335

  13. Inhibitors of propagation of coagulation (factors VIII, IX and XI): a review of current therapeutic practice

    PubMed Central

    Franchini, Massimo; Mannucci, Pier Mannuccio

    2011-01-01

    The management of patients with congenital haemophilia who develop alloantibodies against factors of the propagation phase of blood coagulation, commonly known as inhibitors, is the most important challenge facing haemophilia caregivers at present, as this complication not only compromises the efficacy of replacement therapy but also consumes an enormous amount of economic resources. Development of inhibitors further complicates the clinical course of severe haemophilia, with a prevalence of up to 30% in patients with haemophilia A (factor VIII deficiency) and up to 5% in those with haemophilia B (factor IX deficiency) and haemophilia C (factor XI deficiency). While the short-term goal of treatment of patients who develop alloantibodies is the control of bleeding, the eradication of the inhibitor is the main long-term goal. The management of severe bleeding episodes and the eradication of the autoantibody are also the mainstays of treatment of patients with acquired haemophilia, a rare but life-threatening haemorrhagic condition characterized by the development of inhibitory autoantibodies against coagulation factor VIII. The most recent options available for treating patients with congenital haemophilia complicated by inhibitors and acquired haemophilia because of autoantibodies against factor VIII are summarized in this review article. PMID:21204915

  14. Molecular Phylogeny, Homology Modeling, and Molecular Dynamics Simulation of Race-Specific Bacterial Blight Disease Resistance Protein (xa5) of Rice: A Comparative Agriproteomics Approach

    PubMed Central

    Dehury, Budheswar; Sahu, Mousumi; Sarma, Kishore; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Sharma, Gauri Dutta; Choudhury, Manabendra Dutta

    2013-01-01

    Abstract Rice (Oryza sativa L.), a model plant belonging to the family Poaceae, is a staple food for a majority of the people worldwide. Grown in the tropical and subtropical regions of the world, this important cereal crop is under constant and serious threat from both biotic and abiotic stresses. Among the biotic threats, Xanthomonas oryzae pv. oryzae, causing the damaging bacterial blight disease in rice, is a prominent pathogen. The xa5 gene in the host plant rice confers race-specific resistance to this pathogen. This recessive gene belongs to the Xa gene family of rice and encodes a gamma subunit of transcription factor IIA (TFIIAγ). In view of the importance of this gene in conferring resistance to the devastating disease, we reconstructed the phylogenetic relationship of this gene, developed a three-dimensional protein model, followed by long-term molecular dynamics simulation studies to gain a better understanding of the evolution, structure, and function of xa5. The modeled structure was found to fit well with the small subunit of TFIIA from human, suggesting that it may also act as a small subunit of TFIIA in rice. The model had a stable conformation in response to the atomic flexibility and interaction, when subjected to MD simulation at 20 nano second in aqueous solution. Further structural analysis of xa5 indicated that the protein retained its basic transcription factor function, suggesting that it might govern a novel pathway responsible for bacterial blight resistance. Future molecular docking studies of xa5 underway with its corresponding avirulence gene is expected to shed more direct light into plant–pathogen interactions at the molecular level and thus pave the way for richer agriproteomic insights. PMID:23758479

  15. Monoamine Oxidase Inhibitors Extracted from Tobacco Smoke as Neuroprotective Factors for Potential Treatment of Parkinson's Disease.

    PubMed

    Sari, Youssef; Khalil, Ashraf

    2015-01-01

    Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of mainly the nigrostriatal dopaminergic neurons, which leads to motor dysfunction. Although, most of the drugs are currently used for symptomatic treatment, there are at least three FDA-approved drugs for the treatment of PD that have been suggested preclinically to have neuroprotective effects. Among these drugs are monoamine oxidase (MAO) type B inhibitors such as selegiline and rasagiline, and non-ergot derivative dopamine agonist, pramipexole. In this review article, we focused on the potential uses of non-selective reversible MAO inhibitor, 2,3,6-trimethyl-1,4-naphthoquinone, from flue-cured tobacco leaves extract and two β- carboline alkaloids (harman and norharman) as potent, reversible and non-selective MAO inhibitors for the treatment of PD. In addition, we discussed the potential uses of farnesol as a potent inhibitor of MAO-B and farnesylacetone as a less potent selective MAO-B inhibitor. Furthermore, adducts of 1,2,3,4-tetrahydroisoquinoline have shown to have competitive inhibitory effects for both MAO-A and MAO-B. These inhibitors have potential neuroprotective effects, which might be mediated at least through nerve growth factor, neurotrophin 3, brain derived neurotrophic factor, and glial-cell-line-derived neurotrophic factor. We suggest here the neuroprotective implication of extracted MAO inhibitors from smoke tobacco; however, it is important to note that there are several existing compounds in tobacco smoke that have toxic effects in the brain, these include and not limited to the induction of neuropathological features observed in individuals suffering from Alzheimer's disease and dementia. PMID:25808895

  16. Antithrombin, an Important Inhibitor in Blood Clots.

    PubMed

    Zhu, Ying; Cong, Qing-Wei; Liu, Yue; Wan, Chun-Ling; Yu, Tao; He, Guang; He, Lin; Cai, Lei; Chou, Kuo-Chen

    2016-01-01

    Blood coagulation is healthy and lifesaving because it can stop bleeding. It can, however, be a troublemaker as well, causing serious medical problems including heart attack and stroke. Body has complex blood coagulation cascade to modulate the blood clots. In the environment of plasma, the blood coagulation cascade is regulated by antithrombin, which is deemed one of the most important serine protease inhibitors. It inhibits thrombin; it can inhibit factors IXa and Xa as well. Interestingly, its inhibitory ability will be significantly increased with the existence of heparin. In this minireview paper, we are to summarize the structural features of antithrombin, as well as its heparin binding modes and anti-coagulation mechanisms, in hopes that the discussion and analysis presented in this paper can stimulate new strategies to find more effective approaches or compounds to modulate the antithrombin. PMID:26411319

  17. IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII

    PubMed Central

    Matino, Davide; Gargaro, Marco; Santagostino, Elena; Di Minno, Matteo N.D.; Castaman, Giancarlo; Morfini, Massimo; Rocino, Angiola; Mancuso, Maria E.; Di Minno, Giovanni; Coppola, Antonio; Talesa, Vincenzo N.; Volpi, Claudia; Vacca, Carmine; Orabona, Ciriana; Iannitti, Rossana; Mazzucconi, Maria G.; Santoro, Cristina; Tosti, Antonella; Chiappalupi, Sara; Sorci, Guglielmo; Tagariello, Giuseppe; Belvini, Donata; Radossi, Paolo; Landolfi, Raffaele; Fuchs, Dietmar; Boon, Louis; Pirro, Matteo; Marchesini, Emanuela; Grohmann, Ursula; Puccetti, Paolo; Iorio, Alfonso; Fallarino, Francesca

    2015-01-01

    The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein. PMID:26426076

  18. IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII.

    PubMed

    Matino, Davide; Gargaro, Marco; Santagostino, Elena; Di Minno, Matteo N D; Castaman, Giancarlo; Morfini, Massimo; Rocino, Angiola; Mancuso, Maria E; Di Minno, Giovanni; Coppola, Antonio; Talesa, Vincenzo N; Volpi, Claudia; Vacca, Carmine; Orabona, Ciriana; Iannitti, Rossana; Mazzucconi, Maria G; Santoro, Cristina; Tosti, Antonella; Chiappalupi, Sara; Sorci, Guglielmo; Tagariello, Giuseppe; Belvini, Donata; Radossi, Paolo; Landolfi, Raffaele; Fuchs, Dietmar; Boon, Louis; Pirro, Matteo; Marchesini, Emanuela; Grohmann, Ursula; Puccetti, Paolo; Iorio, Alfonso; Fallarino, Francesca

    2015-10-01

    The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein. PMID:26426076

  19. Identification of inflammatory factor TNFα inhibitor from medicinal herbs.

    PubMed

    Ye, Hong; Wang, Yali; Bennett Jenson, A; Yan, Jun

    2016-04-01

    The inflammatory response is one of the first defenses our body has to fight against potential endangerments. It plays a critical role in host defense, clearing and slowing the infection in the case of microbial invasion. During an inflammatory response, a variety of cytokines are produced by cells and trigger or enhance the specific inflammation response. TNFα, one of these factors, plays a crucial role in many immune and inflammatory processes, such as proliferation, apoptosis, necrosis, and cell survival. It acts in orchestrating the cytokine cascade and the major regulator of inflammatory cytokine production. Abnormality of TNFα signaling leads to many diseases, including rheumatoid arthritis, psoriasis, Crohn's disease, atherosclerosis, and cancer. Due to the importance of TNFα, regulating TNFα activity is a key to treat the related diseases. There is a long history of using medicinal herbs to treat diseases related to inflammation. We searched for an ingredient that has the ability to inhibit TNFα, we examined AO herbal extract, containing 10 individual herbs and most of these herbs have anti-inflammatory activity within humans. We have tested the anti-inflammatory ability of AO herbal extract on mice. Furthermore, we used macrophage cell from young mice and found that AO extract has the ability to reduce the inflammation by inhibiting TNFα level. PMID:26778692

  20. Acquired factor V inhibitor in a patient with mantle cell lymphoma presenting with hematuria followed by thrombosis: a case report

    PubMed Central

    AlJohani, Naif I; Matthews, John H

    2014-01-01

    Acquired factor V inhibitor is a rare hemostatic disorder that presents with hemorrhagic manifestations in the vast majority of patients. Factor V inhibitor may develop through a variety of mechanisms involving development of alloantibodies or autoantibodies specific to Factor V. Autoantibodies, in particular, have been reported in a number of conditions. In this report, we describe a case of acquired factor V inhibitor in a patient with mantle cell lymphoma who presented with hematuria. Seven weeks after diagnosis and successful management, the patient developed deep vein thrombosis in the right lower extremity. The patient’s factor V levels were normalized, and the inhibitor was successfully eradicated using corticosteroids. Here, we discuss this rare disorder, its unusual manifestation, and provide a mini-review of the current literature regarding factor V inhibitors. PMID:24591851

  1. Use of tumor necrosis factor (TNF) inhibitors in patients with HIV/AIDS.

    PubMed

    Gallitano, Stephanie M; McDermott, Laura; Brar, Kanwaljit; Lowenstein, Eve

    2016-05-01

    Patients with HIV and AIDS are living longer because of advancements in antiretroviral therapy. These patients are often susceptible to debilitating inflammatory disorders that are refractory to standard treatment. We discuss the relationship of tumor necrosis factor-alpha and HIV and then review 27 published cases of patients with HIV being treated with tumor necrosis factor-alpha inhibitors. This review is limited because no randomized controlled trials have been performed with this patient population. Regardless, we propose that reliable seropositive patients, who are adherent to medication regimens and frequent monitoring and have failed other treatment modalities, should be considered for treatment with tumor necrosis factor-alpha inhibitors. PMID:26774690

  2. A small-molecule inhibitor of macrophage migration inhibitory factor for the treatment of inflammatory disease.

    PubMed

    Kithcart, Aaron P; Cox, Gina M; Sielecki, Thais; Short, Abigail; Pruitt, James; Papenfuss, Tracey; Shawler, Todd; Gienapp, Ingrid; Satoskar, Abhay R; Whitacre, Caroline C

    2010-11-01

    Multiple sclerosis (MS) is a chronic, debilitating disease of the central nervous system (CNS) characterized by demyelination and axon loss. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has been shown to be elevated in the cerebrospinal fluid of patients during relapses. The purpose of this study was to evaluate a new small-molecule inhibitor of MIF and its ability to reduce the severity of an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We utilized 2 structurally related isoxazolines, which show in vitro inhibition of MIF tautomerase activity. We found that administration of an inhibitor of MIF to mice with established EAE immediately reduced the severity of clinical signs and expanded a population of regulatory T lymphocytes. We also noted that the inhibitor reduced relapses of disease in a relapsing/remitting model of EAE. An analysis of leukocyte migration into the brain revealed that administration of inhibitor reduced entry of these cells. No effects on inflammatory cytokine production or T-cell activation in the periphery were noted. From these studies, we conclude that a small-molecule inhibitor of MIF reduces the severity of EAE and prevents access of immune cells into the CNS, which could be of therapeutic relevance to MS. PMID:20624927

  3. Subsite specificity of anthrax lethal factor and its implications for inhibitor development

    PubMed Central

    Li, Feng; Terzyan, Simon; Tang, Jordan

    2011-01-01

    The lethal factor of Bacillus anthracis is a major factor for lethality of anthrax infection by this bacterium. With the aid of the protective antigen, lethal factor gains excess to the cell cytosol where it manifests toxicity as a metalloprotease. For better understanding of its specificity, we have determined its residue preferences of nineteen amino acids in six subsites (from P3 to P3’) as relative kcat/Km values (specificity constants). These results showed that lethal factor has a broad specificity with preference toward hydrophobic residues, but not charged or branched residues. The most preferred residues in these six subsites are, from P1 to P3’, Trp, Leu, Met, Tyr, Pro, and Leu. The result of residue preference was used to design new substrates with superior hydrolytic characteristics and inhibitors with high potency. For better use of the new findings for inhibitor design, we have modeled the most preferred residues in the active site of lethal factor. The observed interactions provide new insights to future inhibitor designs. PMID:21396916

  4. [Successful direct thrombin inhibitor treatment of a left atrial appendage thrombus developed under rivaroxaban therapy].

    PubMed

    Szegedi, Nándor; Gellér, László; Tahin, Tamás; Merkely, Béla; Széplaki, Gábor

    2016-01-24

    The authors present the history of a 62-year-old man on continuous rivaroxaban therapy who was scheduled for pulmonary vein isolation due to persistent atrial fibrillation. Preoperative transesophageal echocardiography detected the presence of left atrial appendage thrombus. Thrombophilia tests showed that the patient was heterozygous carrier of the methylene-tetrahydrofolate reductase gene mutation. The authors hypothesized that a direct thrombin inhibitor might exert a more appropriate effect against thrombosis in this case and, therefore, a switch to dabigatran was performed. After two months of anticoagulation with the direct thrombin inhibitor and folic acid supplementation the thrombus resolved. The authors underline that thrombus formation may develop in atrial fibrillation even if the patient is adequately treated with rivaroxaban. This case suggests, that methylene-tetrahydrofolate reductase gene mutation may modulate the efficacy of direct Xa factor inhibitors. According to this case history, dabigatran may be an effective therapeutic option in resolving established thrombus. PMID:26772828

  5. Vascular endothelial growth factor inhibitors: investigational therapies for the treatment of psoriasis

    PubMed Central

    Weidemann, Anja K; Crawshaw, Ania A; Byrne, Emily; Young, Helen S

    2013-01-01

    Psoriasis is a common inflammatory autoimmune condition in which environmental factors and genetic predisposition contribute to the development of disease in susceptible individuals. Angiogenesis is known to be a key pathogenic feature of psoriasis. Local and systemic elevation of vascular endothelial growth factor (VEGF)-A has been demonstrated in the skin and plasma of patients with psoriasis and is known to correlate with improvement following some traditional psoriasis treatments. A number of VEGF inhibitors are licensed for the treatment of malignancies and eye disease and isolated case reports suggest that some individuals with psoriasis may improve when exposed to these agents. The small number of cases and lack of unified reporting measures makes it difficult to draw generalizations and underline the heterogeneity of psoriasis as a disease entity. Though not yet licensed for the treatment of psoriasis in humans, experimental data supports the potential of VEGF inhibitors to influence relevant aspects of human cell biology (such as endothelial cell differentiation) and to improve animal models of skin disease. Given the multi-factorial nature of psoriasis it is unlikely that VEGF inhibitors will be effective in all patients, however they have the potential to be a valuable addition to the therapeutic arsenal in selected cases. Current VEGF inhibitors in clinical use are associated with a number of potentially serious side effects including hypertension, left ventricular dysfunction, and gastrointestinal perforation. Such risks require careful consideration in psoriasis populations particularly in light of growing concerns linking psoriasis to increased cardiovascular risk. PMID:24101875

  6. Vascular endothelial growth factor inhibitors: investigational therapies for the treatment of psoriasis.

    PubMed

    Weidemann, Anja K; Crawshaw, Ania A; Byrne, Emily; Young, Helen S

    2013-01-01

    Psoriasis is a common inflammatory autoimmune condition in which environmental factors and genetic predisposition contribute to the development of disease in susceptible individuals. Angiogenesis is known to be a key pathogenic feature of psoriasis. Local and systemic elevation of vascular endothelial growth factor (VEGF)-A has been demonstrated in the skin and plasma of patients with psoriasis and is known to correlate with improvement following some traditional psoriasis treatments. A number of VEGF inhibitors are licensed for the treatment of malignancies and eye disease and isolated case reports suggest that some individuals with psoriasis may improve when exposed to these agents. The small number of cases and lack of unified reporting measures makes it difficult to draw generalizations and underline the heterogeneity of psoriasis as a disease entity. Though not yet licensed for the treatment of psoriasis in humans, experimental data supports the potential of VEGF inhibitors to influence relevant aspects of human cell biology (such as endothelial cell differentiation) and to improve animal models of skin disease. Given the multi-factorial nature of psoriasis it is unlikely that VEGF inhibitors will be effective in all patients, however they have the potential to be a valuable addition to the therapeutic arsenal in selected cases. Current VEGF inhibitors in clinical use are associated with a number of potentially serious side effects including hypertension, left ventricular dysfunction, and gastrointestinal perforation. Such risks require careful consideration in psoriasis populations particularly in light of growing concerns linking psoriasis to increased cardiovascular risk. PMID:24101875

  7. HSP90 inhibitors enhance differentiation and MITF (microphthalmia transcription factor) activity in osteoclast progenitors.

    PubMed

    van der Kraan, A Gabrielle J; Chai, Ryan C C; Singh, Preetinder P; Lang, Benjamin J; Xu, Jiake; Gillespie, Matthew T; Price, John T; Quinn, Julian M W

    2013-04-15

    The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFβ (transforming growth factor β) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity. PMID:23379601

  8. Pituitary Apoplexy After Intravitreal Injection of Vascular Endothelial Growth Factor Inhibitor: A Novel Complication

    PubMed Central

    Kasl, Rebecca A.; Kistka, Heather M.; Turner, Justin H.; Devin, Jessica K.; Chambless, Lola B.

    2015-01-01

    Pituitary adenomas are common in the general population. They can be complicated by intratumoral hemorrhage, otherwise known as apoplexy, which frequently presents with neurologic deficits that may necessitate urgent surgical decompression. Many risk factors for pituitary apoplexy have been suggested in the literature. We present a case of symptomatic apoplexy in a woman following the intravitreal administration of the vascular endothelial growth factor (VEGF) inhibitor ranibizumab. Ophthalmoplegia resolved and visual acuity significantly improved following gross total resection of the tumor via an endoscopic endonasal surgical approach. The association between intravitreal injection of a VEGF inhibitor and pituitary apoplexy has not been previously described, but physicians performing these procedures should be aware of this potential complication. PMID:26623228

  9. Fast rotation of a subkilometer-sized near-Earth object 2011 XA{sub 3}

    SciTech Connect

    Urakawa, Seitaro; Ohtsuka, Katsuhito; Abe, Shinsuke; Ito, Takashi; Nakamura, Tomoki

    2014-05-01

    We present light curve observations and their multiband photometry for near-Earth object (NEO) 2011 XA{sub 3}. The light curve has shown a periodicity of 0.0304 ± 0.0003 days (= 43.8 ± 0.4 minutes). The fast rotation shows that 2011 XA{sub 3} is in a state of tension (i.e., a monolithic asteroid) and cannot be held together by self-gravitation. Moreover, the multiband photometric analysis indicates that the taxonomic class of 2011 XA{sub 3} is S-complex, or V-type. Its estimated effective diameter is 225 ± 97 m (S-complex) and 166 ± 63 m (V-type), respectively. Therefore, 2011 XA{sub 3} is a candidate for the second-largest, fast-rotating, monolithic asteroid. Moreover, the orbital parameters of 2011 XA{sub 3} are apparently similar to those of NEO (3200) Phaethon, but F/B-type. We computed the orbital evolutions of 2011 XA{sub 3} and Phaethon. However, the results of the computation and distinct taxonomy indicate that neither of the asteroids is of common origin.

  10. Clinical Use of Anti-Xa Monitoring in Malignancy-Associated Thrombosis.

    PubMed

    Yentz, Sarah; Onwuemene, Oluwatoyosi A; Stein, Brady L; Cull, Elizabeth H; McMahon, Brandon

    2015-01-01

    Introduction. Low molecular weight heparin (LMWH) is preferred for malignancy-associated venous thromboembolism (VTE). Many providers monitor LMWH with anti-Xa levels, despite little validation on correspondence with patient outcome. Methods. This is a retrospective, single institution study of anti-Xa measurement in malignancy-associated thrombosis. Cases were identified using the Electronic Data Warehouse, and inclusion was confirmed by two independent reviewers. Malignancy type, thrombotic history, measurement rationale and accuracy, clinical context, and management changes were evaluated. Results. 167 cases met inclusion criteria. There was no clear rationale for anti-Xa testing in 56%. Impaired renal function (10%), documented or suspected recurrent thrombosis despite anticoagulation (9%), and bleeding (6%) were the most common reasons for testing. Incorrect measurement occurred in 44%. Renal impairment was not a significant impetus for testing, as 70% had a GFR > 60. BMI > 30 was present in 40%, and 28% had a BMI < 25. Clinical impact was low, as only 11% of patients had management changes. Conclusions. Provider education in accuracy and rationale for anti-Xa testing is needed. Our study illustrates uncertainty of interpretation and clinical impact of routine anti-Xa testing, as management was affected in few patients. It is not yet clear in which clinical context providers should send anti-Xa levels. PMID:26543644

  11. Structure-activity relationships for substrate-based inhibitors of human complement factor B.

    PubMed

    Ruiz-Gómez, Gloria; Lim, Junxian; Halili, Maria A; Le, Giang T; Madala, Praveen K; Abbenante, Giovanni; Fairlie, David P

    2009-10-01

    Human complement is a cascading network of plasma proteins important in immune defense, cooperatively effecting recognition, opsonization, destruction, and removal of pathogens and infected/damaged cells. Overstimulated or unregulated complement activation can result in immunoinflammatory diseases. Key serine proteases in this cascade are difficult to study due to their multiprotein composition, short lifetimes, formation on membranes, or serum circulation as inactive zymogens. Factor B is inactive at pH 7, but a catalytically active serine protease under alkaline conditions, enabling structure-activity relationship studies for 63 substrate-based peptide inhibitors with 4-7 residues and a C-terminal aldehyde. A potent factor B inhibitor was hexpeptide Ac-RLTbaLAR-H (IC(50) 250 nM, pH 9.5), which at pH 7 also blocked formation of membrane attack complex via the "alternative pathway" of complement activation and inhibited human complement mediated lysis of rabbit erythrocytes. Inhibitors of factor B may be valuable probes and drug leads for complement mediated immunity and disease. PMID:19743866

  12. JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

    PubMed Central

    Nishiya, Naoyuki; Sakamoto, Yasumitsu; Oku, Yusuke; Nonaka, Takamasa; Uehara, Yoshimasa

    2015-01-01

    AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR). METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs. RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid

  13. Recent advances in drug design of epidermal growth factor receptor inhibitors.

    PubMed

    Warnault, P; Yasri, A; Coisy-Quivy, M; Chevé, G; Boriès, C; Fauvel, B; Benhida, R

    2013-01-01

    The tyrosine kinase epidermal growth factor receptor (EGFR) has emerged in recent years as a key and validated target of targeted therapies for solid tumors. It plays a central role in oncology since it is involved in many steps of tumor progression such as proliferation, angiogenesis, invasiveness, decreased apoptosis, and loss of differentiation. Recent advances in targeted therapies have demonstrated that tyrosine kinase inhibitors (TKIs), have provided a marked benefit to subsets of patients whose tumors harbor specific genetic abnormalities. However, resistance phenomenon appears rapidly and patients with EGFR mutations acquire resistance to TKI inhibitors decreasing therefore the median time to disease progression to few months. Several strategies were envisioned to overcome this resistance, such as dual-target inhibitors, multitarget and combined therapy. This review summarizes recent advances in TKIs development with special focus on rational strategies for the design of potent EGFR inhibitors including molecular modeling studies based on crystallographic data. Such advances open the way for new research possibilities in modern medicinal chemistry combined to structure-based drug design. PMID:23410174

  14. Response of early active rheumatoid arthritis to tumor necrosis factor inhibitors: evaluation by magnetic resonance imaging.

    PubMed

    Hirose, Wataru; Nishikawa, Kenichiro; Hirose, Masuko; Nanki, Toshihiro; Sugimoto, Hideharu

    2009-01-01

    Inflammatory changes (synovitis and bone marrow edema) and destructive changes (bone erosion) were evaluated by magnetic resonance imaging (MRI) in patients with rheumatoid arthritis (RA), and their relations with disease activity were assessed during treatment with tumor necrosis factor (TNF) inhibitors. Ten patients with early active RA underwent MRI at 0 and 16 weeks of TNF-inhibitor treatment. The carpal bones of the dominant hand were evaluated by the outcome measures in rheumatology clinical trials MRI score for RA. After 16 weeks, the mean disease activity score (DAS 28) decreased significantly from 5.54 to 2.70, while the number of tender joints, number of swollen joints, and inflammatory parameters were also significantly improved. The mean synovitis and marrow edema scores determined by MRI showed a significant decrease from 6.1 to 2.2 and 12.8 to 6.2, respectively, while the annual bone-erosion progression score decreased from 12.6 to 2.0. Although synovitis persisted in some patients, imaging remission was achieved in two patients. In conclusion, TNF-inhibitor therapy achieved an early decrease of disease activity and MRI revealed amelioration of joint destruction. The MRI score for RA is useful for assessing the early response to TNF inhibitors. PMID:18762862

  15. Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer

    PubMed Central

    2014-01-01

    Introduction Although aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2. Methods In vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance. Results Basal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited

  16. Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein.

    PubMed

    Mast, Alan E

    2016-01-01

    Tissue factor (TF) pathway inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response. Alternatively spliced isoforms of TFPI are differentially expressed by endothelial cells and human platelets and plasma. The TFPIβ isoform localizes to the endothelium surface where it is a potent inhibitor of TF-factor VIIa complexes that initiate blood coagulation. The TFPIα isoform is present in platelets. TFPIα contains a stretch of 9 amino acids nearly identical to those found in the B-domain of factor V that are well conserved in mammals. These amino acids provide exosite binding to activated factor V, which allows for TFPIα to inhibit prothrombinase during the initiation phase of blood coagulation. Endogenous inhibition at this point in the coagulation cascade was only recently recognized and has provided a biochemical rationale to explain the pathophysiological mechanisms underlying several clinical disorders. These include the east Texas bleeding disorder that is caused by production of an altered form of factor V with high affinity for TFPI and a paradoxical procoagulant effect of heparins. In addition, these findings have led to ideas for pharmacological targeting of TFPI that may reduce bleeding in hemophilia patients. PMID:26603155

  17. Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

    PubMed Central

    Fjellström, Ola; Akkaya, Sibel; Beisel, Hans-Georg; Eriksson, Per-Olof; Erixon, Karl; Gustafsson, David; Jurva, Ulrik; Kang, Daiwu; Karis, David; Knecht, Wolfgang; Nerme, Viveca; Nilsson, Ingemar; Olsson, Thomas; Redzic, Alma; Roth, Robert; Sandmark, Jenny; Tigerström, Anna; Öster, Linda

    2015-01-01

    Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds. PMID:25629509

  18. Highly predictive support vector machine (SVM) models for anthrax toxin lethal factor (LF) inhibitors.

    PubMed

    Zhang, Xia; Amin, Elizabeth Ambrose

    2016-01-01

    Anthrax is a highly lethal, acute infectious disease caused by the rod-shaped, Gram-positive bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF), a zinc metalloprotease secreted by the bacilli, plays a key role in anthrax pathogenesis and is chiefly responsible for anthrax-related toxemia and host death, partly via inactivation of mitogen-activated protein kinase kinase (MAPKK) enzymes and consequent disruption of key cellular signaling pathways. Antibiotics such as fluoroquinolones are capable of clearing the bacilli but have no effect on LF-mediated toxemia; LF itself therefore remains the preferred target for toxin inactivation. However, currently no LF inhibitor is available on the market as a therapeutic, partly due to the insufficiency of existing LF inhibitor scaffolds in terms of efficacy, selectivity, and toxicity. In the current work, we present novel support vector machine (SVM) models with high prediction accuracy that are designed to rapidly identify potential novel, structurally diverse LF inhibitor chemical matter from compound libraries. These SVM models were trained and validated using 508 compounds with published LF biological activity data and 847 inactive compounds deposited in the Pub Chem BioAssay database. One model, M1, demonstrated particularly favorable selectivity toward highly active compounds by correctly predicting 39 (95.12%) out of 41 nanomolar-level LF inhibitors, 46 (93.88%) out of 49 inactives, and 844 (99.65%) out of 847 Pub Chem inactives in external, unbiased test sets. These models are expected to facilitate the prediction of LF inhibitory activity for existing molecules, as well as identification of novel potential LF inhibitors from large datasets. PMID:26615468

  19. Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

    PubMed Central

    Wong, R L; Gutowski, J K; Katz, M; Goldfarb, R H; Cohen, S

    1987-01-01

    Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADR-containing preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-alpha-tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotinin-conjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These findings are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor we have called ADR may be a protease itself. PMID:3540956

  20. A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration.

    PubMed

    Ban, Hyun Seung; Xu, Xuezhen; Jang, Kusik; Kim, Inhyub; Kim, Bo-Kyung; Lee, Kyeong; Won, Misun

    2016-01-01

    We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells. PMID:27611801

  1. Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

    PubMed

    El-Mowafi, S A; Sineva, E; Alumasa, J N; Nicoloff, H; Tomsho, J W; Ades, S E; Keiler, K C

    2015-01-01

    Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway. PMID:25331704

  2. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

    PubMed

    Kitamura, Seiya; Hvorecny, Kelli L; Niu, Jun; Hammock, Bruce D; Madden, Dean R; Morisseau, Christophe

    2016-05-26

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application. PMID:27120257

  3. NOVEL ATYPICAL PKC INHIBITORS PREVENT VASCULAR ENDOTHELIAL GROWTH FACTOR-INDUCED BLOOD-RETINAL BARRIER DYSFUNCTION

    PubMed Central

    Titchenell, Paul M.; Lin, Cheng-Mao; Keil, Jason M.; Sundstrom, Jeffrey M.; Smith, Charles D.; Antonetti, David A.

    2013-01-01

    SYNOPSIS Pro-inflammatory cytokines and growth factors such as vascular endothelial growth factor (VEGF) contribute to the loss of the blood-retinal barrier (BRB) and subsequent macular edema in various retinal pathologies. VEGF signaling requires conventional PKC (PKCβ) activity; however, PKCβ inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability suggesting the involvement of alternative signaling pathways. Here, we provide evidence for the involvement of atypical protein kinase C (aPKC) signaling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small molecule inhibitors and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. These data suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis and the blood-brain barrier (BBB) in the presence of brain tumors. PMID:22721706

  4. Vascular Endothelial Growth Factor Inhibitor-Induced Hypertension: Basics for Primary Care Providers

    PubMed Central

    Escalante, Carmen P.; Zalpour, Ali

    2011-01-01

    Frequently, primary care providers continue to manage the overall medical care of cancer patients. With newer and often more potent antitumor agents, patients may present to their local physicians with drug-induced toxicities such as hypertension induced by vascular endothelial growth factor (VEGF) inhibitors. It is imperative that these healthcare providers are aware of basic aspects of this drug class, as its use has increased significantly in the last several years. Uncontrolled or malignant hypertension due to these agents should be recognized readily and treated early to prevent more severe outcomes. This overview provides a brief background on the role of VEGF and angiogenesis in tumor metabolism as well as theories of the mechanism of VEGF inhibitors and hypertension. Helpful clinical practice aspects including the types of inhibitors used in the United States and their pharmacologic characteristics will be discussed. Also, diagnosis and treatment of hypertension induced by vascular endothelial growth factors are reviewed. A summary of key aspects of this drug class and hypertension is included. PMID:21629798

  5. Inhibitors of hypoxia-inducible factor 1 block breast cancer metastatic niche formation and lung metastasis.

    PubMed

    Wong, Carmen Chak-Lui; Zhang, Huafeng; Gilkes, Daniele M; Chen, Jasper; Wei, Hong; Chaturvedi, Pallavi; Hubbi, Maimon E; Semenza, Gregg L

    2012-07-01

    Intratumoral hypoxia, a frequent finding in metastatic cancer, results in the activation of hypoxia-inducible factors (HIFs). HIFs are implicated in many steps of breast cancer metastasis, including metastatic niche formation through increased expression of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) proteins, enzymes that remodel collagen at the metastatic site and recruit bone marrow-derived cells (BMDCs) to the metastatic niche. We investigated the effect of two chemically and mechanistically distinct HIF inhibitors, digoxin and acriflavine, on breast cancer metastatic niche formation. Both drugs blocked the hypoxia-induced expression of LOX and LOXL proteins, collagen cross-linking, CD11b⁺ BMDC recruitment, and lung metastasis in an orthotopic breast cancer model. Patients with HIF-1 α-overexpressing breast cancers are at increased risk of metastasis and mortality and our results suggest that such patients may benefit from aggressive therapy that includes a HIF inhibitor. PMID:22231744

  6. Epidermal growth factor receptor tyrosine kinase inhibitors for non-small cell lung cancer

    PubMed Central

    Asami, Kazuhiro; Atagi, Shinji

    2014-01-01

    First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to first-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs. PMID:25302168

  7. Hypoxia inducible factor 1α expression and effects of its inhibitors in canine lymphoma

    PubMed Central

    KAMBAYASHI, Satoshi; IGASE, Masaya; KOBAYASHI, Kosuke; KIMURA, Ayana; SHIMOKAWA MIYAMA, Takako; BABA, Kenji; NOGUCHI, Shunsuke; MIZUNO, Takuya; OKUDA, Masaru

    2015-01-01

    Hypoxic conditions in various cancers are believed to relate with their malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major regulator of the response to low oxygen. In this study, we examined HIF-1α expression in canine lymphoma using cell lines and clinical samples and found that these cells expressed HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol, suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor. Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine lymphoma. PMID:26050843

  8. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

    PubMed Central

    Tucker, Erik I.; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D.; Gailani, Dave; Gruber, András; McCarty, Owen J. T.

    2015-01-01

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  9. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor.

    PubMed

    Puy, Cristina; Tucker, Erik I; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D; Gailani, Dave; Gruber, András; McCarty, Owen J T

    2015-02-26

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  10. Molecular modeling studies of [6,6,5] Tricyclic Fused Oxazolidinones as FXa inhibitors using 3D-QSAR, Topomer CoMFA, molecular docking and molecular dynamics simulations.

    PubMed

    Xu, Cheng; Ren, Yujie

    2015-10-15

    Coagulation factor Xa (Factor Xa, FXa) is a particularly promising target for novel anticoagulant therapy. The first oral factor Xa inhibitor has been approved in the EU and Canada in 2008. In this work, 38 [6,6,5] Tricyclic Fused Oxazolidinones were studied using a combination of molecular modeling techniques including three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, molecular dynamics and Topomer CoMFA (comparative molecular field analysis) were used to build 3D-QSAR models. The results show that the best CoMFA model has q(2)=0.511 and r(2)=0.984, the best CoMSIA (comparative molecular similarity indices analysis) model has q(2)=0.700 and r(2)=0.993 and the Topomer CoMFA analysis has q(2)=0.377 and r(2)=0.886. The results indicated the steric, hydrophobic, H-acceptor and electrostatic fields play key roles in models. Molecular docking and molecular dynamics explored the binding relationship of the ligand and the receptor protein. PMID:26343829

  11. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    PubMed Central

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-01-01

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13. PMID:25697265

  12. Fullerenes and their derivatives as inhibitors of tumor necrosis factor-α with highly promoted affinities.

    PubMed

    Wu, Gaoyin; Gao, Xuejiao J; Jang, Joonkyung; Gao, Xingfa

    2016-07-01

    Tumor necrosis factor-α (TNF-α) is a cell signalling protein involved in systemic inflammation in infectious and other malignant diseases. Physiologically, it plays an important role in regulating host defence, but its overexpression can lead to serious illnesses including cancer, autoimmune disease and inflammatory disease. Gadolinium-based metallofullerenols, e.g., Gd@C82(OH) x (x ≈ 22), are well known for their abundant biological activities with low toxicity experimentally and theoretically; however, their activity in direct TNF-α inhibition has not been explored. In this work, we investigated the inhibiting effects of four types of fullerene-based ligands: fullerenes, fullerenols, metallofullerenes, and metallofullerenols. We reported previously that fullerenes, metallofullerenes and their hydroxylated derivatives (fullerenols) can reside in the same pocket of the TNF-α dimer as that of SPD304-a known inhibitor of TNF-α [He et al. (2005) Science 310:1022, 18]. Ligand docking and binding free energy calculations suggest that, with a similar nonpolar interaction dominated binding pattern, the fullerene-based ligands, C60, C60(OH)12, Gd@C60, C82, C82(OH)12, Gd@C82, Gd@C82(OH)13 and Gd@C82(OH)21, have larger affinity than currently known inhibitors, and could be used to design novel inhibitors of TNF-α in the future. Graphical Abstract Fullerene-material/TNF-α. PMID:27316702

  13. A stonemason with accelerated silicosis in the setting of tumour necrosis factor alpha inhibitor therapy.

    PubMed

    Baird, Timothy; Putt, Michael; Dettrick, Andrew

    2016-09-01

    We present the case of a 26-year-old stonemason with accelerated silicosis in the setting of treatment for psoriasis with the tumour necrosis factor alpha (TNF-alpha) inhibitor adalimumab. Accelerated silicosis is an important occupational lung disease with a poor prognosis and limited treatment options [1]. Although the exact pathogenesis remains unknown, it is suggested that secretion of cytokines, including TNF-alpha, plays a central role in disease progression [1,2]. Importantly, however, TNF-alpha inhibitors, in addition to resulting in an increased risk of infection, are also now being seen to cause interstitial lung disease [3,4]. To our knowledge, this is the first documented patient to develop silicosis whilst on TNF-alpha inhibitor therapy. This case challenges the theory behind TNF-alpha's exact role in the pathogenesis of silicosis and lung fibrosis, highlights the importance of monitoring individuals with both occupational and drug exposures, and illustrates the increasing difficulties physicians face in investigating patients with pulmonary infiltrates and multiple possible aetiologies. PMID:27516887

  14. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors.

    PubMed

    Ji, Hongbin; Zhao, Xiaojun; Yuza, Yuki; Shimamura, Takeshi; Li, Danan; Protopopov, Alexei; Jung, Boonim L; McNamara, Kate; Xia, Huili; Glatt, Karen A; Thomas, Roman K; Sasaki, Hidefumi; Horner, James W; Eck, Michael; Mitchell, Albert; Sun, Yangping; Al-Hashem, Ruqayyah; Bronson, Roderick T; Rabindran, Sridhar K; Discafani, Carolyn M; Maher, Elizabeth; Shapiro, Geoffrey I; Meyerson, Matthew; Wong, Kwok-Kin

    2006-05-16

    The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation. PMID:16672372

  15. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

    PubMed Central

    Wilson, Timothy R.; Fridlyand, Jane; Yan, Yibing; Penuel, Elicia; Burton, Luciana; Chan, Emily; Peng, Jing; Lin, Eva; Wang, Yulei; Sosman, Jeff; Ribas, Antoni; Li, Jiang; Moffat, John; Sutherlin, Daniel P.; Koeppen, Hartmut; Merchant, Mark; Neve, Richard; Settleman, Jeff

    2013-01-01

    Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy1. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance2,3. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase4. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK)5. Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma6 or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-‘addicted’ human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases. PMID:22763448

  16. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors.

    PubMed

    Wilson, Timothy R; Fridlyand, Jane; Yan, Yibing; Penuel, Elicia; Burton, Luciana; Chan, Emily; Peng, Jing; Lin, Eva; Wang, Yulei; Sosman, Jeff; Ribas, Antoni; Li, Jiang; Moffat, John; Sutherlin, Daniel P; Koeppen, Hartmut; Merchant, Mark; Neve, Richard; Settleman, Jeff

    2012-07-26

    Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases. PMID:22763448

  17. Trypsinogen 4 boosts tumor endothelial cells migration through proteolysis of tissue factor pathway inhibitor-2

    PubMed Central

    Ghilardi, Carmen; Silini, Antonietta; Figini, Sara; Anastasia, Alessia; Lupi, Monica; Fruscio, Robert; Giavazzi, Raffaella; Bani, MariaRosa

    2015-01-01

    Proteasescontribute to cancer in many ways, including tumor vascularization and metastasis, and their pharmacological inhibition is a potential anticancer strategy. We report that human endothelial cells (EC) express the trypsinogen 4 isoform of the serine protease 3 (PRSS3), and lack both PRSS2 and PRSS1. Trypsinogen 4 expression was upregulated by the combined action of VEGF-A, FGF-2 and EGF, angiogenic factors representative of the tumor microenvironment. Suppression of trypsinogen 4 expression by siRNA inhibited the angiogenic milieu-induced migration of EC from cancer specimens (tumor-EC), but did not affect EC from normal tissues. We identified tissue factor pathway inhibitor-2 (TFPI-2), a matrix associated inhibitor of cell motility, as the functional target of trypsinogen 4, which cleaved TFPI-2 and removed it from the matrix put down by tumor-EC. Silencing tumor-EC for trypsinogen 4 accumulated TFPI2 in the matrix. Showing that angiogenic factors stimulate trypsinogen 4 expression, which hydrolyses TFPI-2 favoring a pro-migratory situation, our study suggests a new pathway linking tumor microenvironment signals to endothelial cell migration, which is essential for angiogenesis and blood vessel remodeling. Abolishing trypsinogen 4 functions might be an exploitable strategy as anticancer, particularly anti-vascular, therapy. PMID:26318044

  18. 3D/3D registration of coronary CTA and biplane XA reconstructions for improved image guidance

    SciTech Connect

    Dibildox, Gerardo Baka, Nora; Walsum, Theo van; Punt, Mark; Aben, Jean-Paul; Schultz, Carl; Niessen, Wiro

    2014-09-15

    Purpose: The authors aim to improve image guidance during percutaneous coronary interventions of chronic total occlusions (CTO) by providing information obtained from computed tomography angiography (CTA) to the cardiac interventionist. To this end, the authors investigate a method to register a 3D CTA model to biplane reconstructions. Methods: The authors developed a method for registering preoperative coronary CTA with intraoperative biplane x-ray angiography (XA) images via 3D models of the coronary arteries. The models are extracted from the CTA and biplane XA images, and are temporally aligned based on CTA reconstruction phase and XA ECG signals. Rigid spatial alignment is achieved with a robust probabilistic point set registration approach using Gaussian mixture models (GMMs). This approach is extended by including orientation in the Gaussian mixtures and by weighting bifurcation points. The method is evaluated on retrospectively acquired coronary CTA datasets of 23 CTO patients for which biplane XA images are available. Results: The Gaussian mixture model approach achieved a median registration accuracy of 1.7 mm. The extended GMM approach including orientation was not significantly different (P > 0.1) but did improve robustness with regards to the initialization of the 3D models. Conclusions: The authors demonstrated that the GMM approach can effectively be applied to register CTA to biplane XA images for the purpose of improving image guidance in percutaneous coronary interventions.

  19. Intravitreal vascular endothelial growth factor (VEGF) inhibitor injection in unrecognised early pregnancy.

    PubMed

    Kianersi, Farzan; Ghanbari, Heshmatollah; Naderi Beni, Zahra; Naderi Beni, Afsaneh

    2016-10-01

    The use of intravitreal vascular endothelial growth factor (VEGF) inhibitor medications has widened considerably to include indications affecting females of reproductive age. Our patient was inadvertently exposed to bevacizumab within the first trimester when placental growth and fetal organogenesis take place and patient suffered pregnancy loss. There is insufficient information to suggest that such use is safe, nor is there definitive evidence to suggest that it causes harm. We advise that ophthalmologists discuss pregnancy with women of childbearing age undergoing intraocular anti-VEGF injections and in pregnant woman counselling is needed to explain the potential risks and benefits. PMID:27251054

  20. RGS Spectroscopy of the Cygnus Loop XA Knot

    NASA Technical Reports Server (NTRS)

    Gaetz, Terrance J.; Mushotzky, Richard F. (Technical Monitor)

    2003-01-01

    The observations were performed at the end of April 2002, and the data were received in July 2002. Unfortunately, the observations were badly compromised by high levels of background radiation; one the three observations lost entirely. Two replacement observations were scheduled for November 2002, and were only made available in January of 2003. Consequently, we have had little time to grapple with the unusual data analysis challenges. The search for a postdoctoral fellow has been successfully concluded, and Manami Sasaki began working for us in January 2003. She will be supported in part by these funds, and will be working to help understand these data. Examination of the RGS 'Orders' images indicate the presence of broad emission lines (as expected for the diffuse XA knot). However, examination of the 'Spatial' dispersion/cross-dispersion images indicate that the emission is also broad in the cross-dispersion direction. (As a crosscheck, some of the 'Lockman Hole' datasets were also examined as representative 'sky background' datasets; in these, both types of images are relatively flat (outside the calibration source regions). The quicklook plots of the spectra show the expected O VII and O VIII lines, in addition to a complex around 35 Angstroms; the approx. 35 Angstrom line is likely the C V He-beta line at 34.97 Angstrom, but identifying the additional line(s) will require a more careful reduction of the data. Consequently, there is valuable information to be extracted from these data, but it is complicated by diffuse nature of the emission. Because the angular scale is large, we will have to make use of sky background datasets in order to do the background fitting. A color composite image of OM data in the three UV bands was presented at the 'How does the Galaxy Work?' meeting, and compared to optical and X-ray imaging data. Quantitative analysis will require obtaining the effective bandpasses of the UV filters so that the predominant line and continuum

  1. Crystallization and preliminary crystallographic data for a ternary complex between tissue factor, factor VIIa and a BPTI-derived inhibitor

    NASA Astrophysics Data System (ADS)

    Stura, Enrico A.; Ruf, Wolfram; Wilson, Ian A.

    1996-10-01

    The binding of tissue factor (TF) with the serine protease coagulation factor VIIa (VIIa) is the initial trigger for activation of the coagulation protease cascades. In complex with TF, VIIa has profoundly enhanced function in the limited proteolytic activation of the natural substrate factors X and IX. Here we report the screening and identification of crystallization conditions to produce diffraction quality crystals of the complex between TF · VIIa and a potent inhibitor (5L 15) derived from mutagenesis of the bovine pancreatic trypsin inhibitor (BPTI) sequence. The complex crystals were obtained from the soluble extracellular domain of tissue factor, expressed in Escherichia coli as a fusion protein, VIIa expressed in mammalian cells and recombinant 5L15. Because only 1.5 mg of complex were available for this work, a reverse screening based strategy was used in the search and optimization of the crystallization conditions. Two different crystal forms were obtained from polyethylene glycol 4000 and monomethyl polyethylene glycol 2000 with cacodylate buffer at pH 6.5 in the presence of sodium and calcium ions. The addition of magnesium and zinc have profound effects on the crystallization. Both crystal forms are trigonal with cell parameters a = b = 129.3 Å, c = 110.8 Å and a = b = 67.2 Å, c = 314.8 Å diffracting to 7 and 3.2 Å resolution, respectively, each with one molecule in the asymmetric unit. Complete data sets have been collected from each of these forms to the resolution to which the crystals diffract. A structural understanding of the interaction of VIIa with its cofactor TF to form a binary enzyme, and its inhibition by 5L15 will provide a basis for the development of antithrombotic strategies.

  2. Discovery of Novel Human Epidermal Growth Factor Receptor-2 Inhibitors by Structure-based Virtual Screening

    PubMed Central

    Shi, Zheng; Yu, Tian; Sun, Rong; Wang, Shan; Chen, Xiao-Qian; Cheng, Li-Jia; Liu, Rong

    2016-01-01

    Background: Human epidermal growth factor receptor-2 (HER2) is a trans-membrane receptor like protein, and aberrant signaling of HER2 is implicated in many human cancers, such as ovarian cancer, gastric cancer, and prostate cancer, most notably breast cancer. Moreover, it has been in the spotlight in the recent years as a promising new target for therapy of breast cancer. Objective: Since virtual screening has become an integral part of the drug discovery process, it is of great significant to identify novel HER2 inhibitors by structure-based virtual screening. Materials and Methods: In this study, we carried out a series of elegant bioinformatics approaches, such as virtual screening and molecular dynamics (MD) simulations to identify HER2 inhibitors from Food and Drug Administration-approved small molecule drug as potential “new use” drugs. Results: Molecular docking identified top 10 potential drugs which showed spectrum affinity to HER2. Moreover, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) might exert potential inhibitory effects against HER2-targeted anti-breast cancer therapeutics. Conclusion: Together, our findings may provide successful application of virtual screening studies in the lead discovery process, and suggest that our discovered small molecules could be effective HER2 inhibitor candidates for further study. SUMMARY A series of elegant bioinformatics approaches, including virtual screening and molecular dynamics (MD) simulations were took advantage to identify human epidermal growth factor receptor-2 (HER2) inhibitors. Molecular docking recognized top 10 candidate compounds, which showed spectrum affinity to HER2. Further, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) in candidate compounds were identified as potential “new use” drugs against HER2-targeted anti-breast cancer therapeutics. Abbreviations used: HER2: Human epidermal growth factor receptor-2

  3. The toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors.

    PubMed Central

    Kettelhut, I C; Fiers, W; Goldberg, A L

    1987-01-01

    Tumor necrosis factor (TNF) is a macrophage product under active study as an anticancer drug. However, this agent can be very toxic and has been implicated in the pathogenesis of endotoxic shock. After intravenous injection of human recombinant TNF (4 micrograms/g), growing rats showed an unusual constellation of physiological responses, and all died within 2-4 hr. In 1 hr, TNF caused a sharp fall (2.5 degrees C) in body temperature and a large increase in plasma prostaglandin E2 levels. Blood glucose initially increased, but then a profound hypoglycemia developed by 2 hr. The TNF-treated animals also showed diarrhea, cyanosis, and a severe metabolic acidosis. A single injection of the cyclooxygenase inhibitors indomethacin or ibuprofen before the TNF treatment completely prevented the rapid killing and reduced eventual lethality by 70%. These agents blocked prostaglandin E2 production and prevented the hypothermia, changes in blood glucose, acidosis, and other symptoms. Since similar physiological changes have been reported after endotoxin injection, our data support the suggestion that TNF production is a critical factor in the development of septic shock. These findings also indicate that increased production of prostaglandins or thromboxanes is important in endotoxic shock and argue that cyclooxygenase inhibitors should be useful in its therapy. Indomethacin did not block the cytotoxic effects of TNF in vitro on several transformed cell lines (HeLa, Me 180, or L929). Therefore, combined use of TNF with a cyclooxygenase inhibitor may allow safer administration of high doses of this polypeptide to cancer patients. PMID:3108890

  4. Histone Deacetylase Inhibitors: The Epigenetic Therapeutics That Repress Hypoxia-Inducible Factors

    PubMed Central

    Chen, Shuyang; Sang, Nianli

    2011-01-01

    Histone deacetylase inhibitors (HDACIs) have been actively explored as a new generation of chemotherapeutics for cancers, generally known as epigenetic therapeutics. Recent findings indicate that several types of HDACIs repress angiogenesis, a process essential for tumor metabolism and progression. Accumulating evidence supports that this repression is mediated by disrupting the function of hypoxia-inducible factors (HIF-1, HIF-2, and collectively, HIF), which are the master regulators of angiogenesis and cellular adaptation to hypoxia. Since HIF also regulate glucose metabolism, cell survival, microenvironment remodeling, and other alterations commonly required for tumor progression, they are considered as novel targets for cancer chemotherapy. Though the precise biochemical mechanism underlying the HDACI-triggered repression of HIF function remains unclear, potential cellular factors that may link the inhibition of deacetylase activity to the repression of HIF function have been proposed. Here we review published data that inhibitors of type I/II HDACs repress HIF function by either reducing functional HIF-1α levels, or repressing HIF-α transactivation activity. In addition, underlying mechanisms and potential proteins involved in the repression will be discussed. A thorough understanding of HDACI-induced repression of HIF function may facilitate the development of future therapies to either repress or promote angiogenesis for cancer or chronic ischemic disorders, respectively. PMID:21151670

  5. Emerging molecular targets in oncology: clinical potential of MET/hepatocyte growth-factor inhibitors

    PubMed Central

    Smyth, Elizabeth C; Sclafani, Francesco; Cunningham, David

    2014-01-01

    The MET/hepatocyte growth-factor (HGF) signaling pathway plays a key role in the processes of embryogenesis, wound healing, and organ regeneration. Aberrant activation of MET/HGF occurs through multiple mechanisms including gene amplification, mutation, protein overexpression, and abnormal gene splicing interrupting autocrine and paracrine regulatory feedback mechanisms. In many cancers including non-small-cell lung cancer, colorectal, gastric, renal, and hepatocellular cancer, dysregulation of MET may lead to a more aggressive cancer phenotype and may be a negative prognostic indicator. Successful therapeutic targeting of the MET/HGF pathway has been achieved using monoclonal antibodies against the MET receptor and its ligand HGF in addition to MET-specific and multitargeted small-molecule tyrosine-kinase inhibitors with several drugs in late-phase clinical trials including onartuzumab, rilotumumab, tivantinib, and cabozantinib. MET frequently interacts with other key oncogenic tyrosine kinases including epidermal growth-factor receptor (EGFR) and HER-3 and these interactions may be responsible for resistance to anti-EGFR therapies. Similarly, resistance to MET inhibition may be mediated through EGFR activation, or alternatively by increasing levels of MET amplification or acquisition of novel “gatekeeper” mutations. In order to optimize development of effective inhibitors of the MET/HGF pathway clinical trials must be enriched for patients with demonstrable MET-pathway dysregulation for which robustly standardized and validated assays are required. PMID:24959087

  6. Tissue Factor Pathway Inhibitor-2 Gene Polymorphisms Associate With Coronary Atherosclerosis in Chinese Population

    PubMed Central

    Yu, Jia; Liu, Rong-Le; Luo, Xin-Ping; Shi, Hai-ming; Ma, Duan; Pan, Jun-Jie; Ni, Huan-Chun

    2015-01-01

    Abstract Tissue factor pathway inhibitor-2 (TFPI-2) may play critical roles in the pathogenesis of atherosclerosis. In this study, we aimed to investigate the association between TFPI-2 gene polymorphisms and coronary atherosclerosis. Four hundred and seven patients with coronary atherosclerosis and 306 individuals with normal coronary artery were enrolled in the present study. Nine single-nucleotide polymorphisms (SNPs) (rs3763473, rs59805398, rs60215632, rs59999573, rs59740167, rs34489123, rs4517, rs4264, and rs4271) were detected with polymerase chain reaction-direct sequencing method. Severity of coronary atherosclerosis was assessed by Gensini score. After the baseline investigation, patients with coronary atherosclerosis were followed up for incidence of cardiovascular events (CVEs). Eight SNPs were in accordance with the Hardy–Weinberg equilibrium, and 8 haplotypes were constructed based on rs59999573, rs59740167, and rs34489123 after linkage disequilibrium and haplotype analysis. Two SNPs (rs59805398 and rs34489123) and 5 haplotypes correlated with coronary atherosclerosis even after adjustment by Gensini score. At follow-up (median 53 months, range 1–60 months), 85 patients experienced CVE. However, there was no strong association between the gene polymorphisms and the occurrence of CVE. Tissue factor pathway inhibitor-2 gene polymorphisms were associated with coronary atherosclerosis in the Chinese population, suggesting that the information about TFPI-2 gene polymorphisms was useful for assessing the risk of developing coronary atherosclerosis, but there was not enough evidence showing it could predict occurrence of CVE. PMID:26496276

  7. Polymer-conjugated inhibitors of tumor necrosis factor-α for local control of inflammation.

    PubMed

    Washburn, Newell R; Prata, Joseph E; Friedrich, Emily E; Ramadan, Mohamed H; Elder, Allison N; Sun, Liang Tso

    2013-01-01

    Burns, chronic wounds, osteoarthritis, and uveitis are examples of conditions characterized by local, intense inflammatory responses that can impede healing or even further tissue degradation. The most powerful anti-inflammatory drugs available are often administered systemically, but these carry significant side effects and are not compatible for patients that have underlying complications associated with their condition. Conjugation of monoclonal antibodies that neutralize pro-inflammatory cytokines to high molecular weight hydrophilic polymers has been shown to be an effective strategy for local control of inflammation. Lead formulations are based on antibody inhibitors of tumor necrosis factor-α conjugated to hyaluronic acid having molecular weight greater than 1 MDa. This review will discuss fundamental aspects of medical conditions that could be treated with these conjugates and design principles for preparing these cytokine-neutralizing polymer conjugates. Results demonstrating that infliximab, an approved inhibitor of tumor necrosis factor-α, can be incorporated into the conjugates using a broad range of water-soluble polymers are also presented, along with a prospectus for clinical translation. PMID:23903893

  8. Unusual location of tuberculosis in the course of tumor necrosis factor α inhibitor therapy

    PubMed Central

    Brzezicki, Jan; Rymko, Marcin; Jeka, Sławomir

    2015-01-01

    Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis complex mycobacteria. Extrapulmonary tuberculosis usually develops more than two years after infection or many years later. Factors favoring onset of the disease are malnutrition, older age, renal failure, diabetes, cancer, immunosuppression and biological treatment, e.g. tumor necrosis factor α (TNF-α) inhibitors. The paper presents a case of a 56-year-old patient with ankylosing spondylitis treated with infliximab, diagnosed with tuberculosis of the spleen. The unusual location and uncharacteristic symptoms created a lot of diagnostic difficulties, particularly as during qualification for biological treatment tests are performed to exclude infection with Mycobacterium tuberculosis. Pharmacological treatment of tuberculosis is typical, but in the case of tuberculosis of the spleen, splenectomy also is a method of treatment. The decision was made to implement pharmacological treatment, which proved to be effective, so the patient avoided surgery.

  9. Epidermal Growth Factor Receptor Inhibitors: A Review of Cutaneous Adverse Events and Management

    PubMed Central

    Chanprapaph, K.; Vachiramon, V.; Rattanakaemakorn, P.

    2014-01-01

    Epidermal growth factor inhibitors (EGFRI), the first targeted cancer therapy, are currently an essential treatment for many advance-stage epithelial cancers. These agents have the superior ability to target cancers cells and better safety profile compared to conventional chemotherapies. However, cutaneous adverse events are common due to the interference of epidermal growth factor receptor (EGFR) signaling in the skin. Cutaneous toxicities lead to poor compliance, drug cessation, and psychosocial discomfort. This paper summarizes the current knowledge concerning the presentation and management of skin toxicity from EGFRI. The common dermatologic adverse events are papulopustules and xerosis. Less common findings are paronychia, regulatory abnormalities of hair growth, maculopapular rash, mucositis, and postinflammatory hyperpigmentation. Radiation enhances EGFRI rash due to synergistic toxicity. There is a positive correlation between the occurrence and severity of cutaneous adverse effects and tumor response. To date, prophylactic systemic tetracycline and tetracycline class antibiotics have proven to be the most effective treatment regime. PMID:24723942

  10. Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

    PubMed

    Krastel, Philipp; Roggo, Silvio; Schirle, Markus; Ross, Nathan T; Perruccio, Francesca; Aspesi, Peter; Aust, Thomas; Buntin, Kathrin; Estoppey, David; Liechty, Brigitta; Mapa, Felipa; Memmert, Klaus; Miller, Howard; Pan, Xuewen; Riedl, Ralph; Thibaut, Christian; Thomas, Jason; Wagner, Trixie; Weber, Eric; Xie, Xiaobing; Schmitt, Esther K; Hoepfner, Dominic

    2015-08-24

    Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1. PMID:26179970

  11. "XA6" octahedra influencing the arrangement of anionic groups and optical properties in inverse-perovskite [B6O10]XA3 (X = Cl, Br; A = alkali metal).

    PubMed

    Yang, Zhihua; Lei, Bing-Hua; Yang, Bin; Pan, Shilie

    2016-06-01

    Exploring the effect of microscopic units, which set up the perovsikte framework, is of importance for material design. In this study, a series of borate halides with inverse-perovskite structures [B6O10]XA3 (X = Cl, Br; A = alkali metal) have been studied. It was revealed that the distortion and volume of XA6 octahedra influence the arrangement of anionic groups, which leads to the flexibility of the perovskite-related framework and differences in optical properties. Under the structural control scheme, the structure of Rb3B6O10Cl was predicted. The stability of the predicted structure was confirmed by an ab initio density functional theory-based method. The calculation shows Rb3B6O10Cl has a short UV cutoff edge of less than 200 nm, a moderate birefringence and a large second harmonic generation response. PMID:27211304

  12. [Analysis of the Cochrane Review: Direct thrombin inhibitors versus vitamin K antagonists for preventing cerebral or systemic embolism in people with non-valvular atrial fibrillation. Cochrane Database Syst Rev. 2014,3:CD009893].

    PubMed

    Vaz Carneiro, António; Costa, João

    2014-01-01

    Ischemic stroke is one of the most important complications of lone (non-valvular) atrial fibrillation. Its prevention is usually accomplished through oral anticoagulation. Until a few years ago warfarin was the most used agent, but recently two new pharmacologic classes have been introduced for stroke prevention in these patients: oral direct thrombin inhibitors (dabigatran and ximelagatran) and oral factor Xa inhibitors (rivaroxaban, apixaban and edoxaban). In this systematic review, oral direct thrombin inhibitors were compared with warfarin for efficacy and safety. The results indicate that there is no difference in terms of efficacy (except dabigatran 150 mg BID). Oral direct thrombin inhibitors presented less hemorrhages but increased treatment withdrawal due to adverse side-effects (the authors performed post-hoc analyses excluding ximelagatran because this drug was withdrawn from the market owing to safety concerns). There was no difference in terms of mortality between the agents. PMID:24813481

  13. Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5).

    PubMed

    Callahan, James F; Burgess, Joelle L; Fornwald, James A; Gaster, Laramie M; Harling, John D; Harrington, Frank P; Heer, Jag; Kwon, Chet; Lehr, Ruth; Mathur, A; Olson, Barbara A; Weinstock, Joseph; Laping, Nicholas J

    2002-02-28

    Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay. PMID:11855979

  14. Coexpression of heparanase activity, cathepsin L, tissue factor, tissue factor pathway inhibitor, and MMP-9 in proliferative diabetic retinopathy

    PubMed Central

    Siddiquei, Mohammad Mairaj; Nawaz, Mohd Imtiaz; De Hertogh, Gert; Mohammad, Ghulam; Alam, Kaiser; Mousa, Ahmed; Opdenakker, Ghislain

    2016-01-01

    Purpose Heparanase cleaves heparan sulfate side chains of heparan sulfate proteoglycans, activity that is implicated in angiogenesis. Proteolytic cleavage of proheparanase by cathepsin L leads to the formation of catalytically active heparanase. We investigated the expression levels of heparanase enzymatic activity and correlated these with the levels of cathepsin L, the angiogenic factors tissue factor (TF) and matrix metalloproteinase-9 (MMP-9), and the angiostatic factor tissue factor pathway inhibitor (TFPI) in proliferative diabetic retinopathy (PDR). Methods Vitreous samples from 25 patients with PDR and 20 nondiabetic patients and epiretinal membranes from 12 patients with PDR were studied with enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry. Results We observed a significant increase in the expression of heparanase activity in vitreous samples from patients with PDR compared to the nondiabetic controls (p=0.027). Significant positive correlations were found between the levels of heparanase activity and the levels of cathepsin L (r=0.51; p=0.001), TF (r=0.6; p<0.0001), and TFPI (r=0.49; p=0.001). The expression levels of cathepsin L (p=0.019), TF (p<0.0001), TFPI (p<0.0001), and MMP-9 (p=0.029) were significantly higher in the vitreous samples with detected heparanase activity compared to the vitreous samples with undetected heparanase activity. Western blot analysis demonstrated proteolytic cleavage of TFPI in the vitreous samples from patients with PDR. In the epiretinal membranes, cathepsin L, TF, and TFPI were expressed in vascular endothelial cells and CD45-expressing leukocytes. Significant positive correlations were detected between the number of blood vessels that expressed CD31 and the number of blood vessels that expressed TF (r=0.9; p<0.0001) and TFPI (r=0.81; p=0.001). Conclusions The coexpression of these angiogenesis regulatory factors suggests cross-talk between these factors and pathogenesis of PDR

  15. Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage

    PubMed Central

    Bate, Clive; Rumbold, Louis; Williams, Alun

    2007-01-01

    Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902

  16. Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action*

    PubMed Central

    Ouertatani-Sakouhi, Hajer; El-Turk, Farah; Fauvet, Bruno; Cho, Min-Kyu; Pinar Karpinar, Damla; Le Roy, Didier; Dewor, Manfred; Roger, Thierry; Bernhagen, Jürgen; Calandra, Thierry; Zweckstetter, Markus; Lashuel, Hilal A.

    2010-01-01

    Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC50 values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease. PMID:20516071

  17. Discovery of Novel Fibroblast Growth Factor Receptor 1 Kinase Inhibitors by Structure-Based Virtual Screening

    SciTech Connect

    Ravindranathan, K.; Mandiyan, V; Ekkati, A; Bae, J; Schlessinger, J; Jorgensen, W

    2010-01-01

    Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC{sub 50} values of 23 and 50 {micro}M. Initial optimization of 16 led to the more unsaturated 40, which has significantly enhanced potency, 1.9 {micro}M. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

  18. Modulation of influenza vaccine immune responses using an epidermal growth factor receptor kinase inhibitor

    PubMed Central

    Pulit-Penaloza, Joanna A.; Sapkota, Bishu; Stein Esser, E.; Compans, Richard W.; Pollack, Brian P.; Skountzou, Ioanna

    2015-01-01

    Systemic use of epidermal growth factor receptor inhibitors (EGFRIs) has been shown to alter MHC expression and that of several chemokines, and to enhance immune cell recruitment into human skin. We hypothesized that EGFRIs may have value as cutaneous immune response modifiers, and determined the effects of topical application of an irreversible EGFRI on a well-established murine model of influenza vaccination. We found that a single topical application of an EGFRI led to increased levels of antibodies that inhibit influenza mediated hemagglutination and viral cytopathic effects. The topically applied EGFRI significantly enhanced the generation of vaccine-specific IL-4 and IFN-γ producing cells within skin-draining lymph nodes as early as one week following vaccination. The EGFRI/vaccine group showed a twelve-fold reduction in detectable pulmonary viral load four days after infection as compared to the vaccine alone control group. The reduction in the lung viral titers correlated with the survival rate, which demonstrated 100% protection in the EGFRI/vaccine immunized group but only 65% protection in the mice immunized with vaccine alone. These findings are significant because they demonstrate that inhibition of defined signaling pathways within the skin using small molecule kinase inhibitors provides a novel approach to enhance immune responses to vaccines. PMID:26227481

  19. Mitochondrial Inhibitory Factor Protein 1 Functions as an Endogenous Inhibitor for Coupling Factor 6.

    PubMed

    Kawai, Misato; Osanai, Tomohiro; Tanaka, Makoto; Magota, Koji; Tomita, Hirofumi; Okumura, Ken

    2016-07-01

    Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex unlike a proton-mediated clockwise rotation in the mitochondria, resulting in ATP hydrolysis, proton import, and apoptosis. Inhibitory peptide 1 (IF1) inhibits a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis by a clockwise rotation. We tested the hypothesis that IF1 may antagonize the biological action of CF6 in human embryonic kidney 293 cells. We generated mature and immature IF1 expression vectors and those labeled with GFP at the C-terminus. In the immature IF1-GFP overexpressing cells, the mitochondrial network of IF1-GFP was newly found at the plasma membrane after peripheral translocation, whereas in mature IF1-GFP transfected cells, a less punctuate rather homogenous pattern was found in the cytoplasm. IF1 protein was detected in the exosome fraction of culture media, and it was enhanced by mature or immature IF1 transfection. Extracellular ATP hydrolysis was enhanced by CF6, whereas immature or mature IF1 transfection suppressed ATP hydrolysis in response to CF6. Intracellular pH was decreased by CF6 but was unchanged after immature IF1 transfection. CF6-induced increase in apoptotic cells was blocked by immature or mature IF1, being accompanied by protein kinase B (PKB) phosphorylation. IF1 antagonizes the pro-apoptotic action of CF6 by relief of intracellular acidification and resultant phosphorylation of PKB. Given the widespread biological actions of CF6, the physiological and pathological functions of IF1 may be expected to be complex. J. Cell. Biochem. 117: 1680-1687, 2016. © 2015 Wiley Periodicals, Inc. PMID:26659871

  20. Bilateral diaphragmatic paralysis associated with the use of the tumor necrosis factor-alpha inhibitor adalimumab

    PubMed Central

    Martin, Alan William; Rosenblatt, Randall Lee

    2014-01-01

    A 51-year-old woman was referred for evaluation of progressive dyspnea of 3 months— duration. She had received 3 doses of adalimumab for treatment of rheumatoid arthritis prior to the onset of her dyspnea. Her chest examination revealed absent diaphragmatic movement with inspiration. Spirometry showed a severe restrictive defect. Radiologic studies confirmed the diagnosis of bilateral diaphragmatic paralysis. Laboratory and radiologic workup excluded other possible causes of the diagnosis. Adalimumab was discontinued, and she was treated with bilevel positive airway pressure ventilation and intravenous immunoglobulin. Three months later, the diaphragmatic paralysis persisted. This is the second reported case of bilateral diaphragmatic paralysis occurring in a patient who had received adalimumab. Acute neuropathies are rare side effects of tumor necrosis factor-alpha inhibitors. PMID:24688191

  1. Hippuristanol - A potent steroid inhibitor of eukaryotic initiation factor 4A.

    PubMed

    Cencic, Regina; Pelletier, Jerry

    2016-01-01

    Protein synthesis and its regulatory signaling pathways play essential roles in the initiation and maintenance of the cancer phenotype. Insight obtained over the last 3 decades on the mechanisms regulating translation in normal and transformed cells have revealed that perturbed control in cancer cells may offer an Achilles' heel for the development of novel anti-neoplastic agents. Several small molecule inhibitors have been identified and characterized that target translation initiation - more specifically, the rate-limiting step where ribosomes are recruited to mRNA templates. Among these, hippuristanol, a polyhydroxysteroid from the gorgonian Isis hippuris has been found to inhibit translation initiation by blocking the activity of eukaryotic initiation factor (eIF) 4A, an essential RNA helicase involved in this process. Herein, we highlight the biological properties of this compound, its potential development as an anti-cancer agent, and its use to validate eIF4A as an anti-neoplastic target. PMID:27335721

  2. Histone deacetylase inhibitors and transforming growth factor-beta induce 15-hydroxyprostaglandin dehydrogenase expression in human lung adenocarcinoma cells.

    PubMed

    Tong, Min; Ding, Yunfei; Tai, Hsin-Hsiung

    2006-09-14

    Histone deacetylase (HDAC) inhibitors have been actively exploited as potential anticancer agents. To identify gene targets of HDAC inhibitors, we found that HDAC inhibitors such as sodium butyrate, scriptaid, apicidin and oxamflatin induced the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a potential cyclooxygenase-2 (COX-2) antagonist and tumor suppressor, in a time and concentration dependent manner in A549 and H1435 lung adenocarcinoma cells. Detailed analyses indicated that HDAC inhibitors activated the 15-PGDH promoter-luciferase reporter construct in transfected A549 cells. A representative HDAC inhibitor, scriptaid, and its negative structural analog control, nullscript, were further evaluated at the chromatin level. Scriptaid but not nullscript induced a significant accumulation of acetylated histones H3 and H4 which were associated with the 15-PGDH promoter as determined by chromatin immunoprecipitation assay. Transforming growth factor-beta1 (TGF-beta1) also induced the expression of 15-PGDH in a time and concentration dependent manner in A549 and H1435 cells. Induction of 15-PGDH expression by TGF-beta1 was synergistically stimulated by the addition of Wnt3A which was inactive by itself. However, combination of TGF-beta and an HDAC inhibitor, scriptaid, only resulted in an additive effect. Together, our results indicate that 15-PGDH is one of the target genes that HDAC inhibitors and TGF-beta may induce to exhibit tumor suppressive effects. PMID:16844092

  3. Developing an Anti-Xa-Based Anticoagulation Protocol for Patients with Percutaneous Ventricular Assist Devices.

    PubMed

    Sieg, Adam; Mardis, B Andrew; Mardis, Caitlin R; Huber, Michelle R; New, James P; Meadows, Holly B; Cook, Jennifer L; Toole, J Matthew; Uber, Walter E

    2015-01-01

    Because of the complexities associated with anticoagulation in temporary percutaneous ventricular assist device (pVAD) recipients, a lack of standardization exists in their management. This retrospective analysis evaluates current anticoagulation practices at a single center with the aim of identifying an optimal anticoagulation strategy and protocol. Patients were divided into two cohorts based on pVAD implanted (CentriMag (Thoratec; Pleasanton, CA) / TandemHeart (CardiacAssist; Pittsburgh, PA) or Impella (Abiomed, Danvers, MA)), with each group individually analyzed for bleeding and thrombotic complications. Patients in the CentriMag/TandemHeart cohort were subdivided based on the anticoagulation monitoring strategy (activated partial thromboplastin time (aPTT) or antifactor Xa unfractionated heparin (anti-Xa) values). In the CentriMag/TandemHeart cohort, there were five patients with anticoagulation titrated based on anti-Xa values; one patient developed a device thrombosis and a major bleed, whereas another patient experienced major bleeding. Eight patients received an Impella pVAD. Seven total major bleeds in three patients and no thrombotic events were detected. Based on distinct differences between the devices, anti-Xa values, and outcomes, two protocols were created to guide anticoagulation adjustments. However, anticoagulation in patients who require pVAD support is complex with constantly evolving anticoagulation goals. The ideal level of anticoagulation should be individually determined using several coagulation laboratory parameters in concert with hemodynamic changes in the patient's clinical status, the device, and the device cannulation. PMID:26273933

  4. Delirium in the geriatric unit: proton-pump inhibitors and other risk factors

    PubMed Central

    Otremba, Iwona; Wilczyński, Krzysztof; Szewieczek, Jan

    2016-01-01

    Background Delirium remains a major nosocomial complication of hospitalized elderly. Predictive models for delirium may be useful for identification of high-risk patients for implementation of preventive strategies. Objective Evaluate specific factors for development of delirium in a geriatric ward setting. Methods Prospective cross-sectional study comprised 675 consecutive patients aged 79.2±7.7 years (66% women and 34% men), admitted to the subacute geriatric ward of a multiprofile university hospital after exclusion of 113 patients treated with antipsychotic medication because of behavioral disorders before admission. Comprehensive geriatric assessments including a structured interview, physical examination, geriatric functional assessment, blood sampling, ECG, abdominal ultrasound, chest X-ray, Confusion Assessment Method for diagnosis of delirium, Delirium-O-Meter to assess delirium severity, Richmond Agitation-Sedation Scale to assess sedation or agitation, visual analog scale and Doloplus-2 scale to assess pain level were performed. Results Multivariate logistic regression analysis revealed five independent factors associated with development of delirium in geriatric inpatients: transfer between hospital wards (odds ratio [OR] =2.78; confidence interval [CI] =1.54–5.01; P=0.001), preexisting dementia (OR =2.29; CI =1.44–3.65; P<0.001), previous delirium incidents (OR =2.23; CI =1.47–3.38; P<0.001), previous fall incidents (OR =1.76; CI =1.17–2.64; P=0.006), and use of proton-pump inhibitors (OR =1.67; CI =1.11–2.53; P=0.014). Conclusion Transfer between hospital wards, preexisting dementia, previous delirium incidents, previous fall incidents, and use of proton-pump inhibitors are predictive of development of delirium in the geriatric inpatient setting. PMID:27103793

  5. A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggs

    PubMed Central

    Tung, Jeffrey J.; Hansen, David V.; Ban, Kenneth H.; Loktev, Alexander V.; Summers, Matthew K.; Adler, John R.; Jackson, Peter K.

    2005-01-01

    Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCFβTrCP ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein. PMID:15753281

  6. Hydatid cyst surgery complicated by hemorrhage resistant to activated recombinant factor VII, in a hemophiliac A patient with an inhibitor.

    PubMed

    Nabil, Saad; Bentalha, Aziza; Jaiteh, Lamine; El Khorassani, Mohammed; El Koraichi, Alae; El Kettani, Salma E-C

    2016-09-01

    Factor VII is a new coagulation factor replacement therapy. It has permitted the practice of invasive procedures which were up until recently associated with a huge risk of bleeding in patients with hemophilia with inhibitors. Our case illustrates factor replacement therapy failure in a 13-year-old child operated on for hepatic cysts associated with a pelvic cyst. Major bleeding occurred postoperatively requiring several transfusions, an increase in dosage of factor VII, and administration of a heavy dose of factor VIII as a last resort. This case highlights the possibility of failure of factor replacement therapies constituting a life-threatening situation in which alternatives are few. PMID:26761579

  7. Synergistic inhibition with a dual epidermal growth factor receptor/HER-2/neu tyrosine kinase inhibitor and a disintegrin and metalloprotease inhibitor.

    PubMed

    Witters, Lois; Scherle, Peggy; Friedman, Steven; Fridman, Jordan; Caulder, Eian; Newton, Robert; Lipton, Allan

    2008-09-01

    The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-alpha, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu-positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points. PMID:18757423

  8. Hepatocyte growth factor reduces sensitivity to the epidermal growth factor receptor-tyrosine kinase inhibitor, gefitinib, in lung adenocarcinoma cells harboring wild-type EGFR

    PubMed Central

    Yang, Hua; Wang, Rong; Peng, Shunli; Chen, Longhua; Li, Qi; Wang, Wei

    2016-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is an option for lung cancers harboring wild-type EGFR when chemotherapeutic reagents have failed. In this study, we found that the EGFR-TKI, gefitinib, modestly suppressed proliferation of the lung cancer cell lines, A549 and H358, which both harbor wild-type EGFR. Treatment with hepatocyte growth factor (HGF) reduced the sensitivity to gefitinib, whereas sensitivity was restored by treatment with an HGF antibody, a MET inhibitor, or depletion of MET but not ErbB3 gene. Moreover, both PI3K/mTOR inhibitors and MEK inhibitors suppressed proliferation of A549 cells, whereas only PI3K/mTOR inhibitors effectively suppressed cell viability of EGFR mutant PC-9 cells. Our findings suggest that HGF reduced the gefitinib sensitivity through MET and downstream PI3K and MAPK pathways. Combined use of EGFR-TKI and MET inhibitors or inhibition of downstream signaling molecules might be a better second or third line choice for a group of patients with advanced lung cancer harboring wild-type EGFR. PMID:26919104

  9. Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Sutherland, Brian G; Robson, Debra L; Arya, Rigya; Kelly, Karen; Jacobs, René L; Borradaile, Nica M

    2016-09-01

    Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease. PMID:27613825

  10. A new class of isothiocyanate-based irreversible inhibitors of Macrophage Migration Inhibitory Factor (MIF)

    PubMed Central

    Ouertatani-Sakouhi, Hajer; El-Turk, Farah; Fauvet, Bruno; Roger, Thierry; Le Roy, Didier; Karpinar, Damla Pinar; Leng, Lin; Bucala, Richard; Zweckstetter, Markus; Calandra, Thierry; Lashuel, Hilal A.

    2013-01-01

    Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without affecting the oligomerization state of MIF. Different alkyl and arylalkyl ITCs-modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding and biological activity of MIF. Light scattering, analytical ultracentrifugation and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and loss of catalytic activity translated into reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo. PMID:19737008

  11. Microplate-based screening for small molecule inhibitors of neuropilin-2/vascular endothelial growth factor-C interactions.

    PubMed

    Parker, Matthew W; Vander Kooi, Craig W

    2014-05-15

    Vascular endothelial growth factor-C (VEGF-C) is a secreted growth factor essential for lymphangiogenesis. VEGF-C functions in both physiological and pathological lymphangiogenesis, particularly in tumor metastasis, making it an attractive therapeutic target. Members of two families of cell surface receptors transduce VEGF-C signals: neuropilin-2 (Nrp2) and VEGF-receptor (VEGFR)-2/3. Nrp2 is a promising target for inhibition because it is highly expressed in lymphatic vessels. Here we describe a microplate-based assay for discovery of VEGF-C/Nrp2 inhibitors. We optimize this assay for use in screening an inhibitor library and identify three novel Nrp2/VEGF-C binding inhibitors from the National Institutes of Health (NIH) Clinical Collection small molecule library. PMID:24583243

  12. Novel Aurora/vascular endothelial growth factor receptor dual kinase inhibitor as treatment for hepatocellular carcinoma.

    PubMed

    Nakao, Keisuke; Tanaka, Shinji; Miura, Tomoya; Sato, Kota; Matsumura, Satoshi; Aihara, Arihiro; Mitsunori, Yusuke; Ban, Daisuke; Ochiai, Takanori; Kudo, Atsushi; Arii, Shigeki; Tanabe, Minoru

    2015-08-01

    We previously identified Aurora B kinase as the only independent factor predictive of the aggressive recurrence of hepatocellular carcinoma (HCC). In this preclinical study, JNJ-28841072, a novel Aurora/vascular endothelial growth factor receptor dual kinase inhibitor, was evaluated for treatment of HCC. In vitro and in vivo effects of JNJ-28841072 were analyzed using human HCC cell cultures and xenograft models. An orthotopic liver xenograft model was used for the pharmacobiological effects on Aurora kinase and vascularization in hepatic tumors. JNJ-28841072 suppressed in vitro phosphorylation of histone H3 with induction of cell polyploidy and death in a dose-dependent manner (IC50  = 0.8-1.2 μM). In s.c. human HCC xenografts, remarkable inhibition of tumor growth was observed after JNJ-28841072 treatment (P = 0.0005). In orthotopic liver xenografts, the treatment with JNJ-28841072 significantly suppressed in vivo phosphorylation of histone H3 (P = 0.0008), vessel formation (P = 0.018), normoxic area (P = 0.0001), and hepatoma growth (P = 0.038). Our preclinical studies indicate that JNJ-28841072 is a promising novel therapeutic approach for the treatment of HCC. It might be worthy of evaluation in further studies. PMID:26011703

  13. Insulin-like Growth Factor Receptor Inhibitors: Baby or the Bathwater?

    PubMed Central

    2012-01-01

    The success of targeted therapies for cancer is undisputed; strong preclinical evidence has resulted in the approval of several new agents for cancer treatment. The type I insulin-like growth factor receptor (IGF1R) appeared to be one of these promising new targets. Substantial population and preclinical data have all pointed toward this pathway as an important regulator of tumor cell biology. Although early results from clinical trials that targeted the IGF1R showed some evidence of response, larger randomized phase III trials have not shown clear clinical benefit of targeting this pathway in combination with conventional strategies. These disappointing results have resulted in the discontinuation of several anti-IGF1R programs. However, the conduct of these trials has brought to the forefront several important factors that need to be considered in the conduct of future clinical trials. The need to develop biomarkers, a clearer understanding of insulin receptor function, and defining rational combination regimens all require further consideration. In this commentary, the current state of IGF1R inhibitors in cancer therapy is reviewed. PMID:22761272

  14. A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors

    PubMed Central

    2016-01-01

    Human macrophage migration inhibitory factor (MIF) is both a keto–enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein–ligand hydrogen bonding, aryl–aryl, and cation−π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders. PMID:27299179

  15. A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors.

    PubMed

    Cisneros, José A; Robertson, Michael J; Valhondo, Margarita; Jorgensen, William L

    2016-07-13

    Human macrophage migration inhibitory factor (MIF) is both a keto-enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein-ligand hydrogen bonding, aryl-aryl, and cation-π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders. PMID:27299179

  16. Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study

    PubMed Central

    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Shapiro, Amy D.; Lentz, Steven R.; Wicklund, Brian M.; Kelly, Fiona M.; Soucie, J. Michael

    2015-01-01

    Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)<0.5 and positive on 42/42 specimens (100%) with NBU≥2.0 and 43/80 specimens (53.8%) with NBU 0.5–1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays. PMID:23601690

  17. Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

    PubMed Central

    Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

    2014-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

  18. Epidermal growth factor receptor inhibitor protects against abdominal aortic aneurysm in a mouse model.

    PubMed

    Obama, Takashi; Tsuji, Toshiyuki; Kobayashi, Tomonori; Fukuda, Yamato; Takayanagi, Takehiko; Taro, Yoshinori; Kawai, Tatsuo; Forrester, Steven J; Elliott, Katherine J; Choi, Eric; Daugherty, Alan; Rizzo, Victor; Eguchi, Satoru

    2015-05-01

    Angiotensin II (Ang II) has been implicated in the development of abdominal aortic aneurysm (AAA). In vascular smooth muscle cells (VSMC), Ang II activates epidermal growth factor receptor (EGFR) mediating growth promotion. We hypothesized that inhibition of EGFR prevents Ang II-dependent AAA. C57BL/6 mice were co-treated with Ang II and β-aminopropionitrile (BAPN) to induce AAA with or without treatment with EGFR inhibitor, erlotinib. Without erlotinib, 64.3% of mice were dead due to aortic rupture. All surviving mice had AAA associated with EGFR activation. Erlotinib-treated mice did not die and developed far fewer AAA. The maximum diameters of abdominal aortas were significantly shorter with erlotinib treatment. In contrast, both erlotinib-treated and non-treated mice developed hypertension. The erlotinib treatment of abdominal aorta was associated with lack of EGFR activation, endoplasmic reticulum (ER) stress, oxidative stress, interleukin-6 induction and matrix deposition. EGFR activation in AAA was also observed in humans. In conclusion, EGFR inhibition appears to protect mice from AAA formation induced by Ang II plus BAPN. The mechanism seems to involve suppression of vascular EGFR and ER stress. PMID:25531554

  19. Combination of vascular endothelial growth factor inhibitors and laser therapy for diabetic macular oedema: a review.

    PubMed

    Mehta, Hemal; Gillies, Mark C; Fraser-Bell, Samantha

    2016-05-01

    This review provides a perspective on published and ongoing clinical trials of vascular endothelial growth factor inhibitors (anti-VEGF agents) combined with laser therapy for diabetic macular oedema (DMO). Although there was little short-term benefit in combining prompt macular laser with anti-VEGF therapy for centre-involving DMO in the Diabetic Retinopathy Clinical Research Network (DRCRnet) Protocol I study, deferred macular laser was still required in over 40% of study eyes in DRCRnet Protocol T. Macular laser was applied in more than 30% of eyes with centre-involving DMO receiving ranibizumab in the RISE and RIDE studies. For non centre-involving DMO the evidence-base still supports use of focal macular laser alone, although clinicians should be cautious about applying laser too close to the foveal avascular zone with the availability of pharmacotherapy. Ongoing clinical trials are assessing whether selectively targeting areas of peripheral retinal ischaemia with laser reduces the number of anti-VEGF injections to stabilise DMO and whether combining macular micropulse laser with anti-VEGF therapy is beneficial in DMO. PMID:27061760

  20. Natural Nuclear Factor Kappa Beta Inhibitors: Safe Therapeutic Options for Inflammatory Bowel Disease.

    PubMed

    Tambuwala, Murtaza M

    2016-03-01

    Inflammatory bowel disease (IBD) is a chronic and debilitating condition classified as ulcerative colitis and Crohn's disease. IBD usually happens as result of immune dysfunction in the intestinal mucosa resulting in epithelial barrier dysfunction, which leads to exposure of the mucosal immune system to luminal antigenic material. This results in activation of inflammation, which is our bodies natural defense system; however, chronic inflammation leads to barrier dysfunction, which triggers a cycle of inflammation and further barrier dysfunction. This barrier breakdown results in the uncontrolled progression of IBD throughout the intestine. Despite the therapeutic advances made over the last decade, the current first line of treatment of IBD is limited to immunosuppressive and anti-inflammatory drugs, which need to be taken regularly and have significant side effects to the patients. Prolonged inflammation may increase the risk of intestinal malignancy. The role of nuclear factor kappa beta (NF-κβ) has been established in the regulation of innate immunity and inflammation. NF-κβ has also shown to be involved in critical events linking inflammation and cancer development. Recent investigations suggest that the NF-κβ signaling cascade may be the central mediator of gastrointestinal inflammation in IBD and malignancies including esophageal, gastric, and colorectal cancers. In this review, the therapeutic potential of natural NF-κβ inhibitors as safe therapeutic options for the treatment of IBD will be discussed. PMID:26717321

  1. Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2.

    PubMed

    La Venuta, Giuseppe; Wegehingel, Sabine; Sehr, Peter; Müller, Hans-Michael; Dimou, Eleni; Steringer, Julia P; Grotwinkel, Mareike; Hentze, Nikolai; Mayer, Matthias P; Will, David W; Uhrig, Ulrike; Lewis, Joe D; Nickel, Walter

    2016-08-19

    Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis. PMID:27382052

  2. Discovery of Novel Inhibitors of the Tautomerase Activity of Macrophage Migration Inhibitory Factor (MIF).

    PubMed

    Zapatero, Maria Cleofé; Pérez, Paloma; Vázquez, María Jesús; Colmenarejo, Gonzalo; de Los Frailes, Maite; Ramón, Fernando

    2016-06-01

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine associated with multiple diseases, including neurodegenerative disorders. With the ultimate goal of providing novel chemotypes as starting points for development of disease-modifying therapeutics for neurodegeneration, we endeavored to screen the GSK compound collection for MIF inhibitors using a miniaturized, activity-based kinetic assay. The assay monitors the increase in absorbance at 320 nm resulting from keto-to-enol tautomerization of 4-hydroxyphenylpyruvate, a reaction catalyzed by MIF. We ran a full-diversity screen evaluating the inhibitory activity of 1.6 million compounds. Primary hits were confirmed and retested in an orthogonal assay measuring tautomerization of l-dopachrome methyl ester by the decrease in absorbance at 475 nm in kinetic mode. Selected compounds were progressed to medium-throughput mode-of-inhibition studies, which included time dependence, enzyme concentration dependence, and reversibility of their inhibitory effect. With these results and after inspection of the physicochemical properties of compounds, 17 chemotypes were prioritized and progressed to further stages of validation and characterization to better assess their therapeutic potential. PMID:26933127

  3. Tumor necrosis factorinhibitor therapy and fetal risk: A systematic literature review

    PubMed Central

    Marchioni, Renée M; Lichtenstein, Gary R

    2013-01-01

    Tumor necrosis factorinhibitors (anti-TNFs) are effective in the treatment of inflammatory bowel disease (IBD) recalcitrant to conventional medical therapy. As the peak incidence of IBD overlaps with the prime reproductive years, it is crucial to establish pharmacologic regimens for women of childbearing age that achieve effective disease control without posing significant fetal harm. A systematic literature review was performed to identify all human studies with birth outcomes data after maternal exposure to infliximab, adalimumab, or certolizumab pegol within 3 mo of conception or during any trimester of pregnancy. Live births, spontaneous abortions or stillbirths, preterm or premature births, low birth weight or small for gestational age infants, and congenital abnormalities were recorded. Fifty selected references identified 472 pregnancy exposures. The subsequent review includes general information regarding anti-TNF therapy in pregnancy followed by a summary of our findings. The benefits of biologic modalities in optimizing disease control during pregnancy must be weighed against the potential toxicity of drug exposure on the developing fetus. Although promising overall, there is insufficient evidence to prove absolute safety for use of anti-TNFs during pregnancy given the limitations of available data and lack of controlled trials. PMID:23674866

  4. Noncovalent Mutant Selective Epidermal Growth Factor Receptor Inhibitors: A Lead Optimization Case Study.

    PubMed

    Heald, Robert; Bowman, Krista K; Bryan, Marian C; Burdick, Daniel; Chan, Bryan; Chan, Emily; Chen, Yuan; Clausen, Saundra; Dominguez-Fernandez, Belen; Eigenbrot, Charles; Elliott, Richard; Hanan, Emily J; Jackson, Philip; Knight, Jamie; La, Hank; Lainchbury, Michael; Malek, Shiva; Mann, Sam; Merchant, Mark; Mortara, Kyle; Purkey, Hans; Schaefer, Gabriele; Schmidt, Stephen; Seward, Eileen; Sideris, Steve; Shao, Lily; Wang, Shumei; Yeap, Kuen; Yen, Ivana; Yu, Christine; Heffron, Timothy P

    2015-11-25

    Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model. PMID:26455919

  5. Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Ankylosing Spondylitis

    PubMed Central

    Caetano-Lopes, Joana; Vieira-Sousa, Elsa; Campanilho-Marques, Raquel; Ponte, Cristina; Canhão, Helena; Ainola, Mari; Fonseca, João E.

    2015-01-01

    Introduction Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. Methods 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. Results RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-β levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. Conclusion In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli. PMID:26674064

  6. Activating Transcription Factor 3 Expression as a Marker of Response to the Histone Deacetylase Inhibitor Pracinostat.

    PubMed

    Sooraj, Dhanya; Xu, Dakang; Cain, Jason E; Gold, Daniel P; Williams, Bryan R G

    2016-07-01

    Improved treatment strategies are required for bladder cancer due to frequent recurrence of low-grade tumors and poor survival rate from high-grade tumors with current therapies. Histone deacetylase inhibitors (HDACi), approved as single agents for specific lymphomas, have shown promising preclinical results in solid tumors but could benefit from identification of biomarkers for response. Loss of activating transcription factor 3 (ATF3) expression is a feature of bladder tumor progression and correlates with poor survival. We investigated the utility of measuring ATF3 expression as a marker of response to the HDACi pracinostat in bladder cancer models. Pracinostat treatment of bladder cancer cell lines reactivated the expression of ATF3, correlating with significant alteration in proliferative, migratory, and anchorage-dependent growth capacities. Pracinostat also induced growth arrest at the G0-G1 cell-cycle phase, coincident with the activation of tumor suppressor genes. In mouse xenograft bladder cancer models, pracinostat treatment significantly reduced tumor volumes compared with controls, accompanied by reexpression of ATF3 in nonproliferating cells from early to late stage of therapy and in parallel induced antiangiogenesis and apoptosis. Importantly, cells in which ATF3 expression was depleted were less sensitive to pracinostat treatment in vitro, exhibiting significantly higher proliferative and migratory properties. In vivo, control xenograft tumors were significantly more responsive to treatment than ATF3 knockdown xenografts. Thus, reactivation of ATF3 is an important factor in determining sensitivity to pracinostat treatment, both in vitro and in vivo, and could serve as a potential biomarker of response and provide a rationale for therapeutic utility in HDACi-mediated treatments for bladder cancer. Mol Cancer Ther; 15(7); 1726-39. ©2016 AACR. PMID:27196751

  7. The M358R variant of α(1)-proteinase inhibitor inhibits coagulation factor VIIa.

    PubMed

    Sheffield, William P; Bhakta, Varsha

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg-Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg-Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10(2) M(-1)sec(-1). We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. PMID:26797521

  8. Pericardial effusions with tamponade and visceral constriction in patients with rheumatoid arthritis on tumour necrosis factor (TNF)-inhibitor therapy.

    PubMed

    Soh, May Ching; Hart, Hamish H; Corkill, Michael

    2009-04-01

    Tumour necrosis factor-inhibitor (TNF-inhibitor) therapy is increasingly used for the treatment of rheumatoid arthritis. While it is effective for the articular manifestations of rheumatoid arthritis we have reason to believe that it is less effective for extra-articular disease. We present two cases of life-threatening cardiac tamponade in two patients with well-controlled rheumatoid arthritis on adalimumab. An extensive literature search was carried out and three other patients were found. We believe that these cases highlight the need for rheumatologists to be vigilant for extra-articular manifestations of rheumatoid arthritis even in the presence of quiescent joint disease while on TNF-inhibitors. PMID:20374322

  9. N-hydroxybenzimidazole Inhibitors of the Transcription Factor LcrF in Yersinia: Novel Anti-Virulence Agents

    PubMed Central

    Kim, Oak K.; Garrity-Ryan, Lynne K.; Bartlett, Victoria J.; Grier, Mark C.; Verma, Atul K.; Medjanis, Gabriel; Donatelli, Janice E.; Macone, Ann B.; Tanaka, S. Ken; Levy, Stuart B.; Alekshun, Michael N.

    2009-01-01

    LcrF, a Multiple Adaptational Response (MAR) transcription factor, regulates virulence in Yersinia pestis and Yersinia pseudotuberculosis. In a search for small molecule inhibitors of LcrF, an acrylic amide series of N-hydroxybenzimidazoles was synthesized, and the SAR (structure-activity relationship) was examined. Selected test compounds demonstrated inhibitory activity in a primary cell-free LcrF-DNA binding assay as well as in a secondary whole cell assay (Type III secretion system dependent Y. pseudotuberculosis cytotoxicity assay). The inhibitors exhibited no measurable antibacterial activity in vitro, confirming that they do not target bacterial growth. These results demonstrate that N-hydroxybenzimidazole inhibitors, exemplified by 14, 22 and 36, are effective anti-virulence agents, and have the potential to prevent infections caused by Yersinia spp. PMID:19708663

  10. Hemophilia as a defect of the tissue factor pathway of blood coagulation: Effect of factors VIII and IX on factor X activation in a continuous-flow reactor

    SciTech Connect

    Repke, D.; Gemmell, C.H.; Guha, A.; Turitto, V.T.; Nemerson, Y. ); Broze, G.J. Jr. )

    1990-10-01

    The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a wall shear rate of 57, 300, or 1130 sec{sup {minus}1}. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2{endash}-to 3{endash}fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.

  11. 77 FR 17323 - Special Conditions: XtremeAir GmbH, XA42; Acrobatic Category Aerodynamic Stability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-26

    ...These special conditions are issued for the XtremeAir GmbH XA42 airplane. The XA42 airplane has a novel or unusual design feature associated with its static stability. This airplane can perform at the highest level of aerobatic competition. To be competitive, the aircraft was designed with positive and, at some points, neutral stability within its flight envelope. Its lateral and directional......

  12. 76 FR 80829 - Special Conditions: XtremeAir GmbH, XA42; Acrobatic Category Aerodynamic Stability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-27

    ...This action proposes special conditions for the XtremeAir GmbH XA42 airplane. The XA42 airplane has a novel or unusual design feature associated with its static stability. This airplane can perform at the highest level of aerobatic competition. To be competitive, the aircraft was designed with positive and, at some points, neutral stability within its flight envelope. Its lateral and......

  13. The Association Between HLA Class II Alleles and the Occurrence of Factor VIII Inhibitor in Thai Patients with Hemophilia A

    PubMed Central

    Nathalang, Oytip; Sriwanitchrak, Pramote; Sasanakul, Werasak; Chuansumrit, Ampaiwan

    2012-01-01

    Objective: This study aimed to investigate the association between HLA class II alleles and the occurrence of FVIIIinhibitor in Thai hemophilia A patients. Material and Methods: The distribution of HLA-DRB1 alleles and DQB1 alleles in 57 Thai hemophilia A patientsand 36 blood donors as controls was determined using the PCR sequence-specific primer (PCR-SSP) method, and theassociation between the occurrence of factor VIII (FVIII) inhibitor and the presence of certain HLA class II alleles wasinvestigated. Results: The frequency of HLA-DRB1*15 was higher in the hemophilia A patients with and without FVIII inhibitor,whereas that of DRB1*14, DRB1*07, and DQB1*02 was lower in the hemophilia A patients with FVIII inhibitor, ascompared to controls. Interestingly, only the frequency of DRB1*15 was significantly higher in the patients with inhibitorthan in the controls (P = 0.021). Moreover, the frequency of DRB1*15 in the patients with inhibitor was higher than inthose without inhibitor (P = 0.198). Conclusion: The study’s findings show that the DRB1*15 allele might have contributed to the occurrence of inhibitorin the Thai hemophilia A patients; however, additional research using larger samples and high-resolution DRB1 typingis warranted. PMID:24744621

  14. Materials Testing on the DC-X and DC-XA

    NASA Technical Reports Server (NTRS)

    Smith, Dane; Carroll, Carol; Marschall, Jochen; Pallix, Joan

    1997-01-01

    Flight testing of thermal protection materials has been carried out over a two year period on the base heat shield of the Delta Clipper (DC-X and DC-XA), as well on a body flap. The purpose was to use the vehicle as a test bed for materials and more efficient repair or maintenance processes which would be potentially useful for application on new entry vehicles (i.e., X-33, RLV, planetary probes), as well as on the existing space shuttle orbiters. Panels containing Thermal Protection Systems (TPS) and/or structural materials were constructed either at NASA Ames Research Center or at McDonnell Douglas Aerospace (MDA) and attached between two of the four thrusters in the base heat shield of the DC-X or DC-XA. Three different panels were flown on DC-X flights 6, 7, and 8. A total of 7 panels were flown on DC-XA flights 1, 2, and 3. The panels constructed at Ames contained a variety of ceramic TPS including flexible blankets, tiles with high emissivity coatings, lightweight ceramic ablators and other ceramic composites. The MDS test panels consisted primarily of a variety of metallic composites. This report focuses on the ceramic TPS test results.

  15. Comparative nutritional compositions and proteomics analysis of transgenic Xa21 rice seeds compared to conventional rice.

    PubMed

    Gayen, Dipak; Paul, Soumitra; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2016-07-15

    Transgenic rice expressing the Xa21 gene have enhanced resistant to most devastating bacterial blight diseases caused by Xanthomonas oryzae pv. oryzae (Xoo). However, identification of unintended modifications, owing to the genetic modification, is an important aspect of transgenic crop safety assessment. In this study, the nutritional compositions of seeds from transgenic rice plants expressing the Xa21 gene were compared against non-transgenic rice seeds. In addition, to detect any changes in protein translation levels as a result of Xa21 gene expression, rice seed proteome analyses were also performed by two-dimensional gel electrophoresis. No significant differences were found in the nutritional compositions (proximate components, amino acids, minerals, vitamins and anti-nutrients) of the transgenic and non-transgenic rice seeds. Although gel electrophoresis identified 11 proteins that were differentially expressed between the transgenic and non-transgenic seed, only one of these (with a 20-fold up-regulation in the transgenic seed) shows nutrient reservoir activity. No new toxins or allergens were detected in the transgenic seeds. PMID:26948618

  16. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

    PubMed Central

    Vuimo, Tatiana A.; Surov, Stepan S.; Ovsepyan, Ruzanna A.; Korneeva, Vera A.; Vorobiev, Ivan I.; Orlova, Nadezhda A.; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V.; Ataullakhanov, Fazoil I.; Panteleev, Mikhail A.

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  17. Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

    PubMed

    Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

    2016-03-28

    The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches. PMID:26848511

  18. The phylogenetically-related pattern recognition receptors EFR and XA21 recruit similar immune signaling components in monocots and dicots.

    PubMed

    Holton, Nicholas; Nekrasov, Vladimir; Ronald, Pamela C; Zipfel, Cyril

    2015-01-01

    During plant immunity, surface-localized pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs). The transfer of PRRs between plant species is a promising strategy for engineering broad-spectrum disease resistance. Thus, there is a great interest in understanding the mechanisms of PRR-mediated resistance across different plant species. Two well-characterized plant PRRs are the leucine-rich repeat receptor kinases (LRR-RKs) EFR and XA21 from Arabidopsis thaliana (Arabidopsis) and rice, respectively. Interestingly, despite being evolutionary distant, EFR and XA21 are phylogenetically closely related and are both members of the sub-family XII of LRR-RKs that contains numerous potential PRRs. Here, we compared the ability of these related PRRs to engage immune signaling across the monocots-dicots taxonomic divide. Using chimera between Arabidopsis EFR and rice XA21, we show that the kinase domain of the rice XA21 is functional in triggering elf18-induced signaling and quantitative immunity to the bacteria Pseudomonas syringae pv. tomato (Pto) DC3000 and Agrobacterium tumefaciens in Arabidopsis. Furthermore, the EFR:XA21 chimera associates dynamically in a ligand-dependent manner with known components of the EFR complex. Conversely, EFR associates with Arabidopsis orthologues of rice XA21-interacting proteins, which appear to be involved in EFR-mediated signaling and immunity in Arabidopsis. Our work indicates the overall functional conservation of immune components acting downstream of distinct LRR-RK-type PRRs between monocots and dicots. PMID:25607985

  19. The Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots

    PubMed Central

    Holton, Nicholas; Nekrasov, Vladimir; Ronald, Pamela C.; Zipfel, Cyril

    2015-01-01

    During plant immunity, surface-localized pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs). The transfer of PRRs between plant species is a promising strategy for engineering broad-spectrum disease resistance. Thus, there is a great interest in understanding the mechanisms of PRR-mediated resistance across different plant species. Two well-characterized plant PRRs are the leucine-rich repeat receptor kinases (LRR-RKs) EFR and XA21 from Arabidopsis thaliana (Arabidopsis) and rice, respectively. Interestingly, despite being evolutionary distant, EFR and XA21 are phylogenetically closely related and are both members of the sub-family XII of LRR-RKs that contains numerous potential PRRs. Here, we compared the ability of these related PRRs to engage immune signaling across the monocots-dicots taxonomic divide. Using chimera between Arabidopsis EFR and rice XA21, we show that the kinase domain of the rice XA21 is functional in triggering elf18-induced signaling and quantitative immunity to the bacteria Pseudomonas syringae pv. tomato (Pto) DC3000 and Agrobacterium tumefaciens in Arabidopsis. Furthermore, the EFR:XA21 chimera associates dynamically in a ligand-dependent manner with known components of the EFR complex. Conversely, EFR associates with Arabidopsis orthologues of rice XA21-interacting proteins, which appear to be involved in EFR-mediated signaling and immunity in Arabidopsis. Our work indicates the overall functional conservation of immune components acting downstream of distinct LRR-RK-type PRRs between monocots and dicots. PMID:25607985

  20. Use of mTOR inhibitors in the treatment of breast cancer: an evaluation of factors that influence patient outcomes

    PubMed Central

    Jerusalem, Guy; Rorive, Andree; Collignon, Joelle

    2014-01-01

    Many systemic treatment options are available for advanced breast cancer, including endocrine therapy, chemotherapy, anti-human epidermal growth factor receptor 2 (HER2) therapy, and other targeted agents. Recently, everolimus, a mammalian target of rapamycin (mTOR) inhibitor, combined with exemestane, an aromatase inhibitor, has been approved in Europe and the USA for patients suffering from estrogen receptor-positive, HER2-negative advanced breast cancer previously treated by a nonsteroidal aromatase inhibitor, based on the results of BOLERO-2 (Breast cancer trials of OraL EveROlimus). This study showed a statistically significant and clinically meaningful improvement in median progression-free survival. Results concerning the impact on overall survival are expected in the near future. This clinically oriented review focuses on the use of mTOR inhibitors in breast cancer. Results reported with first-generation mTOR inhibitors (ridaforolimus, temsirolimus, everolimus) are discussed. The current and potential role of mTOR inhibitors is reported according to breast cancer subtype (estrogen receptor-positive HER2-negative, triple-negative, and HER2-positive ER-positive/negative disease). Everolimus is currently being evaluated in the adjuvant setting in high-risk estrogen receptor-positive, HER2-negative early breast cancer. Continuing mTOR inhibition or alternatively administering other drugs targeting the phosphatidylinositol-3-kinase/protein kinase B-mTOR pathway after progression on treatments including an mTOR inhibitor is under evaluation. Potential biomarkers to select patients showing a more pronounced benefit are reviewed, but we are not currently using these biomarkers in routine practice. Subgroup analysis of BOLERO 2 has shown that the benefit is consistent in all subgroups and that it is impossible to select patients not benefiting from addition of everolimus to exemestane. Side effects and impact on quality of life are other important issues discussed

  1. Protease Inhibitors in View of Peptide Substrate Databases

    PubMed Central

    2016-01-01

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  2. Protease Inhibitors in View of Peptide Substrate Databases.

    PubMed

    Waldner, Birgit J; Fuchs, Julian E; Schauperl, Michael; Kramer, Christian; Liedl, Klaus R

    2016-06-27

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  3. Antiangiogenic Activity of Alofanib, an Allosteric Inhibitor of Fibroblast Growth Factor Receptor 2.

    PubMed

    Khochenkov, D A; Solomko, E Sch; Peretolchina, N M; Ryabaya, O O; Stepanova, E V

    2015-11-01

    Alofanib is a potential allosteric inhibitor of FGFR2 used in oncology. The inhibitor blocks the extracellular part of the receptor and prevents its binding with the ligand. Alofanib suppressed proliferation of endothelial cells, their migration activity, and ability to form vessellike structures in vitro and significantly decreased the number of microvessels in Matrigel implant and in ovarian cancer (SKOV-3) xenograft in vivo. The results indicate that Alofanib can inhibit angiogenesis. PMID:26597690

  4. QTc interval prolongation with vascular endothelial growth factor receptor tyrosine kinase inhibitors

    PubMed Central

    Ghatalia, P; Je, Y; Kaymakcalan, M D; Sonpavde, G; Choueiri, T K

    2015-01-01

    Background: Multi-targeted vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) are known to cause cardiac toxicity, but the relative risk (RR) of QTc interval prolongation and serious arrhythmias associated with them are not reported. Methods: We conducted a trial-level meta-analysis of randomised phase II and III trials comparing arms with and without a US Food and Drug Administration-approved VEGFR TKI (sunitinib, sorafenib, pazopanib, axitinib, vandetanib, cabozantinib, ponatinib and regorafenib). A total of 6548 patients from 18 trials were selected. Statistical analyses were conducted to calculate the summary incidence, RR and 95% CIs. Results: The RR for all-grade and high-grade QTc prolongation for the TKI vs no TKI arms was 8.66 (95% CI 4.92–15.2, P<0.001) and 2.69 (95% CI 1.33–5.44, P=0.006), respectively, with most of the events being asymptomatic QTc prolongation. Respectively, 4.4% and 0.83% of patients exposed to VEGFR TKI had all-grade and high-grade QTc prolongation. On subgroup analysis, only sunitinib and vandetanib were associated with a statistically significant risk of QTc prolongation, with higher doses of vandetanib associated with a greater risk. The rate of serious arrhythmias including torsades de pointes did not seem to be higher with high-grade QTc prolongation. The risk of QTc prolongation was independent of the duration of therapy. Conclusions: In the largest study to date, we show that VEGFR TKI can be associated with QTc prolongation. Although most cases were of low clinical significance, it is unclear whether the same applies to patients treated off clinical trials. PMID:25349964

  5. Complicated Whipple’s disease and endocarditis following tumor necrosis factor inhibitors

    PubMed Central

    Marth, Thomas

    2014-01-01

    AIM: To test whether treatment with tumor necrosis factor inhibitors (TNFI) is associated with complications of Tropheryma whipplei (T. whipplei) infection. METHODS: Because unexplained arthritis is often the first Whipple’s disease (WD) symptom, patients may undergo treatment with TNFI before diagnosis. This may influence the course of infection with T. whipplei, which causes WD, because host immune defects contribute to the pathogenesis of WD. A literature search and cross referencing identified 19 reports of TNFI treatment prior to WD diagnosis. This case-control study compared clinical data in patients receiving TNFI therapy (group I, n = 41) with patients not receiving TNFI therapy (group II, n = 61). Patients from large reviews served as controls (group III, n = 1059). RESULTS: The rate of endocarditis in patient group I was significantly higher than in patient group II (12.2% in group I vs 1.6% in group II, P < 0.05), and group III (12.2% in group I vs 0.16% in group III, P < 0.01). Other, severe systemic or local WD complications such as pericarditis, fever or specific organ manifestations were increased also in group I as compared to the other patient groups. However, diarrhea and weight loss were somewhat less frequent in patient group I. WD is typically diagnosed with duodenal biopsy and periodic acid Schiff (PAS) staining. PAS-stain as standard diagnostic test had a very high percentage of false negative results (diagnostic failure in 63.6% of cases) in group I. Polymerase chain reaction (PCR) for T. whipplei was more accurate than PAS-stainings (diagnostic accuracy, rate of true positive tests 90.9% for PCR vs 36.4% for PAS, P < 0.01). CONCLUSION: TNFI trigger severe WD complications, particularly endocarditis, and lead to false-negative PAS-tests. In case of TNFI treatment failure, infection with T. whipplei should be considered. PMID:25548618

  6. Pancreatic secretory trypsin inhibitor is a major motogenic and protective factor in human breast milk.

    PubMed

    Marchbank, Tania; Weaver, Gillian; Nilsen-Hamilton, Marit; Playford, Raymond J

    2009-04-01

    Colostrum is the first milk produced after birth and is rich in immunoglobulins and bioactive molecules. We examined whether human colostrum and milk contained pancreatic secretory trypsin inhibitor (PSTI), a peptide of potential relevance for mucosal defense and, using in vitro and in vivo models, determined whether its presence influenced gut integrity and repair. Human milk was collected from individuals over various times from parturition and PSTI concentrations determined with the use of immunoassay. Human milk samples were analyzed for proliferation and promigratory activity (wounded monolayers) and antiapoptotic activity (caspase-3 activity) with the use of intestinal HT29 cells with or without neutralizing antibodies to PSTI and epidermal growth factor (EGF). Rats were restrained and given indomethacin to induce gastric injury. Effect of gavage with human breast milk with or without neutralizing antibodies on amount of injury were compared with animals receiving a commercial formula feed. PSTI is secreted into human milk, with colostrum containing a much higher concentration of PSTI than human milk obtained later. Human milk stimulated migration and proliferation about threefold and reduced indomethacin-induced apoptosis by about 70-80%. Sixty-five percent of the migratory effect of human milk could be removed by immunoneutralization of PSTI. PSTI worked synergistically with EGF in mediating these effects. Gastric damage in rats was reduced by about 75% in the presence of human milk and was more efficacious than the formula feed (P<0.001). Protective effects of the milk were reduced by about 60% by PSTI immunoneutralization. We concluded that PSTI is secreted into human milk at concentrations that have probable pathophysiological relevance. PMID:19147803

  7. Oestrogens Downregulate Tissue Factor Pathway Inhibitor through Oestrogen Response Elements in the 5'-Flanking Region.

    PubMed

    Ali, Huda Omar; Stavik, Benedicte; Myklebust, Christiane Filion; Andersen, Elisabeth; Dahm, Anders E A; Iversen, Nina; Sandset, Per Morten; Skretting, Grethe

    2016-01-01

    Oestrogens influence the pathology and development of hormone-sensitive breast cancers. Tissue factor pathway inhibitor (TFPI) has been shown to be associated with breast cancer pathogenesis. Recently, we found TFPI mRNA levels to be significantly reduced by oestrogens in a breast cancer cell line (MCF7), a process mediated through the oestrogen receptor alpha (ERα). The aim of the present study was to investigate the mechanism(s) by which oestrogens may regulate TFPI at the transcriptional level. The TFPI 5'-flanking region contains three oestrogen response element (ERE) half-sites at positions -845, -769 and -50. Constructs containing the wild type or mutated ERE half-sites of the TFPI 5'-flanking region were generated in a luciferase reporter gene vector and transiently co-transfected with an ERα expression vector into HEK293 cells and subsequently treated with oestrogens. We found that luciferase activity was significantly downregulated after oestrogen stimulation in cells transfected with the wild type construct, an effect that was abolished by mutating either ERE half-sites. Electrophoretic mobility shift assay suggested direct and specific interaction of ERα with the ERE half-sites in the TFPI 5'-flanking region. Chromatin immunoprecipitation showed that ERα was recruited to the region -899 to -578 of the TFPI 5'-flanking region in vivo, where the ERE half-sites -845 and -769 are located. Our results indicate that ERα can interact with all three ERE half-sites in the TFPI 5'-flanking region and thus participate in the repression of oestrogen mediated TFPI transcription in breast cancer cells. PMID:26999742

  8. AZD9291 in epidermal growth factor receptor inhibitor-resistant non-small-cell lung cancer.

    PubMed

    Stinchcombe, Thomas E

    2016-02-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in advanced EGFR mutant non-small cell lung cancer have an objective response rate (ORR) of approximately 60-70% and a median progression free-survival (PFS) of approximately 10-13 months. Studies of tumor biopsies performed after progression on EGFR TKI revealed that 50-60% of EGFR mutant NSCLC developed an EGFR exon 20 T790M mutation as a mechanism of acquired resistance. AZD9291 is a third generation irreversible EGFR TKI with activity against the activating EGFR mutation, the T790M acquired resistance mutation, and relative sparing of the wild-type EGFR. AZD9291 was investigated in a phase I trial with expansion cohorts in patients with disease progression after EGFR TKI. Patients with and without detectable T790M mutations were enrolled in the trial. The ORR in patients with centrally confirmed and without detectable T790M mutations was 61% (95% CI, 52-70%) and 21% (95% CI, 12-34%), respectively. The PFS observed in patients with centrally confirmed and without detectable T790M mutations was 9.6 months (95% CI, 8.3 to not reached) and 2.8 months (95% CI, 2.1-4.3 months), respectively. At the dose for further investigation, 80 mg daily, the rate of all grade 3-5 drug related adverse events was 11%, and the rates of grade 3 diarrhea and rash were 1% and 0%, respectively. The identification of the T790M resistance mutation and the subsequent development of an agent against the mechanism of resistance provide a template for future drug development for acquired resistance to targeted therapy. PMID:26958499

  9. Antitumor effect of dehydroxymethylepoxyquinomicin, a small molecule inhibitor of nuclear factor-κB, on glioblastoma

    PubMed Central

    Fukushima, Tsuyoshi; Kawaguchi, Makiko; Yorita, Kenji; Tanaka, Hiroyuki; Takeshima, Hideo; Umezawa, Kazuo; Kataoka, Hiroaki

    2012-01-01

    Glioblastoma is the most malignant type of brain tumor. Despite recent advances in therapeutic modalities, the prognosis of glioblastoma remains very poor. Recent studies have indicated that RelA/nuclear factor (NF)-κB is consistently activated in human glioblastoma. In this study, we searched for a new treatment modality for glioblastoma, by examining the effects of dehydroxymethylepoxyquinomicin (DHMEQ), a unique small molecule inhibitor of NF-κB. Addition of DHMEQ to cultured human glioblastoma cells inhibited the nuclear translocation of RelA. It also reduced the growth rate of human glioblastoma cells significantly in 6 cell lines and modestly in 3 among 10 cell lines examined. Then, we performed further analyses using 3 sensitive cell lines (U87, U251, and YKG-1). The growth retardation was accompanied by G2/M arrest in vitro. Increased apoptosis was observed in U87 and YKG-1, but not U251 cells after DHMEQ treatment. Then, we tested the efficacy of DHMEQ in chemoprevention through the use of a nude mouse model. Subcutaneous tumors formed by U87 or U251 cells were reduced by ∼40% in size by intraperitoneal administration of DHMEQ started immediately after implantation of the cells. DHMEQ treatment achieved statistically significant improvements in survival curves of mice intracranially implanted with U87 or U251 cells. Histological analysis revealed increased areas of necrosis, increased numbers of collapsed microvessels, decreased nuclear immunoreactivity of RelA, and decreased immunoreactivity of urokinase-type plasminogen activator in the DHMEQ-treated U87 tumor tissues. These results suggest that the targeting of NF-κB by DHMEQ may serve as a promising treatment modality in glioblastoma. PMID:21968049

  10. Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors

    SciTech Connect

    Hagiwara, Hiroki; Saito, Fumiaki; Masaki, Toshihiro; Ikeda, Miki; Nakamura-Ohkuma, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of TSA, one of most potent HDACIs, on myogenesis using the C2C12 skeletal muscle cell line. Black-Right-Pointing-Pointer TSA enhances the expression of myosin heavy chain without affecting DAPC expression. Black-Right-Pointing-Pointer TSA enhances the expression of the early MRFs, Myf5 and MEF2, and suppresses the late MRF, myogenin, after 24 h treatment. Black-Right-Pointing-Pointer TSA enhances the expression of the myogenic repressors, Ids, which inhibit myogenic differentiation. Black-Right-Pointing-Pointer TSA promotes myogenesis by coordinating the expression of MRFs and myogenic repressors. -- Abstract: Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.

  11. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields. PMID:25453359

  12. α-Ketobenzothiazole Serine Protease Inhibitors of Aberrant HGF/c-MET and MSP/RON Kinase Pathway Signaling in Cancer.

    PubMed

    Han, Zhenfu; Harris, Peter K W; Karmakar, Partha; Kim, Tommy; Owusu, Ben Y; Wildman, Scott A; Klampfer, Lidija; Janetka, James W

    2016-03-17

    Upregulation of the HGF and MSP growth-factor processing serine endopeptidases HGFA, matriptase and hepsin is correlated with increased metastasis in multiple tumor types driven by c-MET or RON kinase signaling. We rationally designed P1' α-ketobenzothiazole mechanism-based inhibitors of these proteases. Structure-activity studies are presented, which resulted in the identification of potent inhibitors with differential selectivity. The tetrapeptide inhibitors span the P1-P1' substrate cleavage site via a P1' amide linker off the benzothiazole, occupying the S3' pocket. Optimized inhibitors display sub-nanomolar enzyme inhibition against one, two, or all three of HGFA, matriptase, and hepsin. Several compounds also have good selectivity against the related trypsin-like proteases, thrombin and Factor Xa. Finally, we show that inhibitors block the fibroblast (HGF)-mediated migration of invasive DU145 prostate cancer cells. In addition to prostate cancer, breast, colon, lung, pancreas, gliomas, and multiple myeloma tumors all depend on HGF and MSP for tumor survival and progression. Therefore, these unique inhibitors have potential as new therapeutics for a diverse set of tumor types. PMID:26889658

  13. A common neoepitope is created when the reactive center of C1-inhibitor is cleaved by plasma kallikrein, activated factor XII fragment, C1 esterase, or neutrophil elastase.

    PubMed Central

    de Agostini, A; Patston, P A; Marottoli, V; Carrel, S; Harpel, P C; Schapira, M

    1988-01-01

    The reactive center of C1-inhibitor, a plasma protease inhibitor that belongs to the serpin superfamily, is located on a peptide loop which is highly susceptible to proteolytic cleavage. With plasma kallikrein, C1s and beta-Factor XIIa, this cleavage occurs at the reactive site residue P1 (Arg444); with neutrophil elastase, it takes place near P1, probably at residue P3 (Val442). After these cleavages, C1-inhibitor is inactivated and its conformation is modified. Moreover, in vivo, cleaved C1-inhibitor is removed from the blood stream more rapidly than the intact serpin, which suggests that proteolysis unmasks sites responsible for cellular recognition and the uptake of the cleaved inhibitor. In the study reported here, we show, using an MAb, that an identical neoepitope is created on C1-inhibitor after the cleavage of its exposed loop by plasma kallikrein, C1s, beta-Factor XIIa, and by neutrophil elastase. Images PMID:2457036

  14. Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells.

    PubMed

    Sharma, Bal Krishan; Kolhe, Ravindra; Black, Stephen M; Keller, Jonathan R; Mivechi, Nahid F; Satyanarayana, Ande

    2016-01-01

    Reprograming of metabolism is one of the central hallmarks of cancer. The majority of cancer cells depend on high rates of glycolysis and glutaminolysis for their growth and survival. A number of oncogenes and tumor suppressors have been connected to the regulation of altered glucose and glutamine metabolism in cancer cells. For example, the oncogene c-Myc plays vital roles in cancer cell metabolic adaptation by directly regulating various genes that participate in aerobic glycolysis and glutaminolysis. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays important roles in cell proliferation, differentiation, and cell fate determination. Overexpression of Id1 causes intestinal adenomas and thymic lymphomas in mice, suggesting that Id1 could function as an oncogene. Despite it being an oncogene, whether Id1 plays any prominent role in cancer cell metabolic reprograming is unknown. Here, we demonstrate that Id1 is strongly expressed in human and mouse liver tumors and in hepatocellular carcinoma (HCC) cell lines, whereas its expression is very low or undetectable in normal liver tissues. In HCC cells, Id1 expression is regulated by the MAPK/ERK pathway at the transcriptional level. Knockdown of Id1 suppressed aerobic glycolysis and glutaminolysis, suggesting that Id1 promotes a metabolic shift toward aerobic glycolysis. At the molecular level, Id1 mediates its metabolic effects by regulating the expression levels of c-Myc. Knockdown of Id1 resulted in down-regulation (∼75%) of c-Myc, whereas overexpression of Id1 strongly induced (3-fold) c-Myc levels. Interestingly, knockdown of c-Myc resulted in down-regulation (∼60%) of Id1, suggesting a positive feedback-loop regulatory mechanism between Id1 and c-Myc. Under anaerobic conditions, both Id1 and c-Myc are down-regulated (50-70%), and overexpression of oxygen-insensitive hypoxia-inducible factor 1α (Hif1α) or its downstream target Mxi1 resulted in a significant reduction

  15. Circadian variation of tissue plasminogen activator and its inhibitor, von Willebrand factor antigen, and prostacyclin stimulating factor in men with ischaemic heart disease.

    PubMed Central

    Bridges, A B; McLaren, M; Scott, N A; Pringle, T H; McNeill, G P; Belch, J J

    1993-01-01

    OBJECTIVES--To determine whether plasma concentrations of tissue plasminogen activator antigen, von Willebrand factor antigen, and prostacyclin stimulating factor and plasminogen activator inhibitor activity show circadian variation in men with ischaemic heart disease. DESIGN--Blood samples were obtained every four hours for 24 hours from 10 men with ischaemic heart disease. The men were ambulant from 08:10 until 00:00 when they went to bed and they remained in bed until 08:00 the following morning. PATIENTS--Ten men with positive diagnostic exercise tolerance tests with no significant past history, who were not regularly taking any medical treatment except for glyceryl trinitrate. RESULTS--There was significant circadian variation in plasminogen activator inhibitor activity (p = 0.001) (peak value 04:00 and trough value 20:00), but not in plasma concentrations of tissue plasminogen activator antigen, von Willebrand factor, or prostacyclin stimulating factor. CONCLUSION--Men with ischaemic heart disease showed a significant circadian variation in fibrinolysis. The combination of peak values of plasminogen activator inhibitor activity and failure of plasma concentrations of tissue plasminogen activator antigen to increase in the early morning must predispose to thrombosis at this time. The circadian variation in fibrinolysis may contribute to the increased incidence of myocardial infarction in the morning. PMID:8435236

  16. The development of novel inhibitors of tumor necrosis factor-alpha production based on substituted [5,5]-bicyclic pyrozolones

    SciTech Connect

    Laufersweiler, Matthew; Brugel, Todd; Clark, Michael; Golebiowski, Adam; Bookland, Roger; Laughlin, Steven; Sabat, Mark; Townes, Jennifer; VanRens, John; De, Biswanath; Hsieh, Lily; Heitmeyer, Sandra; Juergens, Karen; Brown, Kimberly; Mekel, Marlene; Walter, Richard; Janusz, Michael

    2010-11-16

    Novel substituted [5,5]-bicyclic pyrzazolones are presented as inhibitors of tumor necrosis factor-{alpha} (TNF-{alpha}) production. Many of these compounds show low nanomolar activity against lipopolysaccaride (LPS)-induced TNF-{alpha} production in THP-1 cells. This class of molecules was co-crystallized with mutated p38, and several analogs showed good oral bioavailability in the rat. Oral activity of these compounds in the rat iodoacetate model for osteoarthritis is discussed.

  17. Overcoming resistance to first/second generation epidermal growth factor receptor tyrosine kinase inhibitors and ALK inhibitors in oncogene-addicted advanced non-small cell lung cancer.

    PubMed

    Romanidou, Ourania; Landi, Lorenza; Cappuzzo, Federico; Califano, Raffaele

    2016-05-01

    Epidermal growth factor receptor (EGFR) activating mutations and anaplastic lymphoma kinase (ALK) gene rearrangement in advanced non-small cell lung cancer (NSCLC) represent the two oncogenic events with an impact on current clinical practice. EGFR tyrosine kinase inhibitors (TKIs) and crizotinib are the standard of care for the treatment of EGFR mutant and ALK gene rearranged advanced NSCLC patients. Unfortunately, despite initial clinical benefit, acquired resistance to EGFR-TKIs or crizotinib usually develops after an average of 10-12 months of treatment. The aim of this review is to describe the mechanisms of resistance to first/second generation EGFR-TKIs and crizotinib. In particular, we focus on strategies to overcome resistance due to secondary EGFR T790M mutation and mutations of the ALK domain. PMID:27239236

  18. Overcoming resistance to first/second generation epidermal growth factor receptor tyrosine kinase inhibitors and ALK inhibitors in oncogene-addicted advanced non-small cell lung cancer

    PubMed Central

    Romanidou, Ourania; Landi, Lorenza; Cappuzzo, Federico; Califano, Raffaele

    2016-01-01

    Epidermal growth factor receptor (EGFR) activating mutations and anaplastic lymphoma kinase (ALK) gene rearrangement in advanced non-small cell lung cancer (NSCLC) represent the two oncogenic events with an impact on current clinical practice. EGFR tyrosine kinase inhibitors (TKIs) and crizotinib are the standard of care for the treatment of EGFR mutant and ALK gene rearranged advanced NSCLC patients. Unfortunately, despite initial clinical benefit, acquired resistance to EGFR-TKIs or crizotinib usually develops after an average of 10–12 months of treatment. The aim of this review is to describe the mechanisms of resistance to first/second generation EGFR-TKIs and crizotinib. In particular, we focus on strategies to overcome resistance due to secondary EGFR T790M mutation and mutations of the ALK domain. PMID:27239236

  19. Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis

    PubMed Central

    Bajaj, Madhu S.; Ogueli, Godwin I.; Kumar, Yogesh; Vadivel, Kanagasabai; Lawson, Gregory; Shanker, Sreejesh; Schmidt, Amy E.; Bajaj, S. Paul

    2011-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R. PMID:21115497

  20. DEPRESSION and SEROTONIN REUPTAKE INHIBITOR TREATMENT AS RISK FACTORS FOR PRETERM BIRTH

    PubMed Central

    Yonkers, Kimberly A.; Norwitz, Errol R.; Smith, Megan V.; Lockwood, Charles J.; Gotman, Nathan; Luchansky, Edward; Lin, Haiqun; Belanger, Kathleen

    2012-01-01

    Background Major depressive disorder as well as the use of serotonin reuptake inhibitors in pregnancy have been associated with preterm birth. Studies that have attempted to separate effects of illness from treatment have been inconclusive. We sought to explore the separate effects of serotonin reuptake inhibitor use and major depressive episodes in pregnancy on risk of preterm birth. Methods We conducted a prospective cohort study of 2793 pregnant women, oversampled for a recent episode of major depression or use of a serotonin reuptake inhibitor. We extracted data on birth outcomes from hospital charts and used binary logistic regression to model preterm birth (<37 weeks’ gestation). We used ordered logistic regression to model early (<34 weeks’ gestation) or late (34-36 weeks) preterm birth, and we used nominal logistic regression to model preterm birth antecedents (spontaneous preterm labor/preterm premature rupture of membranes/preterm for medical indications/term). Results Use of a serotonin reuptake inhibitor, both with (odds ratio=2.1 [95% confidence interval=1.0—4.6]) and without (1.6=[1.0—2.5]) a major depressive episode, was associated with preterm birth. A major depressive episode without serotonin reuptake inhibitor use (1.2; [0.68—2.1]) had no clear effect on preterm risk. None of these exposures was associated with early preterm birth. Use of serotonin reuptake inhibitors in pregnancy was associated with increases in spontaneous but not medically indicated preterm birth. Conclusions Serotonin reuptake inhibitor use increased risk of preterm birth. Although the effect of a major depressive episode alone was unclear, symptomatic women undergoing antidepressant treatment had elevated risk. PMID:22627901

  1. Factor VIII brand and the incidence of factor VIII inhibitors in previously untreated UK children with severe hemophilia A, 2000-2011

    PubMed Central

    Palmer, Benedict P.; Chalmers, Elizabeth A.; Hart, Daniel P.; Liesner, Ri; Rangarajan, Savita; Talks, Katherine; Williams, Michael; Hay, Charles R. M.

    2014-01-01

    The effect of recombinant factor VIII (rFVIII) brand on inhibitor development was investigated in all 407 severe hemophilia A previously untreated patients born in the United Kingdom (UK) between 1 January 2000 and 31 December 2011. Eighty-eight (22%) had been in the RODIN study. Information was extracted from the National Haemophilia Database. Because exposure days (EDs) were not known for some patients, time from first treatment was used as a surrogate for rFVIII exposure. An inhibitor developed in 118 (29%) patients, 60 high and 58 low titer, after a median (interquartile range) of 7.8 (3.3-13.5) months from first exposure and 16 (9-30) EDs. Of 128 patients treated with Kogenate Bayer/Helixate NexGen, 45 (35.2%, 95% confidence interval [CI] 27.4-43.8) developed an inhibitor compared with 42/172 (24.4%, 95% CI 18.6% to 31.4%) with Advate (P = .04). The adjusted hazard ratio (HR) (95% CI) for Kogenate Bayer/Helixate NexGen compared with Advate was 2.14 (1.12-4.10) (P = .02) for high titer and 1.75 (1.11-2.76) (P = .02) for all inhibitors. When excluding UK-RODIN patients, the adjusted HR (95% CI) for high-titer inhibitors was 2.00 (0.93-4.34) (P = .08). ReFacto AF was associated with a higher incidence of all, but not high-titer, inhibitors than Advate. These results will help inform debate around the relative immunogenicity and use of rFVIII brands. PMID:25339360

  2. Acquired Factor XIII Inhibitor in Hospitalized and Perioperative Patients: A Systematic Review of Case Reports and Case Series.

    PubMed

    Tone, Kira J; James, Tyler E; Fergusson, Dean A; Tinmouth, Alan; Tay, Jason; Avey, Marc T; Kilty, Shaun; Lalu, Manoj M

    2016-07-01

    Factor XIII (FXIII) cross-links fibrin monomers to support clot stabilization and wound healing. Acquired FXIII deficiency is caused by autoantibodies that inhibit FXIII and can result in bleeding despite normal routine coagulation test results. Given the rarity of this disease, large clinical studies are not feasible. We therefore conducted a systematic review of case reports and case series of acquired FXIII inhibitor to evaluate potential management and treatment strategies for acquired FXIII inhibitor in hospitalized and/or perioperative patients. A systematic search of MEDLINE, Embase, and Web of Science identified reports of hospitalized and perioperative patients with acquired FXIII deficiency. No restrictions were placed on language or publication type. Article screening and data extraction were performed independently by 2 abstractors. Completeness of reporting was evaluated according to modified elements from the CAse REport (CARE) guidelines. A total of 1028 citations were reviewed, with 36 case reports and 3 case series meeting eligibility criteria (63 patients total). The mean age was 60 (range, 9-87) years with balanced sex representation. At presentation, 48 patients (76%) had intramuscular or subcutaneous bleeding, and 34 patients (54%) had external or surgical bleeding. All cases were diagnosed by initially detecting a FXIII deficiency and then identifying the inhibitor. Clinical improvement in bleeding was seen in patients receiving FXIII concentrate (13/17 patients), cryoprecipitate (5/8), and plasma (10/18). Inhibitor reduction was seen in patients who received rituximab (6/6 patients), plasma exchange (2/2), intravenous immunoglobulin (4/5), steroid (15/20), and cyclophosphamide (10/15). Concurrent initiation of multiple therapies and obvious lack of control comparisons made direct association to outcomes difficult to establish. Outcomes were reported for 55 patients, with 25 patients (45%) having complete inhibitor eradication and 15 patients

  3. F8 gene mutation profile in Indian hemophilia A patients: Identification of 23 novel mutations and factor VIII inhibitor risk association.

    PubMed

    Pinto, Patricia; Ghosh, Kanjaksha; Shetty, Shrimati

    2016-04-01

    'FVIII inhibitors', especially in severe hemophilia A (HA) patients, is a serious adverse effect that complicates their clinical management. Many genetic and non-genetic risk factors have been proposed for FVIII inhibitor development, diverse in different population groups. This is the first study in Indian hemophiliacs that analyzes inhibitor risk in relation to the complete F8 mutation profile, in a case-control study that included 145 Indian severe HA patients, i.e. 69 inhibitor positive (with 18 inhibitor concordant/discordant family members), and 58 inhibitor negative patients, after informed consent. While 53.54% (68/127) index cases were positive for intron 22 or intron 1 inversions, 55 causative F8 mutations were detected in the 59 inversion negative patients, of which 23 were novel mutations (in 24 patients) and 32 were reported earlier (in 35 patients). A higher incidence of mutations, in the C1 and C2 domains in inhibitor positive patients, and in the A1 domain in inhibitor negative patients was observed, though not significantly different. The study suggests that large F8 rearrangements (significantly higher in the inhibitor positive patients) pose the highest risk, while missense mutations (significantly higher in the inhibitor negative patients) pose the lowest risk of inhibitor development in Indian hemophilia A patients. PMID:26897466

  4. Fibroblast Growth Factor (FGF) Receptor/FGF Inhibitors: Novel Targets and Strategies for Optimization of Response of Solid Tumors.

    PubMed

    Hierro, Cinta; Rodon, Jordi; Tabernero, Josep

    2015-12-01

    The fibroblast growth factor receptor (FGFR) pathway plays a major role in several biological processes, from organogenesis to metabolism homeostasis and angiogenesis. Several aberrations, including gene amplifications, point mutations, and chromosomal translocations have been described across solid tumors. Most of these molecular alterations promote multiple steps of carcinogenesis in FGFR oncogene-addicted cells, increasing cell proliferation, angiogenesis, and drug resistance. Data suggest that upregulation of FGFR signaling is a common event in many cancer types. The FGFR pathway thus arises as a potential promising target for cancer treatment. Several FGFR inhibitors are currently under development. Initial preclinical results have translated into limited successful clinical responses when first-generation, nonspecific FGFR inhibitors were evaluated in patients. The future development of selective and unselective FGFR inhibitors will rely on a better understanding of the tissue-specific role of FGFR signaling and identification of biomarkers to select those patients who will benefit the most from these drugs. Further studies are warranted to establish the predictive significance of the different FGFR-aberrations and to incorporate them into clinical algorithms, now that second-generation, selective FGFR inhibitors exist. PMID:26615127

  5. A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

    PubMed Central

    Slingerland, J M; Hengst, L; Pan, C H; Alexander, D; Stampfer, M R; Reed, S I

    1994-01-01

    Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses. Images PMID:8196612

  6. An Acquired Factor VIII Inhibitor in a Patient with HIV and HCV: A Case Presentation and Literature Review

    PubMed Central

    Zeichner, S. B.; Harris, A.; Turner, G.; Francavilla, M.; Lutzky, J.

    2013-01-01

    Introduction. Despite its low incidence, acquired factor VIII inhibitor is the most common autoantibody affecting the clotting cascade. The exact mechanism of acquisition remains unclear, but postpartum patients, those with autoimmune conditions or malignancies, and those with exposure to particular drugs appear most susceptible. There have been several case reports describing acquired FVIII inhibitors in patients receiving interferon alpha for HCV treatment and in patients being treated for HIV. To our knowledge, this is the first case of a patient with HCV and HIV who was not actively receiving treatment for either condition. Case Presentation. A 57-year-old Caucasian male with a history of HIV and HCV was admitted to our hospital for a several day history of progressively worsening right thigh bruising and generalized weakness. CTA of the abdominal arteries revealed large bilateral retroperitoneal hematomas. Laboratory studies revealed the presence of a high titer FVIII inhibitor. Conclusion. Our case of a very rare condition highlights the importance of recognizing and understanding the diagnosis of acquired FVIII inhibitor. Laboratory research and clinical data on the role of newer agents are needed in order to better characterize disease pathogenesis, disease associations, genetic markers, and optimal disease management. PMID:24198984

  7. The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors.

    PubMed Central

    Menard, A; Papini, E; Mock, M; Montecucco, C

    1996-01-01

    The lethal factor of Bacillus anthracis is central to the pathogenesis of anthrax. Its mechanism of action is still unknown. Recently, on the basis of sequence similarities, we suggested that lethal factor might act similarly to leukotriene A4 hydrolase (LTA4), a bifunctional enzyme also endowed with a metallopeptidase activity. Here we show that some inhibitors of the LTA4 hydrolase and metallopeptidase activities of LTA4 hydrolase also affect the cytotoxicity of the anthrax lethal factor on macrophage cell lines, without interfering with the ability of the lethal factor to enter cells. These results support the proposal that anthrax lethal factor might display in the cytosol of intoxicated cells a peptidase activity similar to that of LTA4 hydrolase. PMID:8973585

  8. SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

    PubMed

    Ferreira, Viviana P; Fazito Vale, Vladimir; Pangburn, Michael K; Abdeladhim, Maha; Mendes-Sousa, Antonio Ferreira; Coutinho-Abreu, Iliano V; Rasouli, Manoochehr; Brandt, Elizabeth A; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Pereira, Marcos Horácio; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M C; Gontijo, Nelder F; Collin, Nicolas; Valenzuela, Jesus G

    2016-01-01

    Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

  9. SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

    PubMed Central

    Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

    2016-01-01

    Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

  10. Identification of an Iridium(III)-Based Inhibitor of Tumor Necrosis Factor-α.

    PubMed

    Kang, Tian-Shu; Mao, Zhifeng; Ng, Chan-Tat; Wang, Modi; Wang, Wanhe; Wang, Chunming; Lee, Simon Ming-Yuen; Wang, Yitao; Leung, Chung-Hang; Ma, Dik-Lung

    2016-04-28

    The novel iridium(III) complex 1 was verified as a potent inhibitor of the TNF-α-TNFR protein-protein interaction in vitro and in cellulo. The iridium(III) center plays a critical role in organizing the structure of the bioactive metal complex, as the isolated ligands were found to be completely inactive. Both iridium enantiomers inhibited TNF-α-induced NF-κB activity and TNF-α-TNFR binding. 1 represents a promising scaffold for the further development of more potent organometallic TNF-α inhibitors. PMID:27054262

  11. Association Between Ischemic Stroke and Tumor Necrosis Factor Inhibitor Therapy in Patients With Rheumatoid Arthritis

    PubMed Central

    Low, Audrey S. L.; Lunt, Mark; Mercer, Louise K.; Watson, Kath D.; Dixon, William G.; Symmons, Deborah P. M.

    2016-01-01

    Objective Patients with rheumatoid arthritis (RA) are at an increased risk of ischemic stroke. Tumor necrosis factor inhibitors (TNFi) may influence risk and mortality after ischemic stroke by reducing inflammation. This study was undertaken to examine the association of TNFi with the risk of incident ischemic stroke and with 30‐day and 1‐year mortality after ischemic stroke. Methods Patients with RA starting therapy with TNFi and a biologics‐naive comparator group treated with synthetic disease‐modifying antirheumatic drugs (DMARDs) only were recruited to the British Society for Rheumatology Biologics Register for Rheumatoid Arthritis from 2001 to 2009. Patients were followed up via clinical and patient questionnaires as well as the national death register. Incident strokes were classified as ischemic if brain imaging reports suggested ischemia or if ischemic stroke was reported as the underlying cause of death on a death certificate. Patients with a previous stroke were excluded. Risk of ischemic stroke was compared between patients receiving synthetic DMARDs only and those ever‐exposed to TNFi using a Cox proportional hazards regression model adjusted for potential confounders. Mortality after ischemic stroke was compared between synthetic DMARD–treated patients and TNFi‐treated patients using logistic regression, adjusted for age and sex. Results To April 2010, 127 verified incident ischemic strokes (21 in 3,271 synthetic DMARD–treated patients and 106 in 11,642 TNFi‐treated patients) occurred during 11,973 and 61,226 person‐years of observation, respectively (incidence rate 175 versus 173 per 100,000 person‐years). After adjustment for confounders, there was no association between ever‐exposure to TNFi and ischemic stroke (hazard ratio 0.99 [95% confidence interval (95% CI) 0.54–1.81]). Mortality 30 days or 1 year after ischemic stroke was not associated with concurrent TNFi exposure (odds ratio 0.18 [95% CI 0.03–1.21] and 0.60 [95

  12. The discovery of novel vascular endothelial growth factor receptor tyrosine kinases inhibitors: pharmacophore modeling, virtual screening and docking studies.

    PubMed

    Yu, Hui; Wang, Zhanli; Zhang, Liangren; Zhang, Jufeng; Huang, Qian

    2007-03-01

    We have applied pharmacophore generation, database searching and docking methodologies to discover new structures for the design of vascular endothelial growth factor receptors, the tyrosine kinase insert domain-containing receptor kinase inhibitors. The chemical function based pharmacophore models were built for kinase insert domain-containing receptor kinase inhibitors from a set of 10 known inhibitors using the algorithm HipHop, which is implemented in the CATALYST software. The highest scoring HipHop model consists of four features: one hydrophobic, one hydrogen bond acceptor, one hydrogen bond donor and one ring aromatic function. Using the algorithm CatShape within CATALYST, the bound conformation of 4-amino-furo [2, 3-d] pyrimidine binding to kinase insert domain-containing receptor kinase was used to generate a shape query. A merged shape and hypothesis query that is in an appropriate alignment was then built. The combined shape and hypothesis model was used as a query to search Maybridge database for other potential lead compounds. A total of 39 compounds were retrieved as hits. The hits obtained were docked into kinase insert domain-containing receptor kinase active site. One novel potential lead was proposed based on CATALYST fit value, LigandFit docking scores, and examination of how the hit retain key interactions known to be required for kinase binding. This compound inhibited vascular endothelial growth factor stimulated kinase insert domain-containing receptor phosphorylation in human umbilical vein endothelial cells. PMID:17441906

  13. Immune tolerance therapy in paediatric haemophiliacs with factor VIII inhibitors: 14 years follow-up.

    PubMed

    Kreuz, W; Ehrenforth, S; Funk, M; Auerswald, G; Mentzer, D; Joseph-Steiner, J; Beeg, T; Klarman, D; Scharrer, I; Kornhuber, B

    1995-01-01

    We report our clinical experience in the immune tolerance (IT) therapy of 21 paediatric haemophiliacs with FVIII inhibitor: high responders (16HR) received initially FVIII twice daily at a dosage of 50-300 U/kg/day, 11/16 received a concomitant treatment with activated prothrombin complex concentrate (100-200 U/kg/day). Low responders (five LR) received 20-100 FVIII U/kg every second or third day. Inhibitor elimination was achieved in 19/21 patients in a median time of 4 months in HR and 1.5 months in LR. The outcome and length of time needed to induce IT was significantly correlated with FVIII exposure between the first inhibitor detection and onset of IT therapy and to interruption of IT therapy. For a rapid elimination of FVIII inhibitors it is important to start continuous administration of high-dose FVIII (≥ 100 FVIII U/kg/day) before repeated exposure to FVIII, in order to prevent rebooster effects, prolongation of elimination time, and to reduce expense. PMID:27214218

  14. Metabolic factors, adipose tissue, and plasminogen activator inhibitor-1 levels in Type 2 diabetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasminogen activator inhibitor-1 (PAI-1) production by adipose tissue is increased in obesity, and its circulating levels are high in type 2 diabetes. PAI-1 increases cardiovascular risk by favoring clot stability, interfering with vascular remodeling, or both. We investigated in obese diabetic per...

  15. When are the BET factors the most sensitive to bromodomain inhibitors?

    PubMed Central

    Khochbin, Saadi

    2013-01-01

    The recent publication of two detailed studies of mouse spermatogenesis, either after chemical inhibition of the BET bromodomains, or in the context of genetic alterations of one specific BET member, Brdt, provides the unique opportunity to assess the functional impact of BET bromodomain inhibitors. PMID:23412362

  16. The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity

    PubMed Central

    Caddell, Daniel F.; Wei, Tong; Park, Chang-Jin; Ronald, Pamela C.

    2016-01-01

    Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.

  17. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  18. Human tissue factor pathway inhibitor (TFPI) gene: Complete genomic structure and localization on the genetic map of chromosome 2q

    SciTech Connect

    Enjyoji, Kei-ichi; Emi, Mitsuru; Mukai, Tsunehiro; Imada, Motohiro; Kato, Hisao ); Leppert, M.L.; Lalouel, J.M. Univ. of Utah Medical School, Salt Lake City, UT )

    1993-08-01

    Tissue factor pathway inhibitor (TFPI), a protease inhibitor that circulates in association with plasma lipoproteins (VLDL, LDL and HDL), helps to regulate the extrinsic blood coagulation cascade. The authors have cloned a 125-kb genomic region containing the entire human TFPI gene on six overlapping cosmids and prepared a restriction map of this contig to clarify gene structure. More than half (45 kb) of the 85-kb gene is occupied with 5[prime] noncoding elements: coding begins at exon 3. A HindIII RFLP identified with one cosmid was genotyped in the CEPH panel of 559 reference families. Linkage analysis using markers on human chromosome 2 located the TFPI gene on 2q, 36 cM proximal to D2S43(pYNZ15) and 13 cM distal to the crystalline [gamma]-polypeptide locus CRYGP1(p5G1). 31 refs., 3 figs., 3 tabs.

  19. Prediction of Inhibitory Activity of Epidermal Growth Factor Receptor Inhibitors Using Grid Search-Projection Pursuit Regression Method

    PubMed Central

    Du, Hongying; Hu, Zhide; Bazzoli, Andrea; Zhang, Yang

    2011-01-01

    The epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK) is an important protein target for anti-tumor drug discovery. To identify potential EGFR inhibitors, we conducted a quantitative structure–activity relationship (QSAR) study on the inhibitory activity of a series of quinazoline derivatives against EGFR tyrosine kinase. Two 2D-QSAR models were developed based on the best multi-linear regression (BMLR) and grid-search assisted projection pursuit regression (GS-PPR) methods. The results demonstrate that the inhibitory activity of quinazoline derivatives is strongly correlated with their polarizability, activation energy, mass distribution, connectivity, and branching information. Although the present investigation focused on EGFR, the approach provides a general avenue in the structure-based drug development of different protein receptor inhibitors. PMID:21811593

  20. A Novel Small-molecule Tumor Necrosis Factor α Inhibitor Attenuates Inflammation in a Hepatitis Mouse Model*

    PubMed Central

    Ma, Li; Gong, Haiyan; Zhu, Haiyan; Ji, Qing; Su, Pei; Liu, Peng; Cao, Shannan; Yao, Jianfeng; Jiang, Linlin; Han, Mingzhe; Ma, Xiaotong; Xiong, Dongsheng; Luo, Hongbo R.; Wang, Fei; Zhou, Jiaxi; Xu, Yuanfu

    2014-01-01

    Overexpression of tumor necrosis factor α (TNFα) is a hallmark of many inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and septic shock and hepatitis, making it a potential therapeutic target for clinical interventions. To explore chemical inhibitors against TNFα activity, we applied computer-aided drug design combined with in vitro and cell-based assays and identified a lead chemical compound, (E)-4-(2-(4-chloro-3-nitrophenyl) (named as C87 thereafter), which directly binds to TNFα, potently inhibits TNFα-induced cytotoxicity (IC50 = 8.73 μm) and effectively blocks TNFα-triggered signaling activities. Furthermore, by using a murine acute hepatitis model, we showed that C87 attenuates TNFα-induced inflammation, thereby markedly reducing injuries to the liver and improving animal survival. Thus, our results lead to a novel and highly specific small-molecule TNFα inhibitor, which can be potentially used to treat TNFα-mediated inflammatory diseases. PMID:24634219

  1. Predictive factors of response to mTOR inhibitors in neuroendocrine tumours.

    PubMed

    Zatelli, Maria Chiara; Fanciulli, Giuseppe; Malandrino, Pasqualino; Ramundo, Valeria; Faggiano, Antongiulio; Colao, Annamaria

    2016-03-01

    Medical treatment of neuroendocrine tumours (NETs) has drawn a lot of attention due to the recent demonstration of efficacy of several drugs on progression-free survival, including somatostatin analogs, small tyrosine kinase inhibitors and mTOR inhibitors (or rapalogs). The latter are approved as therapeutic agents in advanced pancreatic NETs and have been demonstrated to be effective in different types of NETs, with variable efficacy due to the development of resistance to treatment. Early detection of patients that may benefit from rapalogs treatment is of paramount importance in order to select the better treatment and avoid ineffective and expensive treatments. Predictive markers for therapeutic response are under intensive investigation, aiming at a tailored patient management and more appropriate resource utilization. This review summarizes the available data on the tissue, circulating and imaging markers that are potentially predictive of rapalog efficacy in NETs. PMID:26666705

  2. A present status of CD-ROM XA and its future - Introduction to "TIBETAN ART in LADAKH" -

    NASA Astrophysics Data System (ADS)

    Saito, Fumio; Nomura, Kounoshin

    The first release of CD-ROM XA was anounced in March, 1989. This anouncement means that the interleaved ADPCM Audio system of the GREEN BOOK is applied to the specifications of CD-ROM. Accordingly it could lead to expanding application areas of CD-ROM. On the other hand within this year the logical format of video and graphics of CD-ROM XA will be probably confirmed. It will open the door to the new technology of CD-ROM, the real multimedia in the future. Hereafter will mention the summary of this release and the introduction of our first trial sample software, "TIBETAN ART in LADAKH" on CD-ROM XA.

  3. Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury

    PubMed Central

    Xie, Jieshi; Yang, Le; Tian, Lei; Li, Weiyang; Yang, Lin; Li, Liying

    2016-01-01

    Macrophage migration inhibitor factor (MIF), a multipotent innate immune mediator, is an upstream component of the inflammatory cascade in diseases such as liver disease. Monocyte chemoattractant protein-1 (MCP-1), a highly representative chemokine, is critical in liver disease pathogenesis. We investigated the role of MIF in regulating hepatocytic MCP-1 expression. MIF and MCP-1 expression were characterized by immunochemistry, RT-PCR, ELISA, and immunoblotting in CCl4-treated mouse liver and isolated hepatocytes. MIF was primarily distributed in hepatocytes, and its expression increased upon acute liver injury. Its expression was also increased in injured hepatocytes, induced by LPS or CCl4, which mimic liver injury in vitro. MIF was expressed earlier than MCP-1, strongly inducing hepatocytic MCP-1 expression. Moreover, the increase in MCP-1 expression induced by MIF was inhibited by CD74- or CD44-specific siRNAs and SB203580, a p38 MAPK inhibitor. Further, CD74 or CD44 deficiency effectively inhibited MIF-induced p38 activation. MIF inhibitor ISO-1 reduced MCP-1 expression and p38 phosphorylation in CCl4-treated mouse liver. Our results showed that MIF regulates MCP-1 expression in hepatocytes of injured liver via CD74, CD44, and p38 MAPK in an autocrine manner, providing compelling information on the role of MIF in liver injury, and implying a new regulatory mechanism for liver inflammation. PMID:27273604

  4. MET inhibitor PHA-665752 suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells

    SciTech Connect

    Liu, Tongxin; Li, Qi; Sun, Quanquan; Zhang, Yuqin; Yang, Hua; Wang, Rong; Chen, Longhua; Wang, Wei

    2014-06-20

    Highlights: • We demonstrated that irradiation induced MET overexpression and activation. • The aberrant MET signal mediated by HGF induced proliferation and radioresistance of NPC cells. • MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. • PHA-665752 suppressed the three downstream pathway of HGF/MET signal in a dose-dependent manner. - Abstract: Although ionizing radiation (IR) has provided considerable improvements in nasopharyngeal carcinoma (NPC), in subsets of patients, radioresistance is still a major problem in the treatment. In this study, we demonstrated that irradiation induced MET overexpression and activation, and the aberrant MET signal mediated by hepatocyte growth factor (HGF) induced radioresistance. We also found that MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. Further investigation indicated that PHA-665752 suppressed the phosphorylation of the Akt, ERK1/2, and STAT3 proteins in a dose-dependent manner. Our data indicated that the combination of IR with a MET inhibitor, such as PHA-665752, might be a promising therapeutic strategy for NPC.

  5. Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury.

    PubMed

    Xie, Jieshi; Yang, Le; Tian, Lei; Li, Weiyang; Yang, Lin; Li, Liying

    2016-01-01

    Macrophage migration inhibitor factor (MIF), a multipotent innate immune mediator, is an upstream component of the inflammatory cascade in diseases such as liver disease. Monocyte chemoattractant protein-1 (MCP-1), a highly representative chemokine, is critical in liver disease pathogenesis. We investigated the role of MIF in regulating hepatocytic MCP-1 expression. MIF and MCP-1 expression were characterized by immunochemistry, RT-PCR, ELISA, and immunoblotting in CCl4-treated mouse liver and isolated hepatocytes. MIF was primarily distributed in hepatocytes, and its expression increased upon acute liver injury. Its expression was also increased in injured hepatocytes, induced by LPS or CCl4, which mimic liver injury in vitro. MIF was expressed earlier than MCP-1, strongly inducing hepatocytic MCP-1 expression. Moreover, the increase in MCP-1 expression induced by MIF was inhibited by CD74- or CD44-specific siRNAs and SB203580, a p38 MAPK inhibitor. Further, CD74 or CD44 deficiency effectively inhibited MIF-induced p38 activation. MIF inhibitor ISO-1 reduced MCP-1 expression and p38 phosphorylation in CCl4-treated mouse liver. Our results showed that MIF regulates MCP-1 expression in hepatocytes of injured liver via CD74, CD44, and p38 MAPK in an autocrine manner, providing compelling information on the role of MIF in liver injury, and implying a new regulatory mechanism for liver inflammation. PMID:27273604

  6. Effects of ketoconazole or rifampin on the pharmacokinetics of tivozanib hydrochloride, a vascular endothelial growth factor receptor tyrosine kinase inhibitor.

    PubMed

    Cotreau, Monette M; Siebers, Nicholas M; Miller, James; Strahs, Andrew L; Slichenmyer, William

    2015-03-01

    The vascular endothelial growth factor (VEGF) pathway is associated with the promotion of endothelial cell proliferation, migration, and survival necessary for angiogenesis. VEGF and its three receptor isoforms are often overexpressed in many human solid tumors. Tivozanib is a potent, selective inhibitor of VEGF receptors 1, 2, and 3, with a long half-life. The purpose of these studies was to evaluate the effect of ketoconazole, a potent inhibitor of CYP3A4, and rifampin, a potent inducer of CYP3A4, on the pharmacokinetics of tivozanib. Two phase I, open-label, 2-period, single-sequence studies evaluated the effect of steady-state ketoconazole (NCT01363778) or rifampin (NCT01363804) on the pharmacokinetic profile, safety, and tolerability of a single oral 1.5-mg dose of tivozanib. Tivozanib was well tolerated in both studies. Steady-state ketoconazole did not cause a clinically significant change in the pharmacokinetics of a single dose of tivozanib; therefore, dosing of tivozanib with a CYP3A4 pathway inhibitor should not cause a clinically significant change in serum tivozanib levels. However, coadministration of tivozanib with rifampin caused a significant decrease in the area under the curve from 0 to infinity and half-life and an increase in clearance of tivozanib, which suggest increased clearance via the enhanced CYP3A4-mediated metabolism of tivozanib. PMID:27128217

  7. Metalloproteinase Inhibitors, Nonantimicrobial Chemically Modified Tetracyclines, and Ilomastat Block Bacillus anthracis Lethal Factor Activity in Viable Cells

    PubMed Central

    Kocer, Salih S.; Walker, Stephen G.; Zerler, Brad; Golub, Lorne M.; Simon, Sanford R.

    2005-01-01

    Lethal toxin, produced by the bacterium Bacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF. All three inhibitors, CMT-300, CMT-308, and Ilomastat, inhibit LF-mediated cleavage of a synthetic peptide substrate based on the N-terminal domain of MEKs. Inhibition of LF-mediated MEK proteolysis by all three agents was also achieved using lysates of the human monocytoid line MonoMac 6 as sources of MAPKKs and visualization of the extent of cleavage after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by detection by Western blotting. Finally, we have demonstrated inhibition of intracellular MEKs in viable human monocytes and MonoMac 6 cells by these agents after incubation of the cells with a reconstituted preparation of recombinant lethal toxin. All three agents are effective inhibitors when incubated with LF prior to exposure to cells, while the CMTs, but not Ilomastat, are also effective when added after LF has already entered the viable cell targets. These results offer promise for strategies to combat effects of the lethal toxin of B. anthracis. PMID:16239558

  8. Evidence of normal functional levels of activated protein C inhibitor in combined Factor V/VIII deficiency disease.

    PubMed Central

    Canfield, W M; Kisiel, W

    1982-01-01

    Human activated protein C (APC) is a plasma serine protease that possesses amidolytic and anticoagulant activity. The rate at which the amidolytic and anticoagulant activity of APC was neutralized in normal plasma was essentially identical to that observed in plasma obtained from four individuals with combined Factor V/VIII deficiency disease. Incubation of radioiodinated APC with either normal human plasma or the combined Factor V/VIII-deficient plasmas resulted in the formation of a stable complex (Mr = 96,000) of the enzyme and a plasma protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pretreatment of the radiolabeled APC with diisopropyl fluorophosphate prevented the formation of the enzyme-protein complex. On the basis of its ability to form a complex with radiolabeled APC, the APC-binding protein was purified to homogeneity from normal human plasma by ammonium sulfate fractionation, heparin-agarose chromatography, and QAE-Sephadex A-50 chromatography. The APC-binding protein (Mr = 54,000) is a glycoprotein, and possesses an amino-terminal sequence of Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp. The amino-terminal sequence of the APC-binding protein exhibited considerable homology with bovine colostrum inhibitor and pancreatic trypsin inhibitor, but no apparent sequence homology with the plasma serine protease inhibitors. Affinity-purified antibody against APC-binding protein immunoprecipitated a complex of radiolabeled APC and native APC-binding protein from normal human plasma. Complex formation was virtually eliminated in plasma immunodepleted of the APC-binding protein. Quantitative electroimmunoassay indicated essentially equal levels of APC-binding protein antigen in normal plasma compared with plasma from four patients with combined Factor V/VIII deficiency disease. Images PMID:6294139

  9. Risk Factors for High-Titer Inhibitor Development in Children with Hemophilia A: Results of a Cohort Study

    PubMed Central

    Heller, Christine; Gutsche, Sven; Holzhauer, Susanne; Kenet, Gili; Kurnik, Karin; Iorio, Alfonso; Nowak-Göttl, Ulrike

    2013-01-01

    Among the discussed risk factors for high-titre inhibitor (HRI) development in patients with hemophilia A (HA) are high dose FVIII replacement therapy and use of recombinant FVIII concentrates (rFVIII). The aim of this study was to evaluate the aforementioned risk factors for HRI development in children with hemophilia A ≤2%. About 288 ascertained PUPs (Israel and Germany) were followed after initial HA diagnosis over 200 exposure days. Inhibitor-free survival, hazard ratios (HR), and 95% confidence intervals (CIs) were calculated. Adjustment was performed for factor VIII concentrates, median single dose over the first three months of treatment, first FVIII administration before the age of three months, presence of risk HA gene mutations, “intensive treatment moments” and “year of birth” (proxy for different treatment periods). HRI occurred in 71/288 children (24.7%). In multivariate analysis adjusted for “year of birth”, underlying risk gene mutations (HR/CI: 2.37/1.40–3.99), FVIII dose, measured per one IU increase per kgbw (HR/CI: 1.05/1.04–1.07), and first FVIII administration before the age of three months showed a significant impact on HR development. The risk of HRI development was similar for recombinant or plasmatic FVIII products. Children at risk should be treated with carefully calculated lower dose regimens, adapted to individual bleeding situations. PMID:24199202

  10. Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

    PubMed

    Xiao, Hongyan; Kovics, Richard; Jackson, Van; Remick, Daniel G

    2004-04-01

    Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation. PMID:15060414

  11. Early eradication of factor VIII inhibitor in patients with congenital hemophilia A by immune tolerance induction with a high dose of immunoglobulin.

    PubMed

    Mizoguchi, Yoko; Furue, Aya; Kagawa, Reiko; Chijimatsu, Ikue; Tomioka, Keita; Shimomura, Maiko; Imanaka, Yusuke; Nishimura, Shiho; Saito, Satoshi; Miki, Mizuka; Ono, Atsushi; Konishi, Nakao; Kawaguchi, Hiroshi; Kobayashi, Masao

    2016-04-01

    The production of factor VIII (FVIII) inhibitory antibodies is a serious problem in patients with hemophilia A. Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors and has been shown to be successful in 70 % of patients with hemophilia A. However, a minority of hemophilia patients present life-long inhibitors. To eliminate such inhibitors, we designed an intravenous immunoglobulin (IVIG) strategy in combination with high dose recombinant FVIII for ITI in hemophilia A children with inhibitors. Four previously untreated patients produced inhibitors within 16 exposures to FVIII. The peak inhibitor titers in these patients ranged from 3 to 14 BU/mL. The patients received ITI combined with IVIG within 1.5 months after the inhibitors were detected. All patients showed a negative titer for inhibitors by 28 days, with no anamnestic responses. The recovery of FVIII in the plasma concentration was normalized within three months after initiation of ITI. An additional course of IVIG administration led to induction of complete tolerance by 20 months after initiation of ITI therapy in all patients. ITI treatment with high-dose FVIII combined with IVIG may be effective for the early elimination of inhibitors. PMID:26830966

  12. Immune-Modulation by Epidermal Growth Factor Receptor Inhibitors: Implication on Anti-Tumor Immunity in Lung Cancer

    PubMed Central

    Herrmann, Amanda C.; Bernatchez, Chantale; Haymaker, Cara; Molldrem, Jeffrey J.; Hong, Waun Ki; Perez-Soler, Roman

    2016-01-01

    Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation. PMID:27467256

  13. Antiestrogen fulvestrant enhances the antiproliferative effects of epidermal growth factor receptor inhibitors in human non-small cell lung cancer

    PubMed Central

    Garon, Edward B.; Pietras, Richard J.; Finn, Richard S.; Kamranpour, Naeimeh; Pitts, Sharon; Márquez-Garbán, Diana C.; Desai, Amrita J.; Dering, Judy; Hosmer, Wylie; von Euw, Erika M.; Dubinett, Steven M.; Slamon, Dennis J.

    2012-01-01

    Introduction Estrogen receptor (ER) signaling and its interaction with epidermal growth factor receptor (EGFR) is a potential therapeutic target in non-small cell lung cancer (NSCLC). To explore cross-communication between ER and EGFR, we have correlated ER pathway gene and protein expression profiles and examined effects of antiestrogens with or without EGFR inhibitors in preclinical models of human NSCLC. Methods We evaluated 54 NSCLC cell lines for growth inhibition with EGFR inhibitors, antiestrogen treatment or the combination. Each line was evaluated for baseline ER pathway protein expression. The majority were also evaluated for baseline ER pathway gene expression. Human NSCLC xenografts were evaluated for effects of inhibition of each pathway either individually or in combination. Results The specific antiestrogen fulvestrant has modest single agent activity in vitro, but in many lines fulvestrant adds to effects of EGFR inhibitors, including synergy in the EGFR mutant, erlotinib-resistant H1975 line. ERα, ERβ, progesterone receptor (PR)-A, PR-B and aromatase proteins are expressed in all lines to varying degrees, with trends towards lower aromatase in more sensitive cell lines. Sensitivity to fulvestrant correlates with greater baseline ERα gene expression. Tumor stability is achieved in human tumor xenografts with either fulvestrant or EGFR inhibitors, but tumors regress significantly when both pathways are inhibited. Conclusions These data provide a rationale for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-EGFR agents in the clinic. Future work should also evaluate dual ER and EGFR inhibition in the setting of secondary resistance to EGFR inhibition. PMID:23399957

  14. Pathogenesis-Related Gene Expression in Rice is Correlated with Developmentally Controlled Xa21-mediated Resistance against Xanthomonas oryzae pv. oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Disease resistance mediated by the resistance gene Xa21 is developmentally controlled in rice. We examined the relationship between pathogenesis related (PR) defense gene expression and Xa21-mediated developmental disease resistance induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1a, OsPR1b, a...

  15. Combination of 4-anilinoquinazoline and rhodanine as novel epidermal growth factor receptor tyrosine kinase inhibitors.

    PubMed

    Li, Si-Ning; Xu, Yun-Yun; Gao, Jia-Yu; Yin, Hong-Ran; Zhang, Shi-Lei; Li, Huan-Qiu

    2015-07-01

    A type of novel rhodanine-based 4-anilinoquinazoline, which designed the combination between quinazoline as the backbone and various substituted biological rhodanine groups as the side chain, have been synthesized, and their antiproliferative activities were also evaluated firstly. These compounds displayed good antiproliferative activity and EGFR-TK inhibitory activity. Among them, compound 8d showed good inhibitory activity (IC50=2.7μM for Hep G2, IC50=3.1μM for A549) and molecular docking of 8d into EGFR TK active site was also performed, this inhibitor well fitting the active site might well explain its excellent inhibitory activity. PMID:26003342

  16. Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type β and epidermal growth factor

    SciTech Connect

    Song, Xiaoling; Thalacker, F.W.; Nilsen-Hamilton, Marit

    2012-04-06

    The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type β (TGFβ) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGFβ regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGFβ also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGFβ pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGFβ are able to promote large and rapid changes in PAI-1 expression.

  17. Effects of the hypoxia-inducible factor-1 inhibitor echinomycin on vascular endothelial growth factor production and apoptosis in human ectopic endometriotic stromal cells.

    PubMed

    Tsuzuki, Tomoko; Okada, Hidetaka; Shindoh, Hisayuu; Shimoi, Kayo; Nishigaki, Akemi; Kanzaki, Hideharu

    2016-04-01

    Recent evidence points to a possible role for hypoxia-inducible factor (HIF)-1 in the pathogenesis and development of endometriosis. The objectives of this study were to investigate the critical role of HIF-1 in endometriosis and the effect of the HIF-1 inhibitor echinomycin on human ectopic endometriotic stromal cells (eESCs). Ectopic endometriotic tissues were obtained from 20 patients, who received an operation for ovarian endometriomas. We examined vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) production, HIF-1 expression, cell proliferation and apoptosis of eESCs. Cobalt chloride (CoCl2) significantly induced expression of HIF-1α protein and VEGF production in a time-dependent manner in eESCs, but reduced SDF-1 production. VEGF production was significantly suppressed by treatment of 100 nM echinomycin without causing cell toxicity, but 0.1-10 nM echinomycin or 100 nM progestin had no significant effect. SDF-1 production was not affected by echinomycin treatment at any dose. Echinomycin inhibited cell proliferation and induced apoptotic cell death of the eESCs, and significantly inhibited expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Echinomycin inhibits VEGF production and induces apoptosis of eESCs by suppression of Bcl-2 and Bcl-xL. These findings suggest the unique therapeutic potential for echinomycin as an inhibitor of HIF-1 activation for endometriosis treatment. PMID:26654708

  18. Detection of Legionella pneumophila serogroup 1 in blood cultures from a patient treated with tumor necrosis factor-alpha inhibitor.

    PubMed

    Kaku, Norihito; Yanagihara, Katsunori; Morinaga, Yoshitomo; Sato, Tsuyoshi; Nakashima, Munetoshi; Sakai, Takahiro; Tominaga, Hiroo; Wakigawa, Fumiko; Nagashima, Seiji; Fukuda, Minoru; Hashiguchi, Kohji; Kohno, Shigeru

    2013-02-01

    A 65-year-old man was admitted to our hospital with a temperature of 39.3 °C, cough, sputum, and pharyngeal discomfort that had persisted for 3 days. He had been treated with methotrexate and adalimumab (a tumor necrosis factor-alpha [TNF-α] inhibitor) for rheumatoid arthritis for 2 years, and he had also been treated with S-1 (tegafur, gimeracil, and oteracil potassium) for pancreatic metastasis of gastric cancer for 2 months. Regardless of the underlying pathologies, his general condition was good and he had worked as an electrician until 2 days before admission. However, his appetite had suddenly decreased from the day before admission, and high fever and hypoxia were also evident upon admission. A chest X-ray and computed tomography scan revealed left pleural effusion and consolidation in both lungs. The pneumonia severity index score was 165 and the risk class was V. Accordingly, we started to treat the pneumonia with a combination of levofloxacin and meropenem. Thereafter, we received positive urinary antigen test findings for Legionella pneumophila. After hospitalization, hypoxia was progressed and hypotension was emerged. Despite the application of appropriate antibiotics, vasopressors, and oxygenation, the patient died 8 h after admission. Even after his death, blood cultures were continued to consider the possibility of bacterial co-infection. Although no bacteria were detected from blood cultures, Gimenez staining revealed pink bacteria in blood culture fluids. Subsequent blood fluid culture in selective medium revealed L. pneumophila serogroup 1. Recently, TNF-α inhibitors have been described as a risk factor for Legionnaires' disease. In consideration of the increased frequency of TNF-α inhibitors, we may need to recognize anew that L. pneumophila might be a pathogen of severe community-acquired pneumonia. PMID:22911089

  19. Histone Deacetylase Inhibitors Inhibit Rhabdomyosarcoma by Reactive Oxygen Species-Dependent Targeting of Specificity Protein Transcription Factors.

    PubMed

    Hedrick, Erik; Crose, Lisa; Linardic, Corinne M; Safe, Stephen

    2015-09-01

    The two major types of rhabdomyosarcoma (RMS) are predominantly diagnosed in children, namely embryonal (ERMS) and alveolar (ARMS) RMS, and patients are treated with cytotoxic drugs, which results in multiple toxic side effects later in life. Therefore, development of innovative chemotherapeutic strategies is imperative, and a recent genomic analysis suggested the potential efficacy of reactive oxygen species (ROS)-inducing agents. Here, we demonstrate the efficacy of the potent histone deacetylase (HDAC) inhibitors, panobinostat and vorinostat, as agents that inhibit RMS tumor growth in vivo, induce apoptosis, and inhibit invasion of RD and Rh30 RMS cell lines. These effects are due to epigenetic repression of cMyc, which leads to decreased expression of cMyc-regulated miRs-17, -20a, and -27a; upregulation of ZBTB4, ZBTB10, and ZBTB34; and subsequent downregulation of Sp transcription factors. We also show that inhibition of RMS cell growth, survival and invasion, and repression of Sp transcription factors by the HDAC inhibitors are independent of histone acetylation but reversible after cotreatment with the antioxidant glutathione. These results show a novel ROS-dependent mechanism of antineoplastic activity for panobinostat and vorinostat that lies outside of their canonical HDAC-inhibitory activity and demonstrates the potential clinical utility for treating RMS patients with ROS-inducing agents. PMID:26162688

  20. Stroke Prevention: Managing Modifiable Risk Factors

    PubMed Central

    Di Legge, Silvia; Koch, Giacomo; Diomedi, Marina; Stanzione, Paolo; Sallustio, Fabrizio

    2012-01-01

    Prevention plays a crucial role in counteracting morbidity and mortality related to ischemic stroke. It has been estimated that 50% of stroke are preventable through control of modifiable risk factors and lifestyle changes. Antihypertensive treatment is recommended for both prevention of recurrent stroke and other vascular events. The use of antiplatelets and statins has been shown to reduce the risk of recurrent stroke and other vascular events. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) are indicated in stroke prevention because they also promote vascular health. Effective secondary-prevention strategies for selected patients include carotid revascularization for high-grade carotid stenosis and vitamin K antagonist treatment for atrial fibrillation. The results of recent clinical trials investigating new anticoagulants (factor Xa inhibitors and direct thrombin inhibitors) clearly indicate alternative strategies in stroke prevention for patients with atrial fibrillation. This paper describes the current landscape and developments in stroke prevention with special reference to medical treatment in secondary prevention of ischemic stroke. PMID:23213626

  1. Four-Week Studies of Oral Hypoxia-Inducible Factor-Prolyl Hydroxylase Inhibitor GSK1278863 for Treatment of Anemia.

    PubMed

    Holdstock, Louis; Meadowcroft, Amy M; Maier, Rayma; Johnson, Brendan M; Jones, Delyth; Rastogi, Anjay; Zeig, Steven; Lepore, John J; Cobitz, Alexander R

    2016-04-01

    Hypoxia-inducible factor prolyl hydroxylase inhibitors stabilize levels of hypoxia-inducible factor that upregulate transcription of multiple genes associated with the response to hypoxia, including production of erythropoietin. We conducted two phase 2a studies to explore the relationship between the dose of the hypoxia-inducible factor-prolyl hydroxylase inhibitor GSK1278863 and hemoglobin response in patients with anemia of CKD (baseline hemoglobin 8.5-11.0 g/dl) not undergoing dialysis and not receiving recombinant human erythropoietin (nondialysis study) and in patients with anemia of CKD (baseline hemoglobin 9.5-12.0 g/dl) on hemodialysis and being treated with stable doses of recombinant human erythropoietin (hemodialysis study). Participants were randomized 1:1:1:1 to a once-daily oral dose of GSK1278863 (0.5 mg, 2 mg, or 5 mg) or control (placebo for the nondialysis study; continuing on recombinant human erythropoietin for the hemodialysis study) for 4 weeks, with a 2-week follow-up. In the nondialysis study, GSK1278863 produced dose-dependent effects on hemoglobin, with the highest dose resulting in a mean increase of 1 g/dl at week 4. In the hemodialysis study, treatment with GSK1278863 in the 5-mg arm maintained mean hemoglobin concentrations after the switch from recombinant human erythropoietin, whereas mean hemoglobin decreased in the lower-dose arms. In both studies, the effects on hemoglobin occurred with elevations in endogenous erythropoietin within the range usually observed in the respective populations and markedly lower than those in the recombinant human erythropoietin control arm in the hemodialysis study, and without clinically significant elevations in plasma vascular endothelial growth factor concentrations. GSK1278863 was generally safe and well tolerated at the doses and duration studied. GSK1278863 may prove an effective alternative for managing anemia of CKD. PMID:26494831

  2. Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

    PubMed Central

    Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

    2016-01-01

    Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation

  3. Sensitivities to various epidermal growth factor receptor-tyrosine kinase inhibitors of uncommon epidermal growth factor receptor mutations L861Q and S768I: What is the optimal epidermal growth factor receptor-tyrosine kinase inhibitor?

    PubMed

    Banno, Eri; Togashi, Yosuke; Nakamura, Yu; Chiba, Masato; Kobayashi, Yoshihisa; Hayashi, Hidetoshi; Terashima, Masato; de Velasco, Marco A; Sakai, Kazuko; Fujita, Yoshihiko; Mitsudomi, Tetsuya; Nishio, Kazuto

    2016-08-01

    Most patients with non-small cell lung cancer (NSCLC) harboring common epidermal growth factor receptor (EGFR) mutations, such as deletions in exon 19 or the L858R mutation in exon 21, respond dramatically to EGFR tyrosine kinase inhibitors (EGFR-TKI), and their sensitivities to various EGFR-TKI have been well characterized. Our previous article showed the in vitro sensitivities of EGFR exon 18 mutations to EGFR-TKI, but little information regarding the sensitivities of other uncommon EGFR mutations is available. First, stable transfectant Ba/F3 cell lines harboring EGFR L858R (Ba/F3-L858R), L861Q (Ba/F3-L861Q) or S768I (Ba/F3-S768I) mutations were created and their drug sensitivities to various EGFR-TKI were examined. Both the Ba/F3-L861Q and Ba/F3-S768I cell lines were less sensitive to erlotinib, compared with the Ba/F3-L858R cell line, but their sensitivities to afatinib were similar to that of the Ba/F3-L858R cell line. The Ba/F3-L861Q cell line was similarly sensitive and the Ba/F3-S768I cell line was less sensitive to osimertinib, compared with the Ba/F3-L858R cell line. The results of western blot analyses were consistent with these sensitivities. Next, similar experiments were also performed using the KYSE270 (L861Q) and KYSE 450 (S768I) cell lines, and their results were compatible with those of the transfectant Ba/F3 cell lines. Our findings suggest that NSCLC harboring the EGFR L861Q mutation might be sensitive to afatinib or osimertinib and that NSCLC harboring the EGFR S768I mutation might be sensitive to afatinib. Overall, afatinib might be the optimal EGFR-TKI against these uncommon EGFR mutations. PMID:27240419

  4. Ring-truncated deguelin derivatives as potent Hypoxia Inducible Factor-1α (HIF-1α) inhibitors.

    PubMed

    Kim, Ho Shin; Hong, Mannkyu; Lee, Su-Chan; Lee, Ho-Young; Suh, Young-Ger; Oh, Dong-Chan; Seo, Ji Hae; Choi, Hoon; Kim, Jun Yong; Kim, Kyu-Won; Kim, Jeong Hun; Kim, Joohwan; Kim, Young-Myeong; Park, So-Jung; Park, Hyun-Ju; Lee, Jeewoo

    2015-11-01

    A series of fluorophenyl and pyridine analogues of 1 and 2 were synthesized as ring-truncated deguelin surrogates and evaluated for their HIF-1α inhibition. Their structure-activity relationship was systematically investigated based on the variation of the linker B-region moiety. Among the inhibitors, compound 25 exhibited potent HIF-1α inhibition in a dose-dependent manner and significant antitumor activity in H1299 with less toxicity than deguelin. It also inhibited in vitro hypoxia-mediated angiogenic processes in HRMECs. The docking study indicates that 25 occupied the C-terminal ATP-binding pocket of HSP90 in a similar mode as 1, which implies that the anticancer and antiangiogenic activities of 25 are derived from HIF-1α destabilization by binding to the C-terminal ATP-binding site of hHSP90. PMID:26457742

  5. Identification of selective covalent inhibitors of platelet activating factor acetylhydrolase 1B2 from the screening of an oxadiazolone-capped peptoid-azapeptoid hybrid library.

    PubMed

    Sarma, Bani Kanta; Liu, Xiaodan; Kodadek, Thomas

    2016-09-01

    A potent and selective inhibitor of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is described. The compound was derived by improvement of a modest affinity primary hit isolated from the screening of a bead-displayed peptoid-azapeptoid hybrid library tethered to an oxadiazolone 'warhead'. The oxadiazolone moiety of the inhibitors was found to react covalently with the active site serine residue of PAFAH1B2. This screening strategy may be useful for the identification of many selective, covalent inhibitors of serine hydrolases. PMID:27160052

  6. Prognostic factors affecting early colectomy in patients with moderate to severe ulcerative colitis treated with calcineurin inhibitors

    PubMed Central

    Ban, Hiromitsu; Bamba, Shigeki; Nishida, Atsushi; Inatomi, Osamu; Shioya, Makoto; Takahashi, Ken-Ichiro; Imaeda, Hirotsugu; Murata, Masaki; Sasaki, Masaya; Tsujikawa, Tomoyuki; Andoh, Akira

    2016-01-01

    Calcineurin inhibitors (CNIs) such as cyclosporine A (CSA) and tacrolimus (FK506) are often used as a second-line drug for steroid-refractory or steroid-dependent patients with ulcerative colitis (UC). The aim of the present study was to determine the prognostic factors for early colectomy. A total of 85 hospitalized patients with UC (CSA, 50 patients; FK506, 35 patients) were enrolled. Colectomy carried out within 60 days of starting CNI therapy was defined as ‘early colectomy’. To assess the prognostic factors affecting early colectomy, clinical practical variables, including the Onodera-prognostic nutritional index (O-PNI): 10xAlb+0.005× (total lymphocyte count), were analyzed. The results demonstrated that the significant factors predicting early colectomy were i) disease severity, ii) immunomodulator-naïve history, iii) lower serum hematocrit, iv) lower serum albumin and v) lower O-PNI. In addition, the significant factors predicting overall colectomy were as follows: i) C7-HRP positivity and ii) >10,000 mg of prednisolone used prior to the initiation of CNI treatment. The combination of hematocrit and O-PNI enhanced the prediction of early colectomy. Clinical variables such as hematocrit and O-PNI were the significant factors predicting colectomy. These results may be used as a guide to predict the outcome of patients with UC in clinical settings.

  7. Molecular predictors of sensitivity to the insulin-like growth factor 1 receptor inhibitor Figitumumab (CP-751,871).

    PubMed

    Pavlicek, Adam; Lira, Maruja E; Lee, Nathan V; Ching, Keith A; Ye, Jingjing; Cao, Joan; Garza, Scott J; Hook, Kenneth E; Ozeck, Mark; Shi, Stephanie T; Yuan, Jing; Zheng, Xianxian; Rejto, Paul A; Kan, Julie L C; Christensen, James G

    2013-12-01

    Figitumumab (CP-751,871), a potent and fully human monoclonal anti-insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors. PMID:24107449

  8. Extracellular Signal-Regulated Kinases Modulate DNA Damage Response - A Contributing Factor to Using MEK Inhibitors in Cancer Therapy

    PubMed Central

    Wei, F; Yan, J; Tang, D

    2011-01-01

    The Raf-MEK-ERK pathway is commonly activated in human cancers, largely attributable to the extracellular signal-regulated kinases (ERKs) being a common downstream target of growth factor receptors, Ras, and Raf. Elevation of these up-stream signals occurs frequently in a variety of malignancies and ERK kinases play critical roles in promoting cell proliferation. Therefore, inhibition of MEK-mediated ERK activation is very appealing in cancer therapy. Consequently, numerous MEK inhibitors have been developed over the years. However, clinical trials have yet to produce overwhelming support for using MEK inhibitors in cancer therapy. Although complex reasons may have contributed to this outcome, an alternative possibility is that the MEK-ERK pathway may not solely provide proliferation signals to malignancies, the central scientific rationale in developing MEK inhibitors for cancer therapy. Recent developments may support this alternative possibility. Accumulating evidence now demonstrated that the MEK-ERK pathway contributes to the proper execution of cellular DNA damage response (DDR), a major pathway of tumor suppression. During DDR, the MEK-ERK pathway is commonly activated, which facilitates the proper activation of DDR checkpoints to prevent cell division. Inhibition of MEK-mediated ERK activation, therefore, compromises checkpoint activation. As a result, cells may continue to proliferate in the presence of DNA lesions, leading to the accumulation of mutations and thereby promoting tumorigenesis. Alternatively, reduction in checkpoint activation may prevent efficient repair of DNA damages, which may cause apoptosis or cell catastrophe, thereby enhancing chemotherapy’s efficacy. This review summarizes our current understanding of the participation of the ERK kinases in DDR. PMID:22087839

  9. Galiellalactone Is a Direct Inhibitor of the Transcription Factor STAT3 in Prostate Cancer Cells*♦

    PubMed Central

    Don-Doncow, Nicholas; Escobar, Zilma; Johansson, Martin; Kjellström, Sven; Garcia, Victor; Munoz, Eduardo; Sterner, Olov; Bjartell, Anders; Hellsten, Rebecka

    2014-01-01

    The transcription factor STAT3 is constitutively active in several malignancies including castration-resistant prostate cancer and has been identified as a promising therapeutic target. The fungal metabolite galiellalactone, a STAT3 signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing active STAT3 and induces apoptosis of prostate cancer stem cell-like cells expressing phosphorylated STAT3 (pSTAT3). However, the molecular mechanism of this STAT3-inhibiting effect by galiellalactone has not been clarified. A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of galiellalactone target proteins. By adding streptavidin-Sepharose beads to GL-biot-treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Confocal microscopy revealed GL-biot in both the cytoplasm and the nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. Galiellalactone inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. Mass spectrometry analysis of recombinant STAT3 protein pretreated with galiellalactone revealed three modified cysteines (Cys-367, Cys-468, and Cys-542). Here we demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer. PMID:24755219

  10. Calpain Inhibitor PD150606 Attenuates Glutamate Induced Spiral Ganglion Neuron Apoptosis through Apoptosis Inducing Factor Pathway In Vitro

    PubMed Central

    Song, Yong-Li; Chen, Xiao-Dong; Mi, Wen-Juan; Wang, Jian; Lin, Ying; Chen, Fu-Quan; Qiu, Jian-Hua

    2015-01-01

    Objective This research aimed to investigate whether glutamate induced spiral ganglion neurons (SGNs) apoptosis through apoptosis inducing factor (AIF) pathway. And verify whether PD150606, a calpain inhibitor could prevent apoptosis by inhibiting cleaving and releasing AIF in mitochondrion. Methods SGNs of postnatal days 0-3 were harvested and cultured in dishes. 20 mM Glu, the caspase inhibitor Z-VAD-FMK and calpain inhibitor PD150606 were added into cultured dishes separately. We used optical microscope and immunofluoresence staining to observe cell morphology and AIF distribution, RT-PCR and Westernblot to analyse AIF and calpain expression in SGNs. TUNEL assay was used to test cell apoptosis. Results Cell morphology and nuclear translocation of AIF were altered in SGNs by 20 mM Glu treated in vitro. The axon of SGN shortened, more apoptosis SGN were observed and the expression of AIF and calpain were up-regulated in Glu-treated group than the normal one (P<0.05). The same experiments were conducted in 20 mM+PD150606 treated group and 20 mM+Z-VAD-FMK group. Obviously AIF were located from cytoplasm to the nuclear and the expressions of AIF and calpain were down-regulated by PD150606 (P<0.05). Positive cells in TUNEL staining decreased after PD150606 treating. However, Z-VAD-FMK had no influence on AIF, calpain expression or cell apoptosis. Conclusion The AIF-related apoptosis pathway is involved in the process of Glu-induced SGN injury. Furthermore, the inhibition of calpain can prevent AIF from releasing the nuclear or inducing SGN apoptosis. PMID:25874633

  11. The impact of disease activity and tumour necrosis factorinhibitor therapy on cytokine levels in juvenile idiopathic arthritis.

    PubMed

    Walters, H M; Pan, N; Lehman, T J A; Adams, A; Kalliolias, G D; Zhu, Y S; Santiago, F; Nguyen, J; Sitaras, L; Cunningham-Rundles, S; Walsh, T J; Toussi, S S

    2016-06-01

    The aim of this study was to evaluate prospectively cytokine levels and disease activity in juvenile idiopathic arthritis (JIA) patients treated with and without tumour necrosis factor (TNF)-α inhibitors. TNF-α inhibitor-naive JIA subjects were followed prospectively for 6 months. Cytokine levels of TNF-α, interleukin (IL)-1β, IL-6, IL-8, IL-10 and IL-17 were measured at baseline for JIA subjects and healthy controls (HCs). Cytokine levels were then measured at four time-points after initiation of TNF-α inhibition for anti-TNF-α-treated (anti-TNF) JIA subjects, and at two subsequent time-points for other JIA (non-TNF) subjects. JIA disease activity by Childhood Health Assessment Questionnaire (CHAQ) disability index/pain score and physician joint count/global assessment was recorded. Sixteen anti-TNF, 31 non-TNF and 16 HCs were analysed. Among JIA subjects, those with higher baseline disease activity (subsequent anti-TNFs) had higher baseline TNF-α, IL-6 and IL-8 than those with lower disease activity (non-TNFs) (P < 0·05). TNF-α and IL-10 increased, and IL-6 and IL-8 no longer remained significantly higher after TNF-α inhibitor initiation in anti-TNF subjects. Subgroup analysis of etanercept versus adalimumab-treated subjects showed that TNF-α and IL-17 increased significantly in etanercept but not adalimumab-treated subjects, despite clinical improvement in both groups of subjects. JIA subjects with increased disease activity at baseline had higher serum proinflammatory cytokines. TNF-α inhibition resulted in suppression of IL-6 and IL-8 in parallel with clinical improvement in all anti-TNF-treated subjects, but was also associated with elevated TNF-α and IL-17 in etanercept-treated subjects. PMID:26934060

  12. Indirect effects of ploidy suggest X chromosome dose, not the X:A ratio, signals sex in Drosophila.

    PubMed

    Erickson, James W; Quintero, Jerome J

    2007-12-01

    In the textbook view, the ratio of X chromosomes to autosome sets, X:A, is the primary signal specifying sexual fate in Drosophila. An alternative idea is that X chromosome number signals sex through the direct actions of several X-encoded signal element (XSE) proteins. In this alternative, the influence of autosome dose on X chromosome counting is largely indirect. Haploids (1X;1A), which possess the male number of X chromosomes but the female X:A of 1.0, and triploid intersexes (XX;AAA), which possess a female dose of two X chromosomes and the ambiguous X:A ratio of 0.67, represent critical tests of these hypotheses. To directly address the effects of ploidy in primary sex determination, we compared the responses of the signal target, the female-specific SxlPe promoter of the switch gene Sex-lethal, in haploid, diploid, and triploid embryos. We found that haploids activate SxlPe because an extra precellular nuclear division elevates total X chromosome numbers and XSE levels beyond those in diploid males. Conversely, triploid embryos cellularize one cycle earlier than diploids, causing premature cessation of SxlPe expression. This prevents XX;AAA embryos from fully engaging the autoregulatory mechanism that maintains subsequent Sxl expression, causing them to develop as sexual mosaics. We conclude that the X:A ratio predicts sexual fate, but does not actively specify it. Instead, the instructive X chromosome signal is more appropriately seen as collective XSE dose in the early embryo. Our findings reiterate that correlations between X:A ratios and cell fates in other organisms need not implicate the value of the ratio as an active signal. PMID:18162044

  13. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway

    PubMed Central

    Herbertz, Stephan; Sawyer, J Scott; Stauber, Anja J; Gueorguieva, Ivelina; Driscoll, Kyla E; Estrem, Shawn T; Cleverly, Ann L; Desaiah, Durisala; Guba, Susan C; Benhadji, Karim A; Slapak, Christopher A; Lahn, Michael M

    2015-01-01

    Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients. PMID:26309397

  14. BIIB021, a synthetic Hsp90 inhibitor, induces mutant ataxin-1 degradation through the activation of heat shock factor 1.

    PubMed

    Ding, Ying; Adachi, Hiroaki; Katsuno, Masahisa; Sahashi, Kentaro; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki; Sobue, Gen

    2016-07-01

    Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract in ataxin-1 (ATXN1). The pathological hallmarks of SCA1 are the loss of cerebellar Purkinje cells and neurons in the brainstem and the presence of nuclear aggregates containing the polyQ-expanded ATXN1 protein. Heat shock protein 90 (Hsp90) inhibitors have been shown to reduce polyQ-induced toxicity. This study was designed to examine the therapeutic effects of BIIB021, a purine-scaffold Hsp90 inhibitor, on the protein homeostasis of polyQ-expanded mutant ATXN1 in a cell culture model of SCA1. Our results demonstrated that BIIB021 activated heat shock factor 1 (HSF1) and suppressed the abnormal accumulation of ATXN1 and its toxicity. The pharmacological degradation of mutant ATXN1 via activated HSF1 was dependent on both the proteasome and autophagy systems. These findings indicate that HSF1 is a key molecule in the regulation of the protein homeostasis of the polyQ-expanded mutant ATXN1 and that Hsp90 has potential as a novel therapeutic target in patients with SCA1. PMID:27058144

  15. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway.

    PubMed

    Herbertz, Stephan; Sawyer, J Scott; Stauber, Anja J; Gueorguieva, Ivelina; Driscoll, Kyla E; Estrem, Shawn T; Cleverly, Ann L; Desaiah, Durisala; Guba, Susan C; Benhadji, Karim A; Slapak, Christopher A; Lahn, Michael M

    2015-01-01

    Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients. PMID:26309397

  16. The evaluation of inhibitive effectiveness of the tumour necrosis factor-α converting enzyme selective inhibitors by HPLC.

    PubMed

    Zhao, Yunbin; Yu, Jin; Gu, Jiuling; Huang, Wei

    2011-04-01

    A novel high-performance liquid chromatography (HPLC) method based on the internal standard method was established for assaying the tumour necrosis factor-α converting enzyme (TACE) activity and matrix metalloprotease-9 (MMP-9) activity, and was used to evaluate the inhibitive effectiveness of inhibitors to TACE and MMP-9. In the assay method for TACE and MMP-9, peptides labelled with the ultraviolet group-Dpa were used as substrates. Alanine-Dpa was synthesised and was used as the internal standard for quantitative analysis. After the peptide substrates were hydrolysed by TACE (MMP-9) for 15 min (25 min) at 37 °C, the amount of remaining substrates were determined by reversed-phased HPLC with UV detection at 353 nm. The relative peak area of the substrate was linearly dependent on the substrate concentration. This method was then applied to determine the 50% inhibitory concentration (IC₅₀) of GM6001 and inhibitor A for both TACE and MMP-9. PMID:21406033

  17. Heat shock factor 1, an inhibitor of non-homologous end joining repair

    PubMed Central

    Kang, Ga-Young; Kim, Eun-Ho; Lee, Hae-June; Gil, Na-Yeon; Cha, Hyuk-Jin; Lee, Yun-Sil

    2015-01-01

    A novel role for HSF1 as an inhibitor of non-homologous end joining (NHEJ) repair activity was identified. HSF1 interacted directly with both of the N-terminal sequences of the Ku70 and Ku86 proteins, which inhibited the endogenous heterodimeric interaction between Ku70 and Ku86. The blocking of the Ku70 and Ku86 interaction by HSF1 induced defective NHEJ repair activity and ultimately activated genomic instability after ionizing radiation (IR), which was similar to effects seen in Ku70 or Ku80 knockout cells. The binding activity between HSF1 and Ku70 or Ku86 was dependent on DNA damage response such as IR exposure, but not on the heat shock mediated transcriptional activation of HSF1. Moreover, the posttranslational modification such as phosphorylation, acetylation and sumoylation of HSF1 did not alter the binding activities of HSF1-Ku70 or HSF1-Ku86. Furthermore, the defect in DNA repair activity by HSF1 was observed regardless of p53 status. Rat mammary tumors derived using dimethylbenz(a)anthracence revealed that high levels of HSF1 expression which correlate with aggressive malignancy, interfered with the binding of Ku70-Ku80. This data suggests that HSF1 interacts with both Ku70 and Ku86 to induce defective NHEJ repair activity and genomic instability, which in turn suggests a novel mechanism of HSF1-mediated cellular carcinogenesis. PMID:26359349

  18. Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli.

    PubMed

    Bønsager, Birgit C; Praetorius-Ibba, Mette; Nielsen, Peter K; Svensson, Birte

    2003-08-01

    Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively. PMID:12880767

  19. Epiderstatin, a new inhibitor of the mitogenic activity induced by epidermal growth factor. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Osada, H; Sonoda, T; Kusakabe, H; Isono, K

    1989-11-01

    Inhibitors of mitogenic activity induced by epidermal growth factor (EGF) were screened from culture broths of soil microorganisms. A strain of actinomycetes has been found to produce a new glutarimide antibiotic named epiderstatin which inhibits the incorporation of [3H]thymidine into quiescent animal cells stimulated by EGF. Taxonomic studies have revealed that the producing strain belongs to a subspecies of Streptomyces pulveraceus, thus the name, Streptomyces pulveraceus subsp. epiderstagenes was given to this strain. The molecular formula (C15H20N2O4) and UV profile (lambda max 295 nm) of the antibiotic are distinct from other known antibiotics. It inhibited the incorporation of [3H]thymidine into quiescent cells stronger than into growing cells. PMID:2584144

  20. Hybrids from 4-anilinoquinazoline and hydroxamic acid as dual inhibitors of vascular endothelial growth factor receptor-2 and histone deacetylase.

    PubMed

    Peng, Fan-Wei; Wu, Ting-Ting; Ren, Zi-Wei; Xue, Jia-Yu; Shi, Lei

    2015-11-15

    A series of hybrids derived from 4-anilinoquinazoline and hydroxamic acid were designed, synthesized, and evaluated as dual inhibitors of vascular endothelia growth factor receptor-2 (VEGFR-2) tyrosine kinase and histone deacetylase (HDAC). Most of these compounds exhibited potent HDAC inhibition and moderate VEGFR-2 inhibition. Among them, compound 6l exhibited the most potent inhibitory activities against VEGFR-2 (IC50=84 nM) and HDAC (IC50=2.8 nM). It also showed the most potent antiproliferative ability against MCF-7, a human breast cancer line, with IC50 of 1.2 μM. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction of compound 6l at the active binding sites of VEGFR-2 and HDAC. PMID:26475519

  1. Hepatitis B and C reactivation with tumor necrosis factor inhibitors: synopsis and interpretation of screening and prophylaxis recommendations.

    PubMed

    Fuchs, Inbal; Abu-Shakra, Mahmoud; Sikuler, Emanuel

    2013-06-01

    Information on reactivation of chronic viral hepatitis infection in patients who are candidates for tumor necrosis factor alpha inhibitors (TNFi) is in a constant state of flux. We retrieved the most updated guidelines (in English) of prominent rheumatological and gastroenterological professional socienties for the mangement of chronic hepatitis B (HBV) and hepatitis C virus (HCV) infection in the context of treatment with TNFi. Subsequently, the major areas of uncertainty and absence of consensus in the guidelines were located and a secondary search for additional studies addressing those areas was performed. Based on our search we formulated a personal interpretation applicable to health care settings with virological laboratories capable of performing viral load measurements, and health systems that can support use of potent nucleoside/tide analogues in well-defined patient populations. PMID:23882898

  2. Data mining PubChem using a support vector machine with the Signature molecular descriptor: classification of factor XIa inhibitors.

    PubMed

    Weis, Derick C; Visco, Donald P; Faulon, Jean-Loup

    2008-11-01

    The amount of high-throughput screening (HTS) data readily available has significantly increased because of the PubChem project (http://pubchem.ncbi.nlm.nih.gov/). There is considerable opportunity for data mining of small molecules for a variety of biological systems using cheminformatic tools and the resources available through PubChem. In this work, we trained a support vector machine (SVM) classifier using the Signature molecular descriptor on factor XIa inhibitor HTS data. The optimal number of Signatures was selected by implementing a feature selection algorithm of highly correlated clusters. Our method included an improvement that allowed clusters to work together for accuracy improvement, where previous methods have scored clusters on an individual basis. The resulting model had a 10-fold cross-validation accuracy of 89%, and additional validation was provided by two independent test sets. We applied the SVM to rapidly predict activity for approximately 12 million compounds also deposited in PubChem. Confidence in these predictions was assessed by considering the number of Signatures within the training set range for a given compound, defined as the overlap metric. To further evaluate compounds identified as active by the SVM, docking studies were performed using AutoDock. A focused database of compounds predicted to be active was obtained with several of the compounds appreciably dissimilar to those used in training the SVM. This focused database is suitable for further study. The data mining technique presented here is not specific to factor XIa inhibitors, and could be applied to other bioassays in PubChem where one is looking to expand the search for small molecules as chemical probes. PMID:18829357

  3. Desensitization and immune tolerance induction in children with severe factor IX deficiency; inhibitors and adverse reactions to replacement therapy: a case-report and literature review.

    PubMed

    Bon, Andrea; Morfini, Massimo; Dini, Alessandro; Mori, Francesca; Barni, Simona; Gianluca, Sottilotta; de Martino, Maurizio; Novembre, Elio

    2015-01-01

    Hemophilia B is a rare X-linked recessive disorder with plasma factor IX (FIX) deficiency. 1-3% of patients treated with exogenous FIX-containing products develop inhibitors (i.e. polyclonal high affinity immunoglobulins) that neutralize the procoagulant activity of a specific coagulation factor. Although the incidence of inhibitors in hemophilia B patients is low, most are "high titer" and frequently associated with the development of severe allergic or anaphylactic reactions. Immune tolerance induction as a strategy for inhibitor eradication was first described in 1984. Unfortunately, the overall reported success of immune tolerance induction in FIX deficiency with inhibitors is approximately 25-40%.We report the case of a 2-year-old boy with hemophilia B severe FIX deficiency (<1%), inhibitor antibodies to FIX development, and a history of adverse reactions to FIX infusions, who underwent a successful desensitization and immune tolerance induction with a daily FIX infusion. With this regimen the inhibitor titer decreased with effective bleeding prevention. PMID:25887512

  4. Thermodynamic and Kinetic Characterization of the Protein Z-dependent Protease Inhibitor (ZPI)-Protein Z Interaction Reveals an Unexpected Role for ZPI Lys-239*

    PubMed Central

    Huang, Xin; Zhou, Jian; Zhou, Aiwu; Olson, Steven T.

    2015-01-01

    The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), circulates in blood as a tight complex with its cofactor, protein Z (PZ), enabling it to function as a rapid inhibitor of membrane-associated factor Xa. Here, we show that N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled K239C ZPI is a sensitive, moderately perturbing reporter of the ZPI-PZ interaction and utilize the labeled ZPI to characterize in-depth the thermodynamics and kinetics of wild-type and variant ZPI-PZ interactions. NBD-labeled K239C ZPI bound PZ with ∼3 nm KD and ∼400% fluorescence enhancement at physiologic pH and ionic strength. The NBD-ZPI-PZ interaction was markedly sensitive to ionic strength and pH but minimally affected by temperature, consistent with the importance of charged interactions. NBD-ZPI-PZ affinity was reduced ∼5-fold by physiologic calcium levels to resemble NBD-ZPI affinity for γ-carboxyglutamic acid/EGF1-domainless PZ. Competitive binding studies with ZPI variants revealed that in addition to previously identified Asp-293 and Tyr-240 hot spot residues, Met-71, Asp-74, and Asp-238 made significant contributions to PZ binding, whereas Lys-239 antagonized binding. Rapid kinetic studies indicated a multistep binding mechanism with diffusion-limited association and slow complex dissociation. ZPI complexation with factor Xa or cleavage decreased ZPI-PZ affinity 2–7-fold by increasing the rate of PZ dissociation. A catalytic role for PZ was supported by the correlation between a decreased rate of PZ dissociation from the K239A ZPI-PZ complex and an impaired ability of PZ to catalyze the K239A ZPI-factor Xa reaction. Together, these results reveal the energetic basis of the ZPI-PZ interaction and suggest an important role for ZPI Lys-239 in PZ catalytic action. PMID:25713144

  5. Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.

    PubMed

    Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J Preben; Andreasen, Peter A; Jensen, Jan K

    2016-07-01

    Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). PMID:27189939

  6. Overexpression of bromodomain factor 3 in Trypanosoma cruzi (TcBDF3) affects differentiation of the parasite and protects it against bromodomain inhibitors.

    PubMed

    Alonso, Victoria Lucia; Ritagliati, Carla; Cribb, Pamela; Cricco, Julia Alejandra; Serra, Esteban Carlos

    2016-06-01

    The bromodomain is the only protein domain known to bind acetylated lysine. In the last few years many bromodomain inhibitors have been developed in order to treat diseases such as cancer caused by aberrant acetylation of lysine residues. We have previously characterized Trypanosoma cruzi bromodomain factor 3 (TcBDF3), a bromodomain with an atypical localization that binds acetylated α-tubulin. In the present work we show that parasites overexpressing TcBDF3 exhibit altered differentiation patterns and are less susceptible to treatment with bromodomain inhibitors. We also demonstrate that recombinant TcBDF3 is able to bind to these inhibitors in vitro in a concentration-dependant manner. In parallel, the overexpression of a mutated version of TcBDF3 negatively affects growth of epimastigotes. Recent results, including the ones presented here, suggest that bromodomain inhibitors can be conceived as a new type of anti-parasitic drug against trypanosomiasis. PMID:27007774

  7. The growth factor inhibitor suramin reduces apoptosis and cell aggregation in protein-free CHO cell batch cultures.

    PubMed

    Zanghi, J A; Renner, W A; Bailey, J E; Fussenegger, M

    2000-01-01

    We have previously shown that Chinese hamster ovary (CHO) cells capable of growing in medium free of exogenous proteins die by apoptosis during all stages of a batch culture (Zanghi et al., 1999). On the basis of the hypothesis that extracellular death factors might be important in apoptosis under these conditions, we examined the effect of the growth factor inhibitor and antitumor agent suramin on CHO cell growth and apoptosis in serum-free culture. Suramin protected against apoptosis during exponential growth, as indicated by the absence of DNA laddering and an increase in cell viability from roughly 70% to above 95%. Suramin also effectively dispersed cell aggregates so that single-cell suspension culture was possible. However, suramin did not protect against apoptosis during the death phase, in contrast to serum, suggesting that antiapoptotic factors in the serum remain to be discovered. The increased viable cell yield following suramin supplementation resulted in a 40% increase in product yield, based on results with cells expressing recombinant secreted alkaline phosphatase. Polysulfated compounds dextran sulfate and polyvinyl sulfate worked nearly as well as suramin in dispersing cell clumps and increasing viable cell yield, which implies that suramin's high sulfate group density may be responsible for its effects in cell culture. In addition, suramin was beneficial for long-term adaptation of CHO cells to protein-free media suspension culture, and the compound was synergistic with insulin in accelerating this adaptation time. PMID:10835230

  8. Investigation of Dose-Dependent Factors Limiting Oral Bioavailability: Case Study With the PI3K-δ Inhibitor.

    PubMed

    Chiang, Po-Chang; Sutherlin, Daniel; Pang, Jodie; Salphati, Laurent

    2016-06-01

    It is understood that a potential issue for drugs with poor aqueous solubility is low oral absorption. If oral exposure issues arise when working with a low solubility drug candidate, the common action is to rely on enabling formulations to solve the issue. However, this approach becomes troublesome in the pre-clinical setting where compound absorption, distribution, metabolism, excretion properties are suboptimal and more factors limiting bioavailability may be at play. A narrow focus on solubility enhancement without a full understanding of compound absorption, distribution, metabolism, excretion properties can produce data that cloak the actual phenomena driving exposure. Compound 1 is a potent and selective PI3Kdelta inhibitor with poor aqueous solubility. In a pharmacokinetic study on dogs, exposure was found to be less than dose-linear. Besides the solubility, further investigations were conducted to identify other factors limiting oral exposure. It was observed that these limiting factors are dose dependent. Results from modeling pharmacokinetic under low-dose conditions suggest that exposure is significantly limited by metabolism and no exposure improvements should be expected from enabled formulations. Furthermore, enabling formulations are expected to exert a beneficial influence at higher doses. An in vivo test was conducted in dogs to verify this phenomenon. PMID:27238480

  9. Is there a role for epidermal growth factor receptor tyrosine kinase inhibitors in epidermal growth factor receptor wild-type non-small cell lung cancer?

    PubMed Central

    Arriola, Edurne; Taus, Álvaro; Casadevall, David

    2015-01-01

    Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a world-wide annual incidence of around 1.3 million. The majority of patients are diagnosed with advanced disease and survival remains poor. However, relevant advances have occurred in recent years through the identification of biomarkers that predict for benefit of therapeutic agents. This is exemplified by the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors for the treatment of EGFR mutant patients. These drugs have also shown efficacy in unselected populations but this point remains controversial. Here we have reviewed the clinical data that demonstrate a small but consistent subgroup of EGFR wild-type patients with NSCLC that obtain a clinical benefit from these drugs. Moreover, we review the biological rationale that may explain this benefit observed in the clinical setting. PMID:26266101

  10. Recombinant activated factor VII in the treatment of bleeds and for the prevention of surgery-related bleeding in congenital haemophilia with inhibitors.

    PubMed

    Santagostino, Elena; Escobar, Miguel; Ozelo, Margareth; Solimeno, Luigi; Arkhammar, Per; Lee, Hye Youn; Rosu, Gabriela; Giangrande, Paul

    2015-06-01

    The availability of recombinant activated factor VII (rFVIIa, eptacog alfa activated) has greatly advanced the care of patients with haemophilia A or B who have developed inhibitors against the infused replacement factor. Recombinant FVIIa is licensed for the on-demand treatment of bleeding episodes and the prevention of bleeding in surgery or invasive procedures in patients with congenital haemophilia with inhibitors. This article attempts to review in detail the extensive evidence of rFVIIa in congenital haemophilia patients with inhibitors. Patients with acute bleeding episodes are best treated on demand at home, to achieve the short- and long-term benefits of rapid bleed control. Key prospective studies have shown that rFVIIa achieves consistently high efficacy rates in the management of acute (including joint) bleeds in inhibitor patients in the home treatment setting. Substantial post-approval data from key registries also support the on-demand efficacy profile of rFVIIa established by the prospective clinical trials. The availability of rFVIIa has allowed major surgery to become a reality for inhibitor patients. Studies in key surgery, including orthopaedic procedures, have found that rFVIIa provides consistently high efficacy rates. Importantly, the wealth of data does not raise any unexpected safety concerns surrounding rFVIIa use; this is likely because rFVIIa is a recombinant product with a localised mechanism of action at the site of vascular injury. In summary, rFVIIa is established as an effective and well-tolerated first-line treatment for on-demand bleeding control and bleed prevention during minor and major (including elective orthopaedic) surgery in inhibitor patients. Use of rFVIIa has been a major step towards narrowing the gap in outcomes between inhibitor patients and non-inhibitor patients. PMID:26073369

  11. Fibroblast growth factor receptor inhibitors as a cancer treatment: from a biologic rationale to medical perspectives.

    PubMed

    Dieci, Maria Vittoria; Arnedos, Monica; Andre, Fabrice; Soria, Jean Charles

    2013-03-01

    The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling pathway plays a fundamental role in many physiologic processes, including embryogenesis, adult tissue homeostasis, and wound healing, by orchestrating angiogenesis. Ligand-independent and ligand-dependent activation have been implicated in a broad range of human malignancies and promote cancer progression in tumors driven by FGF/FGFR oncogenic mutations or amplifications, tumor neoangiogenesis, and targeted treatment resistance, thereby supporting a strong rationale for anti-FGF/FGFR agent development. Efforts are being pursued to develop selective approaches for use against this pathway by optimizing the management of emerging, class-specific toxicity profiles and correctly designing clinical trials to address these different issues. PMID:23418312

  12. MX1013, a dipeptide caspase inhibitor with potent in vivo antiapoptotic activity

    PubMed Central

    Yang, Wu; Guastella, John; Huang, Jin-Cheng; Wang, Yan; Zhang, Li; Xue, Dong; Tran, Minhtam; Woodward, Richard; Kasibhatla, Shailaja; Tseng, Ben; Drewe, John; Cai, Sui Xiong

    2003-01-01

    Caspases play a critical role in apoptosis, and are considered to be key targets for the design of cytoprotective drugs. As part of our antiapoptotic drug-discovery effort, we have synthesized and characterized Z-VD-fmk, MX1013, as a potent, irreversible dipeptide caspase inhibitor. MX1013 inhibits caspases 1, 3, 6, 7, 8, and 9, with IC50 values ranging from 5 to 20 nM. MX1013 is selective for caspases, and is a poor inhibitor of noncaspase proteases, such as cathepsin B, calpain I, or Factor Xa (IC50 values >10 μM). In several cell culture models of apoptosis, including caspase 3 processing, PARP cleavage, and DNA fragmentation, MX1013 is more active than tetrapeptide- and tripeptide-based caspase inhibitors, and blocked apoptosis at concentrations as low as 0.5 μM. MX1013 is more aqueous soluble than tripeptide-based caspase inhibitors such as Z-VAD-fmk. At a dose of 1 mg kg−1 i.v., MX1013 prevented liver damage and the lethality caused by Fas death receptor activation in the anti-Fas mouse-liver apoptosis model, a widely used model of liver failure. At a dose of 20 mg kg−1 (i.v. bolus) followed by i.v. infusion for 6 or 12 h, MX1013 reduced cortical damage by approximately 50% in a model of brain ischemia/reperfusion injury. At a dose of 20 mg kg−1 (i.v. bolus) followed by i.v. infusion for 12 h, MX1013 reduced heart damage by approximately 50% in a model of acute myocardial infarction. Based on these studies, we conclude that MX1013, a dipeptide pan-caspase inhibitor, has a good combination of in vitro and in vivo properties. It has the ability to protect cells from a variety of apoptotic insults, and is systemically active in three animal models of apoptosis, including brain ischemia. PMID:12970077

  13. Axl mediates acquired resistance of head and neck cancer cells to the epidermal growth factor receptor inhibitor erlotinib.

    PubMed

    Giles, Keith M; Kalinowski, Felicity C; Candy, Patrick A; Epis, Michael R; Zhang, Priscilla M; Redfern, Andrew D; Stuart, Lisa M; Goodall, Gregory J; Leedman, Peter J

    2013-11-01

    Elevated expression and activity of the epidermal growth factor receptor (EGFR) is associated with development and progression of head and neck cancer (HNC) and a poor prognosis. Clinical trials with EGFR tyrosine kinase inhibitors (e.g., erlotinib) have been disappointing in HNC. To investigate the mechanisms mediating resistance to these agents, we developed an HNC cell line (HN5-ER) with acquired erlotinib resistance. In contrast to parental HN5 HNC cells, HN5-ER cells exhibited an epithelial-mesenchymal (EMT) phenotype with increased migratory potential, reduced E-cadherin and epithelial-associated microRNAs (miRNA), and elevated vimentin expression. Phosphorylated receptor tyrosine kinase profiling identified Axl activation in HN5-ER cells. Growth and migration of HN5-ER cells were blocked with a specific Axl inhibitor, R428, and R428 resensitized HN5-ER cells to erlotinib. Microarray analysis of HN5-ER cells confirmed the EMT phenotype associated with acquired erlotinib resistance, and identified activation of gene expression associated with cell migration and inflammation pathways. Moreover, increased expression and secretion of interleukin (IL)-6 and IL-8 in HN5-ER cells suggested a role for inflammatory cytokine signaling in EMT and erlotinib resistance. Expression of the tumor suppressor miR-34a was reduced in HN5-ER cells and increasing its expression abrogated Axl expression and reversed erlotinib resistance. Finally, analysis of 302 HNC patients revealed that high tumor Axl mRNA expression was associated with poorer survival (HR = 1.66, P = 0.007). In summary, our results identify Axl as a key mediator of acquired erlotinib resistance in HNC and suggest that therapeutic inhibition of Axl by small molecule drugs or specific miRNAs might overcome anti-EGFR therapy resistance. PMID:24026012

  14. Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation

    SciTech Connect

    Rancourt, Raymond C. Veress, Livia A. Ahmad, Aftab Hendry-Hofer, Tara B. Rioux, Jacqueline S. Garlick, Rhonda B. White, Carl W.

    2013-10-01

    Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. Methods: Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5 mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O{sub 2} saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin–antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. Results: Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. Conclusions: Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury. - Highlights: • TFPI administration to rats after mustard inhalation reduces airway cast formation. • Inhibition of thrombin activation is the likely mechanism for limiting casts. • Rats given TFPI

  15. Tissue expression of human complement inhibitor, decay-accelerating factor, in transgenic pigs. A potential approach for preventing xenograft rejection.

    PubMed

    Rosengard, A M; Cary, N R; Langford, G A; Tucker, A W; Wallwork, J; White, D J

    1995-05-15

    Since complement-mediated hyperacute rejection of xenografts prevents the use of pigs as organ donors to man, the development of transgenic animals expressing species-specific complement inhibitors could provide a strategy for overcoming hyperacute rejection. The complement inhibitor, human decay-accelerating factor (hDAF), prevents the assembly of C3 and C5 convertases. In this article, the first histologic analysis of hDAF expression in pig tissues, specifically expression in endothelial cells of pigs transgenic for hDAF, is described. Twenty-seven transgenic pigs were categorized into 4 groups based on the expression patterns in endothelial, vascular smooth muscle, and squamous epithelial cells of skin biopsy specimens. Skin biopsy specimens permitted evaluation of the pigs without the need to kill them or to perform invasive procedures. Sixteen cases demonstrated endothelial cell staining. Complete necropsy evaluation, available in 14 of the 27 pigs, correlated with the skin biopsy specimen expression of hDAF. The immunoperoxidase data matched identically with the presence of the mRNA transcript in 25 of the 26 cases where RNA data were available. Also, the staining patterns of 6 transgenic pig founders and their 9 offspring (total of 9 founder-offspring pairs) correlated. Since transgenes are variably expressed in different cell types and since tissue lysates represent a melange of cell types, histologic evaluation for protein expression in tissues from transgenic animals will be critical if they are to be bred to become clinical organ donors. In addition to endothelial expression of hDAF, its expression on vascular smooth muscle cells may be important in preventing tissue damage when breaks in the endothelium occur. PMID:7539168

  16. The antiproliferative activity of kinase inhibitors in chronic myeloid leukemia cells is mediated by FOXO transcription factors.

    PubMed

    Pellicano, Francesca; Scott, Mary T; Helgason, G Vignir; Hopcroft, Lisa E M; Allan, Elaine K; Aspinall-O'Dea, Mark; Copland, Mhairi; Pierce, Andrew; Huntly, Brian J P; Whetton, Anthony D; Holyoake, Tessa L

    2014-09-01

    Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network. PMID:24806995

  17. A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

    PubMed Central

    van der Schaar, Hilde M.; Leyssen, Pieter; Thibaut, Hendrik J.; de Palma, Armando; van der Linden, Lonneke; Lanke, Kjerstin H. W.; Lacroix, Céline; Verbeken, Erik; Conrath, Katja; MacLeod, Angus M.; Mitchell, Dale R.; Palmer, Nicholas J.; van de Poël, Hervé; Andrews, Martin

    2013-01-01

    Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIβ in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIβ carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model. PMID:23896472

  18. Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation

    PubMed Central

    Rancourt, Raymond C.; Veress, Livia A.; Ahmad, Aftab; Hendry-Hofer, Tara B.; Rioux, Jacqueline S.; Garlick, Rhonda B.; White, Carl W.

    2013-01-01

    Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. Methods Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5 mg/kg) in vehicle, or vehicle alone, were instilled into the trachea. Arterial O2 saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma analyzed for prothrombin, thrombin-antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. Results Airway obstruction in the form of fibrin-containing casts were evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. Conclusions Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury. PMID:23727623

  19. Drug Repurposing Approach Identifies Inhibitors of the Prototypic Viral Transcription Factor IE2 that Block Human Cytomegalovirus Replication.

    PubMed

    Mercorelli, Beatrice; Luganini, Anna; Nannetti, Giulio; Tabarrini, Oriana; Palù, Giorgio; Gribaudo, Giorgio; Loregian, Arianna

    2016-03-17

    New targets for antiviral strategies are needed against human cytomegalovirus (HCMV), a major human pathogen. A cell-based screen aimed at finding inhibitors of the viral transcription factor Immediate-Early 2 (IE2) was performed in HCMV-infected cells expressing EGFP under the control of an IE2-inducible viral promoter. Screening of a library of bioactive small molecules led to the identification of several compounds able to inhibit EGFP expression and also HCMV replication with potency in the low-micromolar range. Follow-up studies with four selected hits indicated that they all block viral DNA synthesis as well as viral Early and Late gene expression. Furthermore, mechanistic studies confirmed that the compounds specifically act via inhibition of IE2 transactivating activity, thus blocking viral Early gene expression and the progression of virus replication. These results provide proof of concept for identifying small molecules that modulate the activity of a microbial transcription factor to control pathogen replication. PMID:26877023

  20. Trps1 activates a network of secreted Wnt inhibitors and transcription factors crucial to vibrissa follicle morphogenesis

    PubMed Central

    Fantauzzo, Katherine A.; Christiano, Angela M.

    2012-01-01

    Mutations in TRPS1 cause trichorhinophalangeal syndrome types I and III, which are characterized by sparse scalp hair in addition to craniofacial and skeletal abnormalities. Trps1 is a vertebrate transcription factor that contains nine zinc-finger domains, including a GATA-type zinc finger through which it binds DNA. Mice in which the GATA domain of Trps1 has been deleted (Trps1Δgt/Δgt) have a reduced number of pelage follicles and lack vibrissae follicles postnatally. To identify the transcriptional targets of Trps1 in the developing vibrissa follicle, we performed microarray hybridization analysis, comparing expression patterns in the whisker pads of wild-type versus Trps1Δgt/Δgt embryos. We identified a number of transcription factors and Wnt inhibitors among transcripts downregulated in the mutant embryos and several extracellular matrix proteins that were upregulated in the mutant samples, and demonstrated that target gene expression levels were altered in vivo in Trps1Δgt/Δgt vibrissae. Unexpectedly, we discovered that Trps1 can directly bind the promoters of its target genes to activate transcription, expanding upon its established role as a transcriptional repressor. Our findings identify Trps1 as a novel regulator of the Wnt signaling pathway and of early hair follicle progenitors in the developing vibrissa follicle. PMID:22115758

  1. Response of protein C and protein C inhibitor to warfarin therapy in patient with combined deficiency of Factors V and VIII.

    PubMed

    Bern, M M; Suzuki, K; Mann, K; Tracy, P; Hoyer, L; Jensen, W; Gallivan, M; Arkin, C; Davis, G

    1984-12-15

    The role of Protein C in combined factor V/VIII deficiency was examined by reducing the Protein C concentration using warfarin therapy in a patient with the combined deficiency. The factor VIII deficiency was like Hemophilia-A, with deficiency of VIII:C and VIII:C(Ag), but normal VIIIR:Ag and VIIIR:cof. The factor V deficiency was due to loss of the V antigen. During warfarin therapy the Protein C level was reduced, but concentrations of factors V and VIII did not change. Protein C Inhibitor was normal throughout. Thus combined factor V/VIII deficiency is not related to Protein C levels. PMID:6098970

  2. Virtual Screening of Specific Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors from the National Cancer Institute (NCI) Molecular Database

    PubMed Central

    Fan, Cong; Huang, Yan-Xin; Bao, Yong-Li; Sun, Lu-Guo; Wu, Yin; Yu, Chun-Lei; Zhang, Yu; Song, Zhen-Bo; Zheng, Li-Hua; Sun, Ying; Wang, Guan-Nan; Li, Yu-Xin

    2012-01-01

    Insulin-like growth factor 1 receptor (IGF1R) is an attractive drug target for cancer therapy and research on IGF1R inhibitors has had success in clinical trials. A particular challenge in the development of specific IGF1R inhibitors is interference from insulin receptor (IR), which has a nearly identical sequence. A few potent inhibitors that are selective for IGF1R have been discovered experimentally with the aid of computational methods. However, studies on the rapid identification of IGF1R-selective inhibitors using virtual screening and confidence-level inspections of ligands that show different interactions with IGF1R and IR in docking analysis are rare. In this study, we established virtual screening and binding-mode prediction workflows based on benchmark results of IGF1R and several kinase receptors with IGF1R-like structures. We used comprehensive analysis of the known complexes of IGF1R and IR with their binding ligands to screen specific IGF1R inhibitors. Using these workflows, 17 of 139,735 compounds in the NCI (National Cancer Institute) database were identified as potential specific inhibitors of IGF1R. Calculations of the potential of mean force (PMF) with GROMACS were further conducted for three of the identified compounds to assess their binding affinity differences towards IGF1R and IR. PMID:23242155

  3. Platelet derived growth factor inhibitors: A potential therapeutic approach for ocular neovascularization.

    PubMed

    Sadiq, Mohammad Ali; Hanout, Mostafa; Sarwar, Salman; Hassan, Muhammad; Do, Diana V; Nguyen, Quan Dong; Sepah, Yasir Jamal

    2015-01-01

    Retinochoroidal vascular diseases are the leading causes of blindness in the developed world. They include diabetic retinopathy (DR), retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration (AMD), and pathological myopia, among many others. Several different therapies are currently under consideration for the aforementioned disorders. In the following section, agents targeting platelet-derived growth factor (PDGF) are discussed as a potential therapeutic option for retinochoroidal vascular diseases. PDGF plays an important role in the angiogenesis cascade that is activated in retinochoroidal vascular diseases. The mechanism of action, side effects, efficacy, and the potential synergistic role of these agents in combination with other treatment options is discussed. The future of treatment of retinochoroidal vascular diseases, particularly AMD, has become more exciting due to agents such as PDGF antagonists. PMID:26586980

  4. Platelet-Derived Growth Factor Inhibitors: A Potential Therapeutic Approach for Ocular Neovascularization.

    PubMed

    Sadiq, Mohammad Ali; Hanout, Mostafa; Sarwar, Salman; Hassan, Muhammad; Agarwal, Aniruddha; Sepah, Yasir Jamal; Do, Diana V; Nguyen, Quan Dong

    2016-01-01

    Retinochoroidal vascular diseases are the leading causes of blindness in the developed world. They include diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration (AMD), and pathological myopia, among many others. Several different therapies are currently under consideration for the aforementioned disorders. In the following section, agents targeting platelet-derived growth factors (PDGF) are discussed as a potential therapeutic option for retinochoroidal vascular diseases. PDGF play an important role in the angiogenesis cascade that is activated in retinochoroidal vascular diseases. The mechanism of action, side effects, efficacy, and the potential synergistic role of these agents in combination with other treatment options is discussed. The future of treatment of retinochoroidal vascular diseases, particularly neovascular AMD, has become more exciting due to agents like PDGF antagonists. PMID:26501397

  5. Platelet derived growth factor inhibitors: A potential therapeutic approach for ocular neovascularization

    PubMed Central

    Sadiq, Mohammad Ali; Hanout, Mostafa; Sarwar, Salman; Hassan, Muhammad; Do, Diana V.; Nguyen, Quan Dong; Sepah, Yasir Jamal

    2015-01-01

    Retinochoroidal vascular diseases are the leading causes of blindness in the developed world. They include diabetic retinopathy (DR), retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration (AMD), and pathological myopia, among many others. Several different therapies are currently under consideration for the aforementioned disorders. In the following section, agents targeting platelet-derived growth factor (PDGF) are discussed as a potential therapeutic option for retinochoroidal vascular diseases. PDGF plays an important role in the angiogenesis cascade that is activated in retinochoroidal vascular diseases. The mechanism of action, side effects, efficacy, and the potential synergistic role of these agents in combination with other treatment options is discussed. The future of treatment of retinochoroidal vascular diseases, particularly AMD, has become more exciting due to agents such as PDGF antagonists. PMID:26586980

  6. CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis

    PubMed Central

    Citro, Alessandra; Scrivo, Rossana; Martini, Helene; Martire, Carmela; De Marzio, Paolo; Vestri, Anna Rita; Sidney, John; Sette, Alessandro; Barnaba, Vincenzo; Valesini, Guido

    2015-01-01

    CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes) represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factorinhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control). The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factorinhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factorinhibitor therapy to validate in a larger cohort of patients. PMID:26061065

  7. Risk Factors for Nursing Home Placement in Alzheimer's Disease: A Longitudinal Study of Cognition, ADL, Service Utilization, and Cholinesterase Inhibitor Treatment

    ERIC Educational Resources Information Center

    Wattmo, Carina; Wallin, Asa K.; Londos, Elisabet; Minthon, Lennart

    2011-01-01

    Purpose of the Study: To identify risk factors for early nursing home placement (NHP) in Alzheimer's disease (AD), focusing on the impact of longitudinal change in cognition, activities of daily living (ADL), service utilization, and cholinesterase inhibitor treatment (ChEI). Design and Methods: In an open, 3-year, prospective, multicenter study…

  8. Activation of factor X by rat hepatocytes

    SciTech Connect

    Willingham, A.K.; Matschiner, J.T.

    1986-05-01

    Synthesis and secretion of blood coagulation factor X was studied in hepatocytes prepared by perfusion of rat livers with collagenase. Hepatocytes were incubated in the presence of vitamin K and /sup 3/H-leucine for up to 4h at 37/sup 0/C. Factor X was isolated from the incubation medium by immunochemical techniques and analyzed by SDS-PAGE. The recovered /sup 3/H-labeled proteins migrated, after reduction of disulfides, as two polypeptide chains with apparent molecular weights (M/sub r/) of approximately 42,000 and 22,000 representing the heavy and light chains of factor X respectively. The apparent M/sub r/ of the heavy chain was about 10,000 daltons lighter than seen with the heavy chain of factor X isolated from rat plasma and was more characteristic of the heavy chain of factor Xa. When the levels of factor X secreted by hepatocytes were determined by clotting assays, activity was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes (>95% parenchymal cells) the added factor X was rapidly converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium dependent reaction. The physiological significance of a factor X activating enzyme on hepatocyte plasma membranes is not clear.

  9. Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    SciTech Connect

    VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom,Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor,Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.

    2005-09-01

    The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.

  10. Mechanisms of resistance to anti-epidermal growth factor receptor inhibitors in metastatic colorectal cancer.

    PubMed

    Sforza, Vincenzo; Martinelli, Erika; Ciardiello, Fortunato; Gambardella, Valentina; Napolitano, Stefania; Martini, Giulia; Della Corte, Carminia; Cardone, Claudia; Ferrara, Marianna L; Reginelli, Alfonso; Liguori, Giuseppina; Belli, Giulio; Troiani, Teresa

    2016-07-28

    The prognosis of patients with metastatic colorectal cancer (mCRC) remain poor despite the impressive improvement of treatments observed over the last 20 years that led to an increase in median overall survival from 6 mo, with the only best supportive care, to approximately 30 mo with the introduction of active chemotherapy drugs and targeted agents. The monoclonal antibodies (moAbs) cetuximab and panitumumab, directed against the epidermal growth factor receptor (EGFR), undoubtedly represent a major step forward in the treatment of mCRC, given the relevant efficacy in terms of progression-free survival, overall survival, response rate, and quality of life observed in several phase III clinical trials among different lines of treatment. However, the anti-EGFR moAbs were shown only to be effective in a subset of patients. For instance, KRAS and NRAS mutations have been identified as biomarkers of resistance to these drugs, improving the selection of patients who might derive a benefit from these treatments. Nevertheless, several other alterations might affect the response to these drugs, and unfortunately, even the responders eventually become resistant by developing secondary (or acquired) resistance in approximately 13-18 mo. Several studies highlighted that the landscape of responsible alterations of both primary and acquired resistance to anti-EGFR drugs biochemically converge into MEK-ERK and PIK3CA-AKT pathways. In this review, we describe the currently known mechanisms of primary and acquired resistance to anti-EGFR moAbs together with the various strategies evaluated to prevent, overcame or revert them. PMID:27605871

  11. Eugenol: a dual inhibitor of platelet-activating factor and arachidonic acid metabolism.

    PubMed

    Saeed, S A; Simjee, R U; Shamim, G; Gilani, A H

    1995-07-01

    Eugenol is an active principal and responsible for several pharmacological activities of clove oil. We studied the effects of eugenol on human platelet aggregation, arachidonic acid (AA) and platelet-activating factor (PAF) metabolism and in vivo effects on AA and PAF-induced shock in rabbits. Eugenol strongly inhibited PAF-induced platelet aggregation with lesser effect against AA and collegen. The IC(50) values were against AA: 31 ± 0.5; collagen: 64 ± 0.7 and PAF 7 ± 0.2 μM (n=9) respectively. In addition, eugenol stimulated PAF-acetylhydrolase activity suggesting that inhibition of PAF could be due to its inactivation to lyso-PAF. Pretreatment of rabbits with eugenol (50-100 mg/kg) prevented the lethal effects of intravenous PAF (11 μgg/kg) or AA (2 mg/kg) in a dose-dependent fashion. The protective effects of eugenol in the rabbits, however, were more pronounced against PAF-induced mortality (100% protection). In addition, eugenol also inhibited AA metabolism via cyclooxygenase and lipoxygenase pathways in human platelets. Both the production of thromboxane-A(2) and 12-hydroxy-eicosatetraenoic acid was inhibited by eugenol in a concentration-related manner (30-120 μM). In vivo, eugenol (50-100 mg/kg; i.p.) inhibited carrageenan-induced rat paw oedema (P < 0.001). In this test, eugenol was 5 times more potent than aspirin. These results provide evidence that eugenol acts as a dual antagonist of AA and PAF. PMID:23196096

  12. Uveitis Reactivation in Children Treated with Tumor Necrosis FactorInhibitors

    PubMed Central

    Lerman, Melissa A.; Lewen, Michael D.; Kempen, John H.; Mills, Monte D.

    2016-01-01

    PURPOSE To evaluate reactivation of pediatric uveitis during/following treatment with TNF-alpha inhibition (anti-TNFα). DESIGN Retrospective cohort study. METHODS We assessed the incidence of uveitis reactivation in children ≤18 years who had achieved uveitis quiescence under anti-TNFα. Survival analysis was used to calculate reactivation rates while still on (primary outcome), and following discontinuation of (secondary outcome), anti-TNFα. Potential predictive factors were assessed. RESULTS Among 50 children observed to develop quiescence of uveitis under anti-TNFα, 39 met criteria to be “at risk” of the primary (19 for the secondary) outcome. 60% were female, ~half had Juvenile Idiopathic Arthritis, and most were treated with infliximab. Overall, the estimated proportion relapsing within 12 months was 27.8% (95% confidence interval [CI]: 15.9-45.8%); the estimated probability of reactivation was higher following (63.8% [95% CI: 38.9-87.7%]), than before (21.6% [95% CI: 10.8-40.2%]), anti-TNFα discontinuation. Amongst those who discontinued anti-TNFα, the likelihood of reactivation was higher for those treated with adalimumab vs. infliximab (Hazard Ratio [HR] 13.4, p=0.01, 95% CI: 2.2-82.5) and those with older age at uveitis-onset (HR 1.3, p=0.09, 95% CI: 1.0-1.7). The duration of suppression, on medication, did not significantly affect the likelihood of reactivation when quiescence was maintained for ≥1.5 years. CONCLUSIONS Approximately 75% of children remaining on anti-TNFα following achievement of uveitis quiescence remain quiescent at one year. However, most reactivate following anti-TNFα discontinuation. These results suggest that infliximab more often is followed by remission, off medication, than adalimumab. The data do not suggest that maintenance of suppression, for more than 1.5 years decreases the reactivation risk. PMID:25892124

  13. Mechanisms of resistance to anti-epidermal growth factor receptor inhibitors in metastatic colorectal cancer

    PubMed Central

    Sforza, Vincenzo; Martinelli, Erika; Ciardiello, Fortunato; Gambardella, Valentina; Napolitano, Stefania; Martini, Giulia; della Corte, Carminia; Cardone, Claudia; Ferrara, Marianna L; Reginelli, Alfonso; Liguori, Giuseppina; Belli, Giulio; Troiani, Teresa

    2016-01-01

    The prognosis of patients with metastatic colorectal cancer (mCRC) remain poor despite the impressive improvement of treatments observed over the last 20 years that led to an increase in median overall survival from 6 mo, with the only best supportive care, to approximately 30 mo with the introduction of active chemotherapy drugs and targeted agents. The monoclonal antibodies (moAbs) cetuximab and panitumumab, directed against the epidermal growth factor receptor (EGFR), undoubtedly represent a major step forward in the treatment of mCRC, given the relevant efficacy in terms of progression-free survival, overall survival, response rate, and quality of life observed in several phase III clinical trials among different lines of treatment. However, the anti-EGFR moAbs were shown only to be effective in a subset of patients. For instance, KRAS and NRAS mutations have been identified as biomarkers of resistance to these drugs, improving the selection of patients who might derive a benefit from these treatments. Nevertheless, several other alterations might affect the response to these drugs, and unfortunately, even the responders eventually become resistant by developing secondary (or acquired) resistance in approximately 13-18 mo. Several studies highlighted that the landscape of responsible alterations of both primary and acquired resistance to anti-EGFR drugs biochemically converge into MEK-ERK and PIK3CA-AKT pathways. In this review, we describe the currently known mechanisms of primary and acquired resistance to anti-EGFR moAbs together with the various strategies evaluated to prevent, overcame or revert them. PMID:27605871

  14. Systematic Review of Tumor Necrosis Factor Inhibitor Discontinuation Studies in Rheumatoid Arthritis

    PubMed Central

    Navarro-Millán, Iris; Sattui, Sebastian E.; Curtis, Jeffrey R

    2013-01-01

    Background Anti-tumor necrosis factor agents (anti-TNFs) have changed the course of rheumatoid arthritis (RA) for more than a decade. Use of these medications often results in remission, or at least low disease activity (LDA), but at substantial cost. It has been postulated that discontinuation of these medications among RA patients in remission or LDA may be possible without an associated increase in RA disease activity. Objective The goal of this systematic literature review is to summarize published articles regarding discontinuation of anti-TNFs in patients with RA. Methods A systematic literature review was conducted to identify English-language articles indexed in Pubmed from July 1999 through June 2013 reporting results regarding anti-TNF discontinuation in patients with RA. Study designs included observational longitudinal studies and clinical trials. Outcomes had to include one of the following: time to flare after anti-TNF discontinuation, failure to remain in remission, or LDA at the end of the study. Results Ten studies examined discontinuation of anti-TNF therapies in RA. Inclusion criteria varied significantly across studies in terms of disease activity status (remission or LDA) and duration of this disease status (1 year or 1 month) prior to discontinuation being attempted. Results from larger studies (e.g. > 100 patients) suggest that the proportion of patients who discontinued and did not have an increase in disease activity ranged between 24%-81%. In 2 studies that evaluated durability of LDA or remission after anti-TNF discontinuation, the mean time to relapse varied from 15 weeks to 14 months. In studies that analyzed radiographic data, once therapies were reinitiated after an increase in disease activity was detected, patients generally did not experience progression in structural damage. Conclusion Discontinuation of anti-TNF therapy is achievable for many RA patients who start in clinical remission or LDA. However, heterogeneous inclusion

  15. Factors that predict progression-free survival in Chinese lung adenocarcinoma patients treated with epidermal growth factor receptor tyrosine kinase inhibitors

    PubMed Central

    Cui, Shaohua; Xiong, Liwen; Lou, Yuqing; Shi, Huangping; Gu, Aiqin; Zhao, Yizhuo; Chu, Tianqing; Wang, Huimin; Zhang, Wei; Dong, Lili

    2016-01-01

    Background Although first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have shown efficacy in patients with advanced lung cancers, survival predictors with these drugs have not been extensively investigated. This study was performed to explore factors that may predict progression-free survival (PFS) in Chinese lung adenocarcinoma patients treated with EGFR-TKIs. Methods We retrospectively collected clinicopathologic data on 208 patients who received either gefitinib, erlotinib or icotinib, including the patients’ EGFR mutation status and levels of six serum tumor markers [carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA125), squamous cell carcinoma antigen (SCC), cytokeratin-19 fragments (CYFRA21-1) and lactate dehydrogenase (LDH)]. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with PFS. Results At the study cutoff date, 189 (90.9%) of the patients met the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 criteria for progressive disease (PD), while 19 (9.1%) had stable disease (SD). The median PFS of the 208 patients was 12.4 months (95% CI, 11.0–13.8 months). In the multivariate analysis using a Cox proportional hazard model, a non-smoking history [hazard ratio (HR) =2.460; 95% CI, 1.484–4.079; P<0.001], first-line treatment (HR =1.500; 95% CI, 1.062–2.119; P=0.021), and a high pretreatment serum level of CEA (HR =1.424; 95% CI 1.026–1.977; P=0.035) were found to be significant predictors of a longer PFS. Conclusions In Chinese lung adenocarcinoma patients treated with EGFR-TKIs, a non-smoking history, first-line EGFR-TKIs treatment and a high serum level of CEA were independent predictors of a longer PFS along with an EGFR-activating mutation. PMID:26904214

  16. The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats

    PubMed Central

    Krupka, Jennifer; May, Frauke; Weimer, Thomas; Pragst, Ingo; Kleinschnitz, Christoph; Stoll, Guido; Panousis, Con; Dickneite, Gerhard; Nolte, Marc W.

    2016-01-01

    Background and Purpose Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. Methods For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. Results Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. Conclusions With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury. PMID:26815580

  17. Novel hydrazone moiety-bearing aminopyrimidines as selective inhibitors of epidermal growth factor receptor T790M mutant.

    PubMed

    Qin, Mingze; Wang, Tingting; Xu, Boxuan; Ma, Zonghui; Jiang, Nan; Xie, Hongbo; Gong, Ping; Zhao, Yanfang

    2015-11-01

    The epidermal growth factor receptor (EGFR) T790M mutant is found in approximately 50% of clinically acquired resistance to gefitinib among patients with non-small cell lung cancer (NSCLC). Here, a series of novel aminopyrimidines bearing a hydrazone moiety were identified as potent and selective EGFR inhibitors. Compounds 14a, 15g, and 15i potently inhibited all EGFR mutants including EGFR T790M/L858R, EGFR T790M/delE746_A750, and EGFR T790M while they showed weak effects on the wild type (WT) EGFR. In addition, these compounds effectively suppressed proliferation of gefitinib-resistant H1975 (EGFR T790M/L858R) cells but were less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index. Therefore, 14a, 15g, and 15i might be promising candidates to overcome drug resistance mediated by the EGFR T790M mutant. PMID:26451770

  18. A novel fibroblast growth factor receptor 1 inhibitor protects against cartilage degradation in a murine model of osteoarthritis

    PubMed Central

    Xu, Wei; Xie, Yangli; Wang, Quan; Wang, Xiaofeng; Luo, Fengtao; Zhou, Siru; Wang, Zuqiang; Huang, Junlan; Tan, Qiaoyan; Jin, Min; Qi, Huabing; Tang, Junzhou; Chen, Liang; Du, Xiaolan; Zhao, Chengguang; Liang, Guang; Chen, Lin

    2016-01-01

    The attenuated degradation of articular cartilage by cartilage-specific deletion of fibroblast growth factor receptor 1 (FGFR1) in adult mice suggests that FGFR1 is a potential target for treating osteoarthritis (OA). The goal of the current study was to investigate the effect of a novel non-ATP-competitive FGFR1 inhibitor, G141, on the catabolic events in human articular chondrocytes and cartilage explants and on the progression of cartilage degradation in a murine model of OA. G141 was screened and identified via cell-free kinase-inhibition assay. In the in vitro study, G141 decreased the mRNA levels of catabolic markers ADAMTS-5 and MMP-13, the phosphorylation of Erk1/2, JNK and p38 MAPK, and the protein level of MMP-13 in human articular chondrocytes. In the ex vivo study, proteoglycan loss was markedly reduced in G141 treated human cartilage explants. For the in vivo study, intra-articular injection of G141 attenuated the surgical destabilization of the medial meniscus (DMM) induced cartilage destruction and chondrocyte hypertrophy and apoptosis in mice. Our data suggest that pharmacologically antagonize FGFR1 using G141 protects articular cartilage from osteoarthritic changes, and intra-articular injection of G141 is potentially an effective therapy to alleviate OA progression. PMID:27041213

  19. Combining oral contraceptives with a natural nuclear factor-kappa B inhibitor for the treatment of endometriosis-related pain.

    PubMed

    Maia, Hugo; Haddad, Clarice; Casoy, Julio

    2013-01-01

    Endometriosis is a chronic disease in which a persistent state of heightened inflammation is maintained by nuclear factor-kappa B (NF-κB) activation. The progestins present in oral contraceptives are potent inhibitors of NF-κB translocation to cell nuclei, while Pycnogenol® (Pinus pinaster) acts by blocking post-translational events. In this study, the effects of Pycnogenol on pain scores were investigated in patients with endometriosis using oral contraceptives containing either gestodene or drospirenone in extended regimens. Pain scores were determined using a visual analog scale before and after 3 months of treatment. Oral contraceptives, used alone (groups 1 and 3) or in association with Pycnogenol (groups 2 and 4), resulted in significant decreases in pain scores after 3 months of treatment; however, this reduction was significantly greater in the groups using oral contraceptives + Pycnogenol (groups 2 and 4) compared with those using oral contraceptives alone (groups 1 and 3). In the groups using oral contraceptives alone, 50% of patients became pain-free by the end of the third month of treatment. These results suggest that Pycnogenol increases the efficacy of oral contraceptives for the treatment of endometriosis-related pain. PMID:24379702

  20. Cinnamide Derivatives of d‐Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa †

    PubMed Central

    Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Wagner, Stefanie; Audfray, Aymeric; Prestel, Andreas; Möller, Heiko M.; Imberty, Anne

    2015-01-01

    Abstract Pseudomonas aeruginosa is an opportunistic Gram‐negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate‐based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug‐like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d‐mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure–activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure‐based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half‐lives in the 5–20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti‐infectives against this multidrug‐resistant pathogen. PMID:27308201

  1. Protective Effects of the Nuclear Factor Kappa B Inhibitor Pyrrolidine Dithiocarbamate on Experimental Testicular Torsion and Detorsion Injury

    PubMed Central

    Ozden, Hilmi; Guven, Gul; Burukoglu, Dilek; Ustuner, Mehmet Cengiz; Topal, Fatma; Gunes, Hasan Veysi; Ustuner, Derya; Ozbayer, Cansu

    2014-01-01

    Testicular torsion results with the damage of the testis and it is a surgical emergency. Pyrrolidine dithiocarbamate (PDTC) is a low-molecular-weight antioxidant and potent inhibitor of nuclear factor kappa B (NF-κB) activation. In this study, we aimed to investigate the effects of PDTC to testicular torsion-detorsion (T/D) injury. Forty adult male Sprague-Dawley rats were separated into four groups. A sham operation was performed in group I. In group II, torsion is performed 2 hours by 720 degree extravaginally testis. In group III, 4 h reperfusion of the testis was performed after 2 h of testicular torsion. In group IV, after performing the same surgical procedures as in group III, PDTC (100 mg/kg, intravenous's) was administered before 30 min of detorsion. The testes tissue malondialdehyde (MDA), superoxide dismutase (SOD) catalase (CAT) level was evaluated. Histological evaluations were performed after hematoxylin and eosin staining. Testicular tissue MDA levels were the highest in the T/D groups compared with treatment group. Administration of PDTC prevented a further increase in MDA levels. Significant decrease occurred in CAT and SOD levels in treatment group compared with the control group. The rats in the treatment group had normal testicular architecture. The results suggest that PDTC can be a potential protective agent for preventing the biochemical and histological changes related to oxidative stress in testicular injury caused by testis torsion. PMID:25177164

  2. Management of hyperglycemia from epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) targeting T790M-mediated resistance

    PubMed Central

    Ersek, Jennifer L.; Fong, Mei Ka; Sirianno, Lindsey; Story, Ellen S.

    2015-01-01

    Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to small molecule tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and afatinib. Although studies show an increased progression free survival (PFS) with use of EGFR TKIs in the first-line setting, most patients will develop resistance to therapy after the first 8-16 months. T790M is an acquired resistance mutation reported in 60-70% of patients who initially responded to a prior EGFR TKI. Recently, EGFR TKIs targeting T790M have been developed to overcome resistance with positive results in PFS and objective response rate in patients who have had disease progression on at least one TKI. Two EGFR TKIs targeting T790M, AZD9291 and rociletinib, are new active treatment options for NSCLC but differ in adverse effect profiles. Dose-limiting hyperglycemia has been reported with rociletinib and has required dose reduction, an oral antihyperglycemic, or both, without discontinuation of therapy. This suggests that patients may be effectively treated chronically for hyperglycemia associated with EGFR TKIs targeting T790M, however, guidelines for treatment of hyperglycemia in this setting have not been published. We discuss mechanisms of hyperglycemia associated with TKIs and initial management of hyperglycemia, including benefits and limitations of oral antihyperglycemic options, adjustment of therapy based on grade of hyperglycemia, and recommendations for follow-up glucose monitoring. PMID:26629426

  3. The next generation of epidermal growth factor receptor tyrosine kinase inhibitors in the treatment of lung cancer.

    PubMed

    Steuer, Conor E; Khuri, Fadlo R; Ramalingam, Suresh S

    2015-04-15

    The discovery of "driver" genomic alterations in patients with non-small cell lung cancer (NSCLC) has dramatically changed the field of thoracic oncology in recent years. The best understood of these molecular drivers are those involving the epidermal growth factor receptor (EGFR), which when aberrantly activated are integral to the development of a subset of NSCLC tumors. First-generation and second-generation tyrosine kinase inhibitors (TKIs) specific to the activated EGFR have shown significant efficacy and have brought about the era of targeted therapy for NSCLC. The most common resistance mechanism is a threonine-to-methionine substitution (T790M) in exon 20 of the EGFR gene. Although the previous standard of care in patients with EGFR-mutated NSCLC that progressed on initial TKI therapy was chemotherapy, third-generation EGFR TKIs have now been developed and have yielded promising results for this population of patients with NSCLC. This article reviews the emerging data regarding third-generation agents in the treatment of patients with advanced NSCLC. PMID:25521095

  4. Management of hyperglycemia from epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) targeting T790M-mediated resistance.

    PubMed

    Villadolid, Jeryl; Ersek, Jennifer L; Fong, Mei Ka; Sirianno, Lindsey; Story, Ellen S

    2015-10-01

    Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to small molecule tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and afatinib. Although studies show an increased progression free survival (PFS) with use of EGFR TKIs in the first-line setting, most patients will develop resistance to therapy after the first 8-16 months. T790M is an acquired resistance mutation reported in 60-70% of patients who initially responded to a prior EGFR TKI. Recently, EGFR TKIs targeting T790M have been developed to overcome resistance with positive results in PFS and objective response rate in patients who have had disease progression on at least one TKI. Two EGFR TKIs targeting T790M, AZD9291 and rociletinib, are new active treatment options for NSCLC but differ in adverse effect profiles. Dose-limiting hyperglycemia has been reported with rociletinib and has required dose reduction, an oral antihyperglycemic, or both, without discontinuation of therapy. This suggests that patients may be effectively treated chronically for hyperglycemia associated with EGFR TKIs targeting T790M, however, guidelines for treatment of hyperglycemia in this setting have not been published. We discuss mechanisms of hyperglycemia associated with TKIs and initial management of hyperglycemia, including benefits and limitations of oral antihyperglycemic options, adjustment of therapy based on grade of hyperglycemia, and recommendations for follow-up glucose monitoring. PMID:26629426

  5. Cinnamide Derivatives of d-Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa.

    PubMed

    Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Wagner, Stefanie; Audfray, Aymeric; Prestel, Andreas; Möller, Heiko M; Imberty, Anne; Titz, Alexander

    2015-12-01

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate-based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug-like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d-mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure-activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure-based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half-lives in the 5-20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti-infectives against this multidrug-resistant pathogen. PMID:27308201

  6. Increased Bowel Toxicity in Patients Treated With a Vascular Endothelial Growth Factor Inhibitor (VEGFI) After Stereotactic Body Radiation Therapy (SBRT)

    SciTech Connect

    Barney, Brandon M.; Markovic, Svetomir N.; Laack, Nadia N.; Miller, Robert C.; Sarkaria, Jann N.; Macdonald, O. Kenneth; Bauer, Heather J.; Olivier, Kenneth R.

    2013-09-01

    Purpose: Gastrointestinal injury occurs rarely with agents that affect the vascular endothelial growth factor receptor and with abdominal stereotactic body radiation therapy (SBRT). We explored the incidence of serious bowel injury (SBI) in patients treated with SBRT with or without vascular endothelial growth factor inhibitor (VEGFI) therapy. Methods and Materials: Seventy-six patients with 84 primary or metastatic intra-abdominal lesions underwent SBRT (median dose, 50 Gy in 5 fractions). Of the patients, 20 (26%) received VEGFI within 2 years after SBRT (bevacizumab, n=14; sorafenib, n=4; pazopanib, n=1; sunitinib, n=1). The incidence of SBI (Common Terminology Criteria for Adverse Events, version 4.0, grade 3-5 ulceration or perforation) after SBRT was obtained, and the relationship between SBI and VEGFI was examined. Results: In the combined population, 7 patients (9%) had SBI at a median of 4.6 months (range, 3-17 months) from SBRT. All 7 had received VEGFI before SBI and within 13 months of completing SBRT, and 5 received VEGFI within 3 months of SBRT. The 6-month estimate of SBI in the 26 patients receiving VEGFI within 3 months of SBRT was 38%. No SBIs were noted in the 63 patients not receiving VEGFI. The log–rank test showed a significant correlation between SBI and VEGFI within 3 months of SBRT (P=.0006) but not between SBI and radiation therapy bowel dose (P=.20). Conclusions: The combination of SBRT and VEGFI results in a higher risk of SBI than would be expected with either treatment independently. Local therapies other than SBRT may be considered if a patient is likely to receive a VEGFI in the near future.

  7. An in silico approach for identification of novel inhibitors as a potential therapeutics targeting HIV-1 viral infectivity factor.

    PubMed

    Sinha, Chanda; Nischal, Anuradha; Bandaru, Srinivas; Kasera, Priyadarshani; Rajput, Ashish; Nayarisseri, Anuraj; Khattri, Sanjay

    2015-01-01

    Currently available antiviral drugs target the pol-encoded retroviral enzymes or integrases, in addition, inhibitors that target HIV-1 envelope-receptor interactions have also been recently approved. Recent understanding of the interactions between HIV-1 and host restriction factors has provided fresh avenues for development of novel antiviral drugs. For example, viral infectivity factor (Vif) now surfaced as an important therapeutic target in treatment of HIV infection. Vif suppresses A3G antiviral activity by targeting these proteins for polyubiquitination and proteasomal degradation. In the present study we analyzed the inhibitory potential of VEC5 and RN18 to inhibit the Vif-A3G interaction through protein- protein docking studies. Perusal of the study showed that, VEC5 and RN18 though inhibits the interaction however showed sub optimal potential. To overcome this set back, we identified 35 structural analogues of VEC5 and 18 analogues of RN18 through virtual screening approach. Analogue with PubCID 71624757 and 55358204 (AKOS006479723) -structurally akin to VEC5 and RN18 respectively showed much appreciable interaction than their respective parent compound. Evident from Vif-A3G; protein - protein docking studies, analogue PubCID 71624757 demonstrated 1.08 folds better inhibitory potential than its parent compound VEC5 while analogue PubCID 55358204 was 1.15 folds better than RN18. Further these analogues passed drug likeness filters and predicted to be non- toxic. We expect these analogues can be put to pharmacodynamic studies that can pave way the breakthrough in HIV therapeutics. PMID:25579575

  8. Pyrido[2,3-d]pyrimidin-5-ones: A Novel Class of Antiinflammatory Macrophage Colony-Stimulating Factor-1 Receptor Inhibitors

    SciTech Connect

    Huang, Hui; Hutta, Daniel A.; Rinker, James M.; Hu, Huaping; Parsons, William H.; Schubert, Carsten; DesJarlais, Renee L.; Crysler, Carl S.; Chaikin, Margery A.; Donatelli, Robert R.; Chen, Yanmin; Cheng, Deping; Zhou, Zhao; Yurkow, Edward; Manthey, Carl L.; Player, Mark R.

    2010-10-01

    A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound 37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In this model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid arthritis.

  9. Heat shock protein 90 (HSP90) inhibitors activate the heat shock factor 1 (HSF1) stress response pathway and improve glucose regulation in diabetic mice.

    PubMed

    Lee, Jee-Hyung; Gao, Jiaping; Kosinski, Penelope A; Elliman, Stephen J; Hughes, Thomas E; Gromada, Jesper; Kemp, Daniel M

    2013-01-18

    The cytoprotective stress response factor HSF1 regulates the transcription of the chaperone HSP70, which exhibits anti-inflammatory effects and improves insulin sensitivity. We tested the therapeutic potential of this pathway in rodent models of diabetes using pharmacological tools. Activation of the HSF1 pathway was achieved using potent inhibitors of the upstream regulatory protein, HSP90. Treatment with AUY922, a selective HSP90 inhibitor led to robust inhibition of JNK1 phosphorylation, cytoprotection and improved insulin signaling in cells, consistent with effects observed with HSP70 treatment. Chronic dosing with HSP90 inhibitors reversed hyperglycemia in the diabetic db/db mouse model, and improved insulin sensitivity in the diet-induced obese mouse model of insulin resistance, further supporting the concept that the HSF1 pathway is a potentially viable anti-diabetes target. PMID:23261432

  10. Vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors: development and validation of predictive 3-D QSAR models through extensive ligand- and structure-based approaches.

    PubMed

    Ragno, Rino; Ballante, Flavio; Pirolli, Adele; Wickersham, Richard B; Patsilinakos, Alexandros; Hesse, Stéphanie; Perspicace, Enrico; Kirsch, Gilbert

    2015-08-01

    Vascular endothelial growth factor receptor-2, (VEGFR-2), is a key element in angiogenesis, the process by which new blood vessels are formed, and is thus an important pharmaceutical target. Here, 3-D quantitative structure-activity relationship (3-D QSAR) were used to build a quantitative screening and pharmacophore model of the VEGFR-2 receptors for design of inhibitors with improved activities. Most of available experimental data information has been used as training set to derive optimized and fully cross-validated eight mono-probe and a multi-probe quantitative models. Notable is the use of 262 molecules, aligned following both structure-based and ligand-based protocols, as external test set confirming the 3-D QSAR models' predictive capability and their usefulness in design new VEGFR-2 inhibitors. From a survey on literature, this is the first generation of a wide-ranging computational medicinal chemistry application on VEGFR2 inhibitors. PMID:26194852

  11. Histone Deacetylase Inhibitors Enhance the Apoptotic Activity of Insulin-Like Growth Factor Binding Protein-3 by Blocking PKC-Induced IGFBP-3 Degradation

    PubMed Central

    Oh, Seung Hyun; Whang, Young Mi; Min, Hye-Young; Han, Seung Ho; Kang, Ju-Hee; Song, Ki-Hoon; Glisson, Bonnie S.; Kim, Yeul Hong; Lee, Ho-Young

    2012-01-01

    Overexpression of insulin-like growth factor binding protein (IGFBP)-3 induces apoptosis of cancer cells. However, preexisting resistance to IGFBP-3 could limit its antitumor activities. This study characterizes the efficacy and mechanism of the combination of recombinant IGFBP-3 (rIGFBP-3) and HDAC inhibitors to overcome IGFBP-3 resistance in a subset of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cells. The effects of the combination of rIGFBP-3 and a number of HDAC inhibitors on cell proliferation and apoptosis were assessed in vitro and in vivo by using the MTT assay, a flow cytometry-based TUNEL assay, western blot analyses, and the NSCLC xenograft tumor model. Combined treatment with HDAC inhibitors and rIGFBP-3 had synergistic antiproliferative effects accompanied by increased apoptosis rates in a subset of NSCLC and HNSCC cell lines in vitro. Moreover, combined treatment with depsipeptide and rIGFBP-3 completely suppressed tumor growth and increased the apoptosis rate in vivo in H1299 NSCLC xenografts. Evidence suggests that HDAC inhibitors increased the half-life of rIGFBP-3 protein by blocking protein kinase C (PKC)-mediated phosphorylation and degradation of rIGFBP-3. In addition, combined treatment of IGFBP-3 with an HDAC inhibitor facilitates apoptosis through up-regulation of rIGFBP-3 stability and Akt signaling inhibition. The ability of HDAC inhibitors to decrease PKC activation may enhance apoptotic activities of rIGFBP-3 in NSCLC cells in vitro and in vivo. These results indicated that combined treatment with HDAC inhibitor and rIGFBP-3 could be an effective treatment strategy for NSCLC and HNSCC with highly activated PKC. PMID:22362554

  12. Dose Optimization for Single Intradiscal Administration of the Tumor Necrosis FactorInhibitor, Etanercept, in Rat Disc Injury Models

    PubMed Central

    Orita, Sumihisa; Yamauchi, Kazuyo; Suzuki, Takane; Suzuki, Miyako; Sakuma, Yoshihiro; Kubota, Go; Oikawa, Yasuhiro; Sainoh, Takeshi; Sato, Jun; Fujimoto, Kazuki; Shiga, Yasuhiro; Abe, Koki; Kanamoto, Hirohito; Inoue, Masahiro; Kinoshita, Hideyuki; Takahashi, Kazuhisa; Ohtori, Seiji

    2016-01-01

    Study Design Experimental animal study. Purpose We aimed to determine the optimal dose of a single direct injection of the tumor necrosis factor (TNF)-α inhibitor, etanercept, by using the rat model of degenerative intervertebral disc from injury. Overview of Literature The pain-related peptide expression was suppressed in the etanercept (100 µg and 1,000 µg)-administered groups in a dose-dependent manner. Methods The neurotracer FluoroGold (FG) was applied to the surfaces of L4/5 discs to label their innervating dorsal root ganglion (DRG) neurons (n=50). Ten rats were included in the nonpunctured disc sham surgery control group, whereas the other 40 were included in the experimental group in which intervertebral discs were punctured with a 23-gauge needle. Saline or etanercept (10 µg, 100 µg, or 1,000 µg) was injected into the punctured discs (n=10 for each treatment). After 14 days of surgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for calcitonin gene-related peptide (CGRP). The proportion of FG-labeled CGRP-immunoreactive DRG neurons was evaluated in all the groups. Results There were no significant differences between the puncture+saline group and the puncture+10-µg etanercept group (p >0.05). However, a significant decrease in the percentage of FG and CGRP double-positive cells in FG-positive cells was observed in the etanercept (100 µg and 1,000 µg)-administered groups in a dose-dependent manner (p <0.05). Conclusions When a low dose of the TNF-α inhibitor (10 µg of etanercept) was directly administered to the rat intervertebral disc in the rat model of degenerative intervertebral disc from injury, no suppressive effect on the pain-related peptide expression was observed. However, when a higher dose of etanercept (100 µg and 1,000 µg) was administered, the pain-related peptide expression was suppressed in a dose-dependent manner. PMID:27559439

  13. Discovery of selective and noncovalent diaminopyrimidine-based inhibitors of epidermal growth factor receptor containing the T790M resistance mutation.

    PubMed

    Hanan, Emily J; Eigenbrot, Charles; Bryan, Marian C; Burdick, Daniel J; Chan, Bryan K; Chen, Yuan; Dotson, Jennafer; Heald, Robert A; Jackson, Philip S; La, Hank; Lainchbury, Michael D; Malek, Shiva; Purkey, Hans E; Schaefer, Gabriele; Schmidt, Stephen; Seward, Eileen M; Sideris, Steve; Tam, Christine; Wang, Shumei; Yeap, Siew Kuen; Yen, Ivana; Yin, Jianping; Yu, Christine; Zilberleyb, Inna; Heffron, Timothy P

    2014-12-11

    Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties. PMID:25383627

  14. Recognition of insulin-like growth factor (IGF) serum binding proteins by an antibody raised against a specific IGF-inhibitor

    SciTech Connect

    Ooi, G.T.; Herington, A.C.

    1988-10-31

    Evidence suggests that a specific inhibitor of the insulin-like growth factors (IGF) which acts by binding to IGF may be structurally related to the native, MW 150K binding protein (BP) in serum. This has now been examined using a polyclonal antiserum (R8) raised against highly purified inhibitor. Western blotting analysis of inhibitor using R8 gave 4 immunoreactive (ir-) bands (MW 34.5K, 23K, 16K and 12K), the most intense being the MW 16K band, identical to the MW of the inhibitor. Ligand blotting using /sup 125/I-IGF-I indicated specific IGF-binding activity at MW 29K, 26.5K, 16K and 12K, indicating that at least 2 of the ir-bands (16K and 12K) were IGF-BPs. Western blotting of a salt-precipitated fraction of serum gave 8 ir-bands of which 3 (MW 42K, 38K and 34K) were identical with BP bands detected previously by Hossenlopp et al. These immunological crossreactivities indicate that the inhibitor is structurally related to the higher MW IGF-BPs in serum.

  15. Discovery of Selective and Noncovalent Diaminopyrimidine-Based Inhibitors of Epidermal Growth Factor Receptor Containing the T790M Resistance Mutation

    PubMed Central

    2015-01-01

    Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties. PMID:25383627

  16. Tumour necrosis factor inhibitors versus combination intensive therapy with conventional disease modifying anti-rheumatic drugs in established rheumatoid arthritis: TACIT non-inferiority randomised controlled trial

    PubMed Central

    Ibrahim, Fowzia; Farewell, Vern; O’Keeffe, Aidan G; Walker, David; Kelly, Clive; Birrell, Fraser; Chakravarty, Kuntal; Maddison, Peter; Heslin, Margaret; Patel, Anita; Kingsley, Gabrielle H

    2015-01-01

    Objective To determine whether intensive combinations of synthetic disease modifying drugs can achieve similar clinical benefits at lower costs to high cost biologics such as tumour necrosis factor inhibitors in patients with active rheumatoid arthritis resistant to initial methotrexate and other synthetic disease modifying drugs. Design Open label pragmatic randomised multicentre two arm non-inferiority trial over 12 months. Setting 24 rheumatology clinics in England. Participants Patients with rheumatoid arthritis who were eligible for treatment with tumour necrosis factor inhibitors according to current English guidance were randomised to either the tumour necrosis factor inhibitor strategy or the combined disease modifying drug strategy. Interventions Biologic strategy: start tumour necrosis factor inhibitor; second biologic in six month for non-responders. Alternative strategy: start combination of disease modifying drugs; start tumour necrosis factor inhibitors after six months in non-responders. Main outcome measure Primary outcome: reduction in disability at 12 months measured with patient recorded heath assessment questionnaire (range 0.00-3.00) with a 0.22 non-inferiority margin for combination treatment versus the biologic strategy. Secondary outcomes: quality of life, joint damage, disease activity, adverse events, and costs. Intention to treat analysis used multiple imputation methods for missing data. Results 432 patients were screened: 107 were randomised to tumour necrosis factor inhibitors and 101 started taking; 107 were randomised to the combined drug strategy and 104 started taking the drugs. Initial assessments were similar; 16 patients were lost to follow-up (seven with the tumour necrosis factor inhibitor strategy, nine with the combined drug strategy); 42 discontinued the intervention but were followed-up (19 and 23, respectively). The primary outcome showed mean falls in scores on the health assessment questionnaire of −0.30 with the

  17. Spitzer IRS observations of the XA region in the cygnus loop supernova remnant

    SciTech Connect

    Sankrit, Ravi; Bautista, Manuel; Williams, Brian J.; Blair, William P.; Borkowski, Kazimierz J.; Long, Knox S.

    2014-05-20

    We report on spectra of two positions in the XA region of the Cygnus Loop supernova remnant obtained with the InfraRed Spectrograph on the Spitzer Space Telescope. The spectra span the 10-35 μm wavelength range, which contains a number of collisionally excited forbidden lines. These data are supplemented by optical spectra obtained at the Whipple Observatory and an archival UV spectrum from the International Ultraviolet Explorer. Coverage from the UV through the IR provides tests of shock wave models and tight constraints on model parameters. Only lines from high ionization species are detected in the spectrum of a filament on the edge of the remnant. The filament traces a 180 km s{sup –1} shock that has just begun to cool, and the oxygen to neon abundance ratio lies in the normal range found for Galactic H II regions. Lines from both high and low ionization species are detected in the spectrum of the cusp of a shock-cloud interaction, which lies within the remnant boundary. The spectrum of the cusp region is matched by a shock of about 150 km s{sup –1} that has cooled and begun to recombine. The post-shock region has a swept-up column density of about 1.3 × 10{sup 18} cm{sup –2}, and the gas has reached a temperature of 7000-8000 K. The spectrum of the Cusp indicates that roughly half of the refractory silicon and iron atoms have been liberated from the grains. Dust emission is not detected at either position.

  18. Vandetanib (ZD6474), a dual inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinases: current status and future directions.

    PubMed

    Morabito, Alessandro; Piccirillo, Maria Carmela; Falasconi, Fabiano; De Feo, Gianfranco; Del Giudice, Antonia; Bryce, Jane; Di Maio, Massimo; De Maio, Ermelinda; Normanno, Nicola; Perrone, Francesco

    2009-04-01

    Vandetanib is a novel, orally available inhibitor of different intracellular signaling pathways involved in tumor growth, progression, and angiogenesis: vascular endothelial growth factor receptor-2, epidermal growth factor receptor, and REarranged during Transfection tyrosine kinase activity. Phase I clinical trials have shown that vandetanib is well tolerated as a single agent at daily doses < or =300 mg. In the phase II setting, negative results were observed with vandetanib in small cell lung cancer, metastatic breast cancer, and multiple myeloma. In contrast, three randomized phase II studies showed that vandetanib prolonged the progression-free survival (PFS) time of patients with non-small cell lung cancer (NSCLC) as a single agent when compared with gefitinib or when added to chemotherapy. Rash, diarrhea, hypertension, fatigue, and asymptomatic QTc prolongation were the most common adverse events. Antitumor activity was also observed in medullary thyroid cancer. Four randomized phase III clinical trials in NSCLC are exploring the efficacy of vandetanib in combination with docetaxel, the Zactima in cOmbination with Docetaxel In non-small cell lung Cancer (ZODIAC) trial, or with pemetrexed, the Zactima Efficacy with Alimta in Lung cancer (ZEAL) trial, or as a single agent, the Zactima Efficacy when Studied versus Tarceva (ZEST) and the Zactima Efficacy trial for NSCLC Patients with History of EGFR-TKI chemo-Resistance (ZEPHYR) trials. Based on a press release by the sponsor of these trials, the PFS time was longer with vandetanib in the ZODIAC and ZEAL trials; the ZEST trial was negative for its primary superiority analysis, but was successful according to a preplanned noninferiority analysis of PFS. Ongoing phase II and III clinical trials will better define the appropriate schedule, the optimal setting of evaluation, and the safety of long-term use of vandetanib. PMID:19349511

  19. In vitro effects of heparin and tissue factor pathway inhibitor on factor VII assays. possible implications for measurements in vivo after heparin therapy.

    PubMed

    Bladbjerg, E M; Larsen, L F; Ostergaard, P; Jespersen, J

    2000-12-01

    The coagulant activity of blood coagulation factor VII (FVII:C) can be lowered by changes in lifestyle and by therapeutic intervention, e.g. heparin infusion. The question is, however, whether FVII:C determined ex vivo is a valid measure of the FVII activity in vivo. We measured plasma FVII:C, activated FVII (FVIIa), FVII protein (FVII:Ag), tissue factor pathway inhibitor (TFPI), triglycerides, and free fatty acids (FFA) before and 15 min after infusion of a bolus of unfractionated heparin (50 IU/kg body weight) in 12 healthy subjects. Additionally, we conducted in vitro experiments to investigate the effect of unfractionated heparin and TFPI, which is released from the endothelium by heparin, on FVII:C, FVIIa, and FVII:Ag. Heparin infusion decreased triglycerides and increased FFA and TFPI. This was accompanied by significant reductions in FVIIa, FVII:C and FVII:Ag. In vitro, anti-TFPI antibodies increased FVIIa and FVII:C, and heparin reduced FVIIa. The heparinase Hepzyme was unable to abolish the effect of heparin. There were no in vitro effects on FVII:Ag. We conclude that, due to interference by TFPI and heparin in post-heparin plasma, it is impossible to measure the in vivo FVII activity by means of FVII clotting assays. These assays should therefore not be used to measure the coagulation status of patients in heparin therapy, unless extraordinary precautions are taken to eliminate TFPI and heparin effects ex vivo. The observed effect of heparin on FVII:Ag should be investigated further. PMID:11132652

  20. Prognostic tissue biomarker exploration for patients with metastatic renal cell carcinoma receiving vascular endothelial growth factor receptor tyrosine kinase inhibitors.

    PubMed

    Park, Inkeun; Cho, Yong Mee; Lee, Jae-Lyun; Ahn, Jin-Hee; Lee, Dae-Ho

    2016-04-01

    In metastatic renal cell carcinoma (mRCC), the prognostic role of several tumor tissue biomarkers has been evaluated, but the results were controversial. This study aims to verify the prognostic importance of selected tumor tissue biomarkers in patients with mRCC. The clinicopathological features, immunohistochemical staining and scoring for select tissue biomarkers, treatment, and outcome of patients with mRCC treated with vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) between July 2006 and March 2011 at Asan Medical Center in Seoul, South Korea, were reviewed. In total, 123 patients met the inclusion criteria. Most patients had clear-cell carcinoma (107 patients, 87.0 %). First-line VEGFR TKIs were sunitinib (97 patients, 78.9 %), sorafenib (23 patients, 18.7 %), and pazopanib (3 patients, 2.4 %). With a median follow-up period of 60.0 months (95 % confidence interval (CI), 56.3-63.6), median overall survival (OS) and progression-free survival (PFS) were 25.6 months (95 % CI, 19.2-32.0) and 12.2 months (95 % CI, 8.1-16.3), respectively. In the multivariable analysis for OS, carbonic anhydrase IX (CAIX; 47.5 % or less vs. more than 47.5 %, p = 0.014), sarcomatoid change (40 % or less vs. more than 40 %, p < 0.001), tumor necrosis (20 % or less vs. more than 20 %, p = 0.006), and Heng's risk group (good vs. intermediate vs. poor, p = 0.011) were identified as independent prognostic factors. In the multivariable analysis for PFS, CAIX (p < 0.001), phosphatase and tensin homolog (PTEN; 45 % or less vs. more than 45 %, p = 0.004), sarcomatoid change (p = 0.002), and tumor necrosis (p = 0.001) were identified as independent factors affecting PFS. CAIX and PTEN had prognostic importance for mRCC patients receiving first-line VEGFR TKI. Future validation and mechanistic studies are required. PMID:26526582

  1. Safety of Resuming Tumor Necrosis Factor Inhibitors in Ankylosing Spondylitis Patients Concomitant with the Treatment of Active Tuberculosis: A Retrospective Nationwide Registry of the Korean Society of Spondyloarthritis Research

    PubMed Central

    Kim, Hye Won; Kwon, Seong Ryul; Jung, Kyong-Hee; Kim, Seong-Kyu; Baek, Han Joo; Seo, Mi Ryung; Bang, So-Young; Lee, Hye-Soon; Suh, Chang-Hee; Jung, Ju Yang; Son, Chang-Nam; Shim, Seung Cheol; Lee, Sang-Hoon; Lee, Seung-Geun; Lee, Yeon-Ah; Lee, Eun Young; Kim, Tae-Hwan

    2016-01-01

    Backgrounds Patients who develop an active tuberculosis infection during tumor necrosis factor (TNF) inhibitor treatment typically discontinue TNF inhibitor and receive standard anti-tuberculosis treatment. However, there is currently insufficient information on patient outcomes following resumption of TNF inhibitor treatment during ongoing anti- tuberculosis treatment. Our study was designed to investigate the safety of resuming TNF inhibitors in ankylosing spondylitis (AS) patients who developed tuberculosis as a complication of the use of TNF inhibitors. Methods Through the nationwide registry of the Korean Society of Spondyloarthritis Research, 3929 AS patients who were prescribed TNF inhibitors were recruited between June 2003 and June 2014 at fourteen referral hospitals. Clinical information was analyzed about the patients who experienced tuberculosis after exposure to TNF inhibitors. The clinical features of resumers and non-resumers of TNF inhibitors were compared and the outcomes of tuberculosis were surveyed individually. Findings Fifty-six AS patients were treated for tuberculosis associated with TNF inhibitors. Among them, 23 patients resumed TNF inhibitors, and these patients were found to be exposed to TNF inhibitors for a longer period of time and experienced more frequent disease flare-up after discontinuation of TNF inhibitors compared with those who did not resume. Fifteen patients resumed TNF inhibitors during anti-tuberculosis treatment (early resumers) and 8 after completion of anti-tuberculosis treatment (late resumers). Median time to resuming TNF inhibitor from tuberculosis was 3.3 and 9.0 months in the early and late resumers, respectively. Tuberculosis was treated successfully in all resumers and did not relapse in any of them during follow-up (median 33.8 [IQR; 20.8–66.7] months). Conclusions Instances of tuberculosis were treated successfully in our AS patients, even when given concomitantly with TNF inhibitors. We suggest that early

  2. p53 mediates cigarette smoke-induced apoptosis of pulmonary endothelial cells: inhibitory effects of macrophage migration inhibitor factor.

    PubMed

    Damico, Rachel; Simms, Tiffany; Kim, Bo S; Tekeste, Zenar; Amankwan, Henry; Damarla, Mahendra; Hassoun, Paul M

    2011-03-01

    Exposure to cigarette smoke (CS) is the most common cause of emphysema, a debilitating pulmonary disease histopathologically characterized by the irreversible destruction of lung architecture. Mounting evidence links enhanced endothelial apoptosis causally to the development of emphysema. However, the molecular determinants of human endothelial cell apoptosis and survival in response to CS are not fully defined. Such determinants could represent clinically relevant targets for intervention. We show here that CS extract (CSE) triggers the death of human pulmonary macrovascular endothelial cells (HPAECs) through a caspase 9-dependent apoptotic pathway. Exposure to CSE results in the increased expression of p53 in HPAECs. Using the p53 inhibitor, pifithrin-α (PFT-α), and RNA interference (RNAi) directed at p53, we demonstrate that p53 function and expression are required for CSE-mediated apoptosis. The expression of macrophage migration inhibitory factor (MIF), an antiapoptotic cytokine produced by HPAECs, also increases in response to CSE exposure. The addition of recombinant human MIF prevents cell death from exposure to CSE. Further, the suppression of MIF or its receptor/binding partner, Jun activation domain-binding protein 1 (Jab-1), with RNAi enhances the sensitivity of human pulmonary endothelial cells to CSE via a p53-dependent (PFT-α-inhibitable) pathway. Finally, we demonstrate that MIF is a negative regulator of p53 expression in response to CSE, placing MIF upstream of p53 as an antagonist of CSE-induced apoptosis. We conclude that MIF can protect human vascular endothelium from the toxic effects of CSE via the antagonism of p53-mediated apoptosis. PMID:20448056

  3. Fruit Ripening Regulation of α-Mannosidase Expression by the MADS Box Transcription Factor RIPENING INHIBITOR and Ethylene

    PubMed Central

    Irfan, Mohammad; Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-01-01

    α-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression. PMID:26834776

  4. The Complement Inhibitor Factor H Generates an Anti-Inflammatory and Tolerogenic State in Monocyte-Derived Dendritic Cells.

    PubMed

    Olivar, Rut; Luque, Ana; Cárdenas-Brito, Sonia; Naranjo-Gómez, Mar; Blom, Anna M; Borràs, Francesc E; Rodriguez de Córdoba, Santiago; Zipfel, Peter F; Aran, Josep M

    2016-05-15

    The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury, although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation, respectively, attenuating the activity of the C3/C5 convertases and, consequently, avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study, we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly, FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture, resembling immature MoDCs, lower expression of the maturation marker CD83 and the costimulatory molecules CD40, CD80, and CD86, decreased production of key proinflammatory Th1-cytokines (IL-12, TNF-α, IFN-γ, IL-6, and IL-8), and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover, FH-treated MoDCs show low Ag uptake and, when challenged with LPS, display reduced CCR7 expression and chemotactic migration, impaired CD4(+) T cell alloproliferation, inhibition of IFN-γ secretion by the allostimulated T cells, and, conversely, induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus, this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity, transplantation, and autoimmunity. PMID:27076676

  5. Fruit Ripening Regulation of α-Mannosidase Expression by the MADS Box Transcription Factor RIPENING INHIBITOR and Ethylene.

    PubMed

    Irfan, Mohammad; Ghosh, Sumit; Meli, Vijaykumar S; Kumar, Anil; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-01-01

    α-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression. PMID:26834776

  6. 18F-FDG PET/CT for Monitoring Treatment Responses to the Epidermal Growth Factor Receptor Inhibitor Erlotinib

    PubMed Central

    Walter, Franziska; Garon, Edward B.; Reckamp, Karen L.; Figlin, Robert; Phelps, Michael E.; Weber, Wolfgang A.; Czernin, Johannes; Allen-Auerbach, Martin S.

    2016-01-01

    Response rates of unselected non–small cell lung cancer (NSCLC) patients to the epidermal growth factor receptor inhibitor erlotinib are low and range from 10% to 20%. Early response assessments are needed to avoid costs and side effects of inefficient treatments. Here we determined whether early changes in tumor uptake of 18F-FDG can predict progression-free and overall survival in NSCLC patients who are treated with erlotinib. Methods Twenty-two patients (6 men, 16 women; mean age ± SD, 64 ± 13 y) with stage III or stage IV NSCLC who received erlotinib treatment were enrolled prospectively. 18F-FDG PET/CT was performed before the initiation of treatment (n = 22), after 2 wk (n = 22), and after 78 ± 21 d (n = 11). Tumor maximum standardized uptake values were measured for a maximum of 5 lesions for each patient. Tumor responses were classified using modified PET Response Criteria in Solid Tumors (use of maximum standardized uptake values). Median overall survival by Kaplan–Meier analysis was compared between groups using a log-rank test. Results The overall median time to progression was 52 d (95% confidence interval, 47–57 d). The overall median survival time was 131 d (95% confidence interval, 0–351 d). Patients with progressive metabolic disease on early follow-up PET showed a significantly shorter time to progression (47 vs. 119 d; P < 0.001) and overall survival (87 vs. 828 d; P = 0.01) than patients classified as having stable metabolic disease or partial or complete metabolic response. Conclusion These data suggest that 18F-FDG PET/CT performed early after the start of erlotinib treatment can help to identify patients who benefit from this targeted therapy. PMID:22045706

  7. Oestrogens Downregulate Tissue Factor Pathway Inhibitor through Oestrogen Response Elements in the 5’-Flanking Region

    PubMed Central

    Ali, Huda Omar; Stavik, Benedicte; Myklebust, Christiane Filion; Andersen, Elisabeth; Dahm, Anders E. A.; Iversen, Nina; Sandset, Per Morten; Skretting, Grethe

    2016-01-01

    Oestrogens influence the pathology and development of hormone-sensitive breast cancers. Tissue factor pathway inhibitor (TFPI) has been shown to be associated with breast cancer pathogenesis. Recently, we found TFPI mRNA levels to be significantly reduced by oestrogens in a breast cancer cell line (MCF7), a process mediated through the oestrogen receptor alpha (ERα). The aim of the present study was to investigate the mechanism(s) by which oestrogens may regulate TFPI at the transcriptional level. The TFPI 5’-flanking region contains three oestrogen response element (ERE) half-sites at positions -845, -769 and -50. Constructs containing the wild type or mutated ERE half-sites of the TFPI 5’-flanking region were generated in a luciferase reporter gene vector and transiently co-transfected with an ERα expression vector into HEK293 cells and subsequently treated with oestrogens. We found that luciferase activity was significantly downregulated after oestrogen stimulation in cells transfected with the wild type construct, an effect that was abolished by mutating either ERE half-sites. Electrophoretic mobility shift assay suggested direct and specific interaction of ERα with the ERE half-sites in the TFPI 5’-flanking region. Chromatin immunoprecipitation showed that ERα was recruited to the region -899 to -578 of the TFPI 5’-flanking region in vivo, where the ERE half-sites -845 and -769 are located. Our results indicate that ERα can interact with all three ERE half-sites in the TFPI 5’-flanking region and thus participate in the repression of oestrogen mediated TFPI transcription in breast cancer cells. PMID:26999742

  8. Systematic Review of the Risk of Adverse Outcomes Associated with Vascular Endothelial Growth Factor Inhibitors for the Treatment of Cancer

    PubMed Central

    Faruque, Labib Imran; Lin, Meng; Battistella, Marisa; Wiebe, Natasha; Reiman, Tony; Hemmelgarn, Brenda; Thomas, Chandra; Tonelli, Marcello

    2014-01-01

    Background Anti-angiogenic therapy targeted at vascular endothelial growth factor (VEGF) is now used to treat several types of cancer. We did a systematic review of randomized controlled trials (RCTs) to summarize the adverse effects of vascular endothelial growth factor inhibitors (VEGFi), focusing on those with vascular pathogenesis. Methods and Findings We searched MEDLINE, EMBASE and Cochrane Library until April 19, 2012 to identify parallel RCTs comparing a VEGFi with a control among adults with any cancer. We pooled the risk of mortality, vascular events (myocardial infarction, stroke, heart failure, and thromboembolism), hypertension and new proteinuria using random-effects models and calculated unadjusted relative risk (RR). We also did meta-regression and assessed publication bias. We retrieved 83 comparisons from 72 studies (n = 38,078) on 11 different VEGFi from 7901 identified citations. The risk of mortality was significantly lower among VEGFi recipients than controls (pooled RR 0.96, 95% confidence interval [CI] 0.94 to 0.98, I2 = 0%, tau2 = 0; risk difference 2%). Compared to controls, VEGFi recipients had significantly higher risk of myocardial infarction (MI) (RR 3.54, 95% CI 1.61 to 7.80, I2 = 0%, tau2 = 0), arterial thrombotic events (RR 1.80, 95% CI 1.24 to 2.59, I2 = 0%, tau2 = 0); hypertension (RR 3.46, 95% CI 2.89 to 4.15, I2 = 58%, tau2 = 0.16), and new proteinuria (RR 2.51, 95% CI 1.60 to 3.94, I2 = 87%, tau2 = 0.65). The absolute risk difference was 0.8% for MI, 1% for arterial thrombotic events, 15% for hypertension and 12% for new proteinuria. Meta-regression did not suggest any statistically significant modifiers of the association between VEGFi treatment and any of the vascular events. Limitations include heterogeneity across the trials. Conclusions VEGFi increases the risk of MI, hypertension, arterial thromboembolism and proteinuria. The absolute magnitude of the excess risk appears

  9. Limiting the Number of Potential Binding Modes by Introducing Symmetry into Ligands: Structure-Based Design of Inhibitors for Trypsin-Like Serine Proteases.

    PubMed

    Furtmann, Norbert; Häußler, Daniela; Scheidt, Tamara; Stirnberg, Marit; Steinmetzer, Torsten; Bajorath, Jürgen; Gütschow, Michael

    2016-01-11

    In the absence of X-ray data, the exploration of compound binding modes continues to be a challenging task. For structure-based design, specific features of active sites in different targets play a major role in rationalizing ligand binding characteristics. For example, dibasic compounds have been reported as potent inhibitors of various trypsin-like serine proteases, the active sites of which contain several binding pockets that can be targeted by cationic moieties. This results in several possible orientations within the active site, complicating the binding mode prediction of such compounds by docking tools. Therefore, we introduced symmetry in bi- and tribasic compounds to reduce conformational space in docking calculations and to simplify binding mode selection by limiting the number of possible pocket occupations. Asymmetric bisbenzamidines were used as starting points for a multistage and structure-guided optimization. A series of 24 final compounds with either two or three benzamidine substructures was ultimately synthesized and evaluated as inhibitors of five serine proteases, leading to potent symmetric inhibitors for the pharmaceutical drug targets matriptase, matriptase-2, thrombin and factor Xa. This study underlines the relevance of ligand symmetry for chemical biology. PMID:26625703

  10. CHANDRA IMAGING AND SPECTROSCOPY OF THE EASTERN XA REGION OF THE CYGNUS LOOP SUPERNOVA REMNANT

    SciTech Connect

    McEntaffer, R. L.; Brantseg, T.

    2011-04-01

    The XA region of the Cygnus Loop is a bright knot of X-ray emission on the eastern edge of the supernova remnant. The emission results from the interaction of the supernova blast wave with density enhancements at the edge of a precursor formed cavity. However, this interaction is complex given the irregular morphology of the cavity wall. To study the nature and origin of the X-ray emission, we use high spatial resolution images from Chandra. We extract spectra from these images to analyze the physical conditions of the plasma. Our goal is to probe the density of various regions to form a picture of the cavity wall and characterize the interaction between this supernova and the local interstellar medium. We find that a series of regions along the edge of the X-ray emission appears to trace out the location of the cavity wall. The best-fit plasma models result in two temperature component equilibrium models for each region. The low-temperature components have densities that are an order of magnitude higher than the high-temperature components. The high-density plasma may exist in the cavity wall where it equilibrates rapidly and cools efficiently. The low-density plasma is interior to the enhancement and heated further by a reverse shock from the wall. Calculations of shock velocities and timescales since shock heating are consistent with this interpretation. Furthermore, we find a bright knot of emission indicative of a discrete interaction of the blast wave with a high-density cloud in the cavity wall with a size scale {approx}0.1 pc. Aside from this, other extractions made interior to the X-ray edge are confused by line-of-sight projection of various components. Some of these regions show evidence of detecting the cavity wall but their location makes the interpretation difficult. In general, the softer plasmas are well fit at temperatures (kT){approx} 0.11 keV, with harder plasmas at temperatures of (kT){approx} 0.27 keV. All regions displayed consistent metal

  11. Novel Role for γ-Catenin in the Regulation of Cancer Cell Migration via the Induction of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1)*

    PubMed Central

    Sechler, Marybeth; Borowicz, Stanley; Van Scoyk, Michelle; Avasarala, Sreedevi; Zerayesus, Sereke; Edwards, Michael G.; Kumar Karuppusamy Rathinam, Manoj; Zhao, Xiangmin; Wu, Pei-Ying; Tang, Ke; Bikkavilli, Rama Kamesh; Winn, Robert A.

    2015-01-01

    γ-catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors. PMID:25925948

  12. Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1B

    SciTech Connect

    Shia, Steven; Stamos, Jennifer; Kirchhofer, Daniel; Fan, Bin; Wu, Judy; Corpuz, Raquel T.; Santell, Lydia; Lazarus, Robert A.; Eigenbrot, Charles

    2010-07-20

    Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 {angstrom} resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 {angstrom} resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

  13. Challenges of the management of severe hemophilia A with inhibitors: two case reports emphasizing the potential interest of a high-purity human Factor VIII/von Willebrand factor concentrate and individually tailored prophylaxis guided by thrombin-generation test.

    PubMed

    Mathieu, Sophie; Crampe, Carine; Dargaud, Yesim; Lavigne-Lissalde, Géraldine; Escuriola-Ettingshausen, Carmen; Tardy, Brigitte; Meley, Roland; Thouvenin, Sandrine; Stephan, Jean L; Berger, Claire

    2015-12-01

    Severe hemophilia A is an X-linked bleeding disorder. Immune tolerance induction (ITI) is the best strategy of treatment when patients develop inhibitors. The objective is to illustrate the benefit of a high-purity human factor VIII/von Willebrand factor (VWF) concentrate (Octanate) in the management of ITI. We also wanted to raise the potential interest of laboratory assays such as thrombin-generation test (TGT) and epitope mapping. Two patients were treated during ITI, first with a recombinant FVIII and then with plasma-derived factor VIII without success, and, finally, with Octanate. Bypassing agents were used based on the results of TGT. Epitope mapping was performed during ITI therapy. These observations suggest the potential contribution of Octanate in the management of ITI in difficult cases. The use of bypassing agents can be necessary in prophylaxis or to treat bleedings, and may be guided by TGT results. Epitope mapping is used to describe the inhibitor. This article shows a decrease of the inhibitor directed against the C2 domain after initiation of Octanate. A high-purity human factor VIII/von Willebrand factor concentrate (Octanate) may be a valuable therapeutical option for ITI therapy. TGT and epitope mapping could be of help in the management of ITI. PMID:26517064

  14. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics.

    PubMed

    Chung, Clement

    2016-06-01

    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  15. Small Molecular Inhibitor of Transforming Growth Factor-{beta} Protects Against Development of Radiation-Induced Lung Injury

    SciTech Connect

    Anscher, Mitchell S. Thrasher, Bradley; Zgonjanin, Larisa; Rabbani, Zahid N.; Corbley, Michael J.; Fu Kai; Sun Lihong; Lee, W.-C.; Ling, Leona E.; Vujaskovic, Zeljko

    2008-07-01

    Purpose: To determine whether an anti-transforming growth factor-{beta} (TGF-{beta}) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. Methods and Materials: One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7, RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-{beta} were assessed. Results: The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-{beta}1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7g/kg nor for the short-course (3 weeks) SM16 at either dose level. Conclusion: SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-{beta} at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.

  16. Genetic polymorphisms of tumour necrosis factor alpha (TNF-α) promoter gene and response to TNF-α inhibitors in Spanish patients with inflammatory bowel disease.

    PubMed

    López-Hernández, R; Valdés, M; Campillo, J A; Martínez-Garcia, P; Salama, H; Salgado, G; Boix, F; Moya-Quiles, M R; Minguela, A; Sánchez-Torres, A; Miras, M; Garcia, A; Carballo, F; Álvarez-López, M R; Muro, M

    2014-02-01

    Tumour necrosis factor alpha (TNF-α) has an important role in inflammatory response. Alterations in the regulation of TNF-α have been implicated in a variety of inflammatory disorders, including Inflammatory bowel disease (IBD). Indeed, a common treatment for IBD is the use of TNF-α inhibitors. Polymorphisms in the TNF-α promoter region are known to affect the level of gene expression. Our aim was to investigate the influence of these single nucleotide polymorphisms (SNPs) in TNF-α promoter gene play in the risk of IBD in a Spanish population and their individual response to anti-TNF-α treatment. DNA samples from patients with IBD and controls were screened for TNF-α -238G/A (rs361525) and -308G/A (rs1800629) SNPs by PCR-SSOP using a microbeads luminex assay and compared with response to TNF-α inhibitors. There were not statistical differences in -238G/A and -308G/A allele and genotype frequencies between patients. However, we found an increased frequency of -308A allele and -308GA genotype in these nonresponders patients to TNF-α inhibitors with respect to responders patients (Pc < 0.05). This -308GA genotype has been classified as high producer of this cytokine. This fact could actually be interesting to explain the different response of patients with IBD with respect to TNF-α inhibitors. TNF-α promoter gene polymorphism does not seem to play a role in IBD susceptibility, but particular TNF-α genotypes may be involved in the different responses to TNF-α inhibitor treatment in Spanish patients with IBD. PMID:23590430

  17. Development of a comprehensive, validated pharmacophore hypothesis for anthrax toxin lethal factor (LF) inhibitors using genetic algorithms, Pareto scoring, and structural biology.

    PubMed

    Chiu, Ting-Lan; Amin, Elizabeth A

    2012-07-23

    Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF), an 89-kDa zinc hydrolase secreted by the bacilli, is the toxin component chiefly responsible for pathogenesis and has been a popular target for rational and structure-based drug design. Although hundreds of small-molecule compounds have been designed to target the LF active site, relatively few reported inhibitors have exhibited activity in cell-based assays, and no LF inhibitor is currently available to treat or prevent anthrax. This study presents a new pharmacophore map assembly, validated by experiment, designed to rapidly identify and prioritize promising LF inhibitor scaffolds from virtual compound libraries. The new hypothesis incorporates structural information from all five available LF enzyme-inhibitor complexes deposited in the Protein Data Bank (PDB) and is the first LF pharmacophore map reported to date that includes features representing interactions involving all three key subsites of the LF catalytic binding region. In a wide-ranging validation study on all 546 compounds for which published LF biological activity data exist, this model displayed strong selectivity toward nanomolar-level LF inhibitors, successfully identifying 72.1% of existing nanomolar-level compounds in an unbiased test set, while rejecting 100% of weakly active (>100 μM) compounds. In addition to its capabilities as a database searching tool, this comprehensive model points to a number of key design principles and previously unidentified ligand-receptor interactions that are likely to influence compound potency. PMID:22697455

  18. Targeting Vascular Endothelial Growth Factor Receptor 2 and Protein Kinase D1 Related Pathways by a Multiple Kinase Inhibitor in Angiogenesis and Inflammation Related Processes In Vitro

    PubMed Central

    Varga, Attila; Gyulavári, Pál; Greff, Zoltán; Futosi, Krisztina; Németh, Tamás; Simon-Szabó, Laura; Kerekes, Krisztina; Szántai-Kis, Csaba; Brauswetter, Diána; Kokas, Márton; Borbély, Gábor; Erdei, Anna; Mócsai, Attila; Kéri, György; Vántus, Tibor

    2015-01-01

    Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways. PMID:25874616

  19. Dysinosin A: a novel inhibitor of Factor VIIa and thrombin from a new genus and species of Australian sponge of the family Dysideidae.

    PubMed

    Carroll, Anthony R; Pierens, Gregory K; Fechner, Greg; De Almeida Leone, Priscila; Ngo, Anna; Simpson, Moana; Hyde, Edward; Hooper, John N A; Boström, Stig-Lennart; Musil, Djordje; Quinn, Ronald J

    2002-11-13

    A new marine natural product dysinosin A 1 has been isolated from a new genus and species of sponge of the family Dysideidae found near Lizard Island, North Queensland, Australia. Dysinosin A is a potent inhibitor of the blood coagulation cascade factor VIIa and an inhibitor of the serine protease thrombin. Among the distinctive features of dysinosin A are the presence of a 5,6-dihydroxy-octahydroindole-2-carboxylic acid, 3-amino-ethyl 1-N-amidino-Delta-3-pyrroline, a sulfated glyceric acid, and d-leucine, assembled through three peptidic linkages. Dysinosin A inhibited factor VIIa at a Ki of 108 nM and thrombin at a Ki of 452 nM. The identification of the 1-N-amidino-Delta-3-pyrroline and 5,6-dihydroxy-octahydroindole-2-carboxylic acid as P1 and P2 moieties respectively, should pave the way for the design and synthesis of new structure-based inhibitors. PMID:12418859

  20. Rice xa13 Recessive Resistance to Bacterial Blight Is Defeated by Induction of the Disease Susceptibility Gene Os-11N3[W][OA

    PubMed Central

    Antony, Ginny; Zhou, Junhui; Huang, Sheng; Li, Ting; Liu, Bo; White, Frank; Yang, Bing

    2010-01-01

    The rice (Oryza sativa) gene xa13 is a recessive resistance allele of Os-8N3, a member of the NODULIN3 (N3) gene family, located on rice chromosome 8. Os-8N3 is a susceptibility (S) gene for Xanthomonas oryzae pv oryzae, the causal agent of bacterial blight, and the recessive allele is defeated by strains of the pathogen producing any one of the type III effectors AvrXa7, PthXo2, or PthXo3, which are all members of the transcription activator-like (TAL) effector family. Both AvrXa7 and PthXo3 induce the expression of a second member of the N3 gene family, here named Os-11N3. Insertional mutagenesis or RNA-mediated silencing of Os-11N3 resulted in plants with loss of susceptibility specifically to strains of X. oryzae pv oryzae dependent on AvrXa7 or PthXo3 for virulence. We further show that AvrXa7 drives expression of Os-11N3 and that AvrXa7 interacts and binds specifically to an effector binding element within the Os-11N3 promoter, lending support to the predictive models for TAL effector binding specificity. The result indicates that variations in the TAL effector repetitive domains are driven by selection to overcome both dominant and recessive forms of resistance to bacterial blight in rice. The finding that Os-8N3 and Os-11N3 encode closely related proteins also provides evidence that N3 proteins have a specific function in facilitating bacterial blight disease. PMID:21098734

  1. Tankyrase Inhibitor Sensitizes Lung Cancer Cells to Endothelial Growth Factor Receptor (EGFR) Inhibition via Stabilizing Angiomotins and Inhibiting YAP Signaling.

    PubMed

    Wang, Hui; Lu, Bo; Castillo, Johnny; Zhang, Yue; Yang, Zinger; McAllister, Gregory; Lindeman, Alicia; Reece-Hoyes, John; Tallarico, John; Russ, Carsten; Hoffman, Greg; Xu, Wenqing; Schirle, Markus; Cong, Feng

    2016-07-15

    YAP signaling pathway plays critical roles in tissue homeostasis, and aberrant activation of YAP signaling has been implicated in cancers. To identify tractable targets of YAP pathway, we have performed a pathway-based pooled CRISPR screen and identified tankyrase and its associated E3 ligase RNF146 as positive regulators of YAP signaling. Genetic ablation or pharmacological inhibition of tankyrase prominently suppresses YAP activity and YAP target gene expression. Using a proteomic approach, we have identified angiomotin family proteins, which are known negative regulators of YAP signaling, as novel tankyrase substrates. Inhibition of tankyrase or depletion of RNF146 stabilizes angiomotins. Angiomotins physically interact with tankyrase through a highly conserved motif at their N terminus, and mutation of this motif leads to their stabilization. Tankyrase inhibitor-induced stabilization of angiomotins reduces YAP nuclear translocation and decreases downstream YAP signaling. We have further shown that knock-out of YAP sensitizes non-small cell lung cancer to EGFR inhibitor Erlotinib. Tankyrase inhibitor, but not porcupine inhibitor, which blocks Wnt secretion, enhances growth inhibitory activity of Erlotinib. This activity is mediated by YAP inhibition and not Wnt/β-catenin inhibition. Our data suggest that tankyrase inhibition could serve as a novel strategy to suppress YAP signaling for combinatorial targeted therapy. PMID:27231341

  2. Concurrent Intervention With Exercises and Stabilized Tumor Necrosis Factor Inhibitor Therapy Reduced the Disease Activity in Patients With Ankylosing Spondylitis: A Meta-Analysis.

    PubMed

    Liang, Hui; Li, Wen-Rong; Zhang, Hua; Tian, Xu; Wei, Wei; Wang, Chun-Mei

    2015-12-01

    Since the use of tumor necrosis factor (TNF) inhibitor therapy is becoming wider, the effects of concurrent intervention with exercises and stabilized TNF inhibitors therapy in patients with ankylosing spondylitis (AS) are different. The study aimed to objectively evaluate whether concurrent intervention with exercises and stabilized TNF inhibitors can reduce the disease activity in patients with AS. A search from PubMed, Web of Science, EMBASE, and the Cochrane Library was electronically performed to collect studies which compared concurrent intervention with exercise and TNF inhibitor to conventional approach in terms of disease activity in patients with AS published from their inception to June 2015. Studies that measured the Bath Ankylosing Spondylitis Functional Index (BASFI), the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Bath Ankylosing Spondylitis Metrology Index (BASMI), and chest expansion as outcomes were included. Two independent investigators screened the identified articles, extracted the data, and assessed the methodological quality of the included studies. Quantitative analysis was performed with Review Manager (RevMan) software (version 5.3.0). A total of 5 studies comprising 221 participants were included in the study. Meta-analyses showed that concurrent intervention with exercises and stabilized TNF inhibitors therapy significantly reduced the BASMI scores (MD, -0.99; 95% CI, -1.61 to -0.38) and BASDAI scores (MD, -0.58; 95% CI, -1.10 to -0.06), but the BASFI scores (MD, -0.31; 95% CI, -0.76 to 0.15) was not reduced, and chest expansion (MD, 0.80; 95% CI, -0.18 to 1.78) was not increased. Concurrent intervention with exercises and stabilized TNF inhibitors therapy can reduce the disease activity in patients with AS. More randomized controlled trials (RCTs) with high-quality, large-scale, and appropriate follow-up are warranted to further establish the benefit of concurrent intervention with exercises and TNF inhibitors for

  3. Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas' disease vector: gene cloning, expression and characterization of the inhibitor.

    PubMed

    Campos, I T N; Amino, R; Sampaio, C A M; Auerswald, E A; Friedrich, T; Lemaire, H-G; Schenkman, S; Tanaka, A S

    2002-09-01

    This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively). PMID:12213235

  4. Neoadjuvant and adjuvant epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy for lung cancer

    PubMed Central

    Zhai, Haoran; Zhong, Wenzhao; Yang, Xuening

    2015-01-01

    The Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis and the meta-analysis of individual participant data reported by non-small cell lung cancer (NSCLC) Meta-analysis Collaborative Group in neo-adjuvant setting validated respectively that adjuvant and neoadjuvant chemotherapy would significantly improve overall survival (OS) and recurrence-free survival for resectable NSCLC. However, chemotherapy has reached a therapeutic plateau. It has been confirmed that epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeting therapy provides a dramatic response to patients with advanced EGFR-mutation positive NSCLC. Researchers have paid more attention to exploring applications of TKIs to early resectable NSCLCs. Several studies on adjuvant TKI treatment concluded its safety and feasibility. But there existed certain limitations of these studies as inference factors to interpret data accurately: the BR19 study recruited patients among which almost 52% had stage IB and only 15 (3.0%, 15/503) had been confirmed with EGFR-mutant type; retrospective studies performed at Memorial Sloan Kettering Cancer Center (MSKCC) selected EGFR mutant-type NSCLC patients but couldn’t avoid inherent defects inside retrospective researches; the RADIANT study revised endpoints from targeting at EGFR immunohistochemistry (IHC)+ and/or fluorescence in situ hybridization (FISH)+ mutation to only EGFR IHC+ mutation, leading to selective bias; despite that the SELECT study validated efficacy of adjuvant TKI and second round of TKI after resistance occurred, a single-arm clinical trial is not that persuasive in the absence of comparison with chemotherapy. Taking all these limitations into account, CTONG1104 in China and IMPACT in Japan have been conducted and recruiting patients to offer higher level of evidences to explore efficacy of preoperative TKI therapy for early resectable EGFR mutation positive NSCLC patients (confirmed by pathological results of tumor tissue or

  5. Inhibitors of Histone Deacetylase and DNA Methyltransferase Synergistically Activate the Methylated Metallothionein I Promoter by Activating the Transcription Factor MTF-1 and Forming an Open Chromatin Structure

    PubMed Central

    Ghoshal, Kalpana; Datta, Jharna; Majumder, Sarmila; Bai, Shoumei; Dong, Xiaocheng; Parthun, Mark; Jacob, Samson T.

    2002-01-01

    Inhibitors of DNA methyltransferase (Dnmt) and histone deacetylases (HDAC) synergistically activate the methylated metallothionein I gene (MT-I) promoter in mouse lymphosarcoma cells. The cooperative effect of these two classes of inhibitors on MT-I promoter activity was robust following demethylation of only a few CpG dinucleotides by brief exposure to 5-azacytidine (5-AzaC) but persisted even after prolonged treatment with the nucleoside analog. HDAC inhibitors (trichostatin A [TSA] and depsipeptide) either alone or in combination with 5-AzaC did not facilitate demethylation of the MT-I promoter. Treatment of cells with HDAC inhibitors increased accumulation of multiply acetylated forms of H3 and H4 histones that remained unaffected after treatment with 5-AzaC. Chromatin immunoprecipitation (ChIP) assay showed increased association of acetylated histone H4 and lysine 9 (K9)-acetyl H3 with the MT-I promoter after treatment with TSA, which was not affected following treatment with 5-AzaC. In contrast, the association of K9-methyl histone H3 with the MT-I promoter decreased significantly after treatment with 5-AzaC and TSA. ChIP assay with antibodies specific for methyl-CpG binding proteins (MBDs) demonstrated that only methyl-CpG binding protein 2 (MeCP2) was associated with the MT-I promoter, which was significantly enhanced after TSA treatment. Association of histone deacetylase 1 (HDAC1) with the promoter decreased after treatment with TSA or 5-AzaC and was abolished after treatment with both inhibitors. Among the DNA methyltransferases, both Dnmt1 and Dnmt3a were associated with the MT-I promoter in the lymphosarcoma cells, and association of Dnmt1 decreased with time after treatment with 5-AzaC. Treatment of these cells with HDAC inhibitors also increased expression of the MTF-1 (metal transcription factor-1) gene as well as its DNA binding activity. In vivo genomic footprinting studies demonstrated increased occupancy of MTF-1 to metal response elements of

  6. Orally bioavailable pyridine and pyrimidine-based Factor XIa inhibitors: Discovery of the methyl N-phenyl carbamate P2 prime group.

    PubMed

    Corte, James R; Fang, Tianan; Pinto, Donald J P; Orwat, Michael J; Rendina, Alan R; Luettgen, Joseph M; Rossi, Karen A; Wei, Anzhi; Ramamurthy, Vidhyashankar; Myers, Joseph E; Sheriff, Steven; Narayanan, Rangaraj; Harper, Timothy W; Zheng, Joanna J; Li, Yi-Xin; Seiffert, Dietmar A; Wexler, Ruth R; Quan, Mimi L

    2016-05-15

    Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1, and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2 prime group which maintained FXIa activity, reduced the number of H-bond donors, and improved the physicochemical properties compared to the amino indazole P2 prime moiety. Compound (S)-17 was identified as a potent and selective FXIa inhibitor that was orally bioavailable. Replacement of the basic cyclohexyl methyl amine P1 in (S)-17 with the neutral p-chlorophenyltetrazole P1 resulted in the discovery of (S)-24 which showed a significant improvement in oral bioavailability compared to the previously reported imidazole (S)-23. Additional improvements in FXIa binding affinity, while maintaining oral bioavailability, was achieved by replacing the pyridine scaffold with either a regioisomeric pyridine or pyrimidine ring system. PMID:27073051

  7. Case Report of Lichen Planopilaris Occurring in a Pediatric Patient Receiving a Tumor Necrosis Factor α Inhibitor and a Review of the Literature.

    PubMed

    Jayasekera, Prativa S A; Walsh, Maeve L; Hurrell, Daniel; Parslew, Richard A G

    2016-03-01

    A 12-year-old girl with extended oligoarthritis treated with adalimumab presented with a short history of a progressive cutaneous eruption involving the legs and scalp. Physical examination and histologic results were consistent with lichen planopilaris. The adalimumab was discontinued. She received treatment with topical clobetasol propionate and the majority of the lesions resolved. Residual lesions and the extended oligoarthritis were then treated with sulfasalazine. Adalimumab is a tumor necrosis factor α (TNF-α) inhibitor used for the treatment of a variety of immunologically mediated conditions, including lichen planus and lichen planopilaris. TNF-α antagonists have been associated with paradoxical psoriasiform, lichenoid, eczematous, granulomatous, and acneiform eruptions. We detail this case and review the literature of lichenoid reactions secondary to TNF-α inhibitors. PMID:26840781

  8. Development of Nano-Liposomal Formulations of Epidermal Growth Factor Receptor Inhibitors and their Pharmacological Interactions on Drug-Sensitive and Drug-Resistant Cancer Cell Lines

    NASA Astrophysics Data System (ADS)

    Trummer, Brian J.

    A rapidly expanding understanding of molecular derangements in cancer cell function has led to the development of selective, targeted chemotherapeutic agents. Growth factor signal transduction networks are frequently activated in an aberrant fashion, particularly through the activity of receptor tyrosine kinases (RTK). This has spurred an intensive effort to develop receptor tyrosine kinase inhibitors (RTKI) that are targeted to specific receptors, or receptor subfamilies. Chapter 1 reviews the pharmacology, preclinical, and clinical aspects of RTKIs that target the epidermal growth factor receptor (EGFR). EGFR inhibitors demonstrate significant success at inhibiting phosphorylation-based signaling pathways that promote cancer cell proliferation. Additionally RTKIs have physicochemical and structural characteristics that enable them to function as inhibitors of multi-drug resistance transport proteins. Thus EGFR inhibitors and other RTKIs have both on-target and off-target activities that could be beneficial in cancer therapy. However, these agents exert a number of side effects, some of which arise from their hydrophobic nature and large in vivo volume of distribution. Side effects of the EGFR inhibitor gefitinib include skin rash, severe myelotoxicity when combined with certain chemotherapeutic agents, and impairment of the blood brain barrier to xenobiotics. Weighing the preclinical and clinical observations with the EGFR inhibitors, we developed the primary overall hypothesis of this research: that drug-carrier formulations of RTKIs such as the EGFR inhibitors could be developed based on nanoparticulate liposomal carriers. Theoretically, this carrier strategy would ameliorate toxicity and improve the biodistribution and tumor selectivity of these agents. We hypothesized specifically that liposomal formulations could shift the biodistribution of EGFR inhibitors such as gefitinib away from skin, bone marrow, and the blood brain barrier, and toward solid tumors

  9. Indole-3-carbinol enhances the resolution of rat liver fibrosis and stimulates hepatic stellate cell apoptosis by blocking the inhibitor of κB kinase α/inhibitor of κB-α/nuclear factor-κB pathway.

    PubMed

    Ping, Jie; Gao, Ai-mei; Qin, Hai-quan; Wei, Xiao-ning; Bai, Jing; Liu, Lian; Li, Xiao-hai; Li, Rui-wen; Ao, Ying; Wang, Hui

    2011-11-01

    Hepatic stellate cells (HSC) play a pivotal role in liver fibrosis, and the clearance of activated HSC by apoptosis is associated with the resolution of liver fibrosis. The development of strategies that promote this process in a selective way is therefore important. We evaluated the effects of indole-3-carbinol (I3C), a nutritional component derived from vegetables from the Brassica family, on liver fibrosis and HSC apoptosis. The in vivo therapeutic effects of I3C were monitored in three rat models of liver fibrosis induced by porcine serum, bile duct ligation, or multiple hepatotoxic factors, and its proapoptotic effect and molecular mechanism were studied in vitro in HSC-T6, a rat HSC line. The results showed that I3C treatment significantly reduced the number of activated HSC in the livers of rats with liver fibrosis. In histopathology, I3C reduced hepatocyte degeneration and necrosis, accelerated collagen degradation, and promoted the reversal of liver fibrosis. I3C prescribed to HSC-T6 resulted in morphologic alterations typical of apoptosis and DNA cleavage to a nucleosomal ladder. Moreover, I3C significantly increased the HSC-T6 apoptosis rate and the expression ratio of Bax to Bcl-2. High-throughput protein array analysis indicated that the tumor necrosis factor-α/nuclear factor-κB (NF-κB) signal pathway participated in I3C-induced HSC-T6 apoptosis. Western blot and electrophoretic mobility-shift assay confirmed that I3C inhibited the phosphorylation of inhibitor of κB kinase α and inhibitor of κB-α and NF-κB DNA binding activity. In conclusion, I3C could promote the reverse process of liver fibrosis in vivo and induce apoptosis of activated HSC in vitro, which indicates the use of I3C as a potential therapeutic agent in liver fibrosis treatment. PMID:21862660

  10. Occurrence of platelet-activating factor (PAF) and an endogenous inhibitor of platelet aggregation in diffuse cutaneous mastocytosis.

    PubMed Central

    Macpherson, J L; Kemp, A; Rogers, M; Mallet, A I; Toia, R F; Spur, B; Earl, J W; Chesterman, C N; Krilis, S A

    1989-01-01

    We have identified PAF in the blister fluid from a patient with bullous mastocytosis, a rare form of mast-cell disease. We have found a novel endogenous inhibitor of platelet aggregation which obscured the presence of the PAF in unprocessed blister fluid and in ethanol or lipid extracts. The PAF was characterized by the demonstration of chromatographic, mass spectral and biological properties identical to those of authentic PAF. Thus this is the first demonstration of PAF in biological fluid from a patient with mastocytosis. High levels of immunoreactive prostaglandin D2 (PGD2) and histamine were also present in the blister fluid. The interaction between PAF and the inhibitor of platelet aggregation in patients with systemic mastocytosis may provide an explanation for some of the manifestations of the disease, in particular the episodic hypotension, cutaneous flushing and pallor. PMID:2805409

  11. Association between tumour necrosis factorinhibitors and risk of serious infections in people with inflammatory bowel disease: nationwide Danish cohort study

    PubMed Central

    Pasternak, Björn; Friis-Møller, Nina; Andersson, Mikael; Jess, Tine

    2015-01-01

    Objective To investigate whether people with inflammatory bowel disease treated with tumour necrosis factor-α (TNF-α) inhibitors are at increased risk of serious infections. Design Nationwide register based propensity score matched cohort study. Setting Denmark, 2002-12. Participants The background cohort eligible for matching comprised 52 392 people with inflammatory bowel disease, aged 15 to 75 years, of whom 4300 were treated with TNF-α inhibitors. To limit confounding, a two stage matching method was applied; firstly matching on age, sex, disease duration, and inflammatory bowel disease subtype, and secondly matching on propensity scores (1:1 ratio); this yielded 1543 people treated with TNF-α inhibitors and 1543 untreated to be included in the analyses. Main outcome measures The main outcome was any serious infection, defined as a diagnosis of infection associated with hospital admission. Cox regression was used to estimate hazard ratios for two risk periods (90 and 365 days after the start of TNF-α inhibitor treatment). Hazard ratios of site specific serious infections were obtained solely for the 365 days risk period. Results Within the 90 days risk period, 51 cases of infection were observed in users of TNF-α inhibitors (incidence rate 14/100 person years), compared with 33 cases in non-users (9/100 person years), yielding a hazard ratio of 1.63 (95% confidence interval 1.01 to 2.63). Within the risk period of 365 days, the hazard ratio was 1.27 (0.92 to 1.75). In analyses of site specific infections, the hazard ratio was above 2 for several of the subgroups but only reached statistical significance for skin and soft tissue infections (2.51, 1.23 to 5.12). Conclusions This nationwide propensity score matched cohort study suggests an increased risk of serious infections associated with use of TNF-α inhibitors within the first 90 days of starting treatment and a subsequent decline in risk. This calls for increased clinical awareness of potential

  12. Fas-Induced Apoptosis Increases Hepatocyte Tissue Factor Procoagulant Activity In Vitro and In Vivo

    PubMed Central

    Lopez, Michelle; Kopec, Anna K.; Joshi, Nikita; Geddings, Julia E.; Cline, Holly; Towery, Keara L.; Rockwell, Cheryl E.; Mackman, Nigel; Luyendyk, James P.

    2014-01-01

    Hepatocyte (HPC) apoptosis occurs in association with hepatotoxic responses and chronic liver disease, and is coupled to activation of the blood coagulation cascade. HPCs have been shown to express tissue factor (TF), the primary activator of blood coagulation, in a form that lacks procoagulant activity. In this study, we determined the effect of inducing HPC apoptosis on the procoagulant activity of TF. Treatment of primary mouse HPCs with the Fas death receptor agonist (anti-CD95 antibody, Jo2) triggered apoptosis as shown by cleavage of caspase-3, increased caspase-3 proteolytic activity, and cell surface exposure of phosphatidylserine (PS). Jo2-induced apoptosis significantly increased TF-dependent factor Xa generation by HPCs. Moreover, Jo2 treatment was associated with increased levels of microparticle-associated TF procoagulant activity in the culture medium. Pretreatment with a caspase-3 inhibitor significantly reduced Jo2-induced HPC TF activity and prevented the increase in microparticle-associated TF procoagulant activity. Application of the high-affinity PS-binding protein lactadherin inhibited TF-dependent factor Xa generation by Jo2-treated HPCs and dramatically reduced microparticle-associated TF procoagulant activity. Treatment of wild-type mice with a sublethal dose of Jo2 was associated with a robust increase in the activation of coagulation as measured by plasma thrombin-antithrombin (TAT) levels; whereas mice with liver-specific TF deficiency had significantly lower TAT levels. Overall, the results indicate that Fas-initiated, caspase-3-dependent HPC apoptosis increases TF procoagulant activity through a mechanism involving PS externalization. This suggests that activation of liver TF likely contributes to the procoagulant state associated with HPC apoptosis in liver toxicity and disease. PMID:25015658

  13. Programmed Application of Transforming Growth Factor β3 and Rac1 Inhibitor NSC23766 Committed Hyaline Cartilage Differentiation of Adipose-Derived Stem Cells for Osteochondral Defect Repair

    PubMed Central

    Zhu, Shouan; Chen, Pengfei; Wu, Yan; Xiong, Si; Sun, Heng; Xia, Qingqing; Shi, Libing

    2014-01-01

    Hyaline cartilage differentiation is always the challenge with application of stem cells for joint repair. Transforming growth factors (TGFs) and bone morphogenetic proteins can initiate cartilage differentiation but often lead to hypertrophy and calcification, related to abnormal Rac1 activity. In this study, we developed a strategy of programmed application of TGFβ3 and Rac1 inhibitor NSC23766 to commit the hyaline cartilage differentiation of adipose-derived stem cells (ADSCs) for joint cartilage repair. ADSCs were isolated and cultured in a micromass and pellet culture model to evaluate chondrogenic and hypertrophic differentiation. The function of Rac1 was investigated with constitutively active Rac1 mutant and dominant negative Rac1 mutant. The efficacy of ADSCs with programmed application of TGFβ3 and Rac1 inhibitor for cartilage repair was studied in a rat model of osteochondral defects. The results showed that TGFβ3 promoted ADSCs chondro-lineage differentiation and that NSC23766 prevented ADSC-derived chondrocytes from hypertrophy in vitro. The combination of ADSCs, TGFβ3, and NSC23766 promoted quality osteochondral defect repair in rats with much less chondrocytes hypertrophy and significantly higher International Cartilage Repair Society macroscopic and microscopic scores. The findings have illustrated that programmed application of TGFβ3 and Rac1 inhibitor NSC23766 can commit ADSCs to chondro-lineage differentiation and improve the efficacy of ADSCs for cartilage defect repair. These findings suggest a promising stem cell-based strategy for articular cartilage repair. PMID:25154784

  14. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

    PubMed

    Robert, Carine; Nagaria, Pratik K; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J; Cole, Philip A; Rassool, Feyruz V

    2016-06-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells. PMID:27064363

  15. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin

    PubMed Central

    Robert, Carine; Nagaria, Pratik K.; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J.; Cole, Philip A.; Rassool, Feyruz V.

    2016-01-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP “trapping”. Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP “trapping”, which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells. PMID:27064363

  16. HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry

    PubMed Central

    Zhou, Jing; Price, Amanda J.; Halambage, Upul D.; James, Leo C.

    2015-01-01

    ABSTRACT During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4+ T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution. IMPORTANCE The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages—cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid. PMID:26109731

  17. Clinical approaches to treat patients with non-small cell lung cancer and epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance.

    PubMed

    Tartarone, Alfredo; Lerose, Rosa

    2015-10-01

    The discovery of epidermal growth factor receptor activating mutations (EGFR Mut+) has determined a paradigm shift in the treatment of non-small cell lung cancer (NSCLC). In several phase III studies, patients with NSCLC EGFR Mut+ achieved a significantly better progression-free survival when treated with a first- (gefitinib, erlotinib) or second-generation (afatinib) EGFR tyrosine kinase inhibitor (TKI) compared with standard chemotherapy. However, despite these impressive results, most patients with NSCLC EGFR Mut+ develop acquired resistance to TKIs. This review will discuss both the mechanisms of resistance to TKIs and the therapeutic strategies to overcome resistance, including emerging data on third-generation TKIs. PMID:26016841

  18. Synergy of epidermal growth factor receptor kinase inhibitor AG1478 and ErbB2 kinase inhibitor AG879 in human colon carcinoma cells is associated with induction of apoptosis.

    PubMed

    Zhou, Yunfei; Brattain, Michael G

    2005-07-01

    Previous studies have shown that constitutive activation of epidermal growth factor receptor (EGFR) and ErbB2 by elevated autocrine transforming growth factor-alpha (TGF-alpha) expression plays an important role in colon cancer progression. Coexpression of EGFR and ErbB2 is found in a subset of colon cancers and may cooperatively promote cancer cell growth and survival, as heterodimerization is known to provide for diversification of signal transduction. In this study, the EGFR-selective tyrosine kinase inhibitor (TKI) AG1478 inhibited cell growth of an aggressive human colon carcinoma cell line, FET6alphaS26X, which harbors constitutively activated EGFR after stable transfection with TGF-alpha cDNA. However, AG1478 failed to induce apoptosis in FET6alphaS26X cells at concentrations sufficient for cell growth inhibition and complete suppression of EGFR phosphorylation. Similarly, AG879, a selective ErbB2 TKI, was incapable of inducing apoptosis in FET6alphaS26X cells at concentrations sufficient to inhibit cell growth and ErbB2 phosphorylation. To test the hypothesis that targeting both ErbB family members would show better efficacy than targeting the single receptors, combinations of inhibitors at fixed ratios of 1:1, 5:1, and 10:1 of AG1478 and AG879, respectively, were compared with single drugs for inhibition of cell growth. All combinations resulted in synergistic effects as indicated by combination index analysis. Synergistic inhibition was associated with induction of apoptosis as reflected by poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Annexin V staining. Finally, Western blot analysis showed significant inhibition of phosphorylation of both EGFR and ErbB2 by the combination treatment. These data suggest that the strategy to target both EGFR and ErbB2 simultaneously might result in more efficient inhibition of tumor growth than to target single receptor alone. PMID:15994962

  19. Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis.

    PubMed

    Wu, Dan; Gu, Qiuhong; Zhao, Ning; Xia, Fei; Li, Zhiwei

    2015-12-01

    The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a N-terminal carboxybenzyl group (chemically protective group) and a C-terminal hydroxamate moiety (coordinated to the Zn(2+) at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein-peptide affinity predictor and were then tested in vitro with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants Ki of 36 ± 5, 510 ± 46 and 320 ± 26 nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE-inhibitor complex

  20. An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy.

    PubMed

    Ohsawa, Yutaka; Okada, Tadashi; Nishimatsu, Shin-Ichiro; Ishizaki, Masatoshi; Suga, Tomohiro; Fujino, Masahiro; Murakami, Tatsufumi; Uchino, Makoto; Tsuchida, Kunihiro; Noji, Sumihare; Hinohara, Atsushi; Shimizu, Toshiyuki; Shimizu, Kiyoshi; Sunada, Yoshihide

    2012-08-01

    Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3(P104L)) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-β) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-β type I receptor (TβRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-β1, -β2, and -β3 signaling. Here, we show that a TβRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-β1, as well as CAV3(P104L). Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3(P104L) mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-β family members in muscle. These data indicate that both TGF-β-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TβRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-β signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings. PMID:22584670

  1. Assessing the likelihood of new-onset inflammatory bowel disease following tumor necrosis factor-alpha inhibitor therapy for rheumatoid arthritis and juvenile rheumatoid arthritis.

    PubMed

    Krishnan, Asha; Stobaugh, Derrick J; Deepak, Parakkal

    2015-04-01

    The association between inhibition of tumor necrosis factor-alpha (TNF-α) in patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) and the onset of inflammatory bowel disease (IBD) is unclear. We sought to evaluate this association by analyzing adverse events (AEs) reported to the Food and Drug Administration Adverse Event Reporting System (FAERS) with a standardized scoring tool for drug-induced AEs. A search of the FAERS for RA or JRA (January 2003-December 2011) reported with adalimumab, certolizumab pegol, etanercept, golimumab, or infliximab was performed. This dataset was then queried for cases indicating IBD. Full-length reports were accessed using the Freedom of Information Act and organized by age, sex, concomitant medications, co-morbidities, type of TNF-α inhibitor used, and diagnosis/treatment details. The Naranjo score was used to determine whether the drug-induced AEs were definite, probable, possible, or doubtful. There were 158 cases of IBD after TNF-α inhibitor exposure in RA or JRA patients. Use of the Naranjo score revealed that, in a majority of the cases (71.5 %), TNF-α inhibitor exposure was considered a 'possible' cause. A majority of the 'probable cases' in JRA were reported with etanercept (40 patients, 90.91 %). There were no 'definite' cases of anti-TNF-induced IBD. After applying the Naranjo scale, a weak association between new-onset IBD and TNF-α inhibitor therapy in RA patients and a moderately strong association especially with etanercept exposure in JRA patients was observed. However, causality cannot be determined due to limitations of the FAERS and the Naranjo score. PMID:25228459

  2. Activity of the fibroblast growth factor receptor inhibitors dovitinib (TKI258) and NVP-BGJ398 in human endometrial cancer cells.

    PubMed

    Konecny, Gottfried E; Kolarova, Teodora; O'Brien, Neil A; Winterhoff, Boris; Yang, Guorong; Qi, Jingwei; Qi, Zhengdong; Venkatesan, Natarajan; Ayala, Raul; Luo, Tong; Finn, Richard S; Kristof, Jessica; Galderisi, Chad; Porta, Diana Graus; Anderson, Lee; Shi, Michael M; Yovine, Alejandro; Slamon, Dennis J

    2013-05-01

    The recent identification of activating fibroblast growth factor receptor 2 (FGFR2) mutations in endometrial cancer has generated an opportunity for a novel target-based therapy. Here, we explore the therapeutic potential of 2 FGFR inhibitors, the multikinase inhibitor dovitinib (TKI258) and the more selective FGFR inhibitor NVP-BGJ398 for the treatment of endometrial cancer. We examined the effects of both inhibitors on tumor cell growth, FGFR2 signaling, cell cycle, and apoptosis using a panel of 20 molecularly characterized human endometrial cancer cell lines. Anchorage-independent growth was studied using soft agar assays. In vivo studies were conducted using endometrial cancer xenograft models. Cell lines with activating FGFR2 mutations (S252W, N550K) were more sensitive to dovitinib or NVP-BGJ398 when compared with their FGFR2 wild-type counterparts (P = 0.073 and P = 0.021, respectively). Both agents inhibited FGFR2 signaling, induced cell-cycle arrest, and significantly increased apoptosis in FGFR2-mutant lines. In vitro, dovitinib and NVP-BGJ398 were both potent at inhibiting cell growth of FGFR2-mutant endometrial cancer cells, but the activity of dovitinib was less restricted to FGFR2-mutant lines when compared with NVP-BGJ398. In vivo, dovitinib and NVP-BGJ398 significantly inhibited the growth of FGFR2-mutated endometrial cancer xenograft models. In addition, dovitinib showed significant antitumor activity in FGFR2 wild-type endometrial cancer xenograft models including complete tumor regressions in a long-term in vivo study. Dovitinib and NVP-BGJ398 warrant further clinical evaluation in patients with FGFR2-mutated endometrial cancer. Dovitinib may have antitumor activity in endometrial cancer beyond FGFR2-mutated cases and may permit greater flexibility in patient selection. PMID:23443805

  3. Tumor necrosis factor-α-induced apoptosis of gastric cancer MKN28 cells: accelerated degradation of the inhibitor of apoptosis family members.

    PubMed

    Kitagawa, Maki; Shiozaki, Atsushi; Ichikawa, Daisuke; Nakashima, Shingo; Kosuga, Toshiyuki; Konishi, Hirotaka; Komatsu, Shuhei; Fujiwara, Hitoshi; Okamoto, Kazuma; Otsuji, Eigo

    2015-01-15

    The role of the inhibitor of apoptosis (IAP) family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human gastric cancer MKN28 cells was explored. TNF-α induced up-regulation of cIAP2, whereas cycloheximide (CHX) induced down-regulation of XIAP and survivin. Degradation of cIAP1 and XIAP, but not survivin, was accelerated by co-treatment of cells with TNF-α and CHX, and TNF-α-induced up-regulation of cIAP2 was inhibited by BMS-345541 (NF-κB inhibitor). Treatment of MKN28 cells with TNF-α plus CHX induced degradation of survivin and activation of caspase-8 and -3, followed by degradation of cIAP1 and XIAP and apoptosis. Proteasome inhibitors (MG132 and epoxomicin) suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP as well as apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but allowed degradation of survivin, cIAP1 and XIAP. TNF-α receptor 1 and 2 were expressed on MKN28 cells. The magnitude of apoptosis induced by TNF-α plus BMS-345541 was much less than that induced by TNF-α plus CHX. These findings suggest that TNF-α plus CHX-induced apoptosis of gastric cancer MKN28 cells may be caused by accelerated degradation of the IAP family members (survivin, cIAP1, and XIAP), in addition to inhibition of NF-κB-dependent synthesis of anti-apoptotic molecules. PMID:25513960

  4. Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum.

    PubMed

    Tripathi, Jaindra N; Lorenzen, Jim; Bahar, Ofir; Ronald, Pamela; Tripathi, Leena

    2014-08-01

    Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern-recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21-mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar 'Gonja manjaya' (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa. PMID:24612254

  5. Transgenic expression of the rice Xa21 pattern recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

    PubMed Central

    Tripathi, Jaindra Nath; Lorenzen, Jim; Bahar, Ofir; Ronald, Pamela; Tripathi, Leena

    2014-01-01

    Summary Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, bio-control agents or resistant cultivars available to control BXW. Here we take advantage of the robust resistance conferred by the rice pattern recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21 mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar ‘Gonja manjaya’ (AAB) using a rapid bioassay and 12 transgenic plants in the glass house for resistance against Xcm. About fifty percent of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the non-transgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa. PMID:24612254

  6. Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5alpha under the control of the Bacillus BsXA promoter.

    PubMed

    Ruller, Roberto; Rosa, José César; Faça, Victor M; Greene, Lewis J; Ward, Richard J

    2006-01-01

    Xylanase A (XynA) is a class G/11 xylanase secreted by Bacillus subtilis. XynA was purified to homogeneity from B. subtilis strain 168 culture supernatants by ethanol precipitation and cation-exchange chromatography. The DNA fragment encoding the XynA together with the BsXA promoter region was amplified by PCR from B. subtilis 168 genomic DNA, and cloned into the plasmid pT7T3 to give the plasmid pT7BsXA. After transformation of Escherichia coli DH5alpha with pT7BsXA, a 19-fold increase in the levels of the secreted XynA was detected in the supernatant as compared with the B. subtilis culture. Correct post-translation modification of the recombinant protein was confirmed by N-terminal amino acid sequencing and MS analyses. The pH- and temperature-dependences of the native and recombinant proteins were identical, indicating that the pT7BsXA may be useful for the constitutive expression of heterologous protein in E. coli. PMID:15982188

  7. A partial cDNA clone of trypomastigote decay-accelerating factor (T-DAF), a developmentally regulated complement inhibitor of Trypanosoma cruzi, has genetic and functional similarities to the human complement inhibitor DAF.

    PubMed Central

    Tambourgi, D V; Kipnis, T L; da Silva, W D; Joiner, K A; Sher, A; Heath, S; Hall, B F; Ogden, G B

    1993-01-01

    Resistance to complement-mediated lysis in Trypanosoma cruzi is due to the expression of complement-regulatory factors by the virulent developmental forms of this protozoan parasite. An 87- to 93-kDa molecule, which we have termed T-DAF (trypomastigote decay-accelerating factor), is present on the surface of the parasite and inhibits complement activation in a manner functionally similar to the mammalian complement regulatory component, decay-accelerating factor. In this report, we characterized monospecific polyclonal and monoclonal antibodies which were obtained from mice and rabbits immunized with fast protein liquid chromatography-purified T-DAF. These polyclonal antibodies were shown to inhibit T-DAF activity and were capable of inducing lysis of the parasites. Both the polyclonal and monoclonal antibodies were used to screen a cDNA expression library prepared from T. cruzi trypomastigote mRNA. From this library, we obtained a partial lambda gt11 cDNA clone which showed genetic and functional similarity to the human C3 convertase inhibitor DAF (A. Nicholson-Weller, J. Burge, D. T. Fearon, P. F. Weller, and K. F. Austen, J. Immunol. 129:184-189, 1982). Images PMID:7689538

  8. Prospective Evaluation of Weight-Based Prophylactic Enoxaparin Dosing in Critically Ill Trauma Patients: Adequacy of AntiXa Levels Is Improved.

    PubMed

    Nunez, Jade M; Becher, Robert D; Rebo, Gerald J; Farrah, Jason P; Borgerding, Erika M; Stirparo, Joseph J; Lauer, Cynthia; Kilgo, Patrick; Miller, Preston R

    2015-06-01

    Venous thromboembolism (VTE) is a leading cause of death in multisystem trauma patients; the importance of VTE prevention is well recognized. Presently, standard dose enoxaparin (30 mg BID) is used as chemical prophylaxis, regardless of weight or physiologic status. However, evidence suggests decreased bioavailability of enoxaparin in critically ill patients. Therefore, we hypothesized that a weight-based enoxaparin dosing regimen would provide more adequate prophylaxis (as indicated by antifactor Xa levels) for patients in our trauma intensive care unit (TICU).These data were prospectively collected in TICU patients admitted over a 5-month period given twice daily 0.6 mg/kg enoxaparin (actual body weight). Patients were compared with a historical cohort receiving standard dosing. Anti-Xa levels were collected at 11.5 hours (trough, goal ≥ 0.1 IU/mL) after each evening administration. Patient demographics, admission weight, dose, and daily anti-Xa levels were recorded. Patients with renal insufficiency or brain, spine, or spinal cord injury were excluded. Data were collected from 26 patients in the standard-dose group and 37 in the weight-based group. Sixty-four trough anti-Xa measurements were taken in the standard dose group and 74 collected in the weight-based group. Evaluating only levels measured after the third dose, the change in dosing of enoxaparin from 30 to 0.6 mg/kg resulted in an increased percentage of patients with goal antifactor Xa levels from 8 per cent to 61 per cent (P < 0.0001). Examining all troughs, the change in dose resulted in an increase in patients with a goal anti-Xa level from 19 to 59 per cent (P < 0.0001). Weight-based dosing of enoxaparin in trauma ICU patients yields superior results with respect to adequate anti-Xa levels when compared with standard dosing. These findings suggest that weight-based dosing may provide superior VTE prophylaxis in TICU patients. Evaluation of the effects of this dosing paradigm on actual VTE rate is

  9. Impact damage resistance of carbon/epoxy composite tubes for the DC-XA liquid hydrogen feedline

    NASA Technical Reports Server (NTRS)

    Nettles, A. T.

    1995-01-01

    Low-velocity impacts were inflicted upon two elbow sections of carbon/epoxy feedline that are to be a part of the Delta Clipper-XA flight vehicle. A soap-based liquid leak detector solution was used to inspect the impact sites for leaks of pressurized gas that was pumped into the tube. Visual surface damage was noted and recorded for each impact site. After impact testing of each of the two sections of tubes was completed, the damage zones were disected from the tube and cross sectioned through the impact site. These specimens were polished after potting them in epoxy and were examined for microcracking using a fluorescent dye penetrant technique. The results showed that nonvisible damage could cause microcracking, thereby resulting in leaks through the tube wall.

  10. Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

    PubMed Central

    Ménard, Robert; Schoenhofen, Ian C.; Tao, Limei; Aubry, Annie; Bouchard, Patrice; Reid, Christopher W.; Lachance, Paule; Twine, Susan M.; Fulton, Kelly M.; Cui, Qizhi; Hogues, Hervé; Purisima, Enrico O.

    2014-01-01

    Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 μM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 μM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 μM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella. PMID:25267679

  11. Discovery of novel insulin-like growth factor-1 receptor inhibitors with unique time-dependent binding kinetics.

    PubMed

    Jin, Meizhong; Petronella, Brenda A; Cooke, Andy; Kadalbajoo, Mridula; Siu, Kam W; Kleinberg, Andrew; May, Earl W; Gokhale, Prafulla C; Schulz, Ryan; Kahler, Jennifer; Bittner, Mark A; Foreman, Kenneth; Pachter, Jonathan A; Wild, Robert; Epstein, David; Mulvihill, Mark J

    2013-07-11

    This letter describes a series of small molecule inhibitors of IGF-1R with unique time-dependent binding kinetics and slow off-rates. Structure-activity and structure-kinetic relationships were elucidated and guided further optimizations within the series, culminating in compound 2. With an IGF-1R dissociative half-life (t 1/2) of >100 h, compound 2 demonstrated significant and extended PD effects in conjunction with tumor growth inhibition in xenograft models at a remarkably low and intermittent dose, which correlated with the observed in vitro slow off-rate properties. PMID:24900721

  12. Tumor necrosis factor-alpha inhibitor-induced psoriasis or psoriasiform exanthemata: first 120 cases from the literature including a series of six new patients.

    PubMed

    Wollina, Uwe; Hansel, Gesina; Koch, André; Schönlebe, Jaqueline; Köstler, Erich; Haroske, Gunter

    2008-01-01

    Tumor necrosis factor-alpha (TNFalpha) inhibition is effective in the treatment of moderate-to-severe psoriasis. We report on 120 patients from the literature including six new patients (three women and three men) who developed pustular lesions during treatment with TNFalpha inhibitors. We identified 72 women and 36 men (several papers did not specify the gender of patients) with an age range of 13-78 years (mean 42.3 years). The primary diagnoses were rheumatoid arthritis (n = 61), ankylosing spondylitis (n = 21), psoriasis (n = 10), Crohn disease (n = 8), SAPHO (synovitis acne pustulosis hyperostosis osteitis) syndrome (n = 3), psoriatic arthritis (n = 2), and other diagnoses (n = 15). Psoriasis (except palmoplantar pustular type) was the most common adverse effect during anti-TNFalpha treatment (n = 73), followed by palmoplantar pustular psoriasis (n = 37) and psoriasis of the nail (n = 6), sometimes combined in the same patient. Palmoplantar pustulosis and psoriasiform exanthema was the diagnosis in ten patients each. A positive personal history of psoriasis was recorded in 25 patients. A positive family history was noted in eight patients. No data about personal (n = 7) or family history (n = 46) were available in a number of patients. Newly induced psoriasis was diagnosed in 74 patients whereas an exacerbation or aggravation of a pre-existing psoriasis was noted in another 25 patients. All three TNFalpha inhibitors available on the market were involved: infliximab (63 patients), etanercept (37 patients), and adalimumab (26 patients). Several patients were treated with more than a single TFNalpha inhibitor. The timing of cutaneous adverse effects (psoriasis and psoriasiform rash) varied considerably among patients, ranging from after a single application to a delayed response of up to 63 months after initiation of treatment. The mean time to appearance of the cutaneous adverse effect for all TNFalpha inhibitors was 9.5 months. Cessation of the responsible

  13. A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo.

    PubMed

    Olivero, Alan G; Eigenbrot, Charles; Goldsmith, Richard; Robarge, Kirk; Artis, Dean R; Flygare, John; Rawson, Thomas; Sutherlin, Daniel P; Kadkhodayan, Saloumeh; Beresini, Maureen; Elliott, Linda O; DeGuzman, Geralyn G; Banner, David W; Ultsch, Mark; Marzec, Ulla; Hanson, Stephen R; Refino, Canio; Bunting, Stuart; Kirchhofer, Daniel

    2005-03-11

    The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo. PMID:15632123

  14. Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination

    PubMed Central

    2010-01-01

    Background Activating Transcription Factor (ATF) 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC) inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor. Results The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) as well as chromatin immunoprecipitation (ChIP) assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. Conclusion This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity. PMID:20828393

  15. Microenvironment rigidity modulates responses to the HER2 receptor tyrosine kinase inhibitor lapatinib via YAP and TAZ transcription factors